Sample records for a2b antagonist mediate

  1. Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603.

    PubMed

    Goulding, Joelle; May, Lauren T; Hill, Stephen J

    2018-01-01

    Endogenous adenosine A 2B receptors (A 2B AR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A 2B AR antagonist PSB 603 in HEK 293 cells. The A 2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A 2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A 2A AR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E MAX with no significant effect on EC 50 values. Kinetics analysis of the effect of PSB 603 on the A 2B AR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A 2B AR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with boundmore » ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.« less

  3. Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

    PubMed Central

    Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea

    2017-01-01

    NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity. PMID:28760974

  4. AHR-16303B, a novel antagonist of 5-HT2 receptors and voltage-sensitive calcium channels

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Barrett, R.J.; Appell, K.C.; Kilpatrick, B.F.

    1991-01-01

    In vivo and in vitro methods were used to characterize AHR-16303B, a novel compound with antagonistic action at 5-HT2 receptors and voltage-sensitive calcium channels. The 5-HT2 receptor-antagonistic properties of AHR-16303B were demonstrated by inhibition of (a) (3H)ketanserin binding to rat cerebral cortical membranes (IC50 = 165 nM); (b) 5-hydroxytryptamine (5-HT)-induced foot edema in rats (minimum effective dose, (MED) = 0.32 mg/kg orally, p.o.); (c) 5-HT-induced vasopressor responses in spontaneously hypertensive rats (SHR) (ID50 = 0.18 mg/kg intravenously (i.v.), 1.8 mg/kg p.o.), (d) 5-HT-induced antidiuresis in rats (MED = 1 mg/kg p.o.), and (e) platelet aggregation induced by 5-HT + ADPmore » (IC50 = 1.5 mM). The calcium antagonist properties of AHR-16303B were demonstrated by inhibition of (a) (3H)nimodipine binding to voltage-sensitive calcium channels on rabbit skeletal muscle membranes (IC50 = 15 nM), (b) KCl-stimulated calcium flux into cultured PC12 cells (IC50 = 81 nM), and (c) CaCl2-induced contractions of rabbit thoracic aortic strips (pA2 = 8.84). AHR-16303B had little or no effect on binding of radioligands to dopamine2 (DA2) alpha 1, alpha 2, H1, 5-HT1 alpha, beta 2, muscarinic M1, or sigma opioid receptors; had no effect on 5-HT3 receptor-mediated vagal bradycardia; and had only minor negative inotropic, chronotropic, and dromotropic effects on isolated guinea pig atria. In conscious SHR, 30 mg/kg p.o. AHR-16303B completely prevented the vasopressor responses to i.v. 5-HT, and decreased blood pressure (BP) by 24% 3 h after dosing.« less

  5. Diadenosine polyphosphates as antagonists of the endogenous P2Y1 receptor in rat brain capillary endothelial cells of the B7 and B10 clones

    PubMed Central

    Vigne, Paul; Breittmayer, Jean Philippe; Frelin, Christian

    2000-01-01

    Diadenosine polyphosphates (ApnAs, n=2–7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of ApnAs in clones of rat brain capillary endothelial cells that express P2Y1 receptors (B10 cells) or both P2Y1 and P2Y2 receptors (B7 cells).B10 cells responded to Ap3A with rises in intracellular Ca2+ concentration ([Ca2+]i). This response was prevented by adenosine-3′-phosphate-5′-phosphate, an antagonist of P2Y1 receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP.ApnAs inhibited ADP induced increases in [Ca2+]i mediated by P2Y1 receptors by shifting ADP concentration-response curves to larger concentrations. Apparent Ki values were estimated to be 6 μM for Ap4A, 10 μM for Ap5A and 47 μM for Ap6A. Ap2A and Ap3A were much less active.ApnAs were neither agonists nor antagonists of the endogenous P2Y2 receptor in B7 cells.ApnAs are neither agonists nor antagonists of the Gi-coupled, ADP receptor in B10 cells.The results suggest that most actions of ApnAs in B7 and B10 cells can be accounted for by endogenous P2Y1 receptors. Ap4A, Ap5A and Ap6A are specific antagonists of endogenous Ca2+-coupled P2Y1 receptors. PMID:10742308

  6. Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

    PubMed

    Gobeil, F; Charland, S; Filteau, C; Perron, S I; Neugebauer, W; Regoli, D

    1999-03-01

    -To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective

  7. New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding

    NASA Astrophysics Data System (ADS)

    Lupala, Cecylia S.; Gomez-Gutierrez, Patricia; Perez, Juan J.

    2016-01-01

    Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor up-regulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure-activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new

  8. Neutralization of Staphylococcal Enterotoxin B by an Aptamer Antagonist

    PubMed Central

    Wang, Kaiyu; Gan, Longjie; Jiang, Li; Zhang, Xianhui; Yang, Xiangyue; Chen, Min

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a major virulence factor for staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T lymphocytic population, releasing proinflammatory cytokines. Blocking SEB-initiated toxicity may be an effective strategy for treating TSS. Using a process known as systematic evolution of ligands by exponential enrichment (SELEX), we identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd = 64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and cytokine secretion in human peripheral blood mononuclear cells. Moreover, a PEGylated aptamer antagonist significantly reduced mortality in a “double-hit” mouse model of SEB-induced TSS, established via sensitization with d-galactosamine followed by SEB challenge. Therefore, our novel aptamer antagonist may offer potential therapeutic efficacy against SEB-mediated TSS. PMID:25624325

  9. Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.

    PubMed

    Courtin, Noémie; Fotso, Aurélien Fotso; Fautrad, Pierre; Mas, Floriane; Alessi, Marie-Christine; Riteau, Béatrice

    2017-07-01

    Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Tachykinin-mediated respiratory effects in conscious guinea pigs: modulation by NK1 and NK2 receptor antagonists.

    PubMed

    Kudlacz, E M; Logan, D E; Shatzer, S A; Farrell, A M; Baugh, L E

    1993-09-07

    Tachykinins, in particular neurokinin A and substance P, produce a number of airway effects which may contribute to respiratory diseases such as asthma. We examined the ability of aerosolized substance P, neurokinin A or capsaicin to produce respiratory alterations in conscious guinea pigs using modified whole body plethysmography. Substance P-mediated dyspnea and significant respiratory events were inhibited by the NK1 receptor antagonist, CP-96,345. Neurokinin A-mediated respiratory effects were ablated by the NK2 receptor antagonists: MEN 10207, MDL 29,913 and SR 48,968, the latter being the most potent. The peptide-based antagonist, MEN 10207, produced respiratory effects itself suggesting partial agonist activity. The cyclic hexapeptide, MDL 29,913, relaxed airway smooth muscle via mechanisms other than tachykinin antagonism. NK2 but not NK1 receptor antagonists were able to delay the onset of capsaicin-induced dyspnea, although alone they did not usually (in approximately 10% of the animals) eliminate the response. However, when NK2 receptor antagonists were combined with CP-96,345, the incidence of dyspnea induced by capsaicin decreased significantly (40%) suggesting that both tachykinins contribute to dyspnea in this system.

  11. Attenuation of Persistent Experimental Pancreatitis Pain by a Bradykinin B2 Receptor Antagonist

    PubMed Central

    Chen, Qingmin; Vera-Portocarrero, Louis P.; Ossipov, Michael H.; Vardanyan, Marina; Lai, Josephine; Porreca, Frank

    2017-01-01

    Objective The role of bradykinin (BK) receptors in activating and sensitizing peripheral nociceptors is well known. Recently, we showed that spinal dynorphin was pronociceptive through direct or indirect BK receptor activation. Here, we explored the potential role of BK receptors in pain associated with persistent pancreatitis in rats. Methods Experimental pancreatitis and abdominal hypersensitivity were induced by intravenous administrations of dibutyltin dichloride (DBTC). [des-Arg9-Leu8]BK (B1 antagonist) and HOE 140 (B2 antagonist) were given by intraperitoneal or intrathecal injection. Dynorphin antiserum was given intrathecally. Reverse transcription–polymerase chain reaction was used to detect spinal mRNA for BK receptors. Results Dibutyltin dichloride–induced pancreatitis upregulated B1 and B2 mRNA in the thoracic dorsal root ganglion and B2, but not B1, in the pancreas. No changes in spinal B1 or B2 mRNA were observed. Intraperitoneal or intrathecal administration of HOE 140 dose dependently abolished DBTC-induced abdominal hypersensitivity, whereas [des-Arg9-Leu8]BK was without effect by either route of administration. Antiserum to dynorphin (intrathecal) abolished DBTC-induced hypersensitivity. Conclusions These results suggest that blockade of peripheral or spinal BK B2 receptors may be an effective approach for diminishing pain associated with pancreatitis. Moreover, it is suggested that spinal dynorphin may maintain pancreatitis pain through direct or indirect activation of BK B2 receptors in the spinal cord. PMID:20531238

  12. 3-(Fur-2-yl)-10-(2-phenylethyl)-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one, a novel adenosine receptor antagonist with A(2A)-mediated neuroprotective effects.

    PubMed

    Scatena, Alessia; Fornai, Francesco; Trincavelli, Maria Letizia; Taliani, Sabrina; Daniele, Simona; Pugliesi, Isabella; Cosconati, Sandro; Martini, Claudia; Da Settimo, Federico

    2011-09-21

    In this study, compound FTBI (3-(2-furyl)-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one) was selected from a small library of triazinobenzimidazole derivatives as a potent A(2A) adenosine receptor (AR) antagonist and tested for its neuroprotective effects against two different kinds of dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and methamphetamine (METH), in rat PC12 and in human neuroblastoma SH-SY5Y cell lines. FTBI, in a concentration range corresponding to its affinity for A(2A) AR subtype, significantly increased the number of viable PC12 cells after their exposure to METH and, to a similar extent, to MPP+, as demonstrated in both trypan blue exclusion assay and in cytological staining. These neuroprotective effects were also observed with a classical A(2A) AR antagonist, ZM241385, and appeared to be completely counteracted by the AR agonist, NECA, supporting A(2A) ARs are directly involved in FTBI-mediated effects. Similarly, in human SH-SY5Y cells, FTBI was able to prevent cell toxicity induced by MPP+ and METH, showing that this A(2A) AR antagonist has a neuroprotective effect independently by the specific cell model. Altogether these results demonstrate that the A(2A) AR blockade mediates cell protection against neurotoxicity induced by dopaminergic neurotoxins in dopamine containing cells, supporting the potential use of A(2A) AR antagonists in dopaminergic degenerative diseases including Parkinson's disease.

  13. Ranakinestatin-PPF from the skin secretion of the Fukien gold-striped pond frog, Pelophylax plancyi fukienensis: a prototype of a novel class of bradykinin B2 receptor antagonist peptide from ranid frogs.

    PubMed

    Ma, Jie; Luo, Yu; Ge, Lilin; Wang, Lei; Zhou, Mei; Zhang, Yingqi; Duan, Jinao; Chen, Tianbao; Shaw, Chris

    2014-01-01

    The defensive skin secretions of many amphibians are a rich source of bradykinins and bradykinin-related peptides (BRPs). Members of this peptide group are also common components of reptile and arthropod venoms due to their multiple biological functions that include induction of pain, effects on many smooth muscle types, and lowering systemic blood pressure. While most BRPs are bradykinin receptor agonists, some have curiously been found to be exquisite antagonists, such as the maximakinin gene-related peptide, kinestatin-a specific bradykinin B2-receptor antagonist from the skin of the giant fire-bellied toad, Bombina maxima. Here, we describe the identification, structural and functional characterization of a heptadecapeptide (DYTIRTRLHQGLSRKIV), named ranakinestatin-PPF, from the skin of the Chinese ranid frog, Pelophylax plancyi fukienensis, representing a prototype of a novel class of bradykinin B2-receptor specific antagonist. Using a preconstricted preparation of rat tail arterial smooth muscle, a single dose of 10(-6)M of the peptide effectively inhibited the dose-dependent relaxation effect of bradykinin between 10(-11)M and 10(-5)M and subsequently, this effect was pharmacologically-characterized using specific bradykinin B1- (desArg-HOE140) and B2-receptor (HOE140) antagonists; the data from which demonstrated that the antagonism of the novel peptide was mediated through B2-receptors. Ranakinestatin-PPF-thus represents a prototype of an amphibian skin peptide family that functions as a bradykinin B2-receptor antagonist herein demonstrated using mammalian vascular smooth muscle.

  14. Adenosine A(2B) receptor antagonist PSB603 suppresses tumor growth and metastasis by inhibiting induction of regulatory T cells.

    PubMed

    Kaji, Wakako; Tanaka, Satomi; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2014-04-01

    Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A(2B) receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A(2B) receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.

  15. Antagonistic effects of acetylshikonin on LPS-induced NO and PGE2 production in BV2 microglial cells via inhibition of ROS/PI3K/Akt-mediated NF-κB signaling and activation of Nrf2-dependent HO-1.

    PubMed

    Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Lee, Kyoung-Tae; Choi, Yung Hyun; Moon, Sung-Kwon; Kim, Wun-Jae; Kim, Gi-Young

    2015-10-01

    Although acetylshikonin (ACS) is known to have antioxidant and antitumor activities, whether ACS regulates the expression of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated microglial cells remains unclear. In this study, it was found that ACS isolated from Lithospermum erythrorhizon inhibits LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) release by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 microglial cells. Furthermore, ACS reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) and subsequently suppressed iNOS and COX-2 expression. Consistent with these data, ACS attenuated the phosphorylation of PI3K and Akt and suppressed the DNA-binding activity of NF-κB by inducing the generation of reactive oxygen species (ROS) in LPS-stimulated cells. In addition, ACS enhanced heme oxygenase-1 (HO-1) expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation. Zinc protoporphyrin, a specific HO-1 inhibitor, partially attenuated the antagonistic effects of ACS on LPS-induced NO and PGE2 production. By contrast, the presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO and PGE2 production. These data indicate that ACS downregulates proinflammatory mediators such as NO and PGE2 by suppressing PI3K/Akt-dependent NF-κB activity induced by ROS as well as inducing Nrf2-dependent HO-1 activity. Taken together, ACS might be a good candidate to regulate LPS-mediated inflammatory diseases.

  16. Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.

    PubMed

    Li, M; Marubayashi, A; Nakaya, Y; Fukui, K; Arase, S

    2001-12-01

    The mechanism by which minoxidil, an adenosine-triphosphate-sensitive potassium channel opener, induces hypertrichosis remains to be elucidated. Minoxidil has been reported to stimulate the production of vascular endothelial growth factor, a possible promoter of hair growth, in cultured dermal papilla cells. The mechanism of production of vascular endothelial growth factor remains unclear, however. We hypothesize that adenosine serves as a mediator of vascular endothelial growth factor production. Minoxidil-induced increases in levels of intracellular Ca(2+) and vascular endothelial growth factor production in cultured dermal papilla cells were found to be inhibited by 8-sulfophenyl theophylline, a specific antagonist for adenosine receptors, suggesting that dermal papilla cells possess adenosine receptors and sulfonylurea receptors, the latter of which is a well-known target receptor for adenosine-triphosphate-sensitive potassium channel openers. The expression of sulfonylurea receptor 2B and of the adenosine A1, A2A, and A2B receptors was detected in dermal papilla cells by means of reverse transcription polymerase chain reaction analysis. In order to determine which of the adenosine receptor subtypes contribute to minoxidil-induced hair growth, the effects of subtype-specific antagonists for adenosine receptors were investigated. Significant inhibition in increase in intracellular calcium level by minoxidil or adenosine was observed as the result of pretreatment with 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptor, but not by 3,7-dimethyl-1-propargyl-xanthine, an antagonist for adenosine A2 receptor, whereas vascular endothelial growth factor production was blocked by both adenosine A1 and A2 receptor antagonists. These results indicate that the effect of minoxidil is mediated by adenosine, which triggers intracellular signal transduction via both adenosine A1 and A2 receptors, and that the expression of sulfonylurea receptor 2B in

  17. Synthesis and Evaluation of Phenylxanthine Derivatives as Potential Dual A2AR Antagonists/MAO-B Inhibitors for Parkinson's Disease.

    PubMed

    Wang, Xuebao; Han, Chao; Xu, Yong; Wu, Kaiqi; Chen, Shuangya; Hu, Mangsha; Wang, Luyao; Ye, Yun; Ye, Faqing

    2017-06-17

    The aim of this research was to prove the speculation that phenylxanthine (PX) derivatives possess adenosine A2A receptor (A2AR)-blocking properties and to screening and evaluate these PX derivatives as dual A2AR antagonists/MAO-B inhibitors for Parkinson's disease. To explore this hypothesis, two series of PX derivatives were prepared and their antagonism against A2AR and inhibition against MAO-B were determined in vitro. In order to evaluate further the antiparkinsonian properties, pharmacokinetic and haloperidol-induced catalepsy experiments were carried out in vivo. The PX-D and PX-E analogues acted as potent A2AR antagonists with Ki values ranging from 0.27 to 10 μM, and these analogues displayed relatively mild MAO-B inhibition potencies, with inhibitor dissociation constants (Ki values) ranging from 0.25 to 10 μM. Further, the compounds PX-D-P6 and PX-E-P8 displayed efficacious antiparkinsonian properties in haloperidol-induced catalepsy experiments, verifying that these two compounds were potent A2AR antagonists and MAO-B inhibitors. We conclude that PX-D and PX-E analogues are a promising candidate class of dual-acting compounds for treating Parkinson's disease.

  18. Adenosine production by human B cells and B cell–mediated suppression of activated T cells

    PubMed Central

    Saze, Zenichiro; Schuler, Patrick J.; Hong, Chang-Sook; Cheng, Dongmei; Jackson, Edwin K.

    2013-01-01

    Antibody-independent role of B cells in modulating T-cell responses is incompletely understood. Freshly isolated or cultured B cells isolated from the peripheral blood of 30 normal donors were evaluated for CD39 and CD73 coexpression, the ability to produce adenosine 5′-monophosphate (AMP) and adenosine (ADO) in the presence of exogenous adenosine triphosphate (ATP) as well as A1, A2A, A2B, and A3 adenosine receptor (ADOR) expression. Human circulating B cells coexpress ectonucleotidases CD39 and CD73, hydrolyze exogenous ATP to 5′-AMP and ADO, and express messenger RNA for A1R, A2AR, and A3R. 2-chloroadenosine inhibited B-cell proliferation and cytokine expression, and only A3R selective antagonist restored B-cell functions. This suggested that B cells use the A3R for autocrine signaling and self-regulation. Mediated effects on B-cell growth ± ADOR antagonists or agonists were tested in carboxyfluorescein diacetate succinimidyl ester assays. In cocultures, resting B cells upregulated functions of CD4+ and CD8+ T cells. However, in vitro–activated B cells downregulated CD73 expression, mainly produced 5′-AMP, and inhibited T-cell proliferation and cytokine production. These B cells acquire the ability to restrict potentially harmful effects of activated T cells. Thus, B cells emerge as a key regulatory component of T cell–B cell interactions, and their dual regulatory activity is mediated by the products of ATP hydrolysis, 5′-AMP, and ADO. PMID:23678003

  19. Non-selectivity of new bradykinin antagonists for B1 receptors.

    PubMed

    Rhaleb, N E; Gobeil, F; Regoli, D

    1992-01-01

    Two new B1 receptor antagonists, [Hyp3,Thi5,DTic7,Oic8]desArg9-BK and DArg[Hyp3,Thi5,DTic7,Oic8]desArg9-BK were tested in vitro on the rabbit jugular vein and the guinea pig ileum (preparations containing B2 receptors) and on the rabbit aorta (preparation containing B1 receptors) for pharmacological characterization. The results indicate that both compounds are antagonists on both B1 and B2 receptors, are competitive and discriminate between B2A and B2B receptor subtypes.

  20. MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B2 receptor. In vitro pharmacology and molecular characterization.

    PubMed

    Cucchi, Paola; Meini, Stefania; Bressan, Alessandro; Catalani, Claudio; Bellucci, Francesca; Santicioli, Paolo; Lecci, Alessandro; Faiella, Angela; Rotondaro, Luigi; Giuliani, Sandro; Giolitti, Alessandro; Quartara, Laura; Maggi, Carlo Alberto

    2005-12-28

    The pharmacological characterization of the novel nonpeptide antagonist for the B2 receptor, namely MEN16132 (4-(S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B2 receptor has been investigated by means of competition studies at [3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B2 receptor (pKi 10.5), human lung fibroblasts (pKi 10.5), guinea pig airways (pKi 10.0), guinea pig ileum longitudinal smooth muscle (pKi 10.2), or guinea pig cultured colonic myocytes (pKi 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B2 receptor was checked at the human bradykinin B1 receptor (pKi<5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pKB 10.3) and guinea pig (colonic myocytes: pKB 10.3) B2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pKB 9.9) and guinea pig (ileum longitudinal smooth muscle: pKB 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but

  1. Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

    PubMed

    Maggi, C A; Patacchini, R; Giuliani, S; Rovero, P; Dion, S; Regoli, D; Giachetti, A; Meli, A

    1990-07-01

    1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA. Similar pA2 values were obtained after 15 or 90min of incubation with the antagonists. Peptides I, II and III had no inhibitory effect on contractions produced by noradrenaline in the RPA or by carbachol in the HT. 5. Peptides I, II and III showed weak or no antagonistic activity toward the vasodilatator effect of substance P in the dog carotid artery (NK, receptor-mediated

  2. The H(2)-receptor antagonist ranitidine interferes with clopidogrel-mediated P2Y(12) inhibition in platelets.

    PubMed

    Schäfer, Andreas; Flierl, Ulrike; Pförtsch, Stephanie; Seydelmann, Nora; Micka, Jan; Bauersachs, Johann

    2010-10-01

    Use of proton-pump inhibitors (PPIs) is common in patients on dual antiplatelet therapy (DAT). Recent warnings about potential interactions of PPIs with clopidogrel metabolism leading to impaired DAT efficacy has prompted the recommendation of substituting PPIs with H(2)-receptor antagonists such as ranitidine. We investigated whether ranitidine interacts with P2Y(12) inhibition on the platelet level. Blood was collected from 15 patients with stable coronary artery disease, who had undergone elective coronary intervention. Clopidogrel responsiveness was assessed 24h after the administration of a 600mg loading dose using the P2Y(12) specific platelet-reactivity-index (PRI) and light-transmittance aggregometry in the presence and absence of a pharmacologically relevant concentration of the H(2)-receptor antagonist ranitidine (400ng/ml). Adding ranitidine enhanced P2Y(12)-mediated platelet reactivity to ADP assessed by the PRI (mean PRI+/-SEM: before ranitidine 28+/-5%; after ranitidine 37+/-5%, p=0.0025). Similarly, prostaglandin E1 (PGE(1))-mediated inhibition of ADP-induced aggregation was abrogated in the presence of ranitidine (Agg(max)+/-SEM: before PGE(1) 41+/-2%; after PGE(1) 29+/-2%, p<0.01 vs. before PGE(1); after PGE(1)+ranitidine 42+/-2%, p<0.01 vs. after PGE(1)). Exposition of platelets with ranitidine significantly enhanced their responsiveness to ADP and contributed to impaired P2Y(12) inhibition suggesting that ranitidine interacts with clopidogrel efficacy through adenylyl cyclase inhibition on the platelet level. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. Enhanced attention and impulsive action following NMDA receptor GluN2B-selective antagonist pretreatment.

    PubMed

    Higgins, Guy A; Silenieks, Leo B; MacMillan, Cam; Sevo, Julia; Zeeb, Fiona D; Thevarkunnel, Sandy

    2016-09-15

    NMDA GluN2B (NR2B) subtype selective antagonists are currently in clinical development for a variety of indications, including major depression. We previously reported the selective NMDA GluN2B antagonists Ro 63-1908 and traxoprodil, increase premature responding in a 5-choice serial reaction time task (5-CSRTT) suggesting an effect on impulsive action. The present studies extend these investigations to a Go-NoGo and delay discounting task, and the 5-CSRTT under test conditions of both regular (5s) and short (2-5s) multiple ITI (Intertrial interval). Dizocilpine was included for comparison. Both Ro 63-1908 (0.1-1mg/kg SC) and traxoprodil (0.3-3mg/kg SC) increased premature and perseverative responses in both 5-CSRT tasks and improved attention when tested under a short ITI test condition. Ro 63-1908 but not traxoprodil increased motor impulsivity (false alarms) in a Go-NoGo task. Dizocilpine (0.01-0.06mg/kg SC) affected both measures of motor impulsivity and marginally improved attention. In a delay discounting test of impulsive choice, both dizocilpine and Ro 63-1908 decreased impulsive choice (increased choice for the larger, delayed reward), while traxoprodil showed a similar trend. Motor stimulant effects were evident following Ro 63-1908, but not traxoprodil treatment - although no signs of motor stereotypy characteristic of dizocilpine (>0.1mg/kg) were noted. The findings of both NMDA GluN2B antagonists affecting measures of impulsive action and compulsive behavior may underpin emerging evidence to suggest glutamate signaling through the NMDA GluN2B receptor plays an important role in behavioural flexibility. The profiles between Ro 63-1908 and traxoprodil were not identical, perhaps suggesting differences between members of this drug class. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. 2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABA A-ρ1 Receptors

    DOE PAGES

    Xie, A.; Yan, J.; Yue, L.; ...

    2011-08-02

    All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABA(C)R (GABA(A)-rho), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABA(C)R versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABA(C)R(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABA(C)R antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABA(A)R antagonist, SR95531; GABA(B)R antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brownmore » Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABA(C)R in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABA(C)R(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABA(C)R(-/-) mice. Blockade of GABA(A)Rs and GABA(B)Rs, or agonism of GABA(B)Rs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABA(C)R(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABA(B) agonist properties, and further increased by baclofen. The finding that genetic deletion of GABA(C)R, the GABA(C)R antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for CABA(C)R in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABA(C)R antagonists

  5. CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice

    PubMed Central

    Sullivan, Timothy; Miao, Zhenhua; Dairaghi, Daniel J.; Krasinski, Antoni; Wang, Yu; Zhao, Bin N.; Baumgart, Trageen; Ertl, Linda S.; Pennell, Andrew; Seitz, Lisa; Powers, Jay; Zhao, Ruiping; Ungashe, Solomon; Wei, Zheng; Boring, Landin; Tsou, Chia-Lin; Charo, Israel; Schall, Thomas J.; Jaen, Juan C.

    2013-01-01

    Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function. PMID:23986513

  6. The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

    PubMed

    Mølck, Christina; Ryall, James; Failla, Laura M; Coates, Janine L; Pascussi, Jean-Marc; Heath, Joan K; Stewart, Gregory; Hollande, Frédéric

    2016-12-01

    Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A 2b adenosine receptor (A 2b -AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A 2b -AR to the behavior of colorectal cancer cells. The A 2b -AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A 2b -AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. The role of serotonin-2 (5-HT2) and dopamine receptors in the behavioral actions of the 5-HT2A/2C agonist, DOI, and putative 5-HT2C inverse agonist, SR46349B.

    PubMed

    Scarlota, Laura C; Harvey, John A; Aloyo, Vincent J

    2011-02-01

    Atypical antipsychotic efficacy is often attributed to actions at serotonin-2 (5-HT(2)) and dopamine receptors, indicating a potential benefit of understanding the interplay between these systems. Currently, it is known that 5-HT(2) receptors modulate dopamine release, although the role of specific dopamine receptors in 5-HT(2)-mediated behavior is not well understood. We examined the role of 5-HT(2A), 5-HT(2C), and dopamine (D1 and D2) receptors in the behavioral response to a 5-HT(2A/2C) agonist (DOI) and 5-HT(2A/2C) antagonist (SR46349B). Effects were assessed by measuring rabbit head bobs (previously characterized as 5-HT(2A) receptor-mediated) and body shakes (5-HT(2C)-mediated). As expected, DOI produced head bobs and body shakes, and these DOI-elicited behaviors were attenuated by the SR46349B pretreatment. Unexpectedly, SR46349B also induced head bobs when administered alone. However, SR46349B-elicited head bobs are distinguishable from those produced by DOI since the 5-HT(2A) antagonist, ketanserin, only attenuated DOI-elicited head bobs. Conversely, 5-HT(2C) ligands (SB242084 and SB206553) inhibited SR46349B but not DOI-induced head bobs. Furthermore, when administered alone, SB206553 (a 5-HT(2C) inverse agonist) produced head bobs, indicating the behavior can be either 5-HT(2A) or 5-HT(2C) mediated. Next, it was revealed that D1 and D2 receptors play a role in DOI-elicited head bobs, but only D1 receptors are required for SR46349B-elicited head bobs. 5-HT(2A) receptor agonism and 5-HT(2C) inverse agonism produce the same behavior, likely due to similar downstream actions at D1 receptors. Consequently, 5-HT(2C) agonism or D1 agonism may be effective therapies for disorders, such as schizophrenia, currently being treated with 5-HT(2A) antagonists.

  8. Screening of environmental contaminants for ecdysteroid agonist and antagonist activity using the Drosophila melanogaster B(II) cell in vitro assay.

    PubMed

    Dinan, L; Bourne, P; Whiting, P; Dhadialla, T S; Hutchinson, T H

    2001-09-01

    The B(II) bioassay was developed as a rapid and reliable tool for detecting potential insect growth regulators acting as ecdysteroid receptor (ant)agonists. Based on an ecdysteroid-responsive cell line from Drosophila melanogaster, this microplate assay is ideally suited to the evaluation of environmental contaminants as potential endocrine disrupters. Data are presented for about 80 potential environmental contaminants, including industrial chemicals, pesticides, pharmaceuticals, phytoestrogens, and vertebrate steroids, and are compared with data for known (ant)agonists. Apart from androst-4-ene-3,17-dione (a weak antagonist), vertebrate steroids were inactive at concentrations up to 10(-3) M. The vast majority of xenobiotics also showed no (ant)agonist activity. Among the industrial chemicals, antagonistic activity was observed for bisphenol A median effective concentration (EC50) of 1.0 x 10(-4) M and diethylphthalate (EC50 of 2.0 x 10(-3) M). Some organochlorine compounds also showed weak antagonistic activity, including o,p'-dichlorodiphenyldichloroethylene (DDE), p,p'-DDE, dieldrin, and lindane (EC50 of 3.0 x 10(-5) M). For lindane, bisphenol A, and diethylphthalate, activity is not associated with impurities in the samples and, for lindane and bisphenol A at least, the compounds are able to compete with ecdysteroids for the ligand binding site on the receptor complex, albeit at concentrations very much higher than those found in the environment. The only pharmaceutical showing any detectable antagonist activity was 17alpha-ethynylestradiol. In the context of recent publications on potential endocrine disruption in marine and freshwater arthropods, these findings suggest that, for some compounds (e.g., diethylstilbestrol), ecdysteroid receptor-mediated responses are unlikely to be involved in producing chronic effects. The B(II) assay has a potentially valuable role to play in distinguishing between endocrine-mediated, which normally occur at submicromolar

  9. General, kappa, delta and mu opioid receptor antagonists mediate feeding elicited by the GABA-B agonist baclofen in the ventral tegmental area and nucleus accumbens shell in rats: reciprocal and regional interactions.

    PubMed

    Miner, Patricia; Shimonova, Lyudmila; Khaimov, Arthur; Borukhova, Yaffa; Ilyayeva, Ester; Ranaldi, Robert; Bodnar, Richard J

    2012-03-14

    administration in the VTA or NACs was also preceded by administration of NTX (0.1, 1, 5 μg, 0.5 h), BFNA (0.4, 4 μg, 24 h), NBNI (0.6, 6 μg, 0.5 h) or NTI (0.4, 4 μg, 0.5 h) into the other site with intake measured 1, 2 and 4 h after agonist treatment. VTA NTX significantly reduced NACs baclofen-induced feeding. Correspondingly, NACs NTX significantly reduced VTA baclofen-induced feeding, indicating a robust and bidirectional general opioid and GABA-B receptor feeding interaction. Whereas the high, but not low VTA BFNA dose reduced NACs baclofen-induced feeding, NACs BFNA failed to affect VTA baclofen-induced feeding, indicating a unidirectional mu opioid and GABA-B receptor feeding interaction. Whereas VTA NBNI at both doses reduced NACs baclofen-induced feeding, the high, but not low NACs NBNI dose significantly reduced VTA baclofen-induced feeding, indicating a bidirectional kappa opioid and GABA-B receptor feeding interaction. Whereas VTA NTI only transiently reduced NACs baclofen-induced feeding, NACs NTI failed to affect VTA baclofen-induced feeding, indicating a weak unidirectional delta opioid and GABA-B receptor interaction. Whereas administration of NTX or BFNA into the NACs or VTA marginally reduced spontaneous food intake, NBNI or NTI into the same sites failed to alter food intake alone. Therefore, the present study suggests that GABA employs a distributed brain network in mediating its ingestive effects that is dependent upon intact opioid receptor signaling with kappa opioid receptors more involved than mu and delta opioid receptors underlying these regional effects. An alternative hypothesis to be considered is that these effects could be the sum of two independent drug effects (opioid antagonists decreasing and baclofen increasing food intake). Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Signaling via the transcriptionally regulated activin receptor 2B is a novel mediator of neuronal cell death during chicken ciliary ganglion development.

    PubMed

    Koszinowski, S; Buss, K; Kaehlcke, K; Krieglstein, K

    2015-04-01

    The TGF-β ligand superfamily members activin A and BMP control important aspects of embryonic neuronal development and differentiation. Both are known to bind to activin receptor subtypes IIA (ActRIIA) and IIB, while in the avian ciliary ganglion (CG), so far only ActRIIA-expression has been described. We show that the expression of ACVR2B, coding for the ActRIIB, is tightly regulated during CG development and the knockdown of ACVR2B expression leads to a deregulation in the execution of neuronal apoptosis and therefore affects ontogenetic programmed cell death in vivo. While the differentiation of choroid neurons was impeded in the knockdown, pointing toward a reduction in activin A-mediated neural differentiation signaling, naturally occurring neuronal cell death in the CG was not prevented by follistatin treatment. Systemic injections of the BMP antagonist noggin, on the other hand, reduced the number of apoptotic neurons to a similar extent as ACVR2B knockdown. We therefore propose a novel pathway in the regulation of CG neuron ontogenetic programmed cell death, which could be mediated by BMP and signals via the ActRIIB. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Effect of a kinin B2 receptor antagonist on LPS- and cytokine-induced neutrophil migration in rats

    PubMed Central

    Santos, Danielle R; Calixto, João B; Souza, Glória E P

    2003-01-01

    This study examines the involvement of kinins in neutrophil migration into rat subcutaneous air pouches triggered by lipopolysaccharide (LPS), as well as the putative roles played by kinin B1 and B2 receptors, tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and selectins in this response. LPS (5 ng to 10 μg cavity−1) injected into the 6-day-old pouch induced a dose- and time-dependent neutrophil migration which peaked between 4 and 6 h, and was maximal following the dose of 100 ng cavity−1 (saline: 0.46±0.1; LPS: 43±3.70 × 106 cells cavity−1 at 6 h). Bradykinin (BK) (600 nmol) injected into the pouch of saline-treated rats induced only modest neutrophil migration (0.73±0.16 × 106 cells cavity−1). A more robust response to BK (3.2±0.25 × 106 cells cavity−1) was seen in animals pretreated with captopril, but this was still smaller than the responses to IL-1β or TNF-α (15 pmol: 23±2.2 × 106 and 75 pmol: 29.5±2 × 106 cells cavity−1, respectively). Nevertheless, the B1 agonist des-Arg9-BK (600 nmol) failed to induce neutrophil migration. HOE-140 (1 and 2 mg kg−1), a B2 receptor antagonist, reduced LPS-induced neutrophil migration. HOE-140 also reduced the neutrophil migration induced by BK, but had no effect on the migration promoted by IL-1β or TNF-α. des-Arg9-[Leu8]-BK, B1 receptor antagonist was ineffective in changing neutrophil migration caused by any of these stimuli. Neutrophil migration induced by LPS or BK was reduced by interleukin-1 receptor antagonist (IL-1ra) (1 mg kg−1), sheep anti-rat TNF serum (anti-TNF serum) (0.3 ml cavity−1), and the nonspecific selectin inhibitor fucoidin (10 mg kg−1). TNF-α levels in the pouch fluid were increased by LPS or BK injection, peaking at 0.5–1 h and gradually declining thereafter up to 6 h. IL-1β levels increased steadily throughout the 6 h period. HOE-140 markedly inhibited the rise in IL-1β and TNF-α levels in pouch fluid triggered by both stimuli. These

  12. The SnRK2-APC/CTE regulatory module mediates the antagonistic action of gibberellic acid and abscisic acid pathways

    PubMed Central

    Lin, Qibing; Wu, Fuqing; Sheng, Peike; Zhang, Zhe; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Cheng, Zhijun; Wang, Jie; Wang, Haiyang; Wan, Jianmin

    2015-01-01

    Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate many developmental processes and responses to biotic or abiotic stresses in higher plants. However, the molecular mechanism underlying this antagonism is still poorly understood. Here, we show that loss-of-function mutation in rice Tiller Enhancer (TE), an activator of the APC/CTE complex, causes hypersensitivity and hyposensitivity to ABA and GA, respectively. We find that TE physically interacts with ABA receptor OsPYL/RCARs and promotes their degradation by the proteasome. Genetic analysis also shows OsPYL/RCARs act downstream of TE in mediating ABA responses. Conversely, ABA inhibits APC/CTE activity by phosphorylating TE through activating the SNF1-related protein kinases (SnRK2s), which may interrupt the interaction between TE and OsPYL/RCARs and subsequently stabilize OsPYL/RCARs. In contrast, GA can reduce the level of SnRK2s and may promote APC/CTE-mediated degradation of OsPYL/RCARs. Thus, we propose that the SnRK2-APC/CTE regulatory module represents a regulatory hub underlying the antagonistic action of GA and ABA in plants. PMID:26272249

  13. SSTR-Mediated Imaging in Breast Cancer: Is There a Role for Radiolabeled Somatostatin Receptor Antagonists?

    PubMed

    Dalm, Simone U; Haeck, Joost; Doeswijk, Gabriela N; de Blois, Erik; de Jong, Marion; van Deurzen, Carolien H M

    2017-10-01

    Recent studies have shown enhanced tumor targeting by novel somatostatin receptor (SSTR) antagonists compared with clinically widely used agonists. However, these results have been obtained mostly in neuroendocrine tumors, and only limited data are available for cancer types with lower SSTR expression, including breast cancer (BC). To date, two studies have reported higher binding of the antagonist than the agonist in BC, but in both studies only a limited number of cases were evaluated. In this preclinical study, we further investigated whether the application of an SSTR antagonist can improve SSTR-mediated BC imaging in a large panel of BC specimens. We also generated an in vivo BC mouse model and performed SPECT/MRI and biodistribution studies. Methods: Binding of 111 In-DOTA-Tyr 3 -octreotate (SSTR agonist) and 111 In-DOTA-JR11 (SSTR antagonist) to 40 human BC specimens was compared using in vitro autoradiography. SSTR2 immunostaining was performed to confirm SSTR2 expression of the tumor cells. Furthermore, binding of the radiolabeled SSTR agonist and antagonist was analyzed in tissue material from 6 patient-derived xenografts. One patient-derived xenograft, the estrogen receptor-positive model T126, was chosen to generate in vivo mouse models containing orthotopic breast tumors for in vivo SPECT/MRI and biodistribution studies after injection with 177 Lu-DOTA-Tyr 3 -octreotate or 177 Lu-DOTA-JR11. Results: 111 In-DOTA-JR11 binding to human BC tissue was significantly higher than 111 In-DOTA-Tyr 3 -octreotate binding ( P < 0.001). The median ratio of antagonist binding versus agonist binding was 3.39 (interquartile range, 2-5). SSTR2 immunostaining confirmed SSTR2 expression on the tumor cells. SPECT/MRI of the mouse model found better tumor visualization with the antagonist. This result was in line with the significantly higher tumor uptake of the radiolabeled antagonist than of the agonist as measured in biodistribution studies 285 min after radiotracer

  14. Synergistic Antiproliferative Effects of Combined γ-Tocotrienol and PPARγ Antagonist Treatment Are Mediated through PPARγ-Independent Mechanisms in Breast Cancer Cells

    PubMed Central

    Sylvester, Paul W.

    2014-01-01

    Previous findings showed that the anticancer effects of combined γ-tocotrienol and peroxisome proliferator activated receptor γ (PPARγ) antagonist treatment caused a large reduction in PPARγ expression. However, other studies suggest that the antiproliferative effects of γ-tocotrienol and/or PPARγ antagonists are mediated, at least in part, through PPARγ-independent mechanism(s). Studies were conducted to characterize the role of PPARγ in mediating the effects of combined treatment of γ-tocotrienol with PPARγ agonists or antagonists on the growth of PPARγ negative +SA mammary cells and PPARγ-positive and PPARγ-silenced MCF-7 and MDA-MB-231 breast cancer cells. Combined treatment of γ-tocotrienol with PPARγ antagonist decreased, while combined treatment of γ-tocotrienol with PPARγ agonist increased, growth of all cancer cells. However, treatment with high doses of 15d-PGJ2, an endogenous natural ligand for PPARγ, had no effect on cancer cell growth. Western blot and qRT-PCR studies showed that the growth inhibitory effects of combined γ-tocotrienol and PPARγ antagonist treatment decreased cyclooxygenase (COX-2), prostaglandin synthase (PGDS), and prostaglandin D2 (PGD2) synthesis. In conclusion, the anticancer effects of combined γ-tocotrienol and PPARγ antagonists treatment in PPARγ negative/silenced breast cancer cells are mediated through PPARγ-independent mechanisms that are associated with a downregulation in COX-2, PGDS, and PGD2 synthesis. PMID:24729783

  15. Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors.

    PubMed

    Chang, Hung; Yanachkov, Ivan B; Michelson, Alan D; Li, YouFu; Barnard, M R; Wright, George E; Frelinger, Andrew L

    2010-02-01

    Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood. We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors. Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors. Copyright 2009 Elsevier Ltd. All rights reserved.

  16. Agonist and Antagonist Effects of Diadenosine Tetraphosphate, a Platelet Dense Granule Constituent, on Platelet P2Y1, P2Y12 and P2X1 Receptors

    PubMed Central

    Chang, Hung; Yanachkov, Ivan B.; Michelson, Alan D.; Li, YouFu; Barnard, M.R.; Wright, George E.; Frelinger, Andrew L.

    2010-01-01

    Introduction Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) is stored in platelet dense granules, but its effects on platelet function are not well understood. Methods and Results We examined the effects of Ap4A on platelet purinergic receptors P2Y1, P2Y12 and P2X1. Flow cytometry was used to measure the effects of Ap4A in the presence or absence of ADP on: a) P2Y12-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y1-mediated increase in platelet cytosolic Ca2+, and c) P2X1-mediated intraplatelet entry of extracellular Ca2+. ADP-stimulated platelet shape change (P2Y1-mediated) and aggregation (P2Y1- and P2Y12-mediated) were measured optically. Ap4A inhibited 3 µM ADP-induced: a) platelet aggregation (IC50 9.8 ± 2.8 µM), b) P2Y1-mediated shape change, c) P2Y1-mediated increase in platelet cytosolic Ca2+ (IC50 40.8 ± 12.3 µM), and d) P2Y12-mediated decrease in VASP phosphorylation (IC50 >250 µM). In the absence of added ADP, Ap4A had agonist effects on platelet P2X1 and P2Y12, but not P2Y1, receptors. Conclusion Ap4A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y1 and P2Y12 receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X1 and P2Y12 receptors. PMID:19945153

  17. Effect of LF 16-0687MS, a new nonpeptide bradykinin B2 receptor antagonist, in a rat model of closed head trauma.

    PubMed

    Pruneau, D; Chorny, I; Benkovitz, V; Artru, A; Roitblat, L; Shapira, Y

    1999-11-01

    Bradykinin is an endogenous nonapeptide which potently dilates the cerebral vasculature and markedly increases vascular permeability. These effects are mediated by B2 receptors located on the vascular endothelium. Previous experimental studies have shown that blockade of the kallikreinkinin system, which mediates the formation of bradykinin, afforded a reduction of the brain edema that developed following a cryogenic cortical lesion. In the present study, we investigated the effect of LF 16-0687MS, a novel nonpeptide B2 receptor antagonist, on cerebral edema and neurological severity score (NSS) after closed head injury to rats. LF 16-0687MS or its vehicle (NaCl 0.9%) was continuously infused at 10, 30, and 100 microg/kg/min over 23 h starting 1 h after a focal trauma to the left hemisphere was induced using a weight-drop device. The extent of edema formation was evaluated 24 h after trauma from left and right hemispheres samples by measurement of specific gravity and water content. In a separate study, a neurological severity score based on scoring of behavioural and motor functions was evaluated 1 h and over 1 week after trauma. LF 16-0687MS at 100 microg/kg/min markedly reduced the development of brain edema as indicated by a 68% increase in specific gravity (p<0.05) and a 64% decrease of water content (p<0.05) in the left hemisphere. In addition the recovery of neurological function was significantly improved by 100 microg/kg/min LF 16-0687MS from day 3 to day 7 after CHT. In a separate experiment, we also showed that LF 16-0687MS at 100 microg/kg/min given either 1 h before or 30 min after CHT did not affect mean arterial blood pressure. These results show that blockade of bradykinin B2 receptors is an effective approach to reduce cerebral edema and to improve neurological outcome after a focal contusion to the cranium.

  18. Pharmacological significance of the interplay between angiotensin receptors: MAS receptors as putative final mediators of the effects elicited by angiotensin AT1 receptors antagonists.

    PubMed

    Pernomian, Larissa; Pernomian, Laena; Gomes, Mayara S; da Silva, Carlos H T P

    2015-12-15

    The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Function of brain α2B-adrenergic receptor characterized with subtype-selective α2B antagonist and KO mice.

    PubMed

    Luhrs, Lauren; Manlapaz, Cynthia; Kedzie, Karen; Rao, Sandhya; Cabrera-Ghayouri, Sara; Donello, John; Gil, Daniel

    2016-12-17

    Noradrenergic signaling, through the α 2A and α 2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α 2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α 2B receptor in CNS function via the discovery and use of the first subtype-selective α 2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α 2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α 2 subtypes in the brain, we compared α 2B KO, α 2A KO and α 2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α 2B KO mice exhibited increased marble burying and α 2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α 2B KO and α 2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α 2C KO mice increased activity at lower doses relative to either α 2A KO or WT mice. However, α 2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α 2B KO phenotype. These findings suggest that the α 2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and

  20. Classifier ensemble based on feature selection and diversity measures for predicting the affinity of A(2B) adenosine receptor antagonists.

    PubMed

    Bonet, Isis; Franco-Montero, Pedro; Rivero, Virginia; Teijeira, Marta; Borges, Fernanda; Uriarte, Eugenio; Morales Helguera, Aliuska

    2013-12-23

    A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

  1. Synthesis and serotonergic activity of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives: novel antagonists for the vascular 5-HT(1B)-like receptor.

    PubMed

    Moloney, G P; Martin, G R; Mathews, N; Milne, A; Hobbs, H; Dodsworth, S; Sang, P Y; Knight, C; Williams, M; Maxwell, M; Glen, R C

    1999-07-15

    The synthesis and vascular 5-HT(1B)-like receptor activity of a novel series of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives are described. Modifications to the 5-ethylene-linked heterocycle and to substituents on the 2-benzylamide side chain have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B)-like receptor of pK(B) > 7.0, up to 100-fold selectivity over alpha(1)-adrenoceptor affinity and 5-HT(2A) receptor affinity, and which exhibited a favorable pharmacokinetic profile. N-Benzyl-3-[2-(dimethylamino)ethyl]-5-[2-(4,4-dimethyl-2, 5-dioxo-1-imidazolidinyl)ethyl]-1H-indole-2-carboxamide (23) was identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B)-like receptor-mediated agonist activity in the rabbit femoral artery), and competitive vascular 5-HT(1B)-like receptor antagonist with a plasma elimination half-life of approximately 4 h in dog plasma and with good oral bioavailability. The selectivity of compounds from this series for the vascular 5-HT(1B)-like receptors over other receptor subtypes is discussed as well as a proposed mode of binding to the receptor pharmacophore. It has been proposed that the aromatic ring of the 2, N-benzylcarboxamide group can occupy an aromatic binding site rather than the indole ring. The resulting conformation allows an amine-binding site to be occupied by the ethylamine nitrogen and a hydrogen-bonding site to be occupied by one of the hydantoin carbonyls. The electronic nature of the 2,N-benzylcarboxamide aromatic group as well as the size of substituents on this aromatic group is crucial for producing potent and selective antagonists. The structural requirement on the 3-ethylamine side chain incorporating the protonatable nitrogen is achieved by the bulky 2, N-benzylcarboxamide group and its close proximity to the 3-side chain.

  2. Mas-related G protein coupled receptor-X2: A potential new target for modulating mast cell-mediated allergic and inflammatory diseases.

    PubMed

    Ali, Hydar

    2016-12-01

    Mast cells (MCs) are tissue resident immune cells that are best known for their roles in allergic and inflammatory diseases. In addition to the high affinity IgE receptor (FcεRI), MCs express numerous G protein coupled receptors (GPCRs), which are the most common targets of drug therapy. Neurokinin 1 receptor (NK-1R) is expressed on MCs and contributes to IgE and non-IgE-mediated responses in mice. Although NK-1R antagonists are highly effective in modulating experimental allergic and inflammatory responses in mice they lack efficacy in humans. This article reviews recent findings that demonstrate that while neuropeptides (NPs) activate murine MCs via NK-1R and Mas related G protein coupled receptor B2 (MrgprB2), they activate human MCs via Mas-related G protein coupled receptor X2 (MRGPRX2). Interestingly, conventional NK-1R antagonists have off-target activity against mouse MrgprB2 but not human MRGPRX2. These findings suggest that the failure to translate studies with NK-1R antagonists from in vivo mouse studies to the clinic likely reflects their lack of effect on human MRGPRX2. A unique feature of MRGPRX2 that distinguishes it from other GPCRs is that it is activated by a diverse group of ligands that include; neuropeptides, cysteine proteases, antimicrobial peptides and cationic proteins released from activated eosinophils. Thus, the development of small molecule MRGPRX2-specific antagonists or neutralizing antibodies may provide new targets for the treatment of MC-mediated allergic and inflammatory diseases.

  3. Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

    PubMed

    Fattori, Daniela; Rossi, Cristina; Fincham, Christopher I; Berettoni, Marco; Calvani, Federico; Catrambone, Fernando; Felicetti, Patrizia; Gensini, Martina; Terracciano, Rosa; Altamura, Maria; Bressan, Alessandro; Giuliani, Sandro; Maggi, Carlo A; Meini, Stefania; Valenti, Claudio; Quartara, Laura

    2006-06-15

    We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

  4. NOP Receptor Mediates Anti-analgesia Induced by Agonist-Antagonist Opioids

    PubMed Central

    Gear, Robert W.; Bogen, Oliver; Ferrari, Luiz F.; Green, Paul G.; Levine, Jon D.

    2014-01-01

    Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ~90 minutes after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69,593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

  5. Substituted pyrrolidin-2-ones: Centrally acting orexin receptor antagonists promoting sleep. Part 2.

    PubMed

    Sifferlen, Thierry; Boller, Amandine; Chardonneau, Audrey; Cottreel, Emmanuelle; Gatfield, John; Treiber, Alexander; Roch, Catherine; Jenck, Francois; Aissaoui, Hamed; Williams, Jodi T; Brotschi, Christine; Heidmann, Bibia; Siegrist, Romain; Boss, Christoph

    2015-05-01

    Starting from advanced pyrrolidin-2-one lead compounds, this novel series of small-molecule orexin receptor antagonists was further optimized by fine-tuning of the C-3 substitution at the γ-lactam ring. We discuss our design to align in vitro potency with metabolic stability and improved physicochemical/pharmacokinetic properties while avoiding P-glycoprotein-mediated efflux. These investigations led to the identification of the orally active 3-hydroxypyrrolidin-2-one 46, a potent and selective orexin-2 receptor antagonist, that achieved good brain exposure and promoted physiological sleep in rats. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

    PubMed Central

    Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

    2015-01-01

    Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

  7. Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

    PubMed

    Schiffer, Lina; Müller, Anne-Rose; Hobler, Anna; Brixius-Anderko, Simone; Zapp, Josef; Hannemann, Frank; Bernhardt, Rita

    2016-10-01

    Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 2. Synthesis and structure-activity relationships of alpha,alpha-cycloalkylglycine sulfonamides.

    PubMed

    Fattori, Daniela; Rossi, Cristina; Fincham, Christopher I; Caciagli, Valerio; Catrambone, Fernando; D'Andrea, Piero; Felicetti, Patrizia; Gensini, Martina; Marastoni, Elena; Nannicini, Rossano; Paris, Marielle; Terracciano, Rosa; Bressan, Alessandro; Giuliani, Sandro; Maggi, Carlo A; Meini, Stefania; Valenti, Claudio; Quartara, Laura

    2007-02-08

    Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.

  9. Labeling of SR 46349B, a potent and selective 5-HT{sub 2} receptor antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tan, P.; Fowler, J.S.; Ding, Y.S.

    1995-05-01

    SR 46349B is a potent and selective 5-HT{sub 2} receptor antagonist (Kd =1.2 nM) which is currently being evaluated as an antidepressant. We labeled SR46349B with F-18 for PET studies via the nitro-for-fluorine exchange reaction. Among the five nitro-precursors (o-nitroacetophenone) examined for nucleophilic aromatic substitution ({sup 18}F{sup {minus}}, K{sub 2}CO{sub 3}, kryptofix-222, 120{degrees}C, 6 min), only phenol protected ether proceeded well and gave 36.4 {plus_minus} 14.3%(n=19) yield of which was directly hydrolyzed (Hcl, 90{degrees}C, 10 min) to afford. Removal of the nitro-precursor, which was generated in situ during hydrolysis was critical in the purification of the final product and wasmore » accomplished using a combination of C-18 Sep-Pak and silica gel column chromatography. The condensation of {sup 18}F- ketone with Me{sub 2}NCH{sub 2}CH{sub 2}ONH{sub 2}HCl in 2-(2{prime}-methoxyethoxy)ethanol (p-TsOH, 165{degrees}C, 10 min) gave a mixture of [{sup 18}F]SR 46349B and its geometric isomer with ca 1:1 ratio in quantitative yield. [{sup 18}F]SR 46349B was separated from its geometric isomer and other by-products by HPLC [Econosil C-18 semi-prep column, MeOH:MeCN:0.1 MK{sub 2}HPO{sub 4}(27.5:27.5:45), 5 ml/min]. The three step hot synthesis required 170 min and gave a specific activity of 1.14 Ci/{mu}mol, 5% radiochemical yield (EOB) and 96% radiochemical purity.« less

  10. Optimized dosing of a CCR2 antagonist for amplification of vaccine immunity.

    PubMed

    Mitchell, Leah A; Hansen, Ryan J; Beaupre, Adam J; Gustafson, Daniel L; Dow, Steven W

    2013-02-01

    We have recently discovered that inflammatory monocytes recruited to lymph nodes in response to vaccine-induced inflammation can function as potent negative regulators of both humoral and cell-mediated immune responses to vaccination. Monocyte depletion or migration blockade can significantly amplify both antibody titers and cellular immune responses to vaccination with several different antigens in mouse models. Thus, we hypothesized that the use of small molecule CCR2 inhibitors to block monocyte migration into lymph nodes may represent a broadly effective means of amplifying vaccine immunity. To address this question, the role of CCR2 in monocyte recruitment to vaccine draining lymph nodes was initially explored in CCR2-/- mice. Next, a small molecule antagonist of CCR2 (RS102895) was evaluated in mouse vaccination models. Initial studies revealed that a single intraperitoneal dose of RS102895 failed to effectively block monocyte recruitment following vaccination. Pharmacokinetic analysis of RS102895 revealed a short half-life (approximately 1h), and suggested that a multi-dose treatment regimen would be more effective. We found that administration of RS102895 every 6 h resulted in consistent plasma levels of 20 ng/ml or greater, which effectively blocked monocyte migration to lymph nodes following vaccination. Moreover, administration of RS102895 with concurrent vaccination markedly enhanced vaccine responses following immunization against the influenza antigen HA1. We concluded that administration of small molecule CCR2 antagonists such as RS102895 in the immediate post-vaccine period could be used as a novel means of significantly enhancing vaccine immunity. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Discovery of dual orexin receptor antagonists with rat sleep efficacy enabled by expansion of the acetonitrile-assisted/diphosgene-mediated 2,4-dichloropyrimidine synthesis.

    PubMed

    Roecker, Anthony J; Mercer, Swati P; Harrell, C Meacham; Garson, Susan L; Fox, Steven V; Gotter, Anthony L; Prueksaritanont, Thomayant; Cabalu, Tamara D; Cui, Donghui; Lemaire, Wei; Winrow, Christopher J; Renger, John J; Coleman, Paul J

    2014-05-01

    Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX1R and OX2R antagonists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed several compounds in the diazepane amide DORA series with excellent potency and both preclinical and clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi.

    PubMed

    Santicioli, Paolo; Meini, Stefania; Giuliani, Sandro; Lecci, Alessandro; Maggi, Carlo Alberto

    2013-10-24

    In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK 2 receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK 2 receptor agonist [βAla 8 ]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300nM) induced a concentration-dependent rightward shift of the [βAla 8 ]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus indicating a surmountable behaviour. The calculated apparent antagonist potency as pK B value was 8.31±0.05. Ibodutant (0.3-100nM), produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95±4% inhibition), the calculated IC 50 value was 2.98nM (95% c.l. 1.73-5.16nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrate that ibodutant is a potent NK 2 receptor antagonist in guinea pig airways. © 2013 Published by Elsevier B.V.

  13. Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu Xiaohong, E-mail: xuxh63@zjnu.cn; Ye Yinping; Li Tao

    Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) {beta} and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs.more » The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ER{beta} were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17{beta}-estradiol (17{beta}-E{sub 2}) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17{beta}-E{sub 2}. The present results suggest that BPA, like 17{beta}-E{sub 2}, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17{beta}-E{sub 2} when it coexists with 17{beta}-E{sub 2}. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.« less

  14. SCH 206272: a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist.

    PubMed

    Anthes, John C; Chapman, Richard W; Richard, Christian; Eckel, Stephen; Corboz, Michel; Hey, John A; Fernandez, Xiomara; Greenfeder, Scott; McLeod, Robbie; Sehring, Susan; Rizzo, Charles; Crawley, Yvette; Shih, Neng-Yang; Piwinski, John; Reichard, Greg; Ting, Pauline; Carruthers, Nick; Cuss, Francis M; Billah, Motasim; Kreutner, William; Egan, Robert W

    2002-08-23

    Experiments were performed to characterize the pharmacology of SCH 206272 [(R,R)-1'[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4(Z)-(methoxyimino)pentyl]-N-methyl-2-oxo-[1,4'bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK(1), NK(2), and NK(3) receptors. SCH 206272 inhibited binding at human tachykinin NK(1), NK(2), and NK(3) receptors (K(i) = 1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca(2+)](i) mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK(1), NK(2), or NK(3) receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (pK(b) = 7.7 +/- 0.3) induced by the tachykinin NK(1) receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (pK(b = 8.2 +/- 0.3) induced by the tachykinin NK(2) receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (pK(b = 7.6 +/- 0.2), NKA-induced contraction of the bronchus (pK(b) = 7.7 +/- 0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1-10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1-3 mg/kg, p.o.) inhibited NK(1) and NK(2) activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK(1), NK(2), and NK(3) receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease. Copyright 2002 Elsevier

  15. Protective effect of histamine H2 receptor antagonist ranitidine against rotenone-induced apoptosis.

    PubMed

    Park, Hae Jeong; Kim, Hak Jae; Park, Hyun-Kyung; Chung, Joo-Ho

    2009-11-01

    Histamine H(2) receptor antagonists have been reported to improve the motor symptoms of Parkinson's disease (PD) patients and to exert neuroprotective effects. In this study, we investigated the protective effects of the H(2) receptor antagonist ranitidine on rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells, focusing on mitogen-activated protein kinases (MAPKs) and caspases (CASPs)-mediated apoptotic events. Ranitidine blocked the rotenone-induced phosphorylation of c-Jun NH(2)-terminal protein kinase (JNK) and P38 MAPK (P38), and promoted the phosphorylation of extracellular signal-regulated protein kinase (ERK). Ranitidine also prevented the down-regulation of B-cell CLL/lymphoma 2 (BCL2) and the up-regulation of BCL2-associated X protein (BAX) by rotenone. Furthermore, ranitidine not only attenuated rotenone-induced cleavages of CASP9, poly(ADP-ribose) polymerase-1 (PARP) and CASP3, but also suppressed CASP3 enzyme activity. These results indicate that ranitidine protects against rotenone-induced apoptosis, inhibiting phosphorylation of JNK and P38, and activation of CASPs in human dopaminergic SH-SY5Y cells.

  16. Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists.

    PubMed

    Rivier, J; Gulyas, J; Kirby, D; Low, W; Perrin, M H; Kunitake, K; DiGruccio, M; Vaughan, J; Reubi, J C; Waser, B; Koerber, S C; Martinez, V; Wang, L; Taché, Y; Vale, W

    2002-10-10

    We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF(1) and CRF(2) receptors. Predictive methods, physicochemical measurements, and structure-activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin [cyclo(30-33)[DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((12-41))] assume an alpha-helical conformation when interacting with their receptors. We had shown that alpha-helical CRF((9-41)) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion(1) and later for CRF2.(2) More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30-33(3) that would confer high affinity for both CRF(1) and CRF(2)(2,4) in agonists and antagonists of all members of the CRF family.(3) On the other hand, the substitutions that conferred ca. 100-fold CRF(2) selectivity to the antagonist antisauvagine-30 [[DPhe(11),His(12)]sauvagine((11-40))] did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu(32)-Lys(35) side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu(31)-Lys(34) side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF(2). Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31-34)[DPhe(11),His(12),C(alpha)MeLeu(13,39),Nle(17),Glu(31),Lys(34)]Ac-sauvagine((8-40)) (astressin(2)-B) with CRF(2) selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF(2) and CRF(1) in order to confirm that astressin(2)-B could indeed bind to established CRF(2) but not CRF(1) receptor-expressing tissues. Extended duration of action of astressin(2)-B vs that of antisauvagine-30 is demonstrated in

  17. Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-d-Aspartate Receptor Antagonists

    PubMed Central

    Hansen, Kasper B.; Mullasseril, Praseeda; Dawit, Sara; Kurtkaya, Natalie L.; Yuan, Hongjie; Vance, Katie M.; Orr, Anna G.; Kvist, Trine; Ogden, Kevin K.; Le, Phuong; Vellano, Kimberly M.; Lewis, Iestyn; Kurtkaya, Serdar; Du, Yuhong; Qui, Min; Murphy, T. J.; Snyder, James P.; Bräuner-Osborne, Hans

    2010-01-01

    N-Methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism. PMID:20197375

  18. Cholecystokinin type B receptor antagonist PD-136,450 is a partial secretory agonist in the stomach and a full agonist in the pancreas of the rat.

    PubMed Central

    Schmassmann, A; Garner, A; Flogerzi, B; Hasan, M Y; Sanner, M; Varga, L; Halter, F

    1994-01-01

    Gastrin (cholecystokinin type B (CCK-B)) receptor antagonists may help to elucidate the physiological role of gastrin, have therapeutic potential as acid antisecretory drugs, and may be of use as adjuvant therapy for gastrin sensitive tumours. In binding studies, the gastrin receptor antagonist PD-136,450 had at least 1000 fold greater affinity for gastrin (CCK-B) than CCK-A receptors. In this study the biological activity of PD-136,450 was evaluated in conscious and anaesthetised rats. PD-136,450 antagonised gastrin stimulated acid secretion after subcutaneous (IC50: 0.28 mumol/kg; conscious rats) and intravenous (IC50: 0.17 mumol/kg; anaesthetised rats) administration. In basal secreting fistula animals, the compound stimulated acid output to 30 (5)% of the maximal response to gastrin. Stimulant activity was not caused by gastrin release. As an agonist PD-136,450 was about 350 times less potent than gastrin-17 on a molar basis. In addition, PD-136,450 was a powerful agonist of pancreatic secretion in anaesthetised rats. The specific gastrin antagonist L-365,260 inhibited the (partial) agonist activity of PD-136,450 in the stomach and the specific CCK-A receptor antagonist L-364,718 inhibited the agonist activity of PD-136,450 in the pancreas. It is concluded that the agonist effect of PD-136,450 is mediated via interaction with the gastrin (CCK-B) receptor in the stomach and the CCK-A receptor in the pancreas. PMID:8307482

  19. Inhibition of mechanotransducer currents in crayfish sensory neuron by CGS 9343B, a calmodulin antagonist.

    PubMed

    Lin, J H; Rydqvist, B

    2000-05-26

    The effects of CGS 9343B (zaldaride maleate), a calmodulin antagonist, on mechanosensitive channels were examined in crayfish slowly adapting sensory neurons using the two-electrode voltage clamp technique. In addition to its inhibition of voltage-gated Na(+) and K(+) currents, CGS 9343B (<30 microM) blocked reversibly the receptor current in a dose-dependent and voltage-dependent manner with a dissociation constant (K(d)) of 26.8 microM. The time course of the block was 265 s. Within the extension range of 3-30%, the reduction in receptor current was stimulus-independent and the gating mechanisms were not affected. Extracellular Ca(2+) was not necessary for its blocking effects. No changes in passive muscle tension were observed in the presence of 20 microM CGS 9343B. These results suggest that CGS 9343B, as a calmodulin antagonist, can also block mechanosensitive channels, possibly by being incorporated into the lipid membrane and/or interacting with the channel protein.

  20. Stimulant effects of adenosine antagonists on operant behavior: differential actions of selective A2A and A1 antagonists

    PubMed Central

    Randall, Patrick A.; Nunes, Eric J.; Janniere, Simone L.; Stopper, Colin M.; Farrar, Andrew M.; Sager, Thomas N.; Baqi, Younis; Hockemeyer, Jörg; Müller, Christa E.

    2012-01-01

    Rationale Adenosine A2A antagonists can reverse many of the behavioral effects of dopamine antagonists, including actions on instrumental behavior. However, little is known about the effects of selective adenosine antagonists on operant behavior when these drugs are administered alone. Objective The present studies were undertaken to investigate the potential for rate-dependent stimulant effects of both selective and nonselective adenosine antagonists. Methods Six drugs were tested: two nonselective adenosine antagonists (caffeine and theophylline), two adenosine A1 antagonists (DPCPX and CPT), and two adenosine A2A antagonists (istradefylline (KW6002) and MSX-3). Two schedules of reinforcement were employed; a fixed interval 240-s (FI-240 sec) schedule was used to generate low baseline rates of responding and a fixed ratio 20 (FR20) schedule generated high rates. Results Caffeine and theophylline produced rate-dependent effects on lever pressing, increasing responding on the FI-240 sec schedule but decreasing responding on the FR20 schedule. The A2A antagonists MSX-3 and istradefylline increased FI-240 sec lever pressing but did not suppress FR20 lever pressing in the dose range tested. In fact, there was a tendency for istradefylline to increase FR20 responding at a moderate dose. A1 antagonists failed to increase lever pressing rate, but DPCPX decreased FR20 responding at higher doses. Conclusions These results suggest that adenosine A2A antagonists enhance operant response rates, but A1 antagonists do not. The involvement of adenosine A2A receptors in regulating aspects of instrumental response output and behavioral activation may have implications for the treatment of effort-related psychiatric dysfunctions, such as psychomotor slowing and anergia in depression. PMID:21347642

  1. Retinal plasma extravasation in streptozotocin-diabetic rats mediated by kinin B1 and B2 receptors

    PubMed Central

    Abdouh, M; Talbot, S; Couture, R; Hasséssian, H M

    2008-01-01

    Background and purpose: We investigated whether or not kinin receptors play a role in diabetic blood–retinal barrier breakdown, which is a leading cause of vision loss. Experimental approach: Blood–retinal barrier breakdown was quantified using Evans blue, and expression of kinin B1 receptor mRNA was measured using quantitative reverse transcrition-PCR. Diabetic rats (streptozotocin (STZ), 65 mg kg−1) received a single intraocular injection of bradykinin (BK) or des-Arg9-BK, alone, or in combination with antagonists for B1 (des-Arg10-Hoe140, R-715) and/or B2 (Hoe140) receptors, given intraocularly or intravenously (i.v.). Key results: In control rats, BK (0.1–10 nmol) dose-dependently increased plasma extravasation, which was inhibited by Hoe140 (0.2 nmol), whereas des-Arg9-BK (0.1 and 1 nmol) was without effect. B1 receptor mRNA was markedly increased in retinas of diabetic rats, and this was prevented by N-acetyl-L-cysteine (1 g kg−1 day−1 for 7 days). Plasma extravasation in retinas of STZ-diabetic rats was higher than in controls and enhanced by des-Arg9-BK. Response to des-Arg9-BK was inhibited by intraocular or i.v. injection of B1 receptor antagonists. Diabetes-induced plasma extravasation was inhibited only by a combination of des-Arg10-Hoe140 and Hoe 140 (100 nmol kg−1, i.v. 15 min earlier) or by R-715 (1 μmol kg−1, i.v.) injected daily for 7 days. Conclusions and implications: Kinin B1 receptors are upregulated in retinas of STZ-diabetic rats through a mechanism involving oxidative stress. Both kinin B1 and B2 receptors contribute to increased plasma extravasation in diabetic retinopathy. Chronic inhibition of both kinin receptors, possibly with antioxidant adjuvants, may be a novel therapeutic strategy for diabetic retinopathy. PMID:18311190

  2. Randomized, controlled trial of TNF-α antagonist in CTL-mediated severe cutaneous adverse reactions.

    PubMed

    Wang, Chuang-Wei; Yang, Lan-Yan; Chen, Chun-Bing; Ho, Hsin-Chun; Hung, Shuen-Iu; Yang, Chih-Hsun; Chang, Chee-Jen; Su, Shih-Chi; Hui, Rosaline Chung-Yee; Chin, See-Wen; Huang, Li-Fang; Lin, Yang Yu-Wei; Chang, Wei-Yang; Fan, Wen-Lang; Yang, Chin-Yi; Ho, Ji-Chen; Chang, Ya-Ching; Lu, Chun-Wei; Chung, Wen-Hung

    2018-03-01

    Cytotoxic T lymphocyte-mediated (CTL-mediated) severe cutaneous adverse reactions (SCARs), including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), are rare but life-threatening adverse reactions commonly induced by drugs. Although high levels of CTL-associated cytokines, chemokines, or cytotoxic proteins, including TNF-α and granulysin, were observed in SJS-TEN patients in recent studies, the optimal treatment for these diseases remains controversial. We aimed to evaluate the efficacy, safety, and therapeutic mechanism of a TNF-α antagonist in CTL-mediated SCARs. We enrolled 96 patients with SJS-TEN in a randomized trial to compare the effects of the TNF-α antagonist etanercept versus traditional corticosteroids. Etanercept improved clinical outcomes in patients with SJS-TEN. Etanercept decreased the SCORTEN-based predicted mortality rate (predicted and observed rates, 17.7% and 8.3%, respectively). Compared with corticosteroids, etanercept further reduced the skin-healing time in moderate-to-severe SJS-TEN patients (median time for skin healing was 14 and 19 days for etanercept and corticosteroids, respectively; P = 0.010), with a lower incidence of gastrointestinal hemorrhage in all SJS-TEN patients (2.6% for etanercept and 18.2% for corticosteroids; P = 0.03). In the therapeutic mechanism study, etanercept decreased the TNF-α and granulysin secretions in blister fluids and plasma (45.7%-62.5% decrease after treatment; all P < 0.05) and increased the Treg population (2-fold percentage increase after treatment; P = 0.002), which was related to mortality in severe SJS-TEN. The anti-TNF-α biologic agent etanercept serves as an effective alternative for the treatment of CTL-mediated SCARs. ClinicalTrials.gov NCT01276314. Ministry of Science and Technology of Taiwan.

  3. Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB.

    PubMed

    Huang, Zi-Ming; Kang, Jhi-Kai; Chen, Chih-Yu; Tseng, Tz-Hau; Chang, Chien-Wen; Chang, Yung-Chi; Tai, Shyh-Kuan; Hsieh, Shie-Liang; Leu, Chuen-Miin

    2012-06-15

    Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.

  4. Endothelin A receptor antagonists in congestive heart failure: blocking the beast while leaving the beauty untouched?

    PubMed

    Spieker, L E; Noll, G; Ruschitzka, F T; Lüscher, T F

    2001-12-01

    Congestive heart failure (CHF) is a disease process characterized by impaired left ventricular function, increased peripheral and pulmonary vascular resistance and reduced exercise tolerance and dyspnea. Thus, mediators involved in the control of myocardial function and vascular tone may be involved in its pathophysiology. The family of endothelins (ET) consists of four closely related peptides, ET-1, ET-2, ET-3, and ET-4, which cause vasoconstriction, cell proliferation, and myocardial effects through activation of ET(A) receptors. In contrast, endothelial ET(B) receptors mediate vasodilation via release of nitric oxide and prostacyclin. In addition, ET(B) receptors in the lung are a major pathway for the clearance of ET-1 from plasma. Thus, infusion of an ET(A) receptor antagonist into the brachial artery in healthy humans leads to vasodilation whereas infusion of an ET(B) receptor antagonist causes vasoconstriction. ET-1 plasma levels are elevated in CHF and correlate both with the hemodynamic severity and with symptoms. Plasma levels of ET-1 and its precursor, big ET-1, are strong independent predictors of death in patients after myocardial infarction and with CHF. ET-1 contributes to increased systemic and pulmonary vascular resistance, vascular dysfunction, myocardial ischemia, and renal impairment in CHF. Selective ET(A) as well as combined ET(A/B) receptor antagonists have been studied in patients with CHF showing impressive hemodynamic improvements (i.e. reduced peripheral vascular and pulmonary resistance as well as increased cardiac output). These results indicate that ET receptor antagonists indeed have a potential to improve hemodynamics, symptoms, and potentially prognosis of CHF which still carries a high mortality.

  5. Randomized, controlled trial of TNF-α antagonist in CTL-mediated severe cutaneous adverse reactions

    PubMed Central

    Wang, Chuang-Wei; Yang, Lan-Yan; Ho, Hsin-Chun; Hung, Shuen-Iu; Yang, Chih-Hsun; Chang, Chee-Jen; Su, Shih-Chi; Hui, Rosaline Chung-Yee; Chin, See-Wen; Huang, Li-Fang; Lin, Yang Yu-Wei; Chang, Wei-Yang; Fan, Wen-Lang; Yang, Chin-Yi; Ho, Ji-Chen; Chung, Wen-Hung

    2018-01-01

    BACKGROUND. Cytotoxic T lymphocyte–mediated (CTL-mediated) severe cutaneous adverse reactions (SCARs), including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), are rare but life-threatening adverse reactions commonly induced by drugs. Although high levels of CTL-associated cytokines, chemokines, or cytotoxic proteins, including TNF-α and granulysin, were observed in SJS-TEN patients in recent studies, the optimal treatment for these diseases remains controversial. We aimed to evaluate the efficacy, safety, and therapeutic mechanism of a TNF-α antagonist in CTL-mediated SCARs. METHODS. We enrolled 96 patients with SJS-TEN in a randomized trial to compare the effects of the TNF-α antagonist etanercept versus traditional corticosteroids. RESULTS. Etanercept improved clinical outcomes in patients with SJS-TEN. Etanercept decreased the SCORTEN-based predicted mortality rate (predicted and observed rates, 17.7% and 8.3%, respectively). Compared with corticosteroids, etanercept further reduced the skin-healing time in moderate-to-severe SJS-TEN patients (median time for skin healing was 14 and 19 days for etanercept and corticosteroids, respectively; P = 0.010), with a lower incidence of gastrointestinal hemorrhage in all SJS-TEN patients (2.6% for etanercept and 18.2% for corticosteroids; P = 0.03). In the therapeutic mechanism study, etanercept decreased the TNF-α and granulysin secretions in blister fluids and plasma (45.7%–62.5% decrease after treatment; all P < 0.05) and increased the Treg population (2-fold percentage increase after treatment; P = 0.002), which was related to mortality in severe SJS-TEN. CONCLUSIONS. The anti–TNF-α biologic agent etanercept serves as an effective alternative for the treatment of CTL-mediated SCARs. TRIAL REGISTRATION. ClinicalTrials.gov NCT01276314. FUNDING. Ministry of Science and Technology of Taiwan. PMID:29400697

  6. Crystal structure of the adenosine A 2A receptor bound to an antagonist reveals a potential allosteric pocket

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phasemore » diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.« less

  7. MLF1 is a proapoptotic antagonist of HOP complex-mediated survival.

    PubMed

    Sun, Yi; Chao, Jyh-Rong; Xu, Wu; Pourpak, Alan; Boyd, Kelli; Moshiach, Simon; Qi, Guo-Yan; Fu, Amina; Shao, Hua-Rong; Pounds, Stanley; Morris, Stephan W

    2017-04-01

    In the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, anti-apoptotic signals are generated by cleavage and activation of the serine protease HtrA2/OMI by the rhomboid protease PARL upon recruitment of both proteases to inner mitochondrial membrane protein HAX1 (HS1-associated protein X-1). Here we report the negative regulation of the HOP complex by human leukemia-associated myeloid leukemia factor 1 (MLF1). We demonstrate that MLF1 physically and functionally associates with HAX1 and HtrA2. Increased interaction of MLF1 with HAX1 and HtrA2 displaces HtrA2 from the HOP complex and inhibits HtrA2 cleavage and activation, resulting in the apoptotic cell death. Conversely, over-expressed HAX1 neutralizes MLF1's effect and inhibits MLF1-induced apoptosis. Importantly, Mlf1 deletion reverses B- and T-cell lymphopenia and significantly ameliorates the progressive striatal and cerebellar neurodegeneration observed in Hax1 -/- mice, with a doubling of the lifespan of Mlf1 -/- /Hax1 -/- animals compared to Hax1 -/- animals. Collectively, these data indicate that MLF1 serves as a proapoptotic antagonist that interacts with the HOP mitochondrial complex to modulate cell survival. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Chemokine receptor antagonists: part 2.

    PubMed

    Pease, James E; Horuk, Richard

    2009-02-01

    The first part of this two-part review discussed approaches to generating antagonists for some of the CC chemokine receptors, including CCR1, CCR2, CCR3, and CCR4. This second part of the series concludes the review by describing antagonists for CCR5, CCR8, CCR9, CXCR3, CXCR4, and promiscuous antagonists. Chemokine receptor antagonists have found mixed success as therapeutics. Although one antagonist--maraviroc, a CCR5 inhibitor to treat AIDS--has been registered as an approved drug, this is the only success so far. There have been many failures in the clinic and we discuss the idea of promiscuous receptor antagonists as an alternative approach.

  9. Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

    PubMed

    Napier, Susan E; Letourneau, Jeffrey J; Ansari, Nasrin; Auld, Douglas S; Baker, James; Best, Stuart; Campbell-Wan, Leigh; Chan, Ray; Craighead, Mark; Desai, Hema; Ho, Koc-Kan; MacSweeney, Cliona; Milne, Rachel; Richard Morphy, J; Neagu, Irina; Ohlmeyer, Michael H J; Pick, Jack; Presland, Jeremy; Riviello, Chris; Zanetakos, Heather A; Zhao, Jiuqiao; Webb, Maria L

    2011-06-15

    Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Neuronally mediated contraction responses of guinea-pig stomach smooth muscle preparations: modification by benzamide derivatives does not reflect a dopamine antagonist action.

    PubMed

    Costall, B; Naylor, R J; Tan, C C

    1984-06-15

    The actions of the substituted benzamide derivatives metoclopramide, clebopride, YM-09151-2, tiapride, (+)- and (-)-sulpiride and (+)- and (-)-sultopride, and the dopamine antagonists haloperidol and domperidone, were studied on the responses to field stimulation (0.125-10 Hz) of smooth muscle strips taken from cardia, fundus, body and antral regions of the longitudinal and circular muscle of guinea-pig stomach. Field stimulation of the longitudinal strips caused contraction responses which were antagonised by atropine (but not by prazosin, yohimbine, propranolol or methysergide) to indicate a muscarinic cholinergic involvement. Antagonism of the contractions revealed or enhanced relaxation responses mediated via unidentified mechanisms (resistant to cholinergic and adrenergic antagonists). Metoclopramide enhanced the field stimulation-induced contractions of the stomach smooth muscle preparations via atropine sensitive mechanisms but failed to attenuate the field stimulation-induced relaxation responses. Clebopride's action closely followed that of metoclopramide but YM-09151-2 only enhanced the contraction responses of the longitudinal muscle preparations. Other dopamine antagonists, (+)- and (-)-sulpiride, (+)- and (-)-sultopride, tiapride, haloperidol and domperidone failed to facilitate contraction to field stimulation of any stomach tissue. Thus, the actions of metoclopramide, clebopride and YM-09151-2 to facilitate contraction to field stimulation of stomach smooth muscle are mediated via a muscarinic cholinergic mechanism and are not the consequence of an antagonism at any recognisable dopamine receptor.

  11. Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal.

    PubMed

    Maeda, A; Kurosaki, M; Ono, M; Takai, T; Kurosaki, T

    1998-04-20

    Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

  12. Exploring neuroprotective potential of Withania somnifera phytochemicals by inhibition of GluN2B-containing NMDA receptors: An in silico study.

    PubMed

    Kumar, Gaurav; Patnaik, Ranjana

    2016-07-01

    N-methyl-d-aspartate receptors (NMDARs) mediated excitotoxicity has been implicated in multi-neurodegenerative diseases. Due to lack of efficacy and adverse effects of NMDA receptor antagonists, search for herbal remedies that may act as therapeutic agents is an active area of research to combat these diseases. Withania somnifera (WS) is being used for centuries as a nerve tonic and Nootropic agents. The present study targets the in silico evaluation of the neuroprotective efficacy of W. somnifera phytochemicals by inhibition of NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B containing NMDARs. We predict Blood Brain Barrier (BBB) penetration, mutagenicity, drug-likeness and Human Intestinal Absorption properties of 25 WS phytochemicals. Further, molecular docking was performed to know whether these phytochemicals inhibit the GluN2B containing NMDARs or not. The results suggest that Anaferine, Beta-Sitosterol, Withaferin A, Withanolide A, Withanolide B and Withanolide D inhibit GluN2B containing NMDARs through allosteric mode similar to the well-known selective antagonist Ifenprodil. These phytochemicals have potential as an essentially useful oral drug to counter NMDARs mediated excitotoxicity and to treat multi-neurodegenerative diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis

    PubMed Central

    Lagares, David; Ghassemi-Kakroodi, Parisa; Tremblay, Caroline; Santos, Alba; Probst, Clemens K.; Franklin, Alicia; Santos, Daniela M.; Grasberger, Paula; Ahluwalia, Neil; Montesi, Sydney B.; Shea, Barry S.; Black, Katharine E.; Knipe, Rachel; Blati, Meryem; Baron, Murray; Wu, Brian; Fahmi, Hassan; Gandhi, Rajiv; Pardo, Annie; Selman, Moisés; Wu, Jiangping; Pelletier, Jean-Pierre; Martel-Pelletier, Johanne; Tager, Andrew M.; Kapoor, Mohit

    2017-01-01

    Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodelling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however the molecular mediators of myofibroblast activation remain to be fully identified. Here we identify soluble ephrin-B2 as a novel pro-fibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of soluble ephrin-B2 (sEphrin-B2) promotes fibroblast chemotaxis and activation via EphB3/EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a distintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 is induced by transforming growth factor-β1 (TGF-β1), and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1–induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10/sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors Eph3/Eph4, and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases. PMID:29058717

  14. In vitro and in vivo biotransformation of WMS-1410, a potent GluN2B selective NMDA receptor antagonist.

    PubMed

    Falck, Evamaria; Begrow, Frank; Verspohl, Eugen J; Wünsch, Bernhard

    2014-06-01

    Structural modification of the GluN2B selective NMDA receptor antagonist ifenprodil led to the 3-benzazepine WMS-1410 with similar GluN2B affinity but higher receptor selectivity. Herein the in vitro and in vivo biotransformation of WMS-1410 is reported. Incubation of WMS-1410 with rat liver microsomes and different cofactors resulted in four hydroxylated phase I metabolites, two phase II metabolites and five combined phase I/II metabolites. With exception of catechol 4, these metabolites were also identified in the urine of a rat treated with WMS-1410. However the metabolites 7, 8 and 12 clearly show that the catechol metabolite 4 was also formed in vivo. As shown for ifenprodil the phenol of WMS-1410 represents the metabolically most reactive structural element. The biotransformation of WMS-1410 is considerably slower than the biotransformation of ifenprodil indicating a higher metabolic stability. From the viewpoint of metabolic stability the bioisosteric replacement of the phenol of WMS-1410 by a metabolically more stable moiety should be favourable. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Deoxycholic Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Exacerbates DSS-Induced Colitis through Promoting Cathepsin B Release.

    PubMed

    Zhao, Shengnan; Gong, Zizhen; Du, Xixi; Tian, Chunyan; Wang, Lingyu; Zhou, Jiefei; Xu, Congfeng; Chen, Yingwei; Cai, Wei; Wu, Jin

    2018-01-01

    We recently have proved that excessive fecal DCA caused by high-fat diet may serve as an endogenous danger-associated molecular pattern to activate NLRP3 inflammasome and thus contributes to the development of inflammatory bowel disease (IBD). Moreover, the effect of DCA on inflammasome activation is mainly mediated through bile acid receptor sphingosine-1-phosphate receptor 2 (S1PR2); however, the intermediate process remains unclear. Here, we sought to explore the detailed molecular mechanism involved and examine the effect of S1PR2 blockage in a colitis mouse model. In this study, we found that DCA could dose dependently upregulate S1PR2 expression. Meanwhile, DCA-induced NLRP3 inflammasome activation is at least partially achieved through stimulating extracellular regulated protein kinases (ERK) signaling pathway downstream of S1PR2 followed by promoting of lysosomal cathepsin B release. DCA enema significantly aggravated DSS-induced colitis in mice and S1PR2 inhibitor as well as inflammasome inhibition by cathepsin B antagonist substantially reducing the mature IL-1 β production and alleviated colonic inflammation superimposed by DCA. Therefore, our findings suggest that S1PR2/ERK1/2/cathepsin B signaling plays a critical role in triggering inflammasome activation by DCA and S1PR2 may represent a new potential therapeutic target for the management of intestinal inflammation in individuals on a high-fat diet.

  16. A2A Adenosine Receptor Antagonists as Therapeutic Candidates: are they still an interesting challenge?

    PubMed

    Cacciari, Barbara; Federico, Stephanie; Spalluto, Giampiero

    2018-04-22

    In the past decades, many efforts were done to develope ligands for the adenosine receptors, with the purpose to individuate agonists and antagonists affine and selective for each subtypes , named A1, A2A, A2B, and A3. These intense studies allowed a deeper and deeper knowledge of the nature and, moreover, of the pathophysiological roles of all the adenosine receptor subtypes. In particular, the involvment of the A2A adenosine receptor subtype in some physiological mechanisms in the brain, that could be related to important diseases such as the Parkinson's disease, encouraged the research in this field. Particular attention was given to the antagonists endowed with high affinity and selectivity since they could have a real employment in the treatment of Parkinson's disease, and some compounds, such as istradefylline, preladenant and tozadenant, are already studied in clinical trials. Actually, the role of A2A antagonists in Parkinson's disease is becoming contradictory due to contrasting results in the last studies, but, at the same time, new possible employments are emerging for this class of antagonists in cancer pathologies as much interesting to legitimate further efforts in the research of A2A ligands. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Pwp2 mediates UTP-B assembly via two structurally independent domains.

    PubMed

    Boissier, Fanny; Schmidt, Christina Maria; Linnemann, Jan; Fribourg, Sébastien; Perez-Fernandez, Jorge

    2017-06-09

    The SSU processome constitutes a large ribonucleoprotein complex involved in the early steps of ribosome biogenesis. UTP-B is one of the first multi-subunit protein complexes that associates with the pre-ribosomal RNA to form the SSU processome. To understand the molecular basis of the hierarchical assembly of the SSU-processome, we have undergone a structural and functional analysis of the UTP-B subunit Pwp2p. We show that Pwp2p is required for the proper assembly of UTP-B and for a productive association of UTP-B with pre-rRNA. These two functions are mediated by two distinct structural domains. The N-terminal domain of Pwp2p folds into a tandem WD-repeat (tWD) that associates with Utp21p, Utp18p, and Utp6p to form a core complex. The CTDs of Pwp2p and Utp21p mediate the assembly of the heterodimer Utp12p:Utp13p that is required for the stable incorporation of the UTP-B complex in the SSU processome. Finally, we provide evidence suggesting a role of UTP-B as a platform for the binding of assembly factors during the maturation of 20S rRNA precursors.

  18. An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

    PubMed

    Ovigne, J M; Vermot-Desroches, C; Lecron, J C; Portier, M; Lupker, J; Pecceu, F; Wijdenes, J

    1998-06-01

    The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

  19. Unprecedented Therapeutic Potential with a Combination of A2A/NR2B Receptor Antagonists as Observed in the 6-OHDA Lesioned Rat Model of Parkinson's Disease

    PubMed Central

    Michel, Anne; Downey, Patrick; Nicolas, Jean-Marie; Scheller, Dieter

    2014-01-01

    In Parkinson's disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients. PMID:25513815

  20. A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

    PubMed

    Kargl, Julia; Brown, Andrew J; Andersen, Liisa; Dorn, Georg; Schicho, Rudolf; Waldhoer, Maria; Heinemann, Akos

    2013-07-01

    The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

  1. The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jaakola, Veli-Pekka; Griffith, Mark T.; Hanson, Michael A.

    2009-01-15

    The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A{sub 2A} adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extendedmore » conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.« less

  2. Design and Synthesis of a Series of l-trans-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X-ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2S,4R)-4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid.

    PubMed

    Krogsgaard-Larsen, Niels; Delgar, Claudia G; Koch, Karina; Brown, Patricia M G E; Møller, Charlotte; Han, Liwei; Huynh, Tri H V; Hansen, Stinne W; Nielsen, Birgitte; Bowie, Derek; Pickering, Darryl S; Kastrup, Jette Sandholm; Frydenvang, Karla; Bunch, Lennart

    2017-01-12

    Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (K i = 4 μM). In a functional assay, 1b displayed full antagonist activity with IC 50 = 6 ± 2 μM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13-14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure-activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

  3. β 1 Adrenoceptor antagonistic effects of the supposedly selective β 2 adrenoceptor antagonist ICI 118,551 on the positive inotropic effect of adrenaline in murine hearts.

    PubMed

    Pecha, Simon; Flenner, Frederik; Söhren, Klaus-Dieter; Lorenz, Kristina; Eschenhagen, Thomas; Christ, Torsten

    2015-10-01

    Studies on the relative contribution of β 1- and β 2-adrenoceptors (AR) generally employ selective β 1- and β 2-AR antagonists such as CGP 20712A and ICI 118,551, respectively, and assume that antagonism by one of these compounds indicates mediation by the respective AR subtype. Here, we evaluated the β 2-AR-selectivity of ICI 118,551 in ventricular muscle strips of transgenic mice lacking β 1-AR (β 1-KO), β 2-AR (β 2-KO), or both (β 1/β 2-KO). Strips were electrically driven and force development was measured. In wild type (WT), ICI 118,551 (100 nmol/L) shifted the concentration-response curve (CRC) for adrenaline by about 0.5 log units to the right, corresponding to the known affinity of ICI 118,551 to β 1-AR but not to β 2-AR. Conversely, the phosphodiesterase inhibitor rolipram (10 μmol/L) shifted the CRC to the left, but did not enlarge the ICI 118,551 shift, indicating exclusive β 1-AR mediation even when PDE4 is inactive. In line with this, rolipram and ICI 118,551 had similar effects in β 2-KO than in WT. In contrast, β 1-KO did not show any inotropic reaction to adrenaline (+/- rolipram). In WT, the β 1-AR selective antagonist CGP 20712A (100 nmol/L) shifted the CRC for isoprenaline by 2.1 log units, corresponding to the affinity of CGP 20712A to β 1-AR. Rolipram increased the sensitivity to adrenaline independently of the presence of CGP 20712A. We conclude that effects sensitive to the β 2-AR antagonist ICI 118,551 are not necessarily β 2-AR-mediated and CGP 20712A-resistant effects cannot be simply interpreted as β 2-AR-mediated. Catecholamine effects in murine ventricles strictly depend on β 1-AR, even if PDE 4 is blocked.

  4. β1 Adrenoceptor antagonistic effects of the supposedly selective β2 adrenoceptor antagonist ICI 118,551 on the positive inotropic effect of adrenaline in murine hearts

    PubMed Central

    Pecha, Simon; Flenner, Frederik; Söhren, Klaus-Dieter; Lorenz, Kristina; Eschenhagen, Thomas; Christ, Torsten

    2015-01-01

    Studies on the relative contribution of β1- and β2-adrenoceptors (AR) generally employ selective β1- and β2-AR antagonists such as CGP 20712A and ICI 118,551, respectively, and assume that antagonism by one of these compounds indicates mediation by the respective AR subtype. Here, we evaluated the β2-AR-selectivity of ICI 118,551 in ventricular muscle strips of transgenic mice lacking β1-AR (β1-KO), β2-AR (β2-KO), or both (β1/β2-KO). Strips were electrically driven and force development was measured. In wild type (WT), ICI 118,551 (100 nmol/L) shifted the concentration–response curve (CRC) for adrenaline by about 0.5 log units to the right, corresponding to the known affinity of ICI 118,551 to β1-AR but not to β2-AR. Conversely, the phosphodiesterase inhibitor rolipram (10 μmol/L) shifted the CRC to the left, but did not enlarge the ICI 118,551 shift, indicating exclusive β1-AR mediation even when PDE4 is inactive. In line with this, rolipram and ICI 118,551 had similar effects in β2-KO than in WT. In contrast, β1-KO did not show any inotropic reaction to adrenaline (+/− rolipram). In WT, the β1-AR selective antagonist CGP 20712A (100 nmol/L) shifted the CRC for isoprenaline by 2.1 log units, corresponding to the affinity of CGP 20712A to β1-AR. Rolipram increased the sensitivity to adrenaline independently of the presence of CGP 20712A. We conclude that effects sensitive to the β2-AR antagonist ICI 118,551 are not necessarily β2-AR-mediated and CGP 20712A-resistant effects cannot be simply interpreted as β2-AR-mediated. Catecholamine effects in murine ventricles strictly depend on β1-AR, even if PDE 4 is blocked. PMID:26516580

  5. Investigation on Quantitative Structure Activity Relationships and Pharmacophore Modeling of a Series of mGluR2 Antagonists

    PubMed Central

    Zhang, Meng-Qi; Zhang, Xiao-Le; Li, Yan; Fan, Wen-Jia; Wang, Yong-Hua; Hao, Ming; Zhang, Shu-Wei; Ai, Chun-Zhi

    2011-01-01

    MGluR2 is G protein-coupled receptor that is targeted for diseases like anxiety, depression, Parkinson’s disease and schizophrenia. Herein, we report the three-dimensional quantitative structure–activity relationship (3D-QSAR) studies of a series of 1,3-dihydrobenzo[ b][1,4]diazepin-2-one derivatives as mGluR2 antagonists. Two series of models using two different activities of the antagonists against rat mGluR2, which has been shown to be very similar to the human mGluR2, (activity I: inhibition of [3H]-LY354740; activity II: mGluR2 (1S,3R)-ACPD inhibition of forskolin stimulated cAMP.) were derived from datasets composed of 137 and 69 molecules respectively. For activity I study, the best predictive model obtained from CoMFA analysis yielded a Q2 of 0.513, R2 ncv of 0.868, R2 pred = 0.876, while the CoMSIA model yielded a Q2 of 0.450, R2 ncv = 0.899, R2 pred = 0.735. For activity II study, CoMFA model yielded statistics of Q2 = 0.5, R2 ncv = 0.715, R2 pred = 0.723. These results prove the high predictability of the models. Furthermore, a combined analysis between the CoMFA, CoMSIA contour maps shows that: (1) Bulky substituents in R7, R3 and position A benefit activity I of the antagonists, but decrease it when projected in R8 and position B; (2) Hydrophilic groups at position A and B increase both antagonistic activity I and II; (3) Electrostatic field plays an essential rule in the variance of activity II. In search for more potent mGluR2 antagonists, two pharmacophore models were developed separately for the two activities. The first model reveals six pharmacophoric features, namely an aromatic center, two hydrophobic centers, an H-donor atom, an H-acceptor atom and an H-donor site. The second model shares all features of the first one and has an additional acceptor site, a positive N and an aromatic center. These models can be used as guidance for the development of new mGluR2 antagonists of high activity and selectivity. This work is the first report on 3

  6. Investigation on quantitative structure activity relationships and pharmacophore modeling of a series of mGluR2 antagonists.

    PubMed

    Zhang, Meng-Qi; Zhang, Xiao-Le; Li, Yan; Fan, Wen-Jia; Wang, Yong-Hua; Hao, Ming; Zhang, Shu-Wei; Ai, Chun-Zhi

    2011-01-01

    MGluR2 is G protein-coupled receptor that is targeted for diseases like anxiety, depression, Parkinson's disease and schizophrenia. Herein, we report the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a series of 1,3-dihydrobenzo[ b][1,4]diazepin-2-one derivatives as mGluR2 antagonists. Two series of models using two different activities of the antagonists against rat mGluR2, which has been shown to be very similar to the human mGluR2, (activity I: inhibition of [(3)H]-LY354740; activity II: mGluR2 (1S,3R)-ACPD inhibition of forskolin stimulated cAMP.) were derived from datasets composed of 137 and 69 molecules respectively. For activity I study, the best predictive model obtained from CoMFA analysis yielded a Q(2) of 0.513, R(2) (ncv) of 0.868, R(2) (pred) = 0.876, while the CoMSIA model yielded a Q(2) of 0.450, R(2) (ncv) = 0.899, R(2) (pred) = 0.735. For activity II study, CoMFA model yielded statistics of Q(2) = 0.5, R(2) (ncv) = 0.715, R(2) (pred) = 0.723. These results prove the high predictability of the models. Furthermore, a combined analysis between the CoMFA, CoMSIA contour maps shows that: (1) Bulky substituents in R(7), R(3) and position A benefit activity I of the antagonists, but decrease it when projected in R(8) and position B; (2) Hydrophilic groups at position A and B increase both antagonistic activity I and II; (3) Electrostatic field plays an essential rule in the variance of activity II. In search for more potent mGluR2 antagonists, two pharmacophore models were developed separately for the two activities. The first model reveals six pharmacophoric features, namely an aromatic center, two hydrophobic centers, an H-donor atom, an H-acceptor atom and an H-donor site. The second model shares all features of the first one and has an additional acceptor site, a positive N and an aromatic center. These models can be used as guidance for the development of new mGluR2 antagonists of high activity and selectivity

  7. Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

    PubMed

    Thomsen, Morgane; Caine, Simon Barak

    2016-04-05

    Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. [11C]AZ10419096 - a full antagonist PET radioligand for imaging brain 5-HT1B receptors.

    PubMed

    Lindberg, Anton; Nag, Sangram; Schou, Magnus; Takano, Akihiro; Matsumoto, Junya; Amini, Nahid; Elmore, Charles S; Farde, Lars; Pike, Victor W; Halldin, Christer

    2017-11-01

    The serotonergic system is widely present in all regions of the central nervous system (CNS) and plays a key modulatory role in many of its functions. Positron emission tomography (PET) is used to study several serotonin receptors in CNS in vivo. The G-protein coupled receptor 5-HT 1B is mostly present in the occipital cortex and in midbrain and is linked to several psychiatric disorders. There is evidence that agonist PET radioligands for neuroreceptors are more sensitive to endogenous neurotransmitters than antagonists. Our previously developed 5-HT 1B receptor PET radioligand, [ 11 C]AZ10419369, is now considered a partial agonist. In this work we are aiming to develop a full antagonist PET radioligand for imaging brain 5-HT 1B receptors, and evaluate its sensitivity to increased endogenous serotonin concentration. [ 11 C]AZ10419096 was synthesized by rapid methylation of the prepared corresponding N-desmethyl precursor with [ 11 C]methyl triflate. Five PET measurements were performed in cynomolgus monkeys, consisting of two at baseline, one after treatment of a monkey with a 5-HT 1B antagonist, AR-A000002, and two in which fenfluramine was administered during scanning to induce endogenous serotonin release. [ 11 C]AZ10419096 was synthesized in high yield and purity within 30 min, including purification, formulation and sterile filtration. The baseline PET measurements demonstrated [ 11 C]AZ10419096 to have favorable radioligand characteristics, including high specific binding in brain regions that have high 5-HT 1B density, such as occipital cortex and globus pallidus, as well as subsequent rapid elimination from brain and a minor abundance of lipophilic radiometabolites in plasma. AR-A00002 completely blocked radioligand receptor-specific binding. Fenfluramine produced a distinct displacement of radioligand consistent with an expected increase of synaptic endogenous serotonin concentration. [ 11 C]AZ10419096, a full 5-HT 1B antagonist PET radioligand

  9. Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells.

    PubMed

    Lefler, David; Mukhin, Yurii V; Pettus, Tobiah; Leeb-Lundberg, L M Fredrik; Garnovskaya, Maria N; Raymond, John R

    2003-04-01

    Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.

  10. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function

    PubMed Central

    Sato, Kazuya; Feng, Xingmin; Chen, Jichun; Li, Jungang; Muranski, Pawel; Desierto, Marie J.; Keyvanfar, Keyvan; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S.

    2016-01-01

    Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically “fatless” mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation. PMID:26589913

  11. Testing a spin-2 mediator by angular observables in b →s μ+μ-

    NASA Astrophysics Data System (ADS)

    Fajfer, Svjetlana; Melić, Blaženka; Patra, Monalisa

    2018-05-01

    We consider the effects of the spin-2 particle in the b →s μ+μ- transition assuming that the spin-2 particle couples in a flavor-nonuniversal way to b and s quarks and in the leptonic sector couples only to the muons, thereby only contributing to the process b →s μ+μ-. The Bs-B¯s transition gives the strong constraint on the coupling of the spin-2 mediator and b and s quarks, while the observed discrepancy from the standard model prediction for the muon anomalous magnetic moment (g -2 )μ serves to constrain the μ coupling to a spin-2 particle. We find that the spin-2 particle can modify the angular observables in the B →K μ+μ- and B →K*μ+μ- decays and produce effects that do not exist in the standard model. The generated forward-backward asymmetries in these processes can reach 15%, while other observables for these decays receive tiny effects.

  12. Antagonist interaction with the human 5-HT7 receptor mediates the rapid and potent inhibition of non-G-protein-stimulated adenylate cyclase activity: a novel GPCR effect

    PubMed Central

    Klein, MT; Teitler, M

    2011-01-01

    BACKGROUND AND PURPOSE The human 5-hydroxytryptamine7 (h5-HT7) receptor is Gs-coupled and stimulates the production of the intracellular signalling molecule cAMP. Previously, we reported a novel property of the h5-HT7 receptor: pseudo-irreversible antagonists irreversibly inhibit forskolin-stimulated (non-receptor-mediated) cAMP production. Herein, we sought to determine if competitive antagonists also affect forskolin-stimulated activity and if this effect is common among other Gs-coupled receptors. EXPERIMENTAL APPROACH Recombinant cell lines expressing h5-HT7 receptors or other receptors of interest were briefly exposed to antagonists; cAMP production was then stimulated by forskolin and quantified by an immunocompetitive assay. KEY RESULTS In human embryonic kidney 293 cells stably expressing h5-HT7 receptors, all competitive antagonists inhibited nearly 100% of forskolin-stimulated cAMP production. This effect was insensitive to pertussis toxin, that is, not Gi/o-mediated. Potency to inhibit forskolin-stimulated activity strongly correlated with h5-HT7 binding affinity (r2= 0.91), indicating that the antagonists acted through h5-HT7 receptors to inhibit forskolin. Potency and maximal effects of clozapine, a prototypical competitive h5-HT7 antagonist, were unaffected by varying forskolin concentration. Antagonist interaction with h5-HT6, human β1, β2, and β3 adrenoceptors did not inhibit forskolin's activity. CONCLUSIONS AND IMPLICATIONS The inhibition of adenylate cyclase, as measured by forskolin's activity, is an underlying property of antagonist interaction with h5-HT7 receptors; however, this is not a common property of other Gs-coupled receptors. This phenomenon may be involved in the roles played by h5-HT7 receptors in human physiology. Development of h5-HT7 antagonists that do not elicit this effect would aid in the elucidation of its mechanisms and shed light on its possible physiological relevance. PMID:21198551

  13. [Leukotriene antagonists: a new approach in the treatment of asthma].

    PubMed

    Devillier, P; Bessard, G; Advenier, C

    1997-06-01

    Inflammation plays an essential role in the genesis of airflow obstruction and bronchial hyper-reactivity in the early stages of clinical asthma. The treatment of bronchial inflammation has become an essential element in the therapeutic strategy and principally rests on inhaled glucocorticoids. Amongst a number of inflammatory mediators leukotrienes occupy a privileged place by the power of their inflammatory and constrictor effects on bronchial smooth muscles. These properties have justified the clinical development of inhibitors of their synthesis and of specific antagonists to their receptors. Leukotriene antagonists are specific for a sub type of leukotriene receptors C4, D4 and E4 which is implicated in the majority of the bronchial constrictor and inflammatory effects of leukotrienes. The antagonists of Cys-LT1 receptor but also the inhibitors of the leukotriene synthesis exert an additive bronchodilator effect to those of B2 stimulants confirming an efficacious protection vis a vis bronchial provocation tests and above all they improve the clinical scores, lung function and also enable a decrease in the consumption of beta 2 agonists. The marketing of these products represents a major event because it corresponds to the advent of a new therapeutic class. The ease of administration by the oral route, their demonstrated efficacy and their good tolerance profile (in particular for ICI 204.219, and antagonists to Cys-LT1 receptors) are elements which foresee a success for this new asthmatic treatment. However numerous studies, notably comparative studies vis a vis reference treatments will be necessary to define their place in the strategic approach to the treatment of asthma.

  14. A2B Adenosine Receptor–Mediated Induction of IL-6 Promotes CKD

    PubMed Central

    Dai, Yingbo; Zhang, Weiru; Wen, Jiaming; Zhang, Yujin; Kellems, Rodney E.

    2011-01-01

    Chronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, can exacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here, mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase (ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol–modified ADA to reduce adenosine levels and the inhibition of the A2B adenosine receptor (A2BR) attenuated renal fibrosis and dysfunction. Furthermore, activation of A2BR promoted renal fibrosis in both mice infused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). These three mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggesting that they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharmacologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renal fibrosis downstream of A2BR. Taken together, these data suggest that A2BR-mediated induction of IL-6 contributes to renal fibrogenesis and shows potential therapeutic targets for CKD. PMID:21511827

  15. Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a, V2 and V1b receptor ligands.

    PubMed

    Serradeil-Le Gal, C; Wagnon, J; Valette, G; Garcia, G; Pascal, M; Maffrand, J P; Le Fur, G

    2002-01-01

    with ascites and impaired renal function, a 10-day oral treatment with SR121463 (0.5 mg/kg) totally corrected hyponatremia and restored normal urine excretion. This compound also displayed interesting new properties in a rabbit model of ocular hypertension, decreasing intraocular pressure after single or repeated instillation. Thus, V2 receptor blockade could be of interest in several water-retaining diseases such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), liver cirrhosis and congestive heart failure and deserves to be widely explored. Finally, further chemical developments in the oxindole family have led to the first specific and orally active V1b receptor antagonists (with SSR149415 as a representative), an awaited class of drugs with expected therapeutic interest mainly in ACTH-secreting tumors and various emotional diseases such as stress-related disorders, anxiety and depression. However, from the recently described tissue localization for this receptor, we could also speculate on other unexpected uses. In conclusion, the development of AVP receptor antagonists is a field of intensive pharmacological and clinical investigation. Selective and orally active compounds are now available to give new insight into the pathophysiological role of AVP and to provide promising drugs.

  16. The scavenger activity of the human P2X7 receptor differs from P2X7 pore function by insensitivity to antagonists, genetic variation and sodium concentration: Relevance to inflammatory brain diseases.

    PubMed

    Ou, Amber; Gu, Ben J; Wiley, James S

    2018-04-01

    Activation of P2X7 receptors is widely recognised to initiate proinflammatory responses. However P2X7 also has a dual function as a scavenger receptor which is active in the absence of ATP and plasma proteins and may be important in central nervous system (CNS) diseases. Here, we investigated both P2X7 pore formation and its phagocytic function in fresh human monocytes (as a model of microglia) by measuring ATP-induced ethidium dye uptake and fluorescent bead uptake respectively. This was studied in monocytes expressing various polymorphic variants as well as in the presence of different P2X7 antagonists and ionic media. P2X7-mediated phagocytosis was found to account for about half of Latrunculin (or Cytochalasin D)-sensitive bead engulfment by fresh human monocytes. Monocytes harbouring P2X7 Ala348Thr or Glu496Ala polymorphic variants showed increase or loss of ethidium uptake respectively, but these changes in pore formation did not always correspond to the changes in phagocytosis of YG beads. Unlike pore function, P2X7-mediated phagocytosis was not affected by three potent selective P2X7 antagonists and remained identical in Na + and K + media. Taken together, our results show that P2X7 is a scavenger receptor with important function in the CNS but its phagocytic function has features distinct from its pore function. Both P2X7 pore formation and P2X7-mediated phagocytosis should be considered in the design of new P2X7 antagonists for the treatment of CNS diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

    PubMed Central

    Fox, Alyson; Kaur, Satbir; Li, Bifang; Panesar, Moh; Saha, Uma; Davis, Clare; Dragoni, Ilaria; Colley, Sian; Ritchie, Tim; Bevan, Stuart; Burgess, Gillian; McIntyre, Peter

    2005-01-01

    We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (Ki 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (Ki 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB1-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man. PMID:15685199

  18. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

    PubMed Central

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

    2015-01-01

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

  19. Pharmacophore-based virtual screening, biological evaluation and binding mode analysis of a novel protease-activated receptor 2 antagonist

    NASA Astrophysics Data System (ADS)

    Cho, Nam-Chul; Seo, Seoung-Hwan; Kim, Dohee; Shin, Ji-Sun; Ju, Jeongmin; Seong, Jihye; Seo, Seon Hee; Lee, Iiyoun; Lee, Kyung-Tae; Kim, Yun Kyung; No, Kyoung Tai; Pae, Ae Nim

    2016-08-01

    Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe2435.39 is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na+ allosteric site in contrast to PAR2 agonist that showed Na+ relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.

  20. Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.

    PubMed

    Meyer, Sharon A; Jirtle, Randy L

    2013-05-07

    The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.

  1. Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

    PubMed

    Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

    2018-06-01

    The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

  2. A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

    PubMed Central

    Coleman, Carrie B.; McGraw, Jennifer E.; Feldman, Emily R.; Roth, Alexa N.; Keyes, Lisa R.; Grau, Katrina R.; Cochran, Stephanie L.; Waldschmidt, Thomas J.; Liang, Chengyu; Forrest, J. Craig; Tibbetts, Scott A.

    2014-01-01

    Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the

  3. LSD and DOB: interaction with 5-HT2A receptors to inhibit NMDA receptor-mediated transmission in the rat prefrontal cortex.

    PubMed

    Arvanov, V L; Liang, X; Russo, A; Wang, R Y

    1999-09-01

    Both the phenethylamine hallucinogen (-)-1-2, 5-dimethoxy-4-bromophenyl-2-aminopropane (DOB), a selective serotonin 5-HT2A,2C receptor agonist, and the indoleamine hallucinogen D-lysergic acid diethylamide (LSD, which binds to 5-HT1A, 1B, 1D, 1E, 1F, 2A, 2C, 5, 6, 7, dopamine D1 and D2, and alpha1 and alpha2 adrenergic receptors), but not their non-hallucinogenic congeners, inhibited N-methyl-D-aspartate (NMDA)-induced inward current and NMDA receptor-mediated synaptic responses evoked by electrical stimulation of the forceps minor in pyramidal cells of the prefrontal cortical slices. The inhibitory effect of hallucinogens was mimicked by 5-HT in the presence of selective 5-HT1A and 5-HT3 receptor antagonists. The inhibitory action of DOB, LSD and 5-HT on the NMDA transmission was blocked by the 5-HT2A receptor antagonists R-(+)-alpha-(2, 3-dimethoxyphenil)-1-[4-fluorophenylethyl]-4-piperidineme thanol (M100907) and ketanserin. However, at low concentrations, when both LSD and DOB by themselves only partially depressed the NMDA response, they blocked the inhibitory effect of 5-HT, suggesting a partial agonist action. Whereas N-(4-aminobutyl)-5-chloro-2-naphthalenesulphonamide (W-7, a calmodulin antagonist) and N-[2-[[[3-(4'-chlorophenyl)- 2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxy-b enzenesulphonamide phosphate (KN-93, a Ca2+/CaM-KII inhibitor), but not the negative control 2-[N-4'methoxybenzenesulphonyl]amino-N-(4'-chlorophenyl)-2-propeny l-N -methylbenzylamine phosphate (KN-92), blocked the inhibitory action of LSD and DOB, the selective protein kinase C inhibitor chelerythrine was without any effect. We conclude that phenethylamine and indoleamine hallucinogens may exert their hallucinogenic effect by interacting with 5-HT2A receptors via a Ca2+/CaM-KII-dependent signal transduction pathway as partial agonists and modulating the NMDA receptors-mediated sensory, perceptual, affective and cognitive processes.

  4. Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications.

    PubMed

    Lambrecht, G

    2000-11-01

    P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.

  5. Role of humoral mediators in, and influence of a liposomal formulation on, acute amphotericin B nephrotoxicity.

    PubMed

    Sabra, R; Zeinoun, N; Sharaf, L H; Ghali, R; Beshara, G; Serhal, H

    2001-04-01

    The mechanisms responsible for amphotericin B nephrotoxicity remain incompletely understood, but clearly involve reduction in renal blood flow and glomerular filtration rate. Both direct effects of amphotericin B on contractile vascular cells, and indirect effects, due to humoural mediators, have been proposed. This study examines the role of nitric oxide, endothelin and angiotensin II in the acute nephrotoxic effects of amphotericin B in rats, and compares the anti-fungal and nephrotoxic effects of liposomal amphotericin B and amphotericin B-deoxycholate. Anaesthetized rats were given infusions of amphotericin B-deoxycholate in the presence or absence of N-nitro-L-arginine, PD 145065, a non-specific endothelin receptor antagonist, and L-158809, an angiotensin II type I receptor antagonist, or increasing doses of liposomal amphotericin B. Amphotericin B-deoxycholate (0.03 mg/kg/min intravenously) caused a significant 44% reduction in glomerular filtration rate and 65% maximal fall in renal blood flow. N-Nitro-L-arginine-treated rats had a lower renal blood flow and glomerular filtration rate at baseline, but sustained similar reduction of 53% and 75% in these parameters, respectively. PD145065 and L-158809 did not modify these effects either. Increasing doses of liposomal amphotericin B (from 0.01 up to 0.50 mg/kg/min.) induced no change in either glomerular filtration rate or renal blood flow. In vitro susceptibility tests revealed similar potency for liposomal amphotericin B and amphotericin B-deoxycholate in their fungistatic effects and slightly higher potency for amphotericin B-deoxycholate in their fungicidal effect. These results suggest that endogenous endothelin, angiotensin II or nitric oxide systems are not involved in the nephrotoxic effects of amphotericin B. The liposomal amphotericin B results suggest that amphotericin B nephrotoxicity is due to a direct interaction of amphotericin B with renal cells that is prevented by its encapsulation in

  6. 8-Substituted 1,3-dimethyltetrahydropyrazino[2,1-f]purinediones: Water-soluble adenosine receptor antagonists and monoamine oxidase B inhibitors.

    PubMed

    Brunschweiger, Andreas; Koch, Pierre; Schlenk, Miriam; Rafehi, Muhammad; Radjainia, Hamid; Küppers, Petra; Hinz, Sonja; Pineda, Felipe; Wiese, Michael; Hockemeyer, Jörg; Heer, Jag; Denonne, Frédéric; Müller, Christa E

    2016-11-01

    Multitarget approaches, i.e., addressing two or more targets simultaneously with a therapeutic agent, are hypothesized to offer additive therapeutic benefit for the treatment of neurodegenerative diseases. Validated targets for the treatment of Parkinson's disease are, among others, the A 2A adenosine receptor (AR) and the enzyme monoamine oxidase B (MAO-B). Additional blockade of brain A 1 ARs may also be beneficial. We recently described 8-benzyl-substituted tetrahydropyrazino[2,1-f]purinediones as a new lead structure for the development of such multi-target drugs. We have now designed a new series of tetrahydropyrazino[2,1-f]purinediones to extensively explore their structure-activity-relationships. Several compounds blocked human and rat A 1 and A 2A ARs at similar concentrations representing dual A 1 /A 2A antagonists with high selectivity versus the other AR subtypes. Among the best dual A 1 /A 2A AR antagonists were 8-(3-(4-chlorophenyl)propyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (41, K i human A 1 : 65.5nM, A 2A : 230nM; K i rat A 1 : 352nM, A 2A : 316nM) and 1,3-dimethyl-8-((2-(thiophen-2-yl)thiazol-4-yl)methyl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (57, K i human A 1 : 642nM, A 2A : 203nM; K i rat A 1 : 166nM, A 2A : 121nM). Compound 57 was found to be well water-soluble (0.7mg/mL) at a physiological pH value of 7.4. One of the new compounds showed triple-target inhibition: (R)-1,3-dimethyl-8-(2,1,3,4-tetrahydronaphthalen-1-yl)-6,7,8,9-tetrahydropyrazino[2,1-f]purine-2,4(1H,3H)-dione (49) was about equipotent at A 1 and A 2A ARs and at MAO-B (K i human A 1 : 393nM, human A 2A : 595nM, IC 50 human MAO-B: 210nM) thus allowing future in vivo explorations of the intended multi-target approach. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

    PubMed

    Joseph, Lauren; Thomsen, Morgane

    2017-06-30

    Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism.

    PubMed

    Angus, James A; Hughes, Richard J A; Wright, Christine E

    2017-12-01

    Dual endothelin ET A and ET B receptor antagonists are approved therapy for pulmonary artery hypertension (PAH). We hypothesized that ET B receptor-mediated clearance of endothelin-1 at specific vascular sites may compromise this targeted therapy. Concentration-response curves (CRC) to endothelin-1 or the ET B agonist sarafotoxin S6c were constructed, with endothelin receptor antagonists, in various rat and mouse isolated arteries using wire myography or in rat isolated trachea. In rat small mesenteric arteries, bosentan displaced endothelin-1 CRC competitively indicative of ET A receptor antagonism. In rat small pulmonary arteries, bosentan 10 μmol L -1 left-shifted the endothelin-1 CRC, demonstrating potentiation consistent with antagonism of an ET B receptor-mediated endothelin-1 clearance mechanism. Removal of endothelium or L-NAME did not alter the EC 50 or Emax of endothelin-1 nor increase the antagonism by BQ788. In the presence of BQ788 and L-NAME, bosentan displayed ET A receptor antagonism. In rat trachea (ET B ), bosentan was a competitive ET B antagonist against endothelin-1 or sarafotoxin S6c. Modeling showed the importance of dual receptor antagonism where the potency ratio of ET A to ET B antagonism is close to unity. In conclusion, the rat pulmonary artery is an example of a special vascular bed where the resistance to antagonism of endothelin-1 constriction by ET dual antagonists, such as bosentan or the ET B antagonist BQ788, is possibly due to the competition of potentiation of endothelin-1 by blockade of ET B -mediated endothelin-1 clearance located on smooth muscle and antagonism of ET A - and ET B -mediated contraction. This conclusion may have direct application for the efficacy of endothelin-1 antagonists for treating PAH. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  9. Association between ALDH2 and ADH1B polymorphisms, alcohol drinking and gastric cancer: a replication and mediation analysis.

    PubMed

    Ishioka, Kuka; Masaoka, Hiroyuki; Ito, Hidemi; Oze, Isao; Ito, Seiji; Tajika, Masahiro; Shimizu, Yasuhiro; Niwa, Yasumasa; Nakamura, Shigeo; Matsuo, Keitaro

    2018-04-03

    Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms have a strong impact on carcinogenic acetaldehyde accumulation after alcohol drinking. To date, however, evidence for a significant ALDH2-alcohol drinking interaction and a mediation effect of ALDH2/ADH1B through alcohol drinking on gastric cancer have remained unclear. We conducted two case-control studies to validate the interaction and to estimate the mediation effect on gastric cancer. We calculated odds ratios (OR) and 95% confidence intervals (CI) for ALDH2/ADH1B genotypes and alcohol drinking using conditional logistic regression models after adjustment for potential confounding in the HERPACC-2 (697 cases and 1372 controls) and HERPACC-3 studies (678 cases and 678 controls). We also conducted a mediation analysis of the combination of the two studies to assess whether the effects of these polymorphisms operated through alcohol drinking or through other pathways. ALDH2 Lys alleles had a higher risk with increased alcohol consumption compared with ALDH2 Glu/Glu (OR for heavy drinking, 3.57; 95% CI 2.04-6.27; P for trend = 0.007), indicating a significant ALDH2-alcohol drinking interaction (P interaction  = 0.024). The mediation analysis indicated a significant positive direct effect (OR 1.67; 95% CI 1.38-2.03) and a protective indirect effect (OR 0.84; 95% CI 0.76-0.92) of the ALDH2 Lys alleles with the ALDH2-alcohol drinking interaction. No significant association of ADH1B with gastric cancer was observed. The observed ALDH2-alcohol drinking interaction and the direct effect of ALDH2 Lys alleles may suggest the involvement of acetaldehyde in the development of gastric cancer.

  10. Attenuation in rats of impairments of memory by scopolamine, a muscarinic receptor antagonist, by mecamylamine, a nicotinic receptor antagonist.

    PubMed

    Newman, L A; Gold, P E

    2016-03-01

    Scopolamine, a muscarinic antagonist, impairs learning and memory for many tasks, supporting an important role for the cholinergic system in these cognitive functions. The findings are most often interpreted to indicate that a decrease in postsynaptic muscarinic receptor activation mediates the memory impairments. However, scopolamine also results in increased release of acetylcholine in the brain as a result of blocking presynaptic muscarinic receptors. The present experiments assess whether scopolamine-induced increases in acetylcholine release may impair memory by overstimulating postsynaptic cholinergic nicotinic receptors, i.e., by reaching the high end of a nicotinic receptor activation inverted-U dose-response function. Rats tested in a spontaneous alternation task showed dose-dependent working memory deficits with systemic injections of mecamylamine and scopolamine. When an amnestic dose of scopolamine (0.15 mg/kg) was co-administered with a subamnestic dose of mecamylamine (0.25 mg/kg), this dose of mecamylamine significantly attenuated the scopolamine-induced memory impairments. We next assessed the levels of acetylcholine release in the hippocampus in the presence of scopolamine and mecamylamine. Mecamylamine injections resulted in decreased release of acetylcholine, while scopolamine administration caused a large increase in acetylcholine release. These findings indicate that a nicotinic antagonist can attenuate impairments in memory produced by a muscarinic antagonist. The nicotinic antagonist may block excessive activation of nicotinic receptors postsynaptically or attenuate increases in acetylcholine release presynaptically. Either effect of a nicotinic antagonist-to decrease scopolamine-induced increases in acetylcholine output or to decrease postsynaptic acetylcholine receptor activation-may mediate the negative effects on memory of muscarinic antagonists.

  11. A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques.

    PubMed

    Peluffo, M C; Stanley, J; Braeuer, N; Rotgeri, A; Fritzemeier, K-H; Fuhrmann, U; Buchmann, B; Adevai, T; Murphy, M J; Zelinski, M B; Lindenthal, B; Hennebold, J D; Stouffer, R L

    2014-07-01

    and blood lipid profiles. Several mRNAs encoding proteins involved in PGE2 synthesis, metabolism and signaling increase (P < 0.05) in the periovulatory follicle after administration of an ovulatory hCG bolus. PGE2 signaling through PTGER2 induces cumulus cell expansion and production of hyaluronic acid, which are critical events for fertilization. Moreover, chronic administration of a selective PTGER2 antagonist resulted in a significant (P < 0.05 versus vehicle-treated controls) contraceptive effect without altering steroid hormone patterns or menstrual cyclicity during a 5-months contraceptive trial. Fertility recovered as early as 1 month after ending treatment. This is a proof-of-concept study in a non-human primate model. Further investigations are warranted to elucidate the mechanism(s) of PTGER2 antagonist action in the primate ovary. Although PTGER2 antagonist treatment did not produce any obvious undesirable effects, improvements in the mode of administration, as well as the efficacy of these compounds, are necessary to consider such a contraceptive for women. Monitoring as well as improving the efficacy and safety of female contraceptives is an important public health activity. Even though hormonal contraceptives are effective for women, concerns remain regarding their side-effects and long-term use because of the widespread actions of such steroidal products in many tissues. Moreover, some women cannot take hormones for medical reasons. Thus, development of non-hormonal contraceptives for women is warranted. Supported by Bayer HealthCare Pharmaceuticals, The Eunice Kennedy Shriver NICHD Contraceptive Development and Research Center (U54 HD055744), NIH Office of the Director (Oregon National Primate Research Center P51 OD011092), and a Lalor Foundation Postdoctoral Basic Research Fellowship (MCP). The use of the Leica confocal was supported by grant number S10RR024585. Some of the authors (N.B., A.R., K.-H.F., U.F., B.B. and B.L.) are employees of Bayer

  12. Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

    PubMed Central

    Maggi, C. A.; Patacchini, R.; Giuliani, S.; Rovero, P.; Dion, S.; Regoli, D.; Giachetti, A.; Meli, A.

    1990-01-01

    1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2167737

  13. Synthesis and serotonergic activity of variously substituted (3-amido)phenylpiperazine derivatives and benzothiophene-4-piperazine derivatives: novel antagonists for the vascular 5-HT1B receptor.

    PubMed

    Moloney, Gerard P; Garavelas, Agatha; Martin, Graeme R; Maxwell, Miles; Glen, Robert C

    2004-04-01

    The synthesis and vascular 5-HT(1B) receptor activity of a novel series of substituted 3-amido phenylpiperazine and 4-(4-methyl-1-piperazinyl)-1-benzo[b]thiophene derivatives is described. Modifications to the amido linked sidechains of the 3-amidophenyl-piperazine derivatives and to the 2-sidechain of the 1-benzo[b]thiophene derivatives have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B) receptor of pK(B) > 7.0. From the 3-amidophenyl-piperazine series, N-(4-(4-chlorophenyl)thiazol-2-yl-3-(4-methyl-1-piperazinyl)benzamide (30) and from the benzo[b]thiophene-4-piperazine series N-(2-ethylphenyl)-4-(4-methyl-1- piperazinyl)-1-benzo[b]thiophene-2-carboxamide (38) were identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B) receptor mediated agonist activity in the rabbit femoral artery) and competitive vascular 5-HT(1B) receptor antagonist. The affinity of compounds from these two series of compounds for the vascular 5-HT(1B) receptor is discussed as well as a proposed mode of binding to the receptor pharmacophore.

  14. Kinin B1 Receptor Promotes Neurogenic Hypertension Through Activation of Centrally Mediated Mechanisms.

    PubMed

    Sriramula, Srinivas; Lazartigues, Eric

    2017-12-01

    Hypertension is associated with increased activity of the kallikrein-kinin system. Kinin B1 receptor (B1R) activation leads to vasoconstriction and inflammation. Despite evidence supporting a role for the B1R in blood pressure regulation, the mechanisms by which B1R could alter autonomic function and participate in the pathogenesis of hypertension remain unidentified. We sought to explore whether B1R-mediated inflammation contributes to hypertension and investigate the molecular mechanisms involved. In this study, we tested the hypothesis that activation of B1R in the brain is involved in the pathogenesis of hypertension, using the deoxycorticosterone acetate-salt model of neurogenic hypertension in wild-type and B1R knockout mice. Deoxycorticosterone acetate-salt treatment in wild-type mice led to significant increases in B1R mRNA and protein levels and bradykinin levels, enhanced gene expression of carboxypeptidase N supporting an increase in the B1R ligand, associated with enhanced blood pressure, inflammation, sympathoexcitation, autonomic dysfunction, and impaired baroreflex sensitivity, whereas these changes were blunted or prevented in B1R knockout mice. B1R stimulation was further shown to involve activation of the ASK1-JNK-ERK1/2 and NF-κB pathways in the brain. To dismiss potential developmental alterations in knockout mice, we further used B1R blockade selectively in the brain of wild-type mice. Supporting the central origin of this mechanism, intracerebroventricular infusion of a specific B1R antagonist, attenuated the deoxycorticosterone acetate-salt-induced increase in blood pressure in wild-type mice. Our data provide the first evidence of a central role for B1R-mediated inflammatory pathways in the pathogenesis of deoxycorticosterone acetate-salt hypertension and offer novel insights into possible B1R-targeted therapies for the treatment of neurogenic hypertension. © 2017 American Heart Association, Inc.

  15. Novel spirotetracyclic zwitterionic dual H(1)/5-HT(2A) receptor antagonists for the treatment of sleep disorders.

    PubMed

    Gianotti, Massimo; Botta, Maurizio; Brough, Stephen; Carletti, Renzo; Castiglioni, Emiliano; Corti, Corrado; Dal-Cin, Michele; Delle Fratte, Sonia; Korajac, Denana; Lovric, Marija; Merlo, Giancarlo; Mesic, Milan; Pavone, Francesca; Piccoli, Laura; Rast, Slavko; Roscic, Maja; Sava, Anna; Smehil, Mario; Stasi, Luigi; Togninelli, Andrea; Wigglesworth, Mark J

    2010-11-11

    Histamine H(1) and serotonin 5-HT(2A) receptors mediate two different mechanisms involved in sleep regulation: H(1) antagonists are sleep inducers, while 5-HT(2A) antagonists are sleep maintainers. Starting from 9'a, a novel spirotetracyclic compound endowed with good H(1)/5-HT(2A) potency but poor selectivity, very high Cli, and a poor P450 profile, a specific optimization strategy was set up. In particular, we investigated the possibility of introducing appropriate amino acid moieties to optimize the developability profile of the series. Following this zwitterionic approach, we were able to identify several advanced leads (51, 65, and 73) with potent dual H(1)/5-HT(2A) activity and appropriate developability profiles. These compounds exhibited efficacy as hypnotic agents in a rat telemetric sleep model with minimal effective doses in the range 3-10 mg/kg po.

  16. Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

    PubMed

    Shlamkovich, Tomer; Aharon, Lidan; Barton, William A; Papo, Niv

    2017-05-16

    In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

  17. NMDA receptor antagonists inhibit catalepsy induced by either dopamine D1 or D2 receptor antagonists.

    PubMed

    Moore, N A; Blackman, A; Awere, S; Leander, J D

    1993-06-11

    In the present study, we investigated the ability of NMDA receptor antagonists to inhibit catalepsy induced by haloperidol, or SCH23390 and clebopride, selective dopamine D1 and D2 receptor antagonists respectively. Catalepsy was measured by recording the time the animal remained with its forepaws placed over a rod 6 cm above the bench. Pretreatment with either the non-competitive NMDA receptor antagonist, MK-801 (0.25-0.5 mg/kg i.p.) or the competitive antagonist, LY274614 (10-20 mg/kg i.p.) reduced the cataleptic response produced by haloperidol (10 mg/kg), SCH23390 (2.5-10 mg/kp i.p.) or clebopride (5-20 mg/kg i.p.). This demonstrates that NMDA receptor antagonists will reduce both dopamine D1 and D2 receptor antagonist-induced catalepsy. Muscle relaxant doses of chlordiazepoxide (10 mg/kg i.p.) failed to reduce the catalepsy induced by haloperidol, suggesting that the anticataleptic effect of the NMDA receptor antagonists was not due to a non-specific action. These results support the hypothesis that NMDA receptor antagonists may have beneficial effects in disorders involving reduced dopaminergic function, such as Parkinson's disease.

  18. MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist.

    PubMed

    Cialdai, Cecilia; Tramontana, Manuela; Patacchini, Riccardo; Lecci, Alessandro; Catalani, Claudio; Catalioto, Rose-Marie; Meini, Stefania; Valenti, Claudio; Altamura, Maria; Giuliani, Sandro; Maggi, Carlo Alberto

    2006-11-07

    The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2a dose-related and persistent manner colon contractions induced by the selective tachykinin NK2 receptor agonist, [betaAla8]neurokinin A(4-10) (3 nmol/kg i.v.), either after intravenous (ED50 0.18 micromol/kg), intraduodenal (ED50 3.16 micromol/kg) or oral administration (10-30 micromol/kg) without affecting, at 3 micromol/kg, i.v., the colonic contractions produced by the NK1 receptor selective agonist [Sar9]substance P sulfone (3 nmol/kg i.v.). In addition MEN15596 was effective in inhibiting bronchoconstriction produced by i.v. administration of [betaAla8]neurokinin A(4-10). Overall the results indicate that MEN15596 is a potent and selective

  19. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi, Wen-Zhu; Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853; Miao, Yu-Liang

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation ofmore » hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.« less

  20. Modulation of chloroplast movement in the green alga Mougeotia by the Ca2+ ionophore A23187 and by calmodulin antagonists.

    PubMed Central

    Serlin, B S; Roux, S J

    1984-01-01

    The Ca2+ ionophore A23187 can induce chloroplast rotation within a single nonirradiated Mougeotia cell. The induced turning was dependent on the position of ionophore application and Ca2+ in the external medium. The role of calmodulin in mediating light-induced chloroplast rotation in the alga Mougeotia was investigated by using the paired calmodulin-antagonist drugs W5-W7 and W12-W13. In each pair, the antagonist with the greater affinity for calmodulin had the greater inhibitor effect on the phytochrome-controlled light response. These results support the hypothesis that calcium functions as a chemical messenger to couple the stimulus of phytochrome photoactivation with physiological responses in plants. Images PMID:11536594

  1. Impact and benefit of A(2B)-adenosine receptor agonists for the respiratory tract: mucociliary clearance, ciliary beat frequency, trachea muscle tonus and cytokine release.

    PubMed

    Walaschewski, Robin; Begrow, Frank; Verspohl, Eugen J

    2013-01-01

    Adenosine is known to induce a bronchospasm in asthma- and COPD patients. The role of A(2B) receptors was investigated with respect to several parameters of the respiratory tract: tonus of smooth muscle, ciliary beat frequency as measured by high-speed video camera connected to a microscope (both in rats) and mucociliary clearance (MCC; transport of a fluorescent dye using a microdialysis procedure) in mice.  NECA (5'-N-ethylcarboxamidoadenosine) (a non-selective adenosine receptor agonist) was able to acutely induce a contraction, which was reversed to a relaxation after repeated dosing. This relaxation was completely abolished by PSB-1115, an A(2B) receptor antagonist. IL-13 (cytokine) was not involved mediating acute contractility effects. MCC was increased by BAY 60-6583 (A(2B) receptor agonist) and NECA (counteracted by the A(2B) receptor antagonist PSB-1115). Activation of A(2B) adenosine receptors by BAY 60-6583 induced an increase of the ciliary beat frequency, which could be reduced by administration of PSB-1115. Several cytokines were increased by NECA although only some are relevant because they are not blocked by A(2B) receptor antagonism. The A(2B) receptors are involved in airway relaxation, MCC improvement and ciliary beat frequency. A(2B) receptor agonists may be of therapeutic value and should be developed. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

  2. 2'-O-methyl-modified RNAs act as TLR7 antagonists.

    PubMed

    Robbins, Marjorie; Judge, Adam; Liang, Lisa; McClintock, Kevin; Yaworski, Ed; MacLachlan, Ian

    2007-09-01

    RNA molecules such as single-stranded RNA (ssRNA) and small interfering RNA (siRNA) duplexes induce Toll-like receptor (TLR)-mediated immune stimulation after intracellular delivery. We have previously shown that selective incorporation of 2'-O-methyl (2'OMe) residues into siRNA abrogates cytokine production without reduction of gene silencing activity. Here we show that 2'OMe-modified RNA acts as a potent inhibitor of RNA-mediated cytokine induction in both human and murine systems. This activity does not require the direct incorporation of 2'OMe nucleotides into the immunostimulatory RNA or that the 2'OMe nucleotide-containing RNA be annealed as a complementary strand to form a duplex. Our results indicate that 2'OMe RNA acts as a potent antagonist of immunostimulatory RNA. We further show that 2'OMe RNA is able significantly to reduce both interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) induction by the small-molecule TLR7 agonist loxoribine in human peripheral blood mononuclear cells (human PBMCs), in murine Flt3L dendritic cells (Flt3L DCs), and in vivo in mice. These results indicate that 2'OMe-modified RNA may have utility as an inhibitor of TLR7 with potential applications in the treatment of inflammatory and autoimmune diseases that involve TLR7-mediated immune stimulation.

  3. Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

    PubMed

    Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

    1998-08-01

    This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT

  4. Bradykinin B2 receptors play a neuroprotective role in Hypoxia/reoxygenation injury related to pyroptosis pathway.

    PubMed

    Tang, Min; Li, Xia; Liu, Ping; Wang, Jianwen; He, Fangping; Zhu, Xiongchao

    2018-05-27

    Kinins are pro-inflammatory peptides that mediate numerous vascular and pain responses in tissue injury. Kinins exert their biological functions via two G-protein-coupled receptors: bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R). We previously demonstrated the up-regulation of B2R after hypoxia/reoxygenation (H/R) injury in primary cultured cortical neurons. However, the role of B2R in inflammasome-induced pyroptosis remains unknown. We induced H/R neuronal injury in primary cultured cortical neurons harvested from embryonic day 17 brains. Next, we examined the neuroprotective function of B2R in H/R-induced neuronal apoptosis or necrosis using an annexin V FITC/propidium iodide (PI) double-staining technique. The pyroptosis signaling cascade, including caspase-1, IL-1β and IL-18 levels and cleaved gasdermin D (GSDMD) expression was examined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting to explore the underlying molecular mechanism. H/R injury significantly increased B2R protein expression (P<0.05) as well as the percentage of early apoptotic and necrotic or late apoptotic neurons as verified by the annexin V FITC/PI flow cytometric analysis. Bradykinin (BK), a specific B2R agonist, caused a significant decrease in apoptotic neuronal death after H/R injury, while HOE140, a specific B2R antagonist, markedly reduced the neuoprotective effect of B2R. Following H/R injury, BK downregulated the caspase-1, IL-1β and IL-18 levels (P<0.01). In contrast, pretreatment with HOE140 significantly increased caspase-1, IL-1β, and IL-18 levels (P<0.01). Further analysis revealed that GSDMD, a key executioner of pyroptosis, is a target for B2R-mediated inhibition of neuronal pyroptosis. Cleaved GSDMD expression was significantly inhibited by BK pretreatment and significantly enhanced by HOE140 pretreatment (P<0.01). These results indicate that activation of B2R plays an important role in pyroptosis mediated

  5. Structure of a Protein Phosphatase 2A Holoenzyme: Insights into B55-Mediated Tau Dephosphorylation

    PubMed Central

    Xu, Yanhui; Chen, Yu; Zhang, Ping; Jeffrey, Philip D.; Shi, Yigong

    2009-01-01

    Summary Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular physiology. Members of the regulatory B/B55/PR55 family are thought to play a key role in the dephosphorylation of Tau, whose hyperphosphorylation contributes to Alzheimer's disease. The underlying mechanisms of the PP2A-Tau connection remain largely enigmatic. Here, we report the complete reconstitution of a Tau dephosphorylation assay and the crystal structure of a heterotrimeric PP2A holoenzyme involving the regulatory subunit Bα. We show that Bα specifically and markedly facilitates dephosphorylation of the phosphorylated Tau in our reconstituted assay. The Bα subunit comprises a seven-bladed β propeller, with an acidic, substrate-binding groove located in the center of the propeller. The β propeller latches onto the ridge of the PP2A scaffold subunit with the help of a protruding β hairpin arm. Structure-guided mutagenesis studies revealed the underpinnings of PP2A-mediated dephosphorylation of Tau. PMID:18922469

  6. Behavioral effects of gamma-hydroxybutyrate, its precursor gamma-butyrolactone, and GABA(B) receptor agonists: time course and differential antagonism by the GABA(B) receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348).

    PubMed

    Koek, Wouter; Mercer, Susan L; Coop, Andrew; France, Charles P

    2009-09-01

    Gamma-hydroxybutyrate (GHB) is used therapeutically and recreationally. The mechanism by which GHB produces its therapeutic and recreational effects is not entirely clear, although GABA(B) receptors seem to play an important role. This role could be complex, because there are indications that different GABA(B) receptor mechanisms mediate the effects of GHB and the prototypical GABA(B) receptor agonist baclofen. To further explore possible differences in underlying GABA(B) receptor mechanisms, the present study examined the effects of GHB and baclofen on operant responding and their antagonism by the GABA(B) receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348). Pigeons were trained to peck a key for access to food during response periods that started at different times after the beginning of the session. In these pigeons, GHB, its precursor gamma-butyrolactone (GBL), and the GABA(B) receptor agonists baclofen and 3-aminopropyl(methyl)phosphinic acid hydrochloride (SKF97541) decreased the rate of responding in a dose- and time-dependent manner. CGP35348 shifted the dose-response curve of each agonist to the right, but the magnitude of the shift differed among the agonists. Schild analysis yielded a pA(2) value of CGP35348 to antagonize GHB and GBL [i.e., 3.9 (3.7-4.2)] that was different (P = 0.0011) from the pA(2) value to antagonize baclofen and SKF97541 [i.e., 4.5 (4.4-4.7)]. This finding is further evidence that the GABA(B) receptor mechanisms mediating the effects of GHB and prototypical GABA(B) receptor agonists are not identical. A better understanding of the similarities and differences between these mechanisms, and their involvement in the therapeutic effects of GHB and baclofen, could lead to more effective medications with fewer adverse effects.

  7. Attenuation in rats of impairments of memory by scopolamine, a muscarinic receptor antagonist, by mecamylamine, a nicotinic receptor antagonist

    PubMed Central

    Newman, L. A.

    2015-01-01

    Rationale Scopolamine, a muscarinic antagonist, impairs learning and memory for many tasks, supporting an important role for the cholinergic system in these cognitive functions. The findings are most often interpreted to indicate that a decrease in postsynaptic muscarinic receptor activation mediates the memory impairments. However, scopolamine also results in increased release of acetylcholine in the brain as a result of blocking presynaptic muscarinic receptors. Objectives The present experiments assess whether scopolamine-induced increases in acetylcholine release may impair memory by overstimulating postsynaptic cholinergic nicotinic receptors, i.e., by reaching the high end of a nicotinic receptor activation inverted-U dose-response function. Results Rats tested in a spontaneous alternation task showed dose-dependent working memory deficits with systemic injections of mecamylamine and scopolamine. When an amnestic dose of scopolamine (0.15 mg/kg) was co-administered with a subamnestic dose of mecamylamine (0.25 mg/kg), this dose of mecamylamine significantly attenuated the scopolamine-induced memory impairments. We next assessed the levels of acetylcholine release in the hippocampus in the presence of scopolamine and mecamylamine. Mecamylamine injections resulted in decreased release of acetylcholine, while scopolamine administration caused a large increase in acetylcholine release. Conclusions These findings indicate that a nicotinic antagonist can attenuate impairments in memory produced by a muscarinic antagonist. The nicotinic antagonist may block excessive activation of nicotinic receptors postsynaptically or attenuate increases in acetylcholine release presynaptically. Either effect of a nicotinic antagonist—to decrease scopolamine-induced increases in acetylcholine output or to decrease post-synaptic acetylcholine receptor activation—may mediate the negative effects on memory of muscarinic antagonists. PMID:26660295

  8. Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists.

    PubMed

    Rivera, Alicia

    2007-09-01

    Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg.kg(-1).day(-1) intraperitoneally for 14 days), an ET-1 receptor B (ET(B)) antagonist, induced a significant decrease in Gardos channel activity (1.7 +/- 0.1 to 1.0 +/- 0.4 mmol.10(13) cell(-1).h(-1), n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D(50); n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ET(A)) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ET(B)/ET(A) antagonists but not by ET(A) antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ET(B) receptor, leading to Gardos channel modulation in SCD.

  9. Characterization of protoberberine analogs employed as novel human P2X{sub 7} receptor antagonists

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Ga Eun; Lee, Won-Gil; Lee, Song-Yi

    The P2X{sub 7} receptor (P2X{sub 7}R), a member of the ATP-gated ion channel family, is regarded as a promising target for therapy of immune-related diseases including rheumatoid arthritis and chronic pain. A group of novel protoberberine analogs (compounds 3-5), discovered by screening of chemical libraries, was here investigated with respect to their function as P2X{sub 7}R antagonists. Compounds 3-5 non-competitively inhibited BzATP-induced ethidium ion influx into hP2X{sub 7}-expressing HEK293 cells, with IC{sub 50} values of 100-300 nM. This antagonistic action on the channel further confirmed that both BzATP-induced inward currents and Ca{sup 2+} influx were strongly inhibited by compounds 3-5more » in patch-clamp and Ca{sup 2+} influx assays. The antagonists also effectively suppressed downstream signaling of P2X{sub 7} receptors including IL-1{beta} release and phosphorylation of ERK1/2 and p38 proteins in hP2X{sub 7}-expressing HEK293 cells or in differentiated human monocytes (THP-1 cells). Moreover, IL-2 secretion from CD3/CD28-stimulated Jurkat T cell was also dramatically inhibited by the antagonist. These results imply that novel protoberberine analogs may modulate P2X{sub 7} receptor-mediated immune responses by allosteric inhibition of the receptor. - Graphical abstract: Display Omitted« less

  10. A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques

    PubMed Central

    Peluffo, M.C.; Stanley, J.; Braeuer, N.; Rotgeri, A.; Fritzemeier, K.-H.; Fuhrmann, U.; Buchmann, B.; Adevai, T.; Murphy, M.J.; Zelinski, M.B.; Lindenthal, B.; Hennebold, J.D.; Stouffer, R.L.

    2014-01-01

    STUDY QUESTION Can administration of a prostaglandin (PG) E2 receptor 2 (PTGER2) antagonist prevent pregnancy in adult female monkeys by blocking periovulatory events in the follicle without altering menstrual cyclicity or general health? SUMMARY ANSWER This is the first study to demonstrate that a PTGER2 antagonist can serve as an effective non-hormonal contraceptive in primates. WHAT IS KNOWN ALREADY The requirement for PGE2 in ovulation and the release of an oocyte surrounded by expanded cumulus cells (cumulus–oocyte expansion; C-OE) was established through the generation of PTGS2 and PTGER2 null-mutant mice. A critical role for PGE2 in primate ovulation is supported by evidence that intrafollicular injection of indomethacin in rhesus monkeys suppressed follicle rupture, whereas co-injection of PGE2 with indomethacin resulted in ovulation. STUDY DESIGN, SIZE, DURATION First, controlled ovulation protocols were performed in adult, female rhesus monkeys to analyze the mRNA levels for genes encoding PGE2 synthesis and signaling components in the naturally selected pre-ovulatory follicle at different times after the ovulatory hCG stimulus (0, 12, 24, 36 h pre-ovulation; 36 h post-ovulation, n = 3–4/time point). Second, controlled ovarian stimulation cycles were utilized to obtain multiple cumulus–oocyte complexes (COCs) from rhesus monkeys to evaluate the role of PGE2 in C-OE in vitro (n = 3–4 animals/treatment; ≥3 COCs/animal/treatment). Third, adult cycling female cynomolgus macaques were randomly assigned (n = 10/group) to vehicle (control) or PTGER2 antagonist (BAY06) groups to perform a contraceptive trial. After the first treatment cycle, a male of proven fertility was introduced into each group and they remained housed together for the duration of the 5-month contraceptive trial that was followed by a post-treatment reversibility trial. PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative real-time PCR, COC culture and expansion, immunofluorescence

  11. Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A2A antagonists MSX-3 and SCH58261, but not the adenosine A1 antagonist DPCPX.

    PubMed

    Collins, Lyndsey E; Galtieri, Daniel J; Brennum, Lise T; Sager, Thomas N; Hockemeyer, Jörg; Müller, Christa E; Hinman, James R; Chrobak, James J; Salamone, John D

    2010-02-01

    Tremulous jaw movements in rats, which can be induced by dopamine (DA) antagonists, DA depletion, and cholinomimetics, have served as a useful model for studies of tremor. Although adenosine A(2A) antagonists can reduce the tremulous jaw movements induced by DA antagonists and DA depletion, there are conflicting reports about the interaction between adenosine antagonists and cholinomimetic drugs. The present studies investigated the ability of adenosine antagonists to reverse the tremorogenic effect of the muscarinic agonist pilocarpine. While the adenosine A(2A) antagonist MSX-3 was incapable of reversing the tremulous jaw movements induced by the 4.0mg/kg dose of pilocarpine, both MSX-3 and the adenosine A(2A) antagonist SCH58261 reversed the tremulous jaw movements elicited by 0.5mg/kg pilocarpine. Systemic administration of the adenosine A(1) antagonist DPCPX failed to reverse the tremulous jaw movements induced by either an acute 0.5mg/kg dose of the cholinomimetic pilocarpine or the DA D2 antagonist pimozide, indicating that the tremorolytic effects of adenosine antagonists may be receptor subtype specific. Behaviorally active doses of MSX-3 and SCH 58261 showed substantial in vivo occupancy of A(2A) receptors, but DPCPX did not. The results of these studies support the use of adenosine A(2A) antagonists for the treatment of tremor. Copyright 2009 Elsevier Inc. All rights reserved.

  12. Surface-mediated delivery of siRNA from fibrin hydrogels for knockdown of the BMP-2 binding antagonist noggin.

    PubMed

    Kowalczewski, Christine J; Saul, Justin M

    2015-10-01

    Antagonists and inhibitory molecules responsible for maintaining tissue homeostasis can present a significant barrier to healing when tissue engineering/regenerative medicine strategies are employed. One example of this situation is the up-regulation of antagonists such as noggin in response to increasing concentrations of bone morphogenetic protein-2 (BMP-2) present from endogenous bone repair processes or delivered exogenously from biomaterials (synthetic bone grafts). While recombinant human (rh)BMP-2 delivered from synthetic bone grafts has been shown to be an effective alternative to autografts and allografts, the supraphysiological doses of rhBMP-2 have led to clinically-adverse side effects. The high rhBMP-2 dosage may be required, in part, to overcome the presence of antagonists such as noggin. Small interfering RNA (siRNA) is an appealing approach to overcome this problem because it can knock-down antagonists or inhibitory molecules in a temporary manner. Here, we conducted fundamental studies on the delivery of siRNA from material surfaces as a means to knock-down antagonists like noggin. Non-viral cationic lipid (Lipofectamine)-siRNA complexes were delivered from a fibrin hydrogel surface to MC3T3-E1 preosteoblasts that were treated with a supraphysiological dose of rhBMP-2 to achieve noggin mRNA expression levels higher than cells naïve to rhBMP-2. Confocal microscopy and flow cytometry showed intracellular uptake of siRNA in over 98% of MC3T3-E1 cells after 48 h. Doses of 0.5 and 1 μg noggin siRNA were able to significantly reduce noggin mRNA to levels equivalent to those in MC3T3-E1 cells not exposed to rhBMP-2 with no effects on cell viability. Small interfering RNA (siRNA) has been considered for treatment of diseases ranging from Alzheimer's to cancer. However, the ability to use siRNA in conjunction with biomaterials to direct tissue regeneration processes has received relatively little attention. Using the bone morphogenetic protein 2

  13. Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation*

    PubMed Central

    Rea, Matthew; Jiang, Tingting; Eleazer, Rebekah; Eckstein, Meredith; Marshall, Alan G.; Fondufe-Mittendorf, Yvonne N.

    2016-01-01

    Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis. PMID:27169413

  14. Paroxetine-induced reduction of sexual incentive motivation in female rats is not modified by 5-HT1B or 5-HT2C antagonists.

    PubMed

    Kaspersen, Helge; Agmo, Anders

    2012-03-01

    Clinical data show that paroxetine causes sexual dysfunction in a substantial proportion of women taking this compound. This work was conducted to determine whether chronic paroxetine reduces sexual incentive motivation in female rats and whether this compound can modify any aspect of paced mating. The role of the 5-HT(1B) and 5-HT(2C) receptors in any potential effects was also evaluated. Ovariectomized female rats were implanted with osmotic minipumps releasing 10 mg/kg per day of paroxetine or vehicle for 28 days. Tests for sexual incentive motivation and paced mating were performed just before implantation and at regular intervals thereafter. The females were primed with estradiol benzoate (25 μg/rat) and progesterone (1 mg/rat) before each of these tests. On days 25-27 of treatment, the females were injected with the 5-HT(1B) antagonist GR125,743 (5 mg/kg), the 5-HT(2C) antagonist SB206,553 (5 mg/kg) and vehicle in counterbalanced order. Preinjection time was 30 min. Paroxetine reduced sexual incentive motivation on day 20 of treatment without affecting any aspect of paced mating. None of the antagonists modified the inhibitory effect of paroxetine on sexual incentive motivation. In the group chronically treated with vehicle, SB206,553 reduced proceptive behaviors in the paced mating test. No other effect was obtained. The effects of paroxetine seen in female rats are similar to those observed in women, suggesting that disturbances of sexual incentive motivation in rats are predictive of sexual dysfunction in women. The 5-HT(1B) and 5-HT(2C) receptors do not seem to be of any importance for paroxetine's inhibitory effect.

  15. A novel method for determination of aflatoxin B1 mediated by FCLA + BSA

    NASA Astrophysics Data System (ADS)

    Chen, WenLi; Xing, Da

    2005-02-01

    As a chemiluminescence (CL) probe, 3,7-dihydro-6-{4-{2-(N"-(5-fluoresceinyl) thioureido)ethoxy}phenyl}-2-met -hylimi-dazo{1,2-a}pyrazin-3-one dosium salt (FCLA) can sensitively and specifically react with singlet oxygen (1O2 ) and superoxide(O2""). BSA (Bovine Serum Albumin) can enlarge the CL intensity of FCLA to 860%. This report presents a novel method for determination of Aflatoxin B1 (AfB1) mediated by FCLA+BSA. The concentration of AFB1 showed an obvious positive correlation with the CL intensity mediated by FCLA+BSA. This method could measure accurately ng/ml of AfB1 concentration. At the same time, the fluorescence spectrum of FCLA+BSA and FCLA+BSA+AfB1 were measured respectively, which showed that the fluorescence intensity of FCLA+BSA+AfB1 was higher than FCLA+BSA. Comparing the peak value of FCLA, FCLA+BSA and FCLA+BSA+AfB1 had a 6nm Einstein shift (red shift). The study suggested that CL method mediated by FCLA+BSA might be applicable to the determination of AfB1 concentration.

  16. alpha(2)-adrenoceptor antagonist properties of OPC-28326, a novel selective peripheral vasodilator.

    PubMed

    Orito, K; Kishi, M; Imaizumi, T; Nakazawa, T; Hashimoto, A; Mori, T; Kambe, T

    2001-10-01

    1. Antagonistic properties of OPC-28326 ([4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl)] piperidine hydrochloride monohydrate), a selective peripheral vasodilator, were investigated by analysing the data from functional studies in various tissues from the rat and binding studies of the drug to alpha(2)-adrenoceptor subtypes. 2. Using a human recombinant receptor and rat kidney cortex, we found that OPC-28326 displays affinities to alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors with K(i) values of 2040, 285, and 55 nM, respectively. The K(i) values of yohimbine for alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenoceptors were 3.0, 2.0 and 11.0 nM, respectively. 3. B-HT 920, an alpha(2)-adrenoceptor agonist, produced a pressor response via peripheral postsynaptic alpha(2)-adrenoceptor stimulation (thought to be an alpha(2B)-subtype) in a reserpine-pretreated pithed rat preparation. OPC-28326 (3 - 30 mg kg(-1), i.v.) and yohimbine (0.3 - 3 mg kg(-1), i.v.) caused dose-dependent rightward shift in the pressor dose-response curve induced by B-HT 920. The apparent pA(2) values were 1.55 (0.87 - 2.75, 95% confidence interval) and 0.11 (0.06 - 0.21) mg kg(-1), respectively. The potency of OPC-28326 was about 14 times less than that of yohimbine. 4. Clonidine inhibited the tension developed by electrical stimulation, of the rat vas deferens, by its peripheral presynaptic alpha(2A/D)-adrenoceptor action. OPC-28326 (1 - 100 microM) and yohimbine (10 - 1000 nM) caused a rightward shift in the concentration-response curve of clonidine. The pA(2) values were 5.73 (5.54 - 5.91) and 7.92 (7.84 - 8.01), respectively, providing evidence for a potency of OPC-28326 of about 155 times less than that of yohimbine. 5. Mydriasis was induced by brimonidine via stimulation of central alpha(2A/D)-adrenoceptors in anaesthetized rats. Intravenous OPC-28326 had no effect on this action, even at a very high dose of 10 mg kg(-1) i.v., while yohimbine (0.1 - 0.3 mg kg(-1

  17. Effect of bradykinin antagonists on bradykinin-induced plasma extravasation, venoconstriction, prostaglandin E2 release, nociceptor stimulation and contraction of the iris sphincter muscle in the rabbit.

    PubMed Central

    Griesbacher, T.; Lembeck, F.

    1987-01-01

    1 The inhibition of the bradykinin-induced plasma extravasation by six bradykinin (Bk) antagonists was tested on rabbit skin. All of them showed inhibitory effects without an agonistic action in the does used. B4310 (Lys-Lys-3-Hyp-5,8-Thi-7-DPhe-Bk) was the most active antagonist and was therefore used in the subsequent experiments. 2 B4310 (5-500 nM) antagonized the bradykinin-induced reduction of the venous outflow from the rabbit isolated ear in dose-dependent manner without affecting the arterial vasoconstriction induced by angiotensin II. 3 The bradykinin-induced release of prostaglandin E2 (PGE2) from the perfused rabbit ear was reduced by 63% when B4310 (800 nM) was infused before, during and after the bradykinin injection. 4 Bradykinin was injected into the ear artery of anaesthetized rabbits and the reflex hypotensive response was used as indicator of the nociception. The response was antagonized by a local infusion of B4310 (50 and 500 nM). The antagonism was dose-dependent and reversible. The parallel shift of the dose-response curve to bradykinin suggests a competitive inhibition. However, B4310 did not antagonize acetylcholine-induced nociceptor stimulation. 5 B4310 inhibited bradykinin-induced stimulation of the trigeminal nerve which results in a substance P-mediated contraction of the iris sphincter muscle. A pA2 of 7.59 was calculated. B4310 did not inhibit capsaicin-induced contractions. 6 It is concluded that B4310 inhibits specifically five different actions of bradykinin which are related to its possible pathophysiological role. PMID:3479223

  18. MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways

    PubMed Central

    Yu, Dongyin; Lofgren, Julie A.; Osgood, Tao; Robertson, Rebecca; Canon, Jude; Su, Cheng; Jones, Adrie; Zhao, Xiaoning; Deshpande, Chetan; Payton, Marc; Ledell, Jebediah; Hughes, Paul E.; Oliner, Jonathan D.

    2014-01-01

    While MDM2 inhibitors hold great promise as cancer therapeutics, drug resistance will likely limit their efficacy as single agents. To identify drug combinations that might circumvent resistance, we screened for agents that could synergize with MDM2 inhibition in the suppression of cell viability. We observed broad and robust synergy when combining MDM2 antagonists with either MEK or PI3K inhibitors. Synergy was not limited to cell lines harboring MAPK or PI3K pathway mutations, nor did it depend on which node of the PI3K axis was targeted. MDM2 inhibitors also synergized strongly with BH3 mimetics, BCR-ABL antagonists, and HDAC inhibitors. MDM2 inhibitor-mediated synergy with agents targeting these mechanisms was much more prevalent than previously appreciated, implying that clinical translation of these combinations could have far-reaching implications for public health. These findings highlight the importance of combinatorial drug targeting and provide a framework for the rational design of MDM2 inhibitor clinical trials. PMID:24810962

  19. Betacellulin-Induced Beta Cell Proliferation and Regeneration Is Mediated by Activation of ErbB-1 and ErbB-2 Receptors

    PubMed Central

    Oh, Yoon Sin; Shin, Seungjin; Lee, Youn-Jung; Kim, Eung Hwi; Jun, Hee-Sook

    2011-01-01

    Background Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation. Methodology/Principal Findings The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes. Conclusions/Significance These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells. PMID:21897861

  20. Characterization of the tachykinin neurokinin-2 receptor in the human urinary bladder by means of selective receptor antagonists and peptidase inhibitors.

    PubMed

    Giuliani, S; Patacchini, R; Barbanti, G; Turini, D; Rovero, P; Quartara, L; Giachetti, A; Maggi, C A

    1993-11-01

    The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

  1. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

    PubMed Central

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

    2015-01-01

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

  2. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

    PubMed

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

    2015-07-07

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

  3. SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride], a new nonpeptide antagonist of the bradykinin B1 receptor: biochemical and pharmacological characterization.

    PubMed

    Gougat, Jean; Ferrari, Bernard; Sarran, Lionel; Planchenault, Claudine; Poncelet, Martine; Maruani, Jeanne; Alonso, Richard; Cudennec, Annie; Croci, Tiziano; Guagnini, Fabio; Urban-Szabo, Katalin; Martinolle, Jean-Pierre; Soubrié, Philippe; Finance, Olivier; Le Fur, Gérard

    2004-05-01

    The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.

  4. Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

    PubMed

    Park, Seon-Ah; Hong, Bing-Zhe; Ha, Ki-Chan; Kim, Uh-Hyun; Han, Myung-Kwan; Kwak, Yong-Geun

    2017-06-02

    Cyclic ADP-ribose (cADPR) releases Ca 2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca 2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca 2+ ([Ca 2+ ] i ) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca 2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

  5. P2X and P2Y Receptors Mediate Contraction Induced by Electrical Field Stimulation in Feline Esophageal Smooth Muscle.

    PubMed

    Cho, Young Rae; Jang, Hyeon Soon; Kim, Won; Park, Sun Young; Sohn, Uy Dong

    2010-10-01

    It is well-known that electrical field stimulation (EFS)-induced contraction is mediated by a cholinergic mechanism and other neurotransmitters. NO, ATP, calcitonin gene-related peptide (CGRP), and substance P are released by EFS. To investigate the purinergic mechanism involved in the EFS-induced contraction, purinegic receptors antagonists were used. Suramine, a non-selective P2 receptor antagonist, reduced the contraction induced by EFS. NF023 (10(-7)~10(-4) M), a selective P2X antagonist, inhibited the contraction evoked by EFS. Reactive blue (10(-6)~10(-4) M), selective P2Y antagonist, also blocked the contraction in a dose-dependent manner. In addition, P2X agonist α,β-methylene 5'-adenosine triphosphate (αβMeATP, 10(-7)~10(-5) M) potentiated EFS-induced contraction in a dose-dependent manner. P2Y agonist adenosine 5'-[β-thio]diphosphate trilithium salt (ADPβS, 10(-7)~10(-5) M) also potentiated EFS-induced contractions in a dose-dependent manner. Ecto-ATPase activator apyrase (5 and 10 U/ml) reduced EFS-induced contractions. Inversely, 6-N,N-diethyl-D-β,γ-dibromomethylene 5'-triphosphate triammonium (ARL 67156, 10(-4) M) increased EFS-induced contraction. These data suggest that endogenous ATP plays a role in EFS-induced contractions which are mediated through both P2X-receptors and P2Y-receptors stimulation in cat esophageal smooth muscle.

  6. Atypical sympathomimetic drug lerimazoline mediates contractile effects in rat aorta predominantly by 5-HT2A receptors.

    PubMed

    Rizvić, Eldina; Janković, Goran; Kostić-Rajačić, Slađana; Savić, Miroslav M

    2017-08-20

    Lerimazoline is a sympathomimetic drug that belongs to the imidazoline class of compounds, and is used as a nasal decongestant. Studies on lerimazoline are rare, and its pharmacological profile is not completely understood. Here, we analyzed the affinity of lerimazoline for dopamine receptor D2, serotonin 5-HT1A and 5-HT2A receptors and α1-adrenoceptor, and investigated lerimazoline contractile effects in isolated rat thoracic aorta. We also determined the effect of several antagonists on the contractile response to lerimazoline, including prazosin (α1-adrenoceptor antagonist), RX 821002 and rauwolscine (α2-adrenoceptor antagonists), JP 1302 (α2C-adrenoceptor antagonist), methiothepin (non-selective 5-HT receptor antagonist), SB 224289 (5-HT1B receptor antagonist), BRL 15572 (5-HT1D receptor antagonist), and ketanserin (5-HT2A receptor antagonist). Lerimazoline displayed high affinity for the 5-HT1A receptor (Ki = 162.5 nM), similar to the previously reported affinity for the 5-HT1D receptor. Binding affinity estimates (Ki) for α1, 5-HT2A, and D2 receptors were 6656, 4202 and 3437.5 nM, respectively (the literature reported Ki for 5-HT1B receptor is 3480 nM). Lerimazoline caused concentration-dependent contractions in 70% of preparations, varying in the range between 40% and 55% of the maximal contraction elicited by phenylephrine. While prazosin reduced the maximum contractile response to lerimazoline, rauwolscine showed a non-significant trend in reduction of the response. Both ketanserin (10 nM and 1 µM) and methiothepin strongly suppressed the maximum response to lerimazoline. Overall, our results suggest that 5-HT2A and, less distinctly, α1-adrenergic receptors are involved in the lerimazoline-induced contractions, which makes lerimazoline an "atypical" decongestant.

  7. Adenoviral mediated interferon-alpha 2b gene therapy suppresses the pro-angiogenic effect of vascular endothelial growth factor in superficial bladder cancer.

    PubMed

    Adam, Liana; Black, Peter C; Kassouf, Wassim; Eve, Beryl; McConkey, David; Munsell, Mark F; Benedict, William F; Dinney, Colin P N

    2007-05-01

    Intravesical adenovirus mediated interferon-alpha gene transfer has a potent therapeutic effect against superficial human bladder carcinoma xenografts growing in the bladder of athymic nude mice. We determined whether the inhibition of angiogenesis might contribute to the antitumor effect. We treated several human urothelial carcinoma cells with adenovirus mediated interferon-alpha 2b and monitored its effects on the production of angiogenic factors using real-time reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemical analysis and a gel shift based transcription factor array. To assess the role of adenovirus mediated interferon 2b in angiogenic activity we used in vitro invasion assays and evaluated the anti-angiogenic effects of adenovirus mediated interferon gene therapy in an orthotopic murine model of human superficial bladder cancer. In adenovirus mediated interferon-alpha infected 253J B-V cells vascular endothelial growth factor was decreased and anti-angiogenic interferon-gamma inducible protein 10 was up-regulated. In contrast, the addition of as much as 100,000 IU recombinant interferon had no apparent effect on vascular endothelial growth factor production. Conditioned medium derived from adenovirus mediated interferon 2b infected 253J B-V cells greatly decreased the invasive potential of human endothelial cells and down-regulated their matrix metalloproteinase 2 expression compared to controls. Furthermore, adenovirus mediated interferon 2b blocked pro-angiogenic nuclear signals, such as the transcription factors activating protein-1 and 2, stimulating protein-1, nuclear factor kappaB and c-myb. In vivo experiments revealed significant vascular endothelial growth factor down-regulation and decreased tumor vessel density in the adenovirus mediated interferon 2b treated group compared to controls. Treatment with adenovirus mediated interferon 2b increases the angiostatic activity of the bladder cancer microenvironment

  8. Four Proteins Encoded in the gspB-secY2A2 Operon of Streptococcus gordonii Mediate the Intracellular Glycosylation of the Platelet-Binding Protein GspB

    PubMed Central

    Takamatsu, Daisuke; Bensing, Barbara A.; Sullam, Paul M.

    2004-01-01

    Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB. PMID:15489421

  9. Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

    PubMed

    Kepler, Christopher K; Markova, Dessislava Z; Koerner, John D; Mendelis, Joseph; Chen, Chiu-Ming; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

    2015-08-15

    Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by

  10. The P2Y12 Antagonists, 2-Methylthioadenosine 5′-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels*

    PubMed Central

    Srinivasan, Subhashini; Mir, Fozia; Huang, Jin-Sheng; Khasawneh, Fadi T.; Lam, Stephen C.-T.; Le Breton, Guy C.

    2009-01-01

    ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors. PMID:19346255

  11. Cutting Edge: 2B4-Mediated Coinhibition of CD4+ T Cells Underlies Mortality in Experimental Sepsis.

    PubMed

    Chen, Ching-Wen; Mittal, Rohit; Klingensmith, Nathan J; Burd, Eileen M; Terhorst, Cox; Martin, Greg S; Coopersmith, Craig M; Ford, Mandy L

    2017-09-15

    Sepsis is a leading cause of death in the United States, but the mechanisms underlying sepsis-induced immune dysregulation remain poorly understood. 2B4 (CD244, SLAM4) is a cosignaling molecule expressed predominantly on NK cells and memory CD8 + T cells that has been shown to regulate T cell function in models of viral infection and autoimmunity. In this article, we show that 2B4 signaling mediates sepsis lymphocyte dysfunction and mortality. 2B4 expression is increased on CD4 + T cells in septic animals and human patients at early time points. Importantly, genetic loss or pharmacologic inhibition of 2B4 significantly increased survival in a murine cecal ligation and puncture model. Further, CD4-specific conditional knockouts showed that 2B4 functions on CD4 + T cell populations in a cell-intrinsic manner and modulates adaptive and innate immune responses during sepsis. Our results illuminate a novel role for 2B4 coinhibitory signaling on CD4 + T cells in mediating immune dysregulation. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

    PubMed

    Atabey, N; Gao, Y; Yao, Z J; Breckenridge, D; Soon, L; Soriano, J V; Burke, T R; Bottaro, D P

    2001-04-27

    Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

  13. Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

    PubMed Central

    Savory, Joanne G. A.; Hsu, Brian; Laquian, Ian R.; Giffin, Ward; Reich, Terry; Haché, Robert J. G.; Lefebvre, Yvonne A.

    1999-01-01

    Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1− GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export. PMID:9891038

  14. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    PubMed

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies

  15. Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [³H]dopamine and [³H]norepinephrine release.

    PubMed

    Narayanaswami, Vidya; Somkuwar, Sucharita S; Horton, David B; Cassis, Lisa A; Dwoskin, Linda P

    2013-09-01

    Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [³H]nicotine and [³H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [³H]dopamine and [³H]norepinephrine release (IC₅₀: 3.9 ± 1.2 and 2.2 ± 0.7 μM; Imax: 82 ± 3 and 89 ± 6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [³H]nicotine or [³H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of

  16. Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications.

    PubMed

    Sullivan, Timothy J; Dairaghi, Daniel J; Krasinski, Antoni; Miao, Zhenhua; Wang, Yu; Zhao, Bin N; Baumgart, Trageen; Berahovich, Rob; Ertl, Linda S; Pennell, Andrew; Seitz, Lisa; Miao, Shichang; Ungashe, Solomon; Wei, Zheng; Johnson, Dan; Boring, Landin; Tsou, Chia-Lin; Charo, Israel F; Bekker, Pirow; Schall, Thomas J; Jaen, Juan C

    2012-06-05

    The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.

  17. Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild-Type Mice Treated with a Novel Avpr1b Receptor Antagonist

    PubMed Central

    Roper, J A; Craighead, M; O’Carroll, A-M; Lolait, S J

    2010-01-01

    Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. PMID:20846299

  18. Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist.

    PubMed

    Roper, J A; Craighead, M; O'Carroll, A-M; Lolait, S J

    2010-11-01

    Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.

  19. STAT2-dependent immune responses ensure host survival despite the presence of a potent viral antagonist.

    PubMed

    Le-Trilling, Vu Thuy Khanh; Wohlgemuth, Kerstin; Rückborn, Meike U; Jagnjic, Andreja; Maaßen, Fabienne; Timmer, Lejla; Katschinski, Benjamin; Trilling, Mirko

    2018-05-09

    Pathogen encounter induces interferons which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, host and pathogens are situated in a continuous arms race which shapes host evolution towards optimized immune responses and the pathogens towards enhanced immune evasive properties.Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2 which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lacking M27 and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pair in vitro and in vivo In contrast to wt-MCMV, ΔM27-MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g. liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection.Taken together, our study reveals the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary stand-off situation with fatal consequences when the equilibrium is disturbed. IMPORTANCE The host limits viral replication by interferons which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g. cytomegaloviruses, Zika virus, Dengue virus, and several paramyxoviruses). We analyzed infections of mouse CMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate their importance for host and virus in vitro and in

  20. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Mei, E-mail: limeihit@163.com; Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing; Zhang, Dong-Qing

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Romore » 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.« less

  1. The 5-HT2A receptor antagonist M100907 is more effective in counteracting NMDA antagonist- than dopamine agonist-induced hyperactivity in mice.

    PubMed

    Carlsson, M L; Martin, P; Nilsson, M; Sorensen, S M; Carlsson, A; Waters, S; Waters, N

    1999-01-01

    The purpose of the present study was to compare the effectiveness of the selective 5-HT2A antagonist M100907 in different psychosis models. The classical neuroleptic haloperidol was used as reference compound. Two hyperdopaminergia and two hypoglutamatergia mouse models were used. Hyperdopaminergia was produced by the DA releaser d-amphetamine or the DA uptake inhibitor GBR 12909. Hypoglutamatergia was produced by the un-competitive NMDA receptor antagonist MK-801 or the competitive NMDA receptor antagonist D-CPPene. M100907 was found to counteract the locomotor stimulant effects of the NMDA receptor antagonists MK-801 and D-CPPene, but spontaneous locomotion, d-amphetamine- and GBR-12909-induced hyperactivity were not significantly affected. Haloperidol, on the other hand, antagonized both NMDA antagonist- and DA agonist-induced hyperactivity, as well as spontaneous locomotion in the highest dose used. Based on the present and previous results we draw the conclusion that 5-HT2A receptor antagonists are particularly effective against behavioural anomalies resulting from hypoglutamatergia of various origins. The clinical implications of our results and conclusions would be that a 5-HT2A receptor antagonist, due to i a the low side effect liability, could be the preferable treatment strategy in various disorders associated with hypoglutamatergia; such conditions might include schizophrenia, childhood autism and dementia disorders.

  2. Oleamide suppresses lipopolysaccharide-induced expression of iNOS and COX-2 through inhibition of NF-kappaB activation in BV2 murine microglial cells.

    PubMed

    Oh, Young Taek; Lee, Jung Yeon; Lee, Jinhwa; Lee, Ju Hie; Kim, Ja-Eun; Ha, Joohun; Kang, Insug

    2010-05-03

    Oleamide (cis-9-octadecenamide) is an endogenous sleep-inducing fatty acid amide that accumulates in the cerebrospinal fluid of the sleep-deprived animals. Microglia are the major immune cells involved in neuroinflammation causing brain damage during infection, ischemia, and neurodegenerative disease. In this study, we examined the effects of oleamide on LPS-induced production of proinflammatory mediators and the mechanisms involved in BV2 microglia. Oleamide inhibited LPS-induced production of NO and prostaglandin E2 as well as expression of iNOS and COX-2. We showed that oleamide blocked LPS-induced NF-kappaB activation and phosphorylation of inhibitor kappaB kinase (IKK). We also showed that oleamide inhibited LPS-induced phosphorylation of Akt, p38 MAPK, and ERK, activation of PI 3-kinase, and accumulation of reactive oxygen species (ROS). Finally, we showed that a specific antagonist of the CB2 receptor, AM630, blocked the inhibitory effects of oleamide on LPS-induced production of proinflammatory mediators and activation of NF-kappaB. Taken together, our results suggest that oleamide shows an anti-inflammatory effect through inhibition of NF-kappaB activation in LPS-stimulated BV2 microglia. 2010 Elsevier Ireland Ltd. All rights reserved.

  3. Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

    PubMed

    Leibinger, Marco; Andreadaki, Anastasia; Golla, Renate; Levin, Evgeny; Hilla, Alexander M; Diekmann, Heike; Fischer, Dietmar

    2017-07-03

    Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3 S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3β reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3β KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2 T/A ). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2 T/A in GSK3 S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

  4. Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain.

    PubMed

    Abe, Tetsuya; Matsumura, Shinji; Katano, Tayo; Mabuchi, Tamaki; Takagi, Kunio; Xu, Li; Yamamoto, Akitsugu; Hattori, Kotaro; Yagi, Takeshi; Watanabe, Masahiko; Nakazawa, Takanobu; Yamamoto, Tadashi; Mishina, Masayoshi; Nakai, Yoshihide; Ito, Seiji

    2005-09-01

    Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.

  5. Genetic deletion of GPR52 enhances the locomotor-stimulating effect of an adenosine A2A receptor antagonist in mice: A potential role of GPR52 in the function of striatopallidal neurons.

    PubMed

    Nishiyama, Keiji; Suzuki, Hirobumi; Maruyama, Minoru; Yoshihara, Tomoki; Ohta, Hiroyuki

    2017-09-01

    G protein-coupled receptor 52 (GPR52) is largely co-expressed with dopamine D 2 receptor (DRD2) in the striatum and nucleus accumbens, and this expression pattern is similar to that of adenosine A 2A receptor (ADORA2A). GPR52 has been proposed as a therapeutic target for positive symptoms of schizophrenia, based on observations from pharmacological and transgenic mouse studies. However, the physiological role of GPR52 in dopaminergic functions in the basal ganglia remains unclear. Here, we used GPR52 knockout (KO) mice to examine the role of GPR52 in dopamine receptor-mediated and ADORA2A-mediated locomotor activity and dopamine receptor signaling. High expression of GPR52 protein in the striatum, nucleus accumbens, and lateral globus pallidus of wild type (WT) littermates was confirmed by immunohistochemical analysis. GPR52 KO and WT mice exhibited almost identical locomotor responses to the dopamine releaser methamphetamine and the N-methyl-d-aspartate antagonist MK-801. In contrast, the locomotor response to the ADORA2A antagonist istradefylline was significantly augmented in GPR52 KO mice compared to WT mice. Gene expression analysis revealed that striatal expression of DRD2, but not of dopamine D 1 receptor and ADORA2A, was significantly decreased in GPR52 KO mice. Moreover, a significant reduction in the mRNA expression of enkephalin, a marker of the activity of striatopallidal neurons, was observed in the striatum of GPR52 KO mice, suggesting that GPR52 deletion could enhance DRD2 signaling. Taken together, these results imply the physiological relevance of GPR52 in modulating the function of striatopallidal neurons, possibly by interaction of GPR52 with ADORA2A and DRD2. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Drug pharmacokinetics and pharmacodynamics: PET and microdial studies of SR 46349B, a selective 5HT2 antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tan, P.; Dewey, S.L.; Gatley, S.J.

    1994-05-01

    The brain serotonin system is an important molecular target in drug development. SR 46349B is a propenone oxime ether derivative with a high affinity and selectivity for the serotonin 5HT2 receptor (Kd=1.2 nM). We have labeled SR 46349B with carbon-11 via N-methylation of a nor-precursor (supplied by Sanofi Recherche) with C-11 methyl iodide. Purification by HPLC gave [11C]SR 46349B in 98% radiochemical purity with a specific activity of 1.5 Ci/{mu}mol. Serial PET studies were carried out in a baboon for a 60 minute study period with a two hour time interval between studies. The first study was at baseline andmore » the second after pretreatment with altanserin (0.5 mg/kg iv, 30 min prior to [11C]SR 46349B). Carbon-11 peaked at ca. 20 minutes in the frontal, parietal, temporal and occipital cortices where it plateaued for the rest of the study. Cerebellum, thalamus and striatum peaked at ca. 10 minutes and cleared to 62%, 72% and 80% of peak by 60 min. At 60 minutes, the frontal cortex to cerebellum ratio was 1.5. Treatment with altanserin reduced the frontal cortex to cerebellum ratio to 1.0. HPLC of mouse brain homogenate after [11C]SR 46349B showed >94% of the C-11 was parent compound. Microdialysis in freely moving rats after injection of SR 46349B (n=6; 10 mg/kg, ip) showed an average peak increase in extracellular dopamine of 375% which is higher than the 150% effect of altanserin. Spontaneous movements were markedly reduced. The pharmacokinetics of [11C] SR 46349B in cortical areas is consistent with the long term effects of SR 46349B on 5HT2 receptors and the elevations in extracellular dopamine without increased locomotor activity are consistent with serotonin mediated disinhibition of striatal dopamine release via blockade of serotonin receptors.« less

  7. Pharmacological characterization of the cysteinyl-leukotriene antagonists CGP 45715A (iralukast) and CGP 57698 in human airways in vitro

    PubMed Central

    Capra, Valérie; Bolla, Manlio; Angelo Belloni, Pier; Mezzetti, Maurizio; Carlo Folco, G; Nicosia, Simonetta; Enrico Rovati, G

    1998-01-01

    Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. We investigated the pharmacological profile of the cysteinyl-LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. Competition curves vs [3H]-LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]-LTD4; (b) as in all the G-protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2=16.6 nM±36% CV and Ki1= Ki2=5.7 nM±19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. In functional studies: (a) iralukast and CGP 57698 antagonized LTD4-induced contraction of human bronchi, with pA2 values of 7.77±4.3% CV and 8.51±1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. Both antagonists significantly inhibited antigen-induced contraction of human isolated bronchial strips in a concentration-dependent manner, lowering the upper plateau of the anti-IgE curves. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these

  8. Regulation of ERRα Gene Expression by Estrogen Receptor Agonists and Antagonists in SKBR3 Breast Cancer Cells: Differential Molecular Mechanisms Mediated by G Protein-Coupled Receptor GPR30/GPER-1

    PubMed Central

    Li, Yin; Birnbaumer, Lutz; Teng, Christina T.

    2010-01-01

    In selected tissues and cell lines, 17β-estradiol (E2) regulates the expression of estrogen-related receptor α (ERRα), a member of the orphan nuclear receptor family. This effect is thought to be mediated by the estrogen receptor α (ERα). However in the ERα- and ERβ-negative SKBR3 breast cancer cell line, physiological levels of E2 also stimulate ERRα expression. Here, we explored the molecular mechanism that mediates estrogen action in ER-negative breast cancer cells. We observed that E2, the ERα agonist, as well as the ERα antagonists ICI 182,780 and tamoxifen (TAM), a selective ER modulator, stimulate the transcriptional activity of the ERRα gene and increase the production of ERRα protein in SKBR3 cells. Moreover, the ERRα downstream target genes expression and cellular proliferation are also increased. We show further that the G protein-coupled receptor GPR30/GPER-1 (GPER-1) mediates these effects. The GPER-1 specific ligand G-1 mimics the actions of E2, ICI 182,780, and TAM on ERRα expression, and changing the levels of GPER-1 mRNA by overexpression or small interfering RNA knockdown affected the expression of ERRα accordingly. Utilizing inhibitors, we delineate a different downstream pathway for ER agonist and ER antagonist-triggered signaling through GPER-1. We also find differential histone acetylation and transcription factor recruitment at distinct nucleosomes of the ERRα promoter, depending on whether the cells are activated with E2 or with ER antagonists. These findings provide insight into the molecular mechanisms of GPER-1/ERRα-mediated signaling and may be relevant to what happens in breast cancer cells escaping inhibitory control by TAM. PMID:20211987

  9. Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels.

    PubMed

    Ase, Ariel R; Honson, Nicolette S; Zaghdane, Helmi; Pfeifer, Tom A; Séguéla, Philippe

    2015-04-01

    P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Novel Drugs that Target ErbB2

    DTIC Science & Technology

    2011-05-01

    Fig. 3C). 3. Role of cannabinoid receptors BA-induced downregulation of Sp transcription factors was proteasome-independent (Fig. 2) and...cancer cell lines show that cannabinoids (CBs) decrease Sp proteins (data not shown), the effects of CB1 and CB2 receptor antagonists AM251 and AM630...were observed in MDA-MB-453 cells confirming a role for the cannabinoid receptors in mediating the effects of BA on Sp and Sp-regulated genes

  11. Effects of combined administration of 5-HT1A and/or 5-HT1B receptor antagonists and paroxetine or fluoxetine in the forced swimming test in rats.

    PubMed

    Tatarczyńska, Ewa; Kłodzińska, Aleksandra; Chojnacka-Wójcik, Ewa

    2002-01-01

    Clinical data suggest that coadministration of pindolol, a 5-HT1A/5-HT1B/beta-adrenoceptor antagonist, and selective serotonin reuptake inhibitors (SSRIs) may shorten the time of onset of a clinical action and may increase beneficial effects of the therapy of drug-resistant depression. Effects of combined administration of SSRIs and 5-HT receptor ligands are currently evaluated in animal models for the detection of an antidepressant-like activity; however, the obtained results turned out to be inconsistent. The aim of the present study was to investigate effects of a 5-HT1A antagonist (WAY 100635), 5-HT1B antagonists (SB 216641 and GR 127935) or pindolol, given in combination with paroxetine or fluoxetine (SSRIs), in the forced swimming test in rats (Porsolt test). When given alone, paroxetine (10 and 20 mg/kg), fluoxetine (10 and 20 mg/kg), WAY 100635 (0.1 and 1 mg/kg), SB 216641 (2 mg/kg), GR 127935 (10 and 20 mg/kg) and pindolol (4 and 8 mg/kg) did not shorten the immobility time of rats in that test. Interestingly, SB 216641 administered alone at a dose of 4 mg/kg produced a significant reduction of the immobility time in that test. A combination of paroxetine (20 mg/kg) and WAY 100635 or pindolol failed to reveal a significant interaction; on the other hand, when paroxetine was given jointly with SB 216641 (2 mg/kg) or GR 127935 (10 and 20 mg/kg), that combination showed a significant antiimmobility action in the forced swimming test in rats. The active behaviors in that test did not reflect increased general activity because combined administration of both the 5-HT1B antagonists and paroxetine failed to alter the locomotor activity of rats, measured in the open field test. Coadministration of fluoxetine and all the antagonists used did not affect the behavior of rats in the forced swimming test. The obtained results seem to indicate that blockade of 5-HT1B receptors, but not 5-HT1A ones, can facilitate the antidepressant-like effect of paroxetine in the

  12. Orexin OX2 Receptor Antagonists as Sleep Aids.

    PubMed

    Jacobson, Laura H; Chen, Sui; Mir, Sanjida; Hoyer, Daniel

    The discovery of the orexin system represents the single major progress in the sleep field of the last three to four decades. The two orexin peptides and their two receptors play a major role in arousal and sleep/wake cycles. Defects in the orexin system lead to narcolepsy with cataplexy in humans and dogs and can be experimentally reproduced in rodents. At least six orexin receptor antagonists have reached Phase II or Phase III clinical trials in insomnia, five of which are dual orexin receptor antagonists (DORAs) that target both OX 1 and OX 2 receptors (OX 2 Rs). All clinically tested DORAs induce and maintain sleep: suvorexant, recently registered in the USA and Japan for insomnia, represents the first hypnotic principle that acts in a completely different manner from the current standard medications. It is clear, however, that in the clinic, all DORAs promote sleep primarily by increasing rapid eye movement (REM) and are almost devoid of effects on slow-wave (SWS) sleep. At present, there is no consensus on whether the sole promotion of REM sleep has a negative impact in patients suffering from insomnia. However, sleep onset REM (SOREM), which has been documented with DORAs, is clearly an undesirable effect, especially for narcoleptic patients and also in fragile populations (e.g. elderly patients) where REM-associated loss of muscle tone may promote an elevated risk of falls. Debate thus remains as to the ideal orexin agent to achieve a balanced increase in REM and non-rapid eye movement (NREM) sleep. Here, we review the evidence that an OX 2 R antagonist should be at least equivalent, or perhaps superior, to a DORA for the treatment of insomnia. An OX 2 R antagonist may produce more balanced sleep than a DORA. Rodent sleep experiments show that the OX 2 R is the primary target of orexin receptor antagonists in sleep modulation. Furthermore, an OX 2 R antagonist should, in theory, have a lower narcoleptic/cataplexic potential. In the clinic, the situation

  13. Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box.

    PubMed

    Ezcurra, I; Wycliffe, P; Nehlin, L; Ellerström, M; Rask, L

    2000-10-01

    The transcriptional activator ABI3 is a key regulator of gene expression during embryo maturation in crucifers. In monocots, the related VP1 protein regulates the Em promoter synergistically with abscisic acid (ABA). We identified cis-elements in the Brassica napus napin napA promoter mediating regulation by ABI3 and ABA, by analyzing substitution mutation constructs of napA in transgenic tobacco plantlets ectopically expressing ABI3. In transient analysis using particle bombardment of tobacco leaf sections, a tetramer of the distB ABRE (abscisic acid-responsive element) mediated transactivation by ABI3 and ABI3-dependent response to ABA, whereas a tetramer of the composite RY/G complex, containing RY repeats and a G-box, mediated only ABA-independent transactivation by ABI3. Deletion of the conserved B2 and B3 domains of ABI3 abolished transactivation of napA by ABI3. The two domains of ABI3 interact with different cis-elements: B2 is necessary for ABA-independent and ABA-dependent activations through the distB ABRE, whereas B3 interacts with the RY/G complex. Thus B2 mediates the interaction of ABI3 with the protein complex at the ABRE. The regulation of napA by ABI3 differs from Em regulation by VP1, in that the B3 domain of ABI3 is essential for the ABA-dependent regulation of napA.

  14. Agonists and antagonists for P2 receptors

    PubMed Central

    Jacobson, Kenneth A.; Costanzi, Stefano; Joshi, Bhalchandra V.; Besada, Pedro; Shin, Dae Hong; Ko, Hyojin; Ivanov, Andrei A.; Mamedova, Liaman

    2015-01-01

    Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X2/3/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X2/3/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformation-ally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4 nM at the P2Y1 receptor, with >10 000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed. PMID:16805423

  15. The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

    PubMed Central

    Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  16. Panicolytic-like action of bradykinin in the dorsal periaqueductal gray through μ-opioid and B2-kinin receptors.

    PubMed

    Sestile, Caio César; Maraschin, Jhonatan Christian; Gama, Vanessa Scalco; Zangrossi, Hélio; Graeff, Frederico Guilherme; Audi, Elisabeth Aparecida

    2017-09-01

    A wealth of evidence has shown that opioid and kinin systems may control proximal defense in the dorsal periaqueductal gray matter (dPAG), a critical panic-associated area. Studies with drugs that interfere with serotonin-mediated neurotransmission suggest that the μ-opioid receptor (MOR) synergistically interacts with the 5-HT 1A receptor in the dPAG to inhibit escape, a panic-related behavior. A similar inhibitory effect has also been reported after local administration of bradykinin (BK), which is blocked by the non-selective opioid receptor antagonist naloxone. The latter evidence, points to an interaction between BK and opioids in the dPAG. We further explored the existence of this interaction through the dPAG electrical stimulation model of panic. We also investigated whether intra-dPAG injection of captopril, an inhibitor of the angiotensin-converting enzyme (ACE) that also degrades BK, causes a panicolytic-like effect. Our results showed that intra-dPAG injection of BK inhibited escape performance in a dose-dependent way, and this panicolytic-like effect was blocked by the BK type 2 receptor (B2R) antagonist HOE-140, and by the selective MOR antagonist CTOP. Conversely, the panicolytic-like effect caused by local administration of the selective MOR agonist DAMGO was antagonized by pre-treatment with either CTOP or HOE-140, indicating cross-antagonism between MOR and B2R. Finally, intra-dPAG injection of captopril also impaired escape in a dose-dependent way, and this panicolytic-like effect was blocked by pretreatment with HOE-140, suggesting mediation by endogenous BK. The panicolytic-like effect of captopril indicates that the use of ACE inhibitors in the clinical management of panic disorder may be worth exploring. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats.

    PubMed

    Aono, Yuri; Kiguchi, Yuri; Watanabe, Yuriko; Waddington, John L; Saigusa, Tadashi

    2017-11-15

    The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Injections of the selective adenosine A2A antagonist MSX-3 into the nucleus accumbens core attenuate the locomotor suppression induced by haloperidol in rats.

    PubMed

    Ishiwari, Keita; Madson, Lisa J; Farrar, Andrew M; Mingote, Susana M; Valenta, John P; DiGianvittorio, Michael D; Frank, Lauren E; Correa, Merce; Hockemeyer, Jörg; Müller, Christa; Salamone, John D

    2007-03-28

    There is considerable evidence of interactions between adenosine A2A receptors and dopamine D2 receptors in striatal areas, and antagonists of the A2A receptor have been shown to reverse the motor effects of DA antagonists in animal models. The D2 antagonist haloperidol produces parkinsonism in humans, and also induces motor effects in rats, such as suppression of locomotion. The present experiments were conducted to study the ability of the adenosine A2A antagonist MSX-3 to reverse the locomotor effects of acute or subchronic administration of haloperidol in rats. Systemic (i.p.) injections of MSX-3 (2.5-10.0 mg/kg) were capable of attenuating the suppression of locomotion induced by either acute or repeated (i.e., 14 day) administration of 0.5 mg/kg haloperidol. Bilateral infusions of MSX-3 directly into the nucleus accumbens core (2.5 microg or 5.0 microg in 0.5 microl per side) produced a dose-related increase in locomotor activity in rats treated with 0.5 mg/kg haloperidol either acutely or repeatedly. There were no overall significant effects of MSX-3 infused directly into the dorsomedial nucleus accumbens shell or the ventrolateral neostriatum. These results indicate that antagonism of adenosine A2A receptors can attenuate the locomotor suppression produced by DA antagonism, and that this effect may be at least partially mediated by A2A receptors in the nucleus accumbens core. These studies suggest that adenosine and dopamine systems interact to modulate the locomotor and behavioral activation functions of nucleus accumbens core.

  19. Injections of the selective adenosine A2A antagonist MSX-3 into the nucleus accumbens core attenuate the locomotor suppression induced by haloperidol in rats

    PubMed Central

    Ishiwari, Keita; Madson, Lisa J.; Farrar, Andrew M.; Mingote, Susana M.; Valenta, John P.; DiGianvittorio, Michael D.; Frank, Lauren E.; Correa, Merce; Hockemeyer, Jörg; Müller, Christa; Salamone, John D.

    2009-01-01

    There is considerable evidence of interactions between adenosine A2A receptors and dopamine D2 receptors in striatal areas, and antagonists of the A2A receptor have been shown to reverse the motor effects of DA antagonists in animal models. The D2 antagonist haloperidol produces parkinsonism in humans, and also induces motor effects in rats, such as suppression of locomotion. The present experiments were conducted to study the ability of the adenosine A2A antagonist MSX-3 to reverse the locomotor effects of acute or subchronic administration of haloperidol in rats. Systemic (i.p.) injections of MSX-3 (2.5–10.0 mg/kg) were capable of attenuating the suppression of locomotion induced by either acute or repeated (i.e., 14 day) administration of 0.5 mg/kg haloperidol. Bilateral infusions of MSX-3 directly into the nucleus accumbens core (2.5 µg or 5.0 µg in 0.5 µl per side) produced a dose-related increase in locomotor activity in rats treated with 0.5 mg/kg haloperidol either acutely or repeatedly. There were no overall significant effects of MSX-3 infused directly into the dorsomedial nucleus accumbens shell or the ventrolateral neostriatum. These results indicate that antagonism of adenosine A2A receptors can attenuate the locomotor suppression produced by DA antagonism, and that this effect may be at least partially mediated by A2A receptors in the nucleus accumbens core. These studies suggest that adenosine and dopamine systems interact to modulate the locomotor and behavioral activation functions of nucleus accumbens core. PMID:17223207

  20. Tachykinin-induced contraction of the guinea-pig isolated oesophageal mucosa is mediated by NK2 receptors

    PubMed Central

    Kerr, Karen P; Thai, Binh; Coupar, Ian M

    2000-01-01

    The tachykinin receptor present in the guinea-pig oesophageal mucosa that mediates contractile responses of the muscularis mucosae has been characterized, using functional in vitro experiments. The NK1 receptor-selective agonist, [Sar9(O2)Met11]SP and the NK3 receptor-selective agonists, [MePhe7]-NKB and senktide, produced no response at submicromolar concentrations. The NK2 receptor-selective agonists, [Nle10]-NKA(4–10), and GR 64,349 produced concentration-dependent contractile effects with pD2 values of 8.20±0.16 and 8.30±0.15, respectively. The concentration-response curve to the non-selective agonist, NKA (pD2=8.13±0.04) was shifted significantly rightwards only by the NK2 receptor-selective antagonist, GR 159,897 and was unaffected by the NK1 receptor-selective antagonist, SR 140,333 and the NK3 receptor-selective antagonist, SB 222,200. The NK2 receptor-selective antagonist, GR 159,897, exhibited an apparent competitive antagonism against the NK2 receptor-selective agonist, GR 64,349 (apparent pKB value=9.29±0.16) and against the non-selective agonist, NKA (apparent pKB value=8.71±0.19). The NK2 receptor-selective antagonist, SR 48,968 exhibited a non-competitive antagonism against the NK2 receptor-selective agonist, [Nle10]-NKA(4–10). The pKB value was 10.84±0.19. It is concluded that the guinea-pig isolated oesophageal mucosa is a useful preparation for studying the effects of NK2 receptor-selective agonists and antagonists as the contractile responses to various tachykinins are mediated solely by NK2 receptors. PMID:11090121

  1. Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

    PubMed

    Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

    2012-08-01

    Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

  2. Life or death by NFκB, Losartan promotes survival in dy2J/dy2J mouse of MDC1A

    PubMed Central

    Elbaz, M; Yanay, N; Laban, S; Rabie, M; Mitrani-Rosenbaum, S; Nevo, Y

    2015-01-01

    Inflammation and fibrosis are well-defined mechanisms involved in the pathogenesis of the incurable Laminin α2-deficient congenital muscular dystrophy (MDC1A), while apoptosis mechanism is barely discussed. Our previous study showed treatment with Losartan, an angiotensin II type I receptor antagonist, improved muscle strength and reduced fibrosis through transforming growth factor beta (TGF-β) and mitogen-activated protein kinases (MAPK) signaling inhibition in the dy2J/dy2J mouse model of MDC1A. Here we show for the first time that Losartan treatment up-regulates and shifts the nuclear factor kappa B (NFκB) signaling pathway to favor survival versus apoptosis/damage in this animal model. Losartan treatment was associated with significantly increased serum tumor necrosis factor alpha (TNF-α) level, p65 nuclei accumulation, and decreased muscle IκB-β protein level, indicating NFκB activation. Moreover, NFκB anti-apoptotic target genes TNF receptor-associated factor 1 (TRAF1), TNF receptor-associated factor 2 (TRAF2), cellular inhibitor of apoptosis (cIAP2), and Ferritin heavy chain (FTH1) were increased following Losartan treatment. Losartan induced protein expression toward a pro-survival profile as BCL-2 expression levels were increased and Caspase-3 expression levels were decreased. Muscle apoptosis reduction was further confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. Thus, along with TGF-β and MAPK signaling, NFκB serves as an important regulatory pathway which following Losartan treatment promotes survival in the dy2J/dy2J mouse model of MDC1A. PMID:25766329

  3. MDA5 and LGP2: Accomplices and Antagonists of Antiviral Signal Transduction

    PubMed Central

    Rodriguez, Kenny R.; Bruns, Annie M.

    2014-01-01

    Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral transcriptional response that provides an initial barrier to replication and impacts both innate and adaptive immune responses. Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) proteins mediate intracellular virus recognition and are activated by viral RNA ligands to induce antiviral signal transduction. While the mechanisms of RIG-I regulation are already well understood, less is known about the more enigmatic melanoma differentiation-associated 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Emerging evidence suggests that these two RLRs are intimately associated as both accomplices and antagonists of antiviral signal transduction. PMID:24850739

  4. The BRAIN TRIAL: a randomised, placebo controlled trial of a Bradykinin B2 receptor antagonist (Anatibant) in patients with traumatic brain injury.

    PubMed

    Shakur, Haleema; Andrews, Peter; Asser, Toomas; Balica, Laura; Boeriu, Cristian; Quintero, Juan Diego Ciro; Dewan, Yashbir; Druwé, Patrick; Fletcher, Olivia; Frost, Chris; Hartzenberg, Bennie; Mantilla, Jorge Mejia; Murillo-Cabezas, Francisco; Pachl, Jan; Ravi, Ramalingam R; Rätsep, Indrek; Sampaio, Cristina; Singh, Manmohan; Svoboda, Petr; Roberts, Ian

    2009-12-03

    Cerebral oedema is associated with significant neurological damage in patients with traumatic brain injury. Bradykinin is an inflammatory mediator that may contribute to cerebral oedema by increasing the permeability of the blood-brain barrier. We evaluated the safety and effectiveness of the non-peptide bradykinin B2 receptor antagonist Anatibant in the treatment of patients with traumatic brain injury. During the course of the trial, funding was withdrawn by the sponsor. Adults with traumatic brain injury and a Glasgow Coma Scale score of 12 or less, who had a CT scan showing an intracranial abnormality consistent with trauma, and were within eight hours of their injury were randomly allocated to low, medium or high dose Anatibant or to placebo. Outcomes were Serious Adverse Events (SAE), mortality 15 days following injury and in-hospital morbidity assessed by the Glasgow Coma Scale (GCS), the Disability Rating Scale (DRS) and a modified version of the Oxford Handicap Scale (HIREOS). 228 patients out of a planned sample size of 400 patients were randomised. The risk of experiencing one or more SAEs was 26.4% (43/163) in the combined Anatibant treated group, compared to 19.3% (11/57) in the placebo group (relative risk = 1.37; 95% CI 0.76 to 2.46). All cause mortality in the Anatibant treated group was 19% and in the placebo group 15.8% (relative risk 1.20, 95% CI 0.61 to 2.36). The mean GCS at discharge was 12.48 in the Anatibant treated group and 13.0 in the placebo group. Mean DRS was 11.18 Anatibant versus 9.73 placebo, and mean HIREOS was 3.94 Anatibant versus 3.54 placebo. The differences between the mean levels for GCS, DRS and HIREOS in the Anatibant and placebo groups, when adjusted for baseline GCS, showed a non-significant trend for worse outcomes in all three measures. This trial did not reach the planned sample size of 400 patients and consequently, the study power to detect an increase in the risk of serious adverse events was reduced. This trial

  5. Discovery of Fevipiprant (NVP-QAW039), a Potent and Selective DP2 Receptor Antagonist for Treatment of Asthma

    PubMed Central

    2017-01-01

    Further optimization of an initial DP2 receptor antagonist clinical candidate NVP-QAV680 led to the discovery of a follow-up molecule 2-(2-methyl-1-(4-(methylsulfonyl)-2-(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetic acid (compound 11, NVP-QAW039, fevipiprant), which exhibits improved potency on human eosinophils and Th2 cells, together with a longer receptor residence time, and is currently in clinical trials for severe asthma. PMID:28523115

  6. Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the aryl hydrocarbon receptor (AHR)-mediated impairment of immunoglobulin secretion in human primary B cells.

    PubMed

    Zhou, Jiajun; Zhang, Qiang; Henriquez, Joseph E; Crawford, Robert B; Kaminski, Norbert E

    2018-05-31

    The aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing, cell cycle regulation and cell development. In humans, the activation of AHR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AHR-ligand, impairs the secretion of immunoglobulin M (IgM) to suppress humoral immunity. However, the mechanisms bridging the activation of AHR and the impairment of IgM secretion by human primary B cells remain poorly understood. Recent transcriptomic analysis revealed upregulation of lymphocyte-specific protein tyrosine kinase (LCK) in AHR activated human primary B cells. LCK is a well-characterized tyrosine kinase that phosphorylates critical signaling proteins involved in activation and cytokine production in T cells. Conversely, the role of LCK in human primary B cells is not well understood. In the current studies, we have verified the transcriptomic finding by detecting AHR-mediated upregulation of LCK protein in human primary B cells. We also confirmed the role of AHR in the upregulation of LCK by using a specific AHR antagonist, which abolished the AHR-mediated increase of LCK. Furthermore, we have confirmed the role of LCK in the AHR-mediated suppression of IgM by using LCK specific inhibitors, which restored IgM secretion by human B cells in the presence of TCDD. Collectively, the current studies demonstrate a novel role of LCK in IgM secretion and provide new insights into the mechanism for AHR-mediated impairment of immunoglobulin secretion by human primary B cells.

  7. A Novel Aminotetralin-Type Serotonin (5-HT) 2C Receptor-Specific Agonist and 5-HT2A Competitive Antagonist/5-HT2B Inverse Agonist with Preclinical Efficacy for Psychoses

    PubMed Central

    Morgan, Drake; Felsing, Daniel; Kondabolu, Krishnakanth; Rowland, Neil E.; Robertson, Kimberly L.; Sakhuja, Rajeev; Booth, Raymond G.

    2014-01-01

    Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (−)-trans-(2S,4R)-4-(3′[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (−)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (−)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (−)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (−)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (−)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (−)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders. PMID:24563531

  8. Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A2B adenosine receptors.

    PubMed

    Antonioli, Luca; Pellegrini, Carolina; Fornai, Matteo; Tirotta, Erika; Gentile, Daniela; Benvenuti, Laura; Giron, Maria Cecilia; Caputi, Valentina; Marsilio, Ilaria; Orso, Genny; Bernardini, Nunzia; Segnani, Cristina; Ippolito, Chiara; Csóka, Balázs; Németh, Zoltán H; Haskó, György; Scarpignato, Carmelo; Blandizzi, Corrado; Colucci, Rocchina

    2017-12-01

    Adenosine A 2B receptors (A 2B R) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A 2B R in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A 2B R localization was examined by immunofluorescence. The role of A 2B R in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A 2B R were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A 2B R antagonist MRS1754 enhanced electrically induced NK 1 -mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A 2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A 2B R ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A 2B R expression. A 2B R, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

  9. Angiotensin II type 2 receptor (AT2R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons

    PubMed Central

    Anand, U; Facer, P; Yiangou, Y; Sinisi, M; Fox, M; McCarthy, T; Bountra, C; Korchev, YE; Anand, P

    2013-01-01

    Background The angiotensin II (AngII) receptor subtype 2 (AT2R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. Methods We used immunostaining with characterized antibodies to study the localization of AT2R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. Results AT2R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1R antagonist losartan had no effect on capsaicin responses. AT2R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. Conclusions AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting. PMID:23255326

  10. Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

    PubMed

    Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

    2015-11-01

    In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Serotonin 2C receptor antagonist improves fear discrimination and subsequent safety signal recall

    PubMed Central

    Foilb, Allison R.; Christianson, John P.

    2015-01-01

    The capacity to discriminate between safety and danger is fundamental for survival, but is disrupted in individuals with posttraumatic stress disorder (PTSD). Acute stressors cause a release of serotonin (5-HT) in the forebrain, which is one mechanism for enhanced fear and anxiety; these effects are mediated by the 5-HT2C receptor. Using a fear discrimination paradigm where a danger signal conditioned stimulus (CS+) coterminates with a mild footshock and a safety signal (CS-) indicates the absence of shock, we demonstrate that danger/safety discrimination and fear inhibition develops over the course of 4 daily conditioning sessions. Systemic administration of the 5-HT2C receptor antagonist SB 242084 (0.25 or 1.0 mg/kg) prior to conditioning reduced behavioral freezing during conditioning, improved learning and subsequent inhibition of fear by the safety signal. Discrimination was apparent in the first recall test, and discrimination during training was evident after 3 days of conditioning versus 5 days in the vehicle treated controls. These results suggest a novel therapeutic use for 5-HT2C receptor antagonists to improve learning under stressful circumstances. Potential anatomical loci for 5-HT2C receptor modulation of fear discrimination learning and cognitive performance enhancement are discussed. PMID:26344640

  12. Characterization of JNJ-42847922, a Selective Orexin-2 Receptor Antagonist, as a Clinical Candidate for the Treatment of Insomnia.

    PubMed

    Bonaventure, Pascal; Shelton, Jonathan; Yun, Sujin; Nepomuceno, Diane; Sutton, Steven; Aluisio, Leah; Fraser, Ian; Lord, Brian; Shoblock, James; Welty, Natalie; Chaplan, Sandra R; Aguilar, Zuleima; Halter, Robin; Ndifor, Anthony; Koudriakova, Tatiana; Rizzolio, Michele; Letavic, Michael; Carruthers, Nicholas I; Lovenberg, Timothy; Dugovic, Christine

    2015-09-01

    Dual orexin receptor antagonists have been shown to promote sleep in various species, including humans. Emerging research indicates that selective orexin-2 receptor (OX2R) antagonists may offer specificity and a more adequate sleep profile by preserving normal sleep architecture. Here, we characterized JNJ-42847922 ([5-(4,6-dimethyl-pyrimidin-2-yl)-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl]-(2-fluoro-6-[1,2,3]triazol-2-yl-phenyl)-methanone), a high-affinity/potent OX2R antagonist. JNJ-42847922 had an approximate 2-log selectivity ratio versus the human orexin-1 receptor. Ex vivo receptor binding studies demonstrated that JNJ-42847922 quickly occupied OX2R binding sites in the rat brain after oral administration and rapidly cleared from the brain. In rats, single oral administration of JNJ-42847922 (3-30 mg/kg) during the light phase dose dependently reduced the latency to non-rapid eye movement (NREM) sleep and prolonged NREM sleep time in the first 2 hours, whereas REM sleep was minimally affected. The reduced sleep onset and increased sleep duration were maintained upon 7-day repeated dosing (30 mg/kg) with JNJ-42847922, then all sleep parameters returned to baseline levels following discontinuation. Although the compound promoted sleep in wild-type mice, it had no effect in OX2R knockout mice, consistent with a specific OX2R-mediated sleep response. JNJ-42847922 did not increase dopamine release in rat nucleus accumbens or produce place preference in mice after subchronic conditioning, indicating that the compound lacks intrinsic motivational properties in contrast to zolpidem. In a single ascending dose study conducted in healthy subjects, JNJ-42847922 increased somnolence and displayed a favorable pharmacokinetic and safety profile for a sedative/hypnotic, thus emerging as a promising candidate for further clinical development for the treatment of insomnia. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation.

    PubMed

    Reddy, Jay P; Peddibhotla, Sirisha; Bu, Wen; Zhao, Jing; Haricharan, Svasti; Du, Yi-Chieh Nancy; Podsypanina, Katrina; Rosen, Jeffrey M; Donehower, Larry A; Li, Yi

    2010-02-23

    p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

  14. Antidepressant activity of the adenosine A2A receptor antagonist, istradefylline (KW-6002) on learned helplessness in rats.

    PubMed

    Yamada, Koji; Kobayashi, Minoru; Shiozaki, Shizuo; Ohta, Teruko; Mori, Akihisa; Jenner, Peter; Kanda, Tomoyuki

    2014-07-01

    Istradefylline, an adenosine A2A receptor antagonist, improves motor function in animal models of Parkinson's disease (PD) and in patients with PD. In addition, some A2A antagonists exert antidepressant-like activity in rodent models of depression, such as the forced swim and the tail suspension tests. We have investigated the effect of istradefylline on depression-like behaviors using the rat learned helplessness (LH) model. Acute, as well as chronic, oral administration of istradefylline significantly improved the inescapable shock (IES)-induced escape deficit with a degree of efficacy comparable to chronic treatment with the tricyclic antidepressant desipramine and the selective serotonin (5-HT) reuptake inhibitor, fluoxetine. Both the A1/A2A receptor nonspecific antagonist theophylline and the moderately selective antagonist CGS15943, but not the A1 selective antagonist DPCPX, ameliorated the IES-induced escape deficit. The enhancement of escape response by istradefylline was reversed by a local injection of the A2A specific agonist CGS21680 either into the nucleus accumbens, the caudate-putamen, or the paraventricular nucleus of the hypothalamus, but not by the A1 specific agonist R-PIA into the nucleus accumbens. Moreover, neither the 5-HT2A/2C receptor antagonist methysergide or the adrenergic α 2 antagonist yohimbine, nor the β-adrenergic antagonist propranolol, affected the improvement of escape response induced by istradefylline. Istradefylline exerts antidepressant-like effects via modulation of A2A receptor activity which is independent of monoaminergic transmission in the brain. Istradefylline may represent a novel treatment option for depression in PD as well as for the motor symptoms.

  15. Specific LPA receptor subtype mediation of LPA-induced hypertrophy of cardiac myocytes and involvement of Akt and NFkappaB signal pathways.

    PubMed

    Chen, Jinghai; Chen, Yuefeng; Zhu, Weiquan; Han, Yu; Han, Bianmei; Xu, Ruixia; Deng, Linzi; Cai, Yan; Cong, Xiangfeng; Yang, Yuejing; Hu, Shengshou; Chen, Xi

    2008-04-15

    Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.

  16. Discovery of sodium R-(+)-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyrate (elagolix), a potent and orally available nonpeptide antagonist of the human gonadotropin-releasing hormone receptor.

    PubMed

    Chen, Chen; Wu, Dongpei; Guo, Zhiqiang; Xie, Qiu; Reinhart, Greg J; Madan, Ajay; Wen, Jenny; Chen, Takung; Huang, Charles Q; Chen, Mi; Chen, Yongsheng; Tucci, Fabio C; Rowbottom, Martin; Pontillo, Joseph; Zhu, Yun-Fei; Wade, Warren; Saunders, John; Bozigian, Haig; Struthers, R Scott

    2008-12-11

    The discovery of novel uracil phenylethylamines bearing a butyric acid as potent human gonadotropin-releasing hormone receptor (hGnRH-R) antagonists is described. A major focus of this optimization was to improve the CYP3A4 inhibition liability of these uracils while maintaining their GnRH-R potency. R-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyric acid sodium salt, 10b (elagolix), was identified as a potent and selective hGnRH-R antagonist. Oral administration of 10b suppressed luteinizing hormone in castrated macaques. These efforts led to the identification of 10b as a clinical compound for the treatment of endometriosis.

  17. Structure of CC Chemokine Receptor 2 with Orthosteric and Allosteric Antagonists

    PubMed Central

    Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel, Tracy M.

    2016-01-01

    Summary CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human Class A G protein-coupled receptors (GPCRs). CCR2 is expressed on monocytes, immature dendritic cells and T cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL21. CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see ClinicalTrials.gov) in search of therapies that target the CCR2:chemokine axis. To aid drug discovery efforts5, we solved a structure of CCR2 in a ternary complex with an orthosteric (BMS-6816) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in Class A GPCRs to date; this site spatially overlaps the G protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive GPCR structures solved to date. Like other protein:protein interactions, receptor:chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome drug design obstacles. PMID:27926736

  18. DOR(2)-selective but not DOR(1)-selective antagonist abolishes anxiolytic-like effects of the δ opioid receptor agonist KNT-127.

    PubMed

    Sugiyama, Azusa; Nagase, Hiroshi; Oka, Jun-Ichiro; Yamada, Mitsuhiko; Saitoh, Akiyoshi

    2014-04-01

    Recently, we reported that the δ opioid receptor (DOR) agonist KNT-127 produces anxiolytic-like effects in behaving rats. Here, we report on the roles of DOR subtypes ( DOR(1) and DOR(2)) play in mediating KNT-127-induced anxiolytic-like effects. Pretreatment with the DOR(2)-selective antagonist naltriben (NTB; 0.05mg/kg, s.c.) completely abolished KNT-127 (3.0mg/kg, s.c.)-induced anxiolytic-like effects in rats performing the elevated plus-maze task. By contrast, the DOR(1)-selective antagonist 7-benzylidenenaltrexone (BNTX; 0.5mg/kg, s.c.) produced no effect at a dose that completely blocked the antinociceptive effects of KNT-127. These findings were also supported by results from a light/dark test and open-field test. We clearly demonstrated that the DOR(2)-selective antagonist, but not the DOR(1)-selective antagonist, abolishes the anxiolytic-like effects of the DOR agonist KNT-127, suggesting different roles of these DOR subtypes in anxiety. We propose that DOR(2)-selective agonists would be good candidates for future development of anxiolytic drugs. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  19. Identification of two benzopyrroloxazines acting as selective GPER antagonists in breast cancer cells and cancer-associated fibroblasts.

    PubMed

    Maggiolini, Marcello; Santolla, Maria Francesca; Avino, Silvia; Aiello, Francesca; Rosano, Camillo; Garofalo, Antonio; Grande, Fedora

    2015-01-01

    G-protein coupled estrogen receptor (GPER) is involved in numerous intracellular physiological and pathological events including cancer cell migration and proliferation. Its characterization is yet incomplete due to the limited number of specific ligands. Two novel selective GPER antagonists, based on a benzo[b]pyrrolo[1,2-d][1,4]oxazin-4-one structure, have been designed and synthesized. Their binding to the receptor was confirmed by a competition assay, while the antagonist effects were ascertained by their capability to prevent the ligand-stimulated action of GPER. The transcription mediated by the classical estrogen receptor was not influenced, demonstrating selectivity for GPER. These novel compounds may be considered useful leads toward the dissection of the GPER signaling and the development of new pharmacological treatments in breast cancer.

  20. Rap1b Is an Effector of Axin2 Regulating Crosstalk of Signaling Pathways During Skeletal Development.

    PubMed

    Maruyama, Takamitsu; Jiang, Ming; Abbott, Alycia; Yu, H-M Ivy; Huang, Qirong; Chrzanowska-Wodnicka, Magdalena; Chen, Emily I; Hsu, Wei

    2017-09-01

    Recent identification and isolation of suture stem cells capable of long-term self-renewal, clonal expanding, and differentiating demonstrate their essential role in calvarial bone development, homeostasis, and injury repair. These bona fide stem cells express a high level of Axin2 and are able to mediate bone regeneration and repair in a cell autonomous fashion. The importance of Axin2 is further demonstrated by its genetic inactivation in mice causing skeletal deformities resembling craniosynostosis in humans. The fate determination and subsequent differentiation of Axin2+ stem cells are highly orchestrated by a variety of evolutionary conserved signaling pathways including Wnt, FGF, and BMP. These signals are often antagonistic of each other and possess differential effects on osteogenic and chondrogenic cell types. However, the mechanisms underlying the interplay of these signaling transductions remain largely elusive. Here we identify Rap1b acting downstream of Axin2 as a signaling interrogator for FGF and BMP. Genetic analysis reveals that Rap1b is essential for development of craniofacial and body skeletons. Axin2 regulates Rap1b through modulation of canonical BMP signaling. The BMP-mediated activation of Rap1b promotes chondrogenic fate and chondrogenesis. Furthermore, by inhibiting MAPK signaling, Rap1b mediates the antagonizing effect of BMP on FGF to repress osteoblast differentiation. Disruption of Rap1b in mice not only enhances osteoblast differentiation but also impairs chondrocyte differentiation during intramembranous and endochondral ossifications, respectively, leading to severe defects in craniofacial and body skeletons. Our findings reveal a dual role of Rap1b in development of the skeletogenic cell types. Rap1b is critical for balancing the signaling effects of BMP and FGF during skeletal development and disease. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

  1. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    PubMed

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  2. 5-HT2A receptor antagonists improve motor impairments in the MPTP mouse model of Parkinson's disease.

    PubMed

    Ferguson, Marcus C; Nayyar, Tultul; Deutch, Ariel Y; Ansah, Twum A

    2010-01-01

    Clinical observations have suggested that ritanserin, a 5-HT(2A/C) receptor antagonist may reduce motor deficits in persons with Parkinson's Disease (PD). To better understand the potential antiparkinsonian actions of ritanserin, we compared the effects of ritanserin with the selective 5-HT(2A) receptor antagonist M100907 and the selective 5-HT(2C) receptor antagonist SB 206553 on motor impairments in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-treated mice exhibited decreased performance on the beam-walking apparatus. These motor deficits were reversed by acute treatment with L-3,4-dihydroxyphenylalanine (levodopa). Both the mixed 5-HT(2A/C) antagonist ritanserin and the selective 5-HT(2A) antagonist M100907 improved motor performance on the beam-walking apparatus. In contrast, SB 206553 was ineffective in improving the motor deficits in MPTP-treated mice. These data suggest that 5-HT(2A) receptor antagonists may represent a novel approach to ameliorate motor symptoms of Parkinson's disease. Published by Elsevier Ltd.

  3. 5-HT2A receptor antagonists improve motor impairments in the MPTP mouse model of Parkinson's disease

    PubMed Central

    Ferguson, Marcus C.; Nayyar, Tultul; Deutch, Ariel Y.; Ansah, Twum A.

    2010-01-01

    Clinical observations have suggested that ritanserin, a 5-HT2A/C receptor antagonist may reduce motor deficits in persons with Parkinson's Disease (PD). To better understand the potential antiparkinsonian actions of ritanserin, we compared the effects of ritanserin with the selective 5-HT2A receptor antagonist M100907 and the selective 5-HT2C receptor antagonist SB 206553 on motor impairments in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-treated mice exhibited decreased performance on the beam-walking apparatus. These motor deficits were reversed by acute treatment with L-3,4-dihydroxyphenylalanine (levodopa). Both the mixed 5-HT2A/C antagonist ritanserin and the selective 5-HT2A antagonist M100907 improved motor performance on the beam-walking apparatus. In contrast, SB 206553 was ineffective in improving the motor deficits in MPTP-treated mice. These data suggest that 5-HT2A receptor antagonists may represent a novel approach to ameliorate motor symptoms of Parkinson's disease. PMID:20361986

  4. Could the 5-HT1B receptor inverse agonism affect learning consolidation?

    PubMed

    Meneses, A

    2001-03-01

    Diverse evidence indicates that, the 5-HT system might play a role in learning and memory, since it occurs in brain areas mediating such processes and 5-HT drugs modulate them. Hence in this work, in order to explore further 5-HT involvement on learning and memory 5-HT1B receptors' role is investigated. Evidence indicates that SB-224289 (a 5-HT1B receptor inverse agonist) post-training injection facilitated learning consolidation in an associative autoshaping learning task, this effect was partially reversed by GR 127935 (a 5-HT1B/1D receptor antagonist), but unaffected by MDL 100907 (a 5-HT2A receptor antagonist) or ketanserin (a 5-HT1D/2A/7 receptor antagonist) at low doses. Moreover, SB-224289 antagonized the learning deficit produced by TFMPP (a 5-HT1A/1B/1D/2A/2C receptor agonist), GR 46611 (a 5-HT1A/1B/1D receptor agonist), mCPP (a 5-HT2A/2C/3/7 receptor agonist/antagonist) or GR 127935 (at low dose). SB-224289 did not alter the 8-OH-DPAT (a 5-HT1A/7 receptor agonist) learning facilitatory effect. SB-224289 eliminated the deficit learning produced by the anticholinergic muscarinic scopolamine or the glutamatergic antagonist dizocilpine. Administration of both, GR 127935 (5mg/kg) plus ketanserin (0.01 mg/kg) did not modify learning consolidation; nevertheless, when ketanserin dose was increased (0.1-1.0mg/kg) and SB-224289 dose was maintained constant, a learning facilitation effect was observed. Notably, SB-224289 at 1.0mg/kg potentiated a subeffective dose of the 5-HT1B/1D receptor agonist/antagonist mixed GR 127935, which facilitated learning consolidation and this effect was abolished by ketanserin at a higher dose. Collectively, the data confirm and extend the earlier findings with GR 127935 and the effects of non-selective 5-HT(1B) receptor agonists. Clearly 5-HT1B agonists induced a learning deficit which can be reversed with SB-224289. Perhaps more importantly, SB-224289 enhances learning consolidation when given alone and can reverse the deficits

  5. ENMD-1068, a protease-activated receptor 2 antagonist, inhibits the development of endometriosis in a mouse model.

    PubMed

    Wang, Yifeng; Lin, Min; Weng, Huinan; Wang, Xuefeng; Yang, Li; Liu, Fenghua

    2014-06-01

    Protease-activated receptor 2 plays an important role in the pathogenesis of endometriosis. We studied the effect of ENMD-1068, a protease-activated receptor 2 antagonist, on the development of endometriosis in a noninvasive fluorescent mouse model. A red fluorescent protein-expressing xenograft model of human endometriosis was created in nude mice. After endometriosis induction, the mice were injected intraperitoneally with either 25 mg/kg or 50 mg/kg ENMD-1068 or with 200 μL of the vehicle control daily for 5 days. The endometriotic lesions that developed in the mice were then counted, measured, and collected. The lesions were assessed for the production of interleukin 6 and monocyte chemotactic protein-1 by enzyme-linked immunosorbent assays and evaluated for the activation of nuclear factor-κB and the expression of vascular endothelial growth factor by immunohistochemical analyses. Cell proliferation and apoptosis were assessed by immunohistochemistry for Ki-67 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. ENMD-1068 dose-dependently inhibited the development of endometriotic lesions (P < .05) without apparent toxicity to various organs of the treated mice. Consistently, ENMD-1068 dose-dependently inhibited the expression of interleukin 6 and nuclear factor-κB (P < .05) and cell proliferation (P < .05) in the lesions, as well as increased the percentage of apoptotic cells (P < .05). ENMD-1068 reduced the levels of monocyte chemotactic protein-1 and vascular endothelial growth factor in the lesions (P < .05), but not in a dose-dependent manner. Our study suggests that ENMD-1068 is effective in suppressing the growth of endometriosis, which might be attributed to the drug's antiangiogenic and antiinflammatory activities. Copyright © 2014 Mosby, Inc. All rights reserved.

  6. Baclofen, a GABABR Agonist, Ameliorates Immune-Complex Mediated Acute Lung Injury by Modulating Pro-Inflammatory Mediators

    PubMed Central

    Jin, Shunying; Merchant, Michael L.; Ritzenthaler, Jeffrey D.; McLeish, Kenneth R.; Lederer, Eleanor D.; Torres-Gonzalez, Edilson; Fraig, Mostafa; Barati, Michelle T.; Lentsch, Alex B.; Roman, Jesse; Klein, Jon B.; Rane, Madhavi J.

    2015-01-01

    Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a

  7. H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor.

    PubMed

    Zeng, Zhiwen; Wang, Dejun; Gaur, Uma; Rifang, Liao; Wang, Haitao; Zheng, Wenhua

    2017-09-12

    Impairment of insulin-like growth factor I (IGF-I) signaling plays an important role in the development of neurodegeneration. In the present study, we investigated the effect of H 2 O 2 on the survival signaling of IGF-1 and its underlying mechanisms in human neuronal cells SH-SY5Y. Our results showed that IGF-1 promoted cell survival and stimulated phosphorylation of IGF-1R as well as its downstream targets like AKT and ERK1/2 in these cells. Meanwhile, these effects of IGF-1 were abolished by H 2 O 2 at 200μM concentration which did not cause any significant toxicity to cells itself in our experiments. Moreover, studies using various glutamate receptor subtype antagonists displayed that N-methyl-D -aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) blocked the effects of H 2 O 2 , whereas other glutamate receptor subtype antagonists, such as non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), metabolic glutamate receptor antagonists LY341495 and CPCCOEt, had no effect. Further studies revealed that NR2B-containing NMDARs are responsible for these effects as its effects were blocked by pharmacological inhibitor Ro25-698 or specific siRNA for NR2B, but not NR2A. Finally, our data also showed that Ca 2+ influx contributes to the effects of H 2 O 2 . Similar results were obtained in primary cultured cortical neurons. Taken together, the results from the present study suggested that H 2 O 2 attenuated IGF-1R tyrosine phosphorylation and its survival signaling properties via NR2B containing NMDA receptors and Ca 2+ influx in SH-SY5Y cells. Therefore, NMDAR antagonists, especially NR2B-selective ones, combined with IGF-1 may serve as an alternative therapeutic agent for oxidative stress related neurodegenerative disease.

  8. Tachykinins mediate contraction of the human lower esophageal sphincter in vitro via activation of NK2 receptors.

    PubMed

    Huber, O; Bertrand, C; Bunnett, N W; Pellegrini, C A; Nadel, J A; Nakazato, P; Debas, H T; Geppetti, P

    1993-08-03

    The contractile response to natural tachykinins and selective peptide agonists for tachykinin receptors was studied in strips of circular smooth muscle of human lower esophageal sphincter in vitro. The effects of phosphoramidon, which inhibits neutral endopeptidase (EC.3.4.24.11) and of the non-peptide compounds, SR 48968 and CP-96,345, which selectively block NK1 and NK2 receptors, respectively, were also investigated. Substance P, neurokinin A and neurokinin B produced a concentration-dependent contractile response. The rank order of potency was neurokinin A > neurokinin B > substance P. Phosphoramidon (1 microM) potentiated the response to substance P without changing the order of potency of natural tachykinins. The NK2-selective agonist, ([ beta Ala8]neurokinin A-(4-10)), produced a concentration-dependent contraction. The NK1 ([Sar9,Met(O2)11]substance P, 1 microM) and NK3 ([MePhe7]neurokinin B, 1 microM) selective agonists, however, did not exert any contractile effect. The selective NK2 antagonist, SR 48968, potently inhibited in a concentration-dependent (10 nM-1 microM) manner the response to neurokinin A, without affecting the response to carbachol. The selective NK1 antagonist, CP-96,345 (1 microM), did not affect the response to neurokinin A. These results indicate that tachykinins contract the circular muscle of human lower esophageal sphincter, and that this effect is mediated by NK2 receptor stimulation. Moreover, a phosphoramidon-sensitive mechanism plays a role in the regulation of the response to substance P.

  9. SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.

    PubMed

    Duan, Chaojun; Li, Minghua; Rui, Liangyou

    2004-10-15

    Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

  10. Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

    PubMed Central

    Kozaki, Tatsuya; Komano, Jun; Kanbayashi, Daiki; Takahama, Michihiro; Misawa, Takuma; Satoh, Takashi; Takeuchi, Osamu; Kawai, Taro; Shimizu, Shigeomi; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

    2017-01-01

    The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage. PMID:28213497

  11. The adenosine A2A antagonist MSX-3 reverses the effort-related effects of dopamine blockade: differential interaction with D1 and D2 family antagonists.

    PubMed

    Worden, Lila T; Shahriari, Mona; Farrar, Andrew M; Sink, Kelly S; Hockemeyer, Jörg; Müller, Christa E; Salamone, John D

    2009-04-01

    Brain dopamine (DA) participates in the modulation of instrumental behavior, including aspects of behavioral activation and effort-related choice behavior. Rats with impaired DA transmission reallocate their behavior away from food-seeking behaviors that have high response requirements, and instead select less effortful alternatives. Although accumbens DA is considered a critical component of the brain circuitry regulating effort-related choice behavior, emerging evidence demonstrates a role for adenosine A(2A) receptors. Adenosine A(2A) receptor antagonism has been shown to reverse the effects of DA antagonism. The present experiments were conducted to determine if this effect was dependent upon the subtype of DA receptor that was antagonized to produce the changes in effort-related choice. The adenosine A(2A) receptor antagonist MSX-3 (0.5-2.0 mg/kg IP) was assessed for its ability to reverse the effects of the D1 family antagonist SCH39166 (ecopipam; 0.2 mg/kg IP) and the D2 family antagonist eticlopride (0.08 mg/kg IP), using a concurrent lever pressing/chow feeding procedure. MSX-3 produced a substantial dose-related reversal of the effects of eticlopride on lever pressing and chow intake. At the highest dose of MSX-3, there was a complete reversal of the effects of eticlopride on lever pressing. In contrast, MSX-3 produced only a minimal attenuation of the effects of SCH39166, as measured by regression and effect size analyses. The greater ability of MSX-3 to reverse the effects of D2 vs. D1 blockade may be related to the colocalization of D2 and adenosine A(2A) receptors on the same population of striatal neurons.

  12. Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation

    PubMed Central

    Reddy, Jay P.; Peddibhotla, Sirisha; Bu, Wen; Zhao, Jing; Haricharan, Svasti; Du, Yi-Chieh Nancy; Podsypanina, Katrina; Rosen, Jeffrey M.; Donehower, Larry A.; Li, Yi

    2010-01-01

    p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention. PMID:20133707

  13. Bradykinin B1 and B2 receptors, tumour necrosis factor α and inflammatory hyperalgesia

    PubMed Central

    Poole, S; Lorenzetti, B B; Cunha, J M; Cunha, F Q; Ferreira, S H

    1999-01-01

    The effects of BK agonists and antagonists, and other hyperalgesic/antihyperalgesic drugs were measured (3 h after injection of hyperalgesic drugs) in a model of mechanical hyperalgesia (the end-point of which was indicated by a brief apnoea, the retraction of the head and forepaws, and muscular tremor). DALBK inhibited responses to carrageenin, bradykinin, DABK, and kallidin. Responses to kallidin and DABK were inhibited by indomethacin or atenolol and abolished by the combination of indomethacin+atenolol. DALBK or HOE 140, given 30 min before, but not 2 h after, carrageenin, BK, DABK and kallidin reduced hyperalgesic responses to these agents. A small dose of DABK+a small dose of BK evoked a response similar to the response to a much larger dose of DABK or BK, given alone. Responses to BK were antagonized by HOE 140 whereas DALBK antagonized only responses to larger doses of BK. The combination of a small dose of DALBK with a small dose of HOE 140 abolished the response to BK. The hyperalgesic response to LPS (1 μg) was inhibited by DALBK or HOE 140 and abolished by DALBK+HOE 140. The hyperalgesic response to LPS (5 μg) was not antagonized by DALBK+HOE 140. These data suggest: (a) a predominant role for B2 receptors in mediating hyperalgesic responses to BK and to drugs that stimulate BK release, and (b) activation of the hyperalgesic cytokine cascade independently of both B1 and B2 receptors if the hyperalgesic stimulus is of sufficient magnitude. PMID:10188975

  14. Antagonistic Actions of HLH/bHLH Proteins Are Involved in Grain Length and Weight in Rice

    PubMed Central

    Heang, Dany; Sassa, Hidenori

    2012-01-01

    Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1. PMID:22363621

  15. Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic Schwann cells of the frog neuromuscular junction.

    PubMed Central

    Robitaille, R; Jahromi, B S; Charlton, M P

    1997-01-01

    1. Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo-3 and fluorescence microscopy. 2. Nicotinic antagonists had no effect on Ca2+ responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine-M induced Ca2+ signals in PSCs. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine. 4. The general muscarinic antagonists atropine, quinuclidinyl benzilate and N-methyl-scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20,000-fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non-specific blocker, gallamine (1-10 microM) was effective in blocking muscarine-induced Ca2+ responses. 5. In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine. 6. The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post-translational modification prevented antagonist binding. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:9365908

  16. B1 Cell IgE Impedes Mast Cell-Mediated Enhancement of Parasite Expulsion through B2 IgE Blockade.

    PubMed

    Martin, Rebecca K; Damle, Sheela R; Valentine, Yolander A; Zellner, Matthew P; James, Briana N; Lownik, Joseph C; Luker, Andrea J; Davis, Elijah H; DeMeules, Martha M; Khandjian, Laura M; Finkelman, Fred D; Urban, Joseph F; Conrad, Daniel H

    2018-02-13

    Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1 -/- mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Human T Cell Leukemia Virus Type 2 Tax-Mediated NF-κB Activation Involves a Mechanism Independent of Tax Conjugation to Ubiquitin and SUMO

    PubMed Central

    Journo, Chloé; Bonnet, Amandine; Favre-Bonvin, Arnaud; Turpin, Jocelyn; Vinera, Jennifer; Côté, Emilie; Chevalier, Sébastien Alain; Kfoury, Youmna; Bazarbachi, Ali

    2013-01-01

    Permanent activation of the NF-κB pathway by the human T cell leukemia virus type 1 (HTLV-1) Tax (Tax1) viral transactivator is a key event in the process of HTLV-1-induced T lymphocyte immortalization and leukemogenesis. Although encoding a Tax transactivator (Tax2) that activates the canonical NF-κB pathway, HTLV-2 does not cause leukemia. These distinct pathological outcomes might be related, at least in part, to distinct NF-κB activation mechanisms. Tax1 has been shown to be both ubiquitinated and SUMOylated, and these two modifications were originally proposed to be required for Tax1-mediated NF-κB activation. Tax1 ubiquitination allows recruitment of the IKK-γ/NEMO regulatory subunit of the IKK complex together with Tax1 into centrosome/Golgi-associated cytoplasmic structures, followed by activation of the IKK complex and RelA/p65 nuclear translocation. Herein, we compared the ubiquitination, SUMOylation, and acetylation patterns of Tax2 and Tax1. We show that, in contrast to Tax1, Tax2 conjugation to endogenous ubiquitin and SUMO is barely detectable while both proteins are acetylated. Importantly, Tax2 is neither polyubiquitinated on lysine residues nor ubiquitinated on its N-terminal residue. Consistent with these observations, Tax2 conjugation to ubiquitin and Tax2-mediated NF-κB activation is not affected by overexpression of the E2 conjugating enzyme Ubc13. We further demonstrate that a nonubiquitinable, non-SUMOylable, and nonacetylable Tax2 mutant retains a significant ability to activate transcription from a NF-κB-dependent promoter after partial activation of the IKK complex and induction of RelA/p65 nuclear translocation. Finally, we also show that Tax2 does not interact with TRAF6, a protein that was shown to positively regulate Tax1-mediated activation of the NF-κB pathway. PMID:23135727

  18. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zheng, Yi; Qin, Ling; Zacarías, Natalia V. Ortiz

    CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, heremore » we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.« less

  19. Root Bending Is Antagonistically Affected by Hypoxia and ERF-Mediated Transcription via Auxin Signaling1[OPEN

    PubMed Central

    Eysholdt-Derzsó, Emese

    2017-01-01

    When plants encounter soil water logging or flooding, roots are the first organs to be confronted with reduced gas diffusion resulting in limited oxygen supply. Since roots do not generate photosynthetic oxygen, they are rapidly faced with oxygen shortage rendering roots particularly prone to damage. While metabolic adaptations to low oxygen conditions, which ensure basic energy supply, have been well characterized, adaptation of root growth and development have received less attention. In this study, we show that hypoxic conditions cause the primary root to grow sidewise in a low oxygen environment, possibly to escape soil patches with reduced oxygen availability. This growth behavior is reversible in that gravitropic growth resumes when seedlings are returned to normoxic conditions. Hypoxic root bending is inhibited by the group VII ethylene response factor (ERFVII) RAP2.12, as rap2.12-1 seedlings show exaggerated primary root bending. Furthermore, overexpression of the ERFVII member HRE2 inhibits root bending, suggesting that primary root growth direction at hypoxic conditions is antagonistically regulated by hypoxia and hypoxia-activated ERFVIIs. Root bending is preceded by the establishment of an auxin gradient across the root tip as quantified with DII-VENUS and is synergistically enhanced by hypoxia and the auxin transport inhibitor naphthylphthalamic acid. The protein abundance of the auxin efflux carrier PIN2 is reduced at hypoxic conditions, a response that is suppressed by RAP2.12 overexpression, suggesting antagonistic control of auxin flux by hypoxia and ERFVII. Taken together, we show that hypoxia triggers an escape response of the primary root that is controlled by ERFVII activity and mediated by auxin signaling in the root tip. PMID:28698356

  20. Effects of the bradykinin antagonist B4310 on smooth muscles and blood pressure in the rat, and its enzymatic degradation.

    PubMed Central

    Griesbacher, T.; Lembeck, F.; Saria, A.

    1989-01-01

    1. Six competitive bradykinin (Bk) antagonists were tested for their agonistic properties on the rat uterus. Five of these peptides showed agonistic effects only at concentrations at least two orders of magnitude higher than those of bradykinin. 2. The antagonistic potency of Lys-Lys-3-Hyp-5,8-Thi-7-DPhe-Bk (B4310) in the rat uterus (pA2 = 7.24) and in the rat duodenum (pA2 = 7.31) was very similar to that determined in an earlier study for the antagonism of the bradykinin-induced stimulation of the trigeminal nerve in the rabbit iris sphincter muscle preparation (pA2 = 7.59). 3. The fall in mean arterial blood pressure induced by i.a. injections of bradykinin was greatly reduced during an i.a. infusion of B4310, but not 10 min thereafter, which indicates a rapid inactivation of B4310 in vivo. Bacitracin possibly interferes with the enzymatic cleavage of B4310 but seems to have no effect on the degradation of bradykinin. 4. An i.a. infusion of captopril greatly enhanced the potency of bradykinin in inducing a fall in arterial blood pressure, confirming the important role of angiotensin converting enzyme in the cleavage of bradykinin. However, the design of this experiment did not allow conclusions about the effect of captopril on the degradation of B4310. 5. B4310 incubated with rat lung tissue disappeared from the incubation medium within a few minutes, i.e. as fast as bradykinin, which explains its short duration of action in vivo. Captopril partially inhibited the cleavage of both bradykinin and B4310.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2655805

  1. Tachykinin antagonists and capsaicin-induced contraction of the rat isolated urinary bladder: evidence for tachykinin-mediated cotransmission.

    PubMed

    Maggi, C A; Patacchini, R; Santicioli, P; Giuliani, S

    1991-06-01

    1. The possible involvement of tachykinins (TKs) in the contraction produced by capsaicin in the rat isolated urinary bladder was addressed on the hypothesis that co-release of substance P (SP) and neurokinin A (NKA) occurs from sensory nerve terminals. 2. A low concentration of SP (30 nM) produced a rapid contraction which faded to baseline within 10 min. A low concentration of NKA (10 nM) produced a slowly developing contraction which was still evident at 10 min. Capsaicin (1 microM) produced a rapid phasic response and a tonic response (late response to capsaicin). Co-administration of SP and NKA mimicked the response to capsaicin more than each TK alone. 3. Fading of the response to SP was not caused by receptor desensitization and was partially prevented by peptidase inhibitors. 4. Spantide (3 microM) selectively antagonized the SP-induced contraction while L-659,877 (3-10 microM) or MEN 10,376 (10-30 microM) which are NK2 receptor selective antagonists selectively blocked the response to NKA. Co-administration of spantide and L-659,877 inhibited the response to both SP and NKA by an amount not greater than that produced by each antagonist alone. 5. Spantide selectively reduced the peak response to capsaicin, while leaving the late response unaffected. L-659,877 (3 microM) and MEN 10,376 (10 microM) selectively inhibited the late response to capsaicin while, at higher concentrations, also reduced the peak response to capsaicin. Co-administration of spantide and L-659,877 reduced the peak response to capsaicin more than that produced by each antagonist alone. 6. Bombesin (10 nM) produced a tonic contraction similar to that induced by NKA. The response to bombesin was not affected by spantide, L-659,877 or MEN 10,376. 7 P2. purinoceptor desensitization by repeated administration of alpha,betal-methylene ATP depressed the twitch response to electrical stimulation of postganglionic nerves but did not affect the peak or the late response to capsaicin. 8. We

  2. Past, present and future of A2A adenosine receptor antagonists in the therapy of Parkinson’s disease

    PubMed Central

    Armentero, Marie Therese; Pinna, Annalisa; Ferré, Sergi; Lanciego, José Luis; Müller, Christa E.; Franco, Rafael

    2011-01-01

    Several selective antagonists for adenosine A2A receptors (A2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson’s disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D2 and adenosine A2A receptors in the basal ganglia. At present it is believed that A2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson’s patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized anti-parkinsonian drug therapy, namely the existence of receptor (hetero)dimers/oligomers of G protein-coupled receptors, a topic currently the focus of intense debate within the scientific community. Dopamine D2 receptors (D2Rs) expressed in the striatum are known to form heteromers with A2A adenosine receptors. Thus, the development of heteromer-specific A2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs. PMID:21810444

  3. MDMA-induced loss of parvalbumin interneurons within the dentate gyrus is mediated by 5HT2A and NMDA receptors.

    PubMed

    Collins, Stuart A; Gudelsky, Gary A; Yamamoto, Bryan K

    2015-08-15

    MDMA is a widely abused psychostimulant which causes a rapid and robust release of the monoaminergic neurotransmitters dopamine and serotonin. Recently, it was shown that MDMA increases extracellular glutamate concentrations in the dorsal hippocampus, which is dependent on serotonin release and 5HT2A/2C receptor activation. The increased extracellular glutamate concentration coincides with a loss of parvalbumin-immunoreactive (PV-IR) interneurons of the dentate gyrus region. Given the known susceptibility of PV interneurons to excitotoxicity, we examined whether MDMA-induced increases in extracellular glutamate in the dentate gyrus are necessary for the loss of PV cells in rats. Extracellular glutamate concentrations increased in the dentate gyrus during systemic and local administration of MDMA. Administration of the NMDA receptor antagonist, MK-801, during systemic injections of MDMA, prevented the loss of PV-IR interneurons seen 10 days after MDMA exposure. Local administration of MDL100907, a selective 5HT2A receptor antagonist, prevented the increases in glutamate caused by reverse dialysis of MDMA directly into the dentate gyrus and prevented the reduction of PV-IR. These findings provide evidence that MDMA causes decreases in PV within the dentate gyrus through a 5HT2A receptor-mediated increase in glutamate and subsequent NMDA receptor activation. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. The adenosine A2A antagonist MSX-3 reverses the effort-related effects of dopamine blockade: differential interaction with D1 and D2 family antagonists

    PubMed Central

    Worden, Lila T.; Shahriari, Mona; Farrar, Andrew M.; Sink, Kelly S.; Hockemeyer, Jörg; Müller, Christa E.

    2010-01-01

    Rationale Brain dopamine (DA) participates in the modulation of instrumental behavior, including aspects of behavioral activation and effort-related choice behavior. Rats with impaired DA transmission reallocate their behavior away from food-seeking behaviors that have high response requirements, and instead select less effortful alternatives. Although accumbens DA is considered a critical component of the brain circuitry regulating effort-related choice behavior, emerging evidence demonstrates a role for adenosine A2A receptors. Objective Adenosine A2A receptor antagonism has been shown to reverse the effects of DA antagonism. The present experiments were conducted to determine if this effect was dependent upon the subtype of DA receptor that was antagonized to produce the changes in effort-related choice. Materials and methods The adenosine A2A receptor antagonist MSX-3 (0.5–2.0 mg/kg IP) was assessed for its ability to reverse the effects of the D1 family antagonist SCH39166 (ecopipam; 0.2 mg/kg IP) and the D2 family antagonist eticlopride (0.08 mg/kg IP), using a concurrent lever pressing/chow feeding procedure. Results MSX-3 produced a substantial dose-related reversal of the effects of eticlopride on lever pressing and chow intake. At the highest dose of MSX-3, there was a complete reversal of the effects of eticlopride on lever pressing. In contrast, MSX-3 produced only a minimal attenuation of the effects of SCH39166, as measured by regression and effect size analyses. Conclusions The greater ability of MSX-3 to reverse the effects of D2 vs. D1 blockade may be related to the colocalization of D2 and adenosine A2A receptors on the same population of striatal neurons. PMID:19048234

  5. T-dependent activation of resting B cells mediated by concanavalin A.

    PubMed

    Ratcliffe, M J; Julius, M H

    1984-03-01

    In cultures containing long-term cultured lines of antigen-specific helper T (Th) cells, normal unprimed B cells and concanavalin A (Con A), induction of B cells to immunoglobulin secretion and DNA synthesis was observed. The plaque-forming cell (PFC) response was large (frequently greater than 75 000 PFC/10(6) input B cells) demonstrating the polyspecific nature of the response. Con A-mediated maturation and induction to DNA synthesis of responding B cells was completely Th cell dependent and inhibited with methyl-alpha-D-mannoside. Both resting and blasted B cells, separated by Percoll density centrifugation, were induced to DNA synthesis and immunoglobulin secretion. Responses were completely unrestricted by the B cell major histocompatibility complex, even at the level of the resting B cell. The polyclonal nature of the response taken together with the Con A-mediated bypassing of T cell specificity and restricting haplotype indicates that this response is analogous to lectin-mediated cytotoxicity.

  6. REPRESSOR OF ULTRAVIOLET-B PHOTOMORPHOGENESIS function allows efficient phototropin mediated ultraviolet-B phototropism in etiolated seedlings.

    PubMed

    Vanhaelewyn, Lucas; Schumacher, Paolo; Poelman, Dirk; Fankhauser, Christian; Van Der Straeten, Dominique; Vandenbussche, Filip

    2016-11-01

    Ultraviolet B (UV-B) light is a part of the solar radiation which has significant effects on plant morphology, even at low doses. In Arabidopsis, many of these morphological changes have been attributed to a specific UV-B receptor, UV resistance locus 8 (UVR8). Recent findings showed that next to phototropin regulated phototropism, UVR8 mediated signaling is able of inducing directional bending towards UV-B light in etiolated seedlings of Arabidopsis, in a phototropin independent manner. In this study, kinetic analysis of phototropic bending was used to evaluate the relative contribution of each of these pathways in UV-B mediated phototropism. Diminishing UV-B light intensity favors the importance of phototropins. Molecular and genetic analyses suggest that UV-B is capable of inducing phototropin signaling relying on phototropin kinase activity and regulation of NPH3. Moreover, enhanced UVR8 responses in the UV-B hypersensitive rup1rup2 mutants interferes with the fast phototropin mediated phototropism. Together the data suggest that phototropins are the most important receptors for UV-B induced phototropism in etiolated seedlings, and a RUP mediated negative feedback pathway prevents UVR8 signaling to interfere with the phototropin dependent response. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling.

    PubMed

    Vecchio, Elizabeth A; Chuo, Chung Hui; Baltos, Jo-Anne; Ford, Leigh; Scammells, Peter J; Wang, Bing H; Christopoulos, Arthur; White, Paul J; May, Lauren T

    2016-10-01

    We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Novel therapy in Parkinson's disease: adenosine A(2A) receptor antagonists.

    PubMed

    Szabó, Nikoletta; Kincses, Zsigmond Tamás; Vécsei, László

    2011-04-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder. To date, most of the currently available therapies in PD target the dopaminergic system and none of these therapeutic approaches have been proven to modify the course of the disease. To various extents, these drugs can also cause motor and non-motor complications. A novel target, the adenosine A(2A) receptor (AA2AR), was recently identified, blockade of which may alleviate Parkinsonian symptoms, reduce motor fluctuations and potentially afford neuroprotection. This review is based on a PubMed search covering the relationship of the adenosine receptors and PD. The role of the AA2AR is reviewed and the results of preclinical investigations of antagonists are assessed. A synopsis of current drug development is provided, with a special focus on the pharmacokinetics and relevant clinical trials. The localization of the AA2AR in the central nervous system, the ultra structural localization and the molecular mechanism of its action reveal the potential importance of the AA2AR in movement disorders. The theoretical background and experimental data indicate that AA2AR antagonists may have a potential therapeutic effect in Parkinson's disease. More importantly, the putative neuroprotective effect needs further investigation.

  9. Effectiveness of vasopressin V2 receptor antagonists OPC-31260 and OPC-41061 on polycystic kidney disease development in the PCK rat.

    PubMed

    Wang, Xiaofang; Gattone, Vincent; Harris, Peter C; Torres, Vicente E

    2005-04-01

    cAMP plays a major role in cystogenesis. Recent in vitro studies suggested that cAMP stimulates B-Raf/ERK activation and proliferation of cyst-derived cells in a Ca(2+) inhibitable, Ras-dependent manner. OPC-31260, a vasopressin V2 receptor (VPV2) antagonist, was shown to lower renal cAMP and inhibit renal disease development and progression in models orthologous to human cystic diseases. Here it is shown that OPC-41061, an antagonist chosen for its potency and selectivity for human VPV2, is effective in PCK rats. PCK kidneys have increased Ras-GTP and phosphorylated ERK levels and 95-kD/68-kD B-Raf ratios, changes that are corrected by the administration of OPC-31260 or OPC-41061. These results support the importance of cAMP in the pathogenesis of polycystic kidney disease, confirm the effectiveness of a VPV2 antagonist to be used in clinical trials for this disease, and suggest that OPC-31260 and OPC-41061 inhibit Ras/mitogen-activated protein kinase signaling in polycystic kidneys.

  10. Prevention of neutrophil extravasation by α2-adrenoceptor-mediated endothelial stabilization.

    PubMed

    Herrera-García, Ada María; Domínguez-Luis, María Jesús; Arce-Franco, María; Armas-González, Estefanía; Álvarez de La Rosa, Diego; Machado, José David; Pec, Martina K; Feria, Manuel; Barreiro, Olga; Sánchez-Madrid, Francisco; Díaz-González, Federico

    2014-09-15

    Adrenergic receptors are expressed on the surface of inflammation-mediating cells, but their potential role in the regulation of the inflammatory response is still poorly understood. The objectives of this work were to study the effects of α2-adrenergic agonists on the inflammatory response in vivo and to determine their mechanism of action. In two mouse models of inflammation, zymosan air pouch and thioglycolate-induced peritonitis models, the i.m. treatment with xylazine or UK14304, two α2-adrenergic agonists, reduced neutrophil migration by 60%. The α2-adrenergic antagonist RX821002 abrogated this effect. In flow cytometry experiments, the basal surface expression of L-selectin and CD11b was modified neither in murine nor in human neutrophils upon α2-agonist treatment. Similar experiments in HUVEC showed that UK14304 prevented the activation-dependent upregulation of ICAM-1. In contrast, UK14304 augmented electrical resistance and reduced macromolecular transport through a confluent HUVEC monolayer. In flow chamber experiments, under postcapillary venule-like flow conditions, the pretreatment of HUVECs, but not neutrophils, with α2-agonists decreased transendothelial migration, without affecting neutrophil rolling. Interestingly, α2-agonists prevented the TNF-α-mediated decrease in expression of the adherens junctional molecules, VE-cadherin, β-catenin, and plakoglobin, and reduced the ICAM-1-mediated phosphorylation of VE-cadherin by immunofluorescence and confocal analysis and Western blot analysis, respectively. These findings indicate that α2-adrenoceptors trigger signals that protect the integrity of endothelial adherens junctions during the inflammatory response, thus pointing at the vascular endothelium as a therapeutic target for the management of inflammatory processes in humans. Copyright © 2014 by The American Association of Immunologists, Inc.

  11. Behavioural Assessment of the A2a/NR2B Combination in the Unilateral 6-OHDA-Lesioned Rat Model: A New Method to Examine the Therapeutic Potential of Non-Dopaminergic Drugs

    PubMed Central

    Michel, Anne; Downey, Patrick; Van Damme, Xavier; De Wolf, Catherine; Schwarting, Rainer; Scheller, Dieter

    2015-01-01

    In Parkinson’s disease (PD), dopaminergic therapies are often associated with the development of motor complications. Attention has therefore been focused on the use of non-dopaminergic drugs. This study developed a new behavioural method capable of demonstrating the added value of combining adenosinergic and glutamatergic receptor antagonists in unilateral 6-OHDA lesioned rats. Rats were dosed orally with Tozadenant, a selective A2A receptor antagonist, and three different doses of Radiprodil, an NR2B-selective NMDA receptor antagonist. The drugs were given alone or in combination and rats were placed in an open-field for behavioural monitoring. Video recordings were automatically analysed. Five different behaviours were scored: distance traveled, ipsi- and contraversive turns, body position, and space occupancy. The results show that A2A or NR2B receptor antagonists given alone or in combination did not produce enhanced turning as observed with an active dose of L-Dopa/benserazide. Instead the treated rats maintained a straight body position, were able to shift from one direction to the other and occupied a significantly larger space in the arena. The highest “Tozadenant/Radiprodil” dose combination significantly increased all five behavioural parameters recorded compared to rats treated with vehicle or the same doses of the drugs alone. Our data suggest that the A2A/NR2B antagonist combination may be able to stimulate motor activity to a similar level as that achieved by L-Dopa but in the absence of the side-effects that are associated with dopaminergic hyperstimulation. If these results translate into the clinic, this combination could represent an alternative symptomatic treatment option for PD. PMID:26322641

  12. From Chemotherapy-Induced Emesis to Neuroprotection: Therapeutic Opportunities for 5-HT3 Receptor Antagonists.

    PubMed

    Fakhfouri, Gohar; Mousavizadeh, Kazem; Mehr, Sharam Ejtemaei; Dehpour, Ahmad Reza; Zirak, Mohammad Reza; Ghia, Jean-Eric; Rahimian, Reza

    2015-12-01

    5-HT3 receptor antagonists are extensively used as efficacious agents in counteracting chemotherapy-induced emesis. Recent investigations have shed light on other potential effects (analgesic, anxiolytic, and anti-psychotic). Some studies have reported neuroprotective properties for the 5-HT3 receptor antagonists in vitro and in vivo. When administered to Aβ-challenged rat cortical neurons, 5-HT3 receptor antagonists substantially abated apoptosis, elevation of cytosolic Ca(2), glutamate release, reactive oxygen species (ROS) generation, and caspase-3 activity. In addition, in vivo studies show that 5-HT3 receptor antagonists possess, alongside their anti-emetic effects, notable immunomodulatory properties in CNS. We found that pretreatment with tropisetron significantly improved neurological deficits and diminished leukocyte transmigration into the brain, TNF-α level, and brain infarction in a murine model of embolic stroke. Our recent investigation revealed that tropisetron protects against Aβ-induced neurotoxicity in vivo through both 5-HT3 receptor-dependent and -independent pathways. Tropisetron, in vitro, was found to be an efficacious inhibitor of the signaling pathway leading to the activation of pro-inflammatory NF-κB, a transcription factor pivotal to the upregulation of several neuroinflammatory mediators in brain. This mini review summarizes novel evidence concerning effects of 5-HT3 antagonists and their possible mechanisms of action in ameliorating neurodegenerative diseases including Alzheimer, multiple sclerosis, and stroke. Further, we discuss some newly synthesized 5-HT3 receptor antagonists with dual properties of 5-HT3 receptor blockade/alpha-7 nicotinic receptor activator and their potential in management of memory impairment. Since 5-HT3 receptor antagonists possess a large therapeutic window, they can constitute a scaffold for design and synthesis of new neuroprotective medications.

  13. SETDB1 HISTONE METHYLTRANSFERASE REGULATES MOOD-RELATED BEHAVIORS AND EXPRESSION OF THE NMDA RECEPTOR SUBUNIT NR2B

    PubMed Central

    Jiang, Yan; Jakovcevski, Mira; Bharadwaj, Rahul; Connor, Caroline; Schroeder, Frederick A.; Lin, Cong L.; Straubhaar, Juerg; Martin, Gilles; Akbarian, Schahram

    2010-01-01

    Histone methyltransferases specific for the histone H3-lysine 9 (H3K9) residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to less than 1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture (“3C”) and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30Kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wildtype mice, systemic treatment with the NR2B antagonist, Ro-256981, and hippocampal siRNA-mediated NR2B/Grin2b knockdown, resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations. PMID:20505083

  14. Inhibition of PTP1B Restores IRS1-Mediated Hepatic Insulin Signaling in IRS2-Deficient Mice

    PubMed Central

    González-Rodríguez, Águeda; Gutierrez, Jose A. Mas; Sanz-González, Silvia; Ros, Manuel; Burks, Deborah J.; Valverde, Ángela M.

    2010-01-01

    OBJECTIVE Mice with complete deletion of insulin receptor substrate 2 (IRS2) develop hyperglycemia, impaired hepatic insulin signaling, and elevated gluconeogenesis, whereas mice deficient for protein tyrosine phosphatase (PTP)1B display an opposing hepatic phenotype characterized by increased sensitivity to insulin. To define the relationship between these two signaling pathways in the regulation of liver metabolism, we used genetic and pharmacological approaches to study the effects of inhibiting PTP1B on hepatic insulin signaling and expression of gluconeogenic enzymes in IRS2−/− mice. RESEARCH DESIGN AND METHODS We analyzed glucose homeostasis and insulin signaling in liver and isolated hepatocytes from IRS2−/− and IRS2−/−/PTP1B−/− mice. Additionally, hepatic insulin signaling was assessed in control and IRS2−/− mice treated with resveratrol, an antioxidant present in red wine. RESULTS In livers of hyperglycemic IRS2−/− mice, the expression levels of PTP1B and its association with the insulin receptor (IR) were increased. The absence of PTP1B in the double-mutant mice restored hepatic IRS1-mediated phosphatidylinositol (PI) 3-kinase/Akt/Foxo1 signaling. Moreover, resveratrol treatment of hyperglycemic IRS2−/− mice decreased hepatic PTP1B mRNA and inhibited PTP1B activity, thereby restoring IRS1-mediated PI 3-kinase/Akt/Foxo1 signaling and peripheral insulin sensitivity. CONCLUSIONS By regulating the phosphorylation state of IR, PTB1B determines sensitivity to insulin in liver and exerts a unique role in the interplay between IRS1 and IRS2 in the modulation of hepatic insulin action. PMID:20028942

  15. High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

    PubMed Central

    Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

    2010-01-01

    Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

  16. Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation.

    PubMed

    Inabe, Kazunori; Miyawaki, Toshio; Longnecker, Richard; Matsukura, Hiroyoshi; Tsukada, Satoshi; Kurosaki, Tomohiro

    2002-03-13

    Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.

  17. Pseudolaric acid B attenuates atherosclerosis progression and inflammation by suppressing PPARγ-mediated NF-κB activation.

    PubMed

    Li, Tan; Wang, Wei; Li, Yu-Xiu; Li, Xiao; Ji, Wen-Jie; Ma, Yong-Qiang; Chen, Hong; Zhao, Ji-Hong; Zhou, Xin

    2018-06-01

    Atherosclerosis is a progressive disease of large arteries characterized with chronic inflammation and aberrant immune response. Pseudolaric acid B (PB) has been found to exert multiple effects by inhibiting inflammatory response. However, there is no comprehensive assessment of the effects of PB on atherosclerosis using relevant in vivo and in vitro models. Male ApoE -/- mice were treated with PB orally with a high fat diet (HFD) to clarify its anti-atherosclerotic activities. RAW264.7 macrophage line, a well-accepted cell model of atherosclerosis, was used to investigate anti-inflammatory effects and molecular mechanisms of PB. PB significantly attenuated atherosclerotic lesions by modulating plasma lipid profiles as well as inhibiting inflammatory responses in macrophages of atherosclerotic mice. Meanwhile, PB markedly suppressed the expression of pro-inflammatory cytokines, and regulated cholesterol efflux related genes in oxidative low density lipoprotein (ox-LDL)-loaded macrophages. The cellular uptake of Dil-labeled ox-LDL was significantly inhibited by PB either. Moreover, the ability of PB to suppress nuclear factor kappa B (NF-κB) and activate peroxisome proliferator-activated receptor gamma (PPARγ) was confirmed using luciferase reporter assays. Conversely, the selective PPARγ antagonist GW9662 reversed the influence of PB in macrophages. Together, these findings indicate that PB exerts its protective effects on atherosclerosis by inhibiting macrophage-mediated inflammatory response and cellular ox-LDL uptake, and promoting cholesterol efflux by suppressing NF-κB activation PPARγ-dependently. Therefore, PB may be a promising agent for inflammatory and atherosclerotic diseases. Copyright © 2018. Published by Elsevier B.V.

  18. Antagonist wear of monolithic zirconia crowns after 2 years.

    PubMed

    Lohbauer, Ulrich; Reich, Sven

    2017-05-01

    The aim of this study was to evaluate the amount of wear on the antagonist occlusal surfaces of clinically placed monolithic zirconia premolar and molar crowns (LAVA Plus, 3M ESPE). Fourteen in situ monolithic zirconia crowns and their opposing antagonists (n = 26) are the subject of an ongoing clinical trial and have been clinically examined at baseline and after 24 months. Silicone impressions were taken and epoxy replicas produced for qualitative SEM analysis and quantitative analysis using optical profilometry. Based on the baseline replicas, the follow-up situation has been scanned and digitally matched with the initial topography in order to calculate the mean volume loss (in mm 3 ) as well as the mean maximum vertical loss (in mm) after 2 years in service. The mean volume loss for enamel antagonist contacts (n = 7) was measured to 0.361 mm 3 and the mean of the maximum vertical loss to 0.204 mm. The mean volume loss for pure ceramic contacts (n = 10) was measured to 0.333 mm 3 and the mean of the maximum vertical loss to 0.145 mm. The wear rates on enamel contacts were not significantly different from those measured on ceramic antagonists. Based on the limitations of this study, it can be concluded for the monolithic zirconia material LAVA Plus that the measured wear rates are in consensus with other in vivo studies on ceramic restorations. Further, that no significant difference was found between natural enamel antagonists and ceramic restorations as antagonists. The monolithic zirconia restorations do not seem to be affected by wear within the first 2 years. The monolithic zirconia crowns (LAVA Plus) show acceptable antagonist wear rates after 2 years in situ, regardless of natural enamel or ceramics as antagonist materials.

  19. The effect of adenosine A(2A) receptor antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of VMAT2.

    PubMed

    Gołembiowska, Krystyna; Dziubina, Anna

    2012-08-01

    It has been shown that a decreased vesicular monoamine transporter (VMAT2) function and the disruption of dopamine (DA) storage is an early contributor to oxidative damage of dopamine neurons in Parkinson's disease (PD). In our previous study, we demonstrated that adenosine A(2A) receptor antagonists suppressed oxidative stress in 6-hydroxydopamine-treated rats suggesting that this effect may account for neuroprotective properties of drugs. In the present study, rats were injected with reserpine (10 mg/kg sc) and 18 h later the effect of the adenosine A(2A) receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on extracellular DA, glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis. By disrupting VMAT2 function, reserpine depleted DA stores, and increased glutamate and hydroxyl radical levels in the rat striatum. CSC (1 mg/kg) but not ZM 241385 (3 mg/kg) increased extracellular DA level and production of hydroxyl radical in reserpinised rats. Both antagonists decreased the reserpine-induced increase in extracellular glutamate. L-3,4-Dihydroxyphenylalanine (L-DOPA) (25 mg/kg) significantly enhanced extracellular DA, had no effect on reserpine-induced hydroxyl radical production and decreased extracellular glutamate concentration. CSC but not ZM 241385 given jointly with L-DOPA increased the effect of L-DOPA on extracellular DA and augmented the reserpine-induced hydroxyl radical production. CSC and ZM 241385 did not influence extracellular glutamate level, which was decreased by L-DOPA. It seems that by decreasing the MAO-dependent DA metabolism rate, CSC raised cytosolic DA and by DA autoxidation, it induced hydroxyl radical overproduction. Thus, the methylxanthine A(2A) receptor antagonists bearing properties of MAO-B inhibitor, like CSC, may cause a risk of oxidative stress resulting from dysfunctional DA storage

  20. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats

    PubMed Central

    Blacktop, Jordan M.; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A.; Mantsch, John R.

    2015-01-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 hrs/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5” duration, average every 40 sec; range 10–70 sec) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 µg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. PMID:26596556

  1. Oxytocin and Vasopressin Agonists and Antagonists as Research Tools and Potential Therapeutics

    PubMed Central

    Manning, M; Misicka, A; Olma, A; Bankowski, K; Stoev, S; Chini, B; Durroux, T; Mouillac, B; Corbani, M; Guillon, G

    2012-01-01

    We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V1a, V1b and V2 receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V1b receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V1a agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V2/V1a antagonist, conivaptan and the V2 antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V1a, V1b and V2 antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences. PMID:22375852

  2. Histone Methyltransferase NSD2/MMSET Mediates Constitutive NF-κB Signaling for Cancer Cell Proliferation, Survival, and Tumor Growth via a Feed-Forward Loop

    PubMed Central

    Yang, Ping; Guo, Linlang; Duan, Zhijian J.; Tepper, Clifford G.; Xue, Ling; Chen, Xinbin; Kung, Hsing-Jien; Gao, Allen C.

    2012-01-01

    Constitutive NF-κB activation by proinflammatory cytokines plays a major role in cancer progression. However, the underlying mechanism is still unclear. We report here that histone methyltransferase NSD2 (also known as MMSET or WHSC1), a target of bromodomain protein ANCCA/ATAD2, acts as a strong coactivator of NF-κB by directly interacting with NF-κB for activation of target genes, including those for interleukin-6 (IL-6), IL-8, vascular endothelial growth factor A (VEGFA), cyclin D, Bcl-2, and survivin, in castration-resistant prostate cancer (CRPC) cells. NSD2 is recruited to the target gene promoters upon induction and mediates NF-κB activation-associated elevation of histone H3K36me2 and H3K36me3 marks at the promoter, which involves its methylase activity. Interestingly, we found that NSD2 is also critical for cytokine-induced recruitment of NF-κB and acetyltransferase p300 and histone hyperacetylation. Importantly, NSD2 is overexpressed in prostate cancer tumors, and its overexpression correlates with NF-κB activation. Furthermore, NSD2 expression is strongly induced by tumor necrosis factor alpha (TNF-α) and IL-6 via NF-κB and plays a crucial role in tumor growth. These results identify NSD2 to be a key chromatin regulator of NF-κB and mediator of the cytokine autocrine loop for constitutive NF-κB activation and emphasize the important roles played by NSD2 in cancer cell proliferation and survival and tumor growth. PMID:22645312

  3. Aflatoxin B1 and M1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

    PubMed Central

    Loi, Martina; Fanelli, Francesca; Zucca, Paolo; Liuzzi, Vania C.; Quintieri, Laura; Cimmarusti, Maria T.; Monaci, Linda; Haidukowski, Miriam; Logrieco, Antonio F.; Sanjust, Enrico; Mulè, Giuseppina

    2016-01-01

    Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB1 and AFM1. PMID:27563923

  4. Glutamate Receptor Antagonists as Fast-Acting Therapeutic Alternatives for the Treatment of Depression: Ketamine and Other Compounds

    PubMed Central

    Niciu, Mark J.; Henter, Ioline D.; Luckenbaugh, David A.; Zarate, Carlos A.; Charney, Dennis S.

    2014-01-01

    The N-methyl-d-aspartate (NMDA) receptor antagonist ketamine has rapid and potent antidepressant effects in treatment-resistant major depressive disorder and bipolar depression. These effects are in direct contrast to the more modest effects seen after weeks of treatment with classic monoaminergic antidepressants. Numerous open-label and case studies similarly validate ketamine’s antidepressant properties. These clinical findings have been reverse-translated into preclinical models in an effort to elucidate ketamine’s antidepressant mechanism of action, and three important targets have been identified: mammalian target of rapamycin (mTOR), eukaryotic elongation factor 2 (eEF2), and glycogen synthase kinase-3 (GSK-3). Current clinical and preclinical research is focused on (a) prolonging/maintaining ketamine’s antidepressant effects, (b) developing more selective NMDA receptor antagonists free of ketamine’s adverse effects, and (c) identifying predictor, mediator/moderator, and treatment response biomarkers of ketamine’s antidepressant effects. PMID:24392693

  5. Pharmacology of JB-9315, a new selective histamine H2-receptor antagonist.

    PubMed

    Palacios, B; Montero, M J; Sevilla, M A; San Román, L

    1998-02-01

    1. The histamine H2-receptor antagonistic activity and antisecretory and antiulcer effects of JB-9315 were studied in comparison with the standard H2 blocker ranitidine. 2. In vitro, JB-9315 is a competitive antagonist of histamine H2 receptors in the isolated, spontaneously beating guinea-pig right atrium, with a pA2 value of 7.30 relative to a value of 7.36 for ranitidine. JB-9315 was specific for the histamine H2 receptor because, at high concentration, it did not affect histamine- or acetylcholine-induced contractions in guinea-pig isolated ileum or rat isolated duodenum, respectively. 3. JB-9315 dose dependently inhibited histamine-, pentagastrin- or carbachol-stimulated acid secretion and basal secretion in the perfused stomach preparation of the anesthetized rat. In the pylorus-ligated rat after intraperitoneal administration, total acid output over 4 h was inhibited by JB-9315 with an ID50 of 32.8 mg/kg, confirming its H2-receptor antagonist properties. 4. JB-9315 showed antiulcer activity against cold stress plus indomethacin-induced lesions with an ID50 of 6.8 mg/kg. 5. JB-9315, 50 and 100 mg/kg, inhibited macroscopic gastric hemorrhagic lesions induced by ethanol. In contrast, ranitidine (50 mg/kg) failed to reduce these lesions. 6. These results indicate that JB-9315 is a new antiulcer drug that exerts a cytoprotective effect in addition to its gastric antisecretory activity.

  6. INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Min, Joong Won; Kim, Kwang Il; Kim, Hyun-Ah

    2013-10-11

    Highlights: •HIF-1α-regulated INPP4B enhances glycolysis. •INPP4B regulates aerobic glycolysis by inducing HK2 via Akt-mTOR pathway. •Blockage of INPP4B and HK2 sensitizes radioresistant laryngeal cancer cells to radiation and anticancer drug. •INPP4B is associated with HK2 in human laryngeal cancer tissues. -- Abstract: Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B andmore » this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.« less

  7. Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma.

    PubMed

    Koch, Raphael; Demant, Martin; Aung, Thiha; Diering, Nina; Cicholas, Anna; Chapuy, Bjoern; Wenzel, Dirk; Lahmann, Marlen; Güntsch, Annemarie; Kiecke, Christina; Becker, Sabrina; Hupfeld, Timo; Venkataramani, Vivek; Ziepert, Marita; Opitz, Lennart; Klapper, Wolfram; Trümper, Lorenz; Wulf, Gerald G

    2014-04-03

    Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor.

  8. 6-[N,S-dimethyl-N'-cyanothioureidomethyl]-6,11-dihydro-5H- dibenz[b,e]azepine hydrochloride (Fran 12): a histamine and 5-hydroxytryptamine antagonist with pressor properties.

    PubMed

    Law, S C; Guyett, F J; King, R G; Boura, A L; Jackson, W R; Hodgson, W C

    1992-01-01

    We have synthesized and examined some of the pharmacological properties of 6-[N,S-dimethyl-N'-cyanoisothioureidomethyl]-6,11-dihydro-5H- dibenz(b,e)azepine hydrochloride (Fran 12), a derivative of 6-methylaminomethyl-6,11-dihydro-5H- dibenz[b,e,]azepine. In the guinea-pig isolated ileum, Fran 12 (10(-7)-10(-5) M) caused parallel rightward shifts of the concentration-response curves to histamine. A Schild plot gave a pA2 of 7.48, with a slope not significantly different from -1.0. In the rat stomach fundus strip and in endothelium-denuded aortic rings, Fran 12 inhibited contractile responses to 5-hydroxytryptamine in a non-competitive manner. In both chloralose-anaesthetized and pithed rats, it inhibited pressor responses to 5-hydroxytryptamine. It had no effect on depressor responses to 5-hydroxytryptamine in anaesthetized rats. In pithed rats, Fran 12 (0.25-2 mg/kg, i.v.) produced dose-dependent increases in blood pressure. These were not inhibited by i.v. phentolamine, prazosin, yohimbine, propranolol, methysergide, pentolinium or atropine but were inhibited by verapamil. These results indicate that Fran 12 is a histamine and 5-hydroxytryptamine antagonist which also exerts pressor effects via a peripheral action. The pressor action does not appear to be mediated via effects on alpha 1- or alpha 2-adrenoceptors, muscarinic or nicotinic cholinoceptors or 5-hydroxytryptamine receptors, although calcium channel activation may play a role.

  9. Isobutyrylshikonin inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in BV2 microglial cells by suppressing the PI3K/Akt-mediated nuclear transcription factor-κB pathway.

    PubMed

    Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Lee, Kyoung-Tae; Kang, Chang-Hee; Dilshara, Matharage Gayani; Lee, Hak-Ju; Choi, Yung Hyun; Choi, Il-Whan; Kim, Gi-Young

    2014-12-01

    Microglia are important macrophages to defend against pathogens in the central nervous system (CNS); however, persistent or acute inflammation of microglia lead to CNS disorders via neuronal cell death. Therefore, we theorized that a good strategy for the treatment of CNS disorders would be to target inflammatory mediators from microglia in disease. Consequently, we investigated whether isobutyrylshikonin (IBS) attenuates the production of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Treatment with IBS inhibited the secretion of NO and prostaglandin E2 (as well as the expression of their key regulatory genes), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Isobutyrylshikonin also suppressed LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB), by inhibiting the nuclear translocation of p50 and p65 in addition to blocking the phosphorylation and degradation of IκBα. Pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, showed the down-regulation of LPS-induced iNOS and COX-2 messenger RNA by suppressing NF-κB activity. This indirectly suggests that IBS-mediated NF-κB inhibition is the main signaling pathway involved in the inhibition of iNOS and COX-2 expression. In addition, IBS attenuated LPS-induced phosphorylation of PI3K and Akt, which are upstream molecules of NF-κB, in LPS-stimulated BV2 microglial cells. The functional aspects of the PI3K/Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor that attenuated LPS-induced iNOS and COX-2 expression by suppressing NF-κB activity. These data suggest that an IBS-mediated anti-inflammatory effect may be involved in suppressing the PI3K/Akt-mediated NF-κB signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Discovery of spiropiperidine-based potent and selective Orexin-2 receptor antagonists.

    PubMed

    Fujimoto, Tatsuhiko; Tomata, Yoshihide; Kunitomo, Jun; Hirozane, Mariko; Marui, Shogo

    2011-11-01

    To generate novel human Orexin-2 Receptor (OX2R) antagonists, a spiropiperidine based scaffold was designed and a SAR study was carried out. Compound 4f possessed the highest OX2R antagonistic activity with an IC(50) value of 3nM with 450-fold selectivity against Orexin-1 Receptor (OX1R). Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

    PubMed

    Müller, C E; Maurinsh, J; Sauer, R

    2000-01-01

    The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

  12. Ghrelin accelerates wound healing through GHS-R1a-mediated MAPK-NF-κB/GR signaling pathways in combined radiation and burn injury in rats.

    PubMed

    Liu, Cong; Huang, Jiawei; Li, Hong; Yang, Zhangyou; Zeng, Yiping; Liu, Jing; Hao, Yuhui; Li, Rong

    2016-06-07

    The therapeutic effect of ghrelin on wound healing was assessed using a rat model of combined radiation and burn injury (CRBI). Rat ghrelin, anti-rat tumor necrosis factor (TNF) α polyclonal antibody (PcAb), or selective antagonists of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and growth hormone secretagogue receptor (GHS-R) 1a (SB203580, SP600125, and [D-Lys3]-GHRP-6, respectively), were administered for seven consecutive days. Levels of various signaling molecules were assessed in isolated rat peritoneal macrophages. The results showed that serum ghrelin levels and levels of macrophage glucocorticoid receptor (GR) decreased, while phosphorylation of p38MAPK, JNK, and p65 nuclear factor (NF) κB increased. Ghrelin inhibited the serum induction of proinflammatory mediators, especially TNF-α, and promoted wound healing in a dose-dependent manner. Ghrelin treatment decreased phosphorylation of p38MAPK, JNK, and p65NF-κB, and increased GR levels in the presence of GHS-R1a. SB203580 or co-administration of SB203580 and SP600125 decreased TNF-α level, which may have contributed to the inactivation of p65NF-κB and increase in GR expression, as confirmed by western blotting. In conclusion, ghrelin enhances wound recovery in CRBI rats, possibly by decreasing the induction of TNF-α or other proinflammatory mediators that are involved in the regulation of GHS-R1a-mediated MAPK-NF-κB/GR signaling pathways.

  13. [(35)S]-GTPgammaS autoradiography reveals alpha(2) adrenoceptor-mediated G-protein activation in amygdala and lateral septum.

    PubMed

    Newman-Tancredi, A; Chaput, C; Touzard, M; Millan, M J

    2000-04-03

    alpha(2)-adrenoceptor-mediated G-protein activation was examined by [(35)S]-GTPgammaS autoradiography. In alpha(2)-adrenoceptor-rich regions (amygdala, lateral septum), noradrenaline stimulated [(35)S]-GTPgammaS binding. These actions were abolished by the selective alpha(2) antagonist, atipamezole. Conversely, in caudate nucleus, which expresses few alpha(2) receptors, noradrenaline-induced stimulation was not inhibited by atipamezole, suggesting that it is not mediated by alpha(2)-adrenoceptors.

  14. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structuremore » of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.« less

  15. Accelerated habit formation following amphetamine exposure is reversed by D1, but enhanced by D2, receptor antagonists

    PubMed Central

    Nelson, Andrew J. D.; Killcross, Simon

    2013-01-01

    Repeated exposure to the psychostimulant amphetamine has been shown to disrupt goal-directed instrumental actions and promote the early and abnormal development of goal-insensitive habitual responding (Nelson and Killcross, 2006). To investigate the neuropharmacological specificity of this effect as well as restore goal-directed responding in animals with pre-training amphetamine exposure, animals were treated with the non-selective dopamine antagonist α-flupenthixol, the selective D1 antagonist SCH 23390 or the selective D2 antagonist eticlopride, prior to instrumental training (three sessions). Subsequently, the reinforcer was paired with LiCL-induced gastric-malaise and animals were given a test of goal-sensitivity both in extinction and reacquisition. The effect of these dopaminergic antagonists on the sensitivity of lever press performance to outcome devaluation was assessed in animals with pre-training exposure to amphetamine (Experiments 1A–C) or in non-sensitized animals (Experiment 2). Both α-flupenthixol and SCH23390 reversed accelerated habit formation following amphetamine sensitization. However, eticlopride appeared to enhance this effect and render instrumental performance compulsive as these animals were unable to inhibit responding both in extinction and reacquisition, even though a consumption test confirmed they had acquired an aversion to the reinforcer. These findings demonstrate that amphetamine induced-disruption of goal-directed behavior is mediated by activity at distinct dopamine receptor subtypes and may represent a putative model of the neurochemical processes involved in the loss of voluntary control over behavior. PMID:23720609

  16. Identification of a Potent Tryptophan-based TRPM8 Antagonist With in vivo Analgesic Activity.

    PubMed

    Bertamino, Alessia; Iraci, Nunzio; Ostacolo, Carmine; Ambrosino, Paolo; Musella, Simona; Di Sarno, Veronica; Ciaglia, Tania; Pepe, Giacomo; Sala, Marina; Soldovieri, Maria Virginia; Mosca, Ilaria; Gonzalez-Rodriguez, Sara; Fernández-Carvajal, Asia; Ferrer-Montiel, Antonio; Novellino, Ettore; Taglialatela, Maurizio; Campiglia, Pietro; Gomez-Monterrey, Isabel M

    2018-06-25

    TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of N,N'-dibenzyl tryptamine 1, a potent TRPM8 antagonist, were prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably-transfected HEK293 cells. The tryptophan derivative 14 was identified as a potent (IC 50 0.2±0.2 nM) and selective TRPM8 antagonist. In vivo, 14 showed significant target coverage in both an icilin-induced WDS (at 1-30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1-1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1-4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain.

  17. Contribution of B2 receptors for bradykinin in Arthus reaction-induced plasma extravasation in wild-type or B2 transgenic knockout mice

    PubMed Central

    Samadfam, R; Teixeira, C; Bkaily, G; Sirois, P; de Brum-Fernandes, A; D'Orleans-Juste, P

    2000-01-01

    The aim of the present study was to investigate the contribution of bradykinin (BK) B1 and B2 receptors in a model of type III hypersensitivity, the reverse passive Arthus reaction (RPA), in wild-type mice and transgenic B2 knockout littermates.BK (10 μg mouse−1) or bovine serum albumin (0.5 mg mouse−1) induced a sustained Evans blue extravasation for more than 80 min in naive or rabbit anti-bovine serum albumin-treated mice (RPA model), respectively. The response to the two stimuli was prevented by the B2 receptor antagonist, HOE-140, but not by [Leu8]desArg9-BK (B1 receptor antagonist).In contrast to the wild-type littermates, RPA and bradykinin were unable to trigger an increase in plasma extravasation in B2 knockout mice.Furthermore, endothelin-1 (5 μg mouse−1) and a selective NK-1 receptor agonist [Sar9,Met (O2)11]-SP (20 μg mouse−1), triggered a significant increase in peritoneal plasma extravasation in both wild-type and B2 knockout animals.A pretreatment with indomethacin (200 μg mouse−1) significantly reduced the RPA-induced but not the BK-induced increase in Evans blue extravasation. Furthermore, RPA, but not BK, triggered a significant indomethacin-sensitive increase in peritoneal prostaglandin E2 content.Our results suggest a pivotal role for B2 receptors in the mechanism of plasma extravasation which occurs during the reverse passive Arthus reaction in the mouse. Moreover, our results suggest an important contribution of prostanoids in the plasma leakage mechanisms triggered by RPA but not by bradykinin. PMID:10780980

  18. Benzimidazoles as benzamide replacements within cyclohexane-based CC chemokine receptor 2 (CCR2) antagonists.

    PubMed

    Cherney, Robert J; Mo, Ruowei; Meyer, Dayton T; Pechulis, Anthony D; Guaciaro, Michael A; Lo, Yvonne C; Yang, Gengjie; Miller, Persymphonie B; Scherle, Peggy A; Zhao, Qihong; Cvijic, Mary Ellen; Barrish, Joel C; Decicco, Carl P; Carter, Percy H

    2012-10-01

    We describe the design, synthesis, and evaluation of benzimidazoles as benzamide replacements within a series of trisubstituted cyclohexane CCR2 antagonists. 7-Trifluoromethylbenzimidazoles displayed potent binding and functional antagonism of CCR2 while being selective over CCR3. These benzimidazoles were also incorporated into lactam-containing antagonists, thus completely eliminating the customary bis-amide. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B.

    PubMed

    Lennemann, Nicholas J; Coyne, Carolyn B

    2017-02-01

    The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.

  20. Photoreceptor-mediated bending towards UV-B in Arabidopsis.

    PubMed

    Vandenbussche, Filip; Tilbrook, Kimberley; Fierro, Ana Carolina; Marchal, Kathleen; Poelman, Dirk; Van Der Straeten, Dominique; Ulm, Roman

    2014-06-01

    Plants reorient their growth towards light to optimize photosynthetic light capture--a process known as phototropism. Phototropins are the photoreceptors essential for phototropic growth towards blue and ultraviolet-A (UV-A) light. Here we detail a phototropic response towards UV-B in etiolated Arabidopsis seedlings. We report that early differential growth is mediated by phototropins but clear phototropic bending to UV-B is maintained in phot1 phot2 double mutants. We further show that this phototropin-independent phototropic response to UV-B requires the UV-B photoreceptor UVR8. Broad UV-B-mediated repression of auxin-responsive genes suggests that UVR8 regulates directional bending by affecting auxin signaling. Kinetic analysis shows that UVR8-dependent directional bending occurs later than the phototropin response. We conclude that plants may use the full short-wavelength spectrum of sunlight to efficiently reorient photosynthetic tissue with incoming light. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

  1. TrkB signalling pathway mediates the protective effects of exercise in the diabetic rat retina.

    PubMed

    Allen, Rachael S; Hanif, Adam M; Gogniat, Marissa A; Prall, Brian C; Haider, Raza; Aung, Moe H; Prunty, Megan C; Mees, Lukas M; Coulter, Monica M; Motz, Cara T; Boatright, Jeffrey H; Pardue, Machelle T

    2018-05-01

    Diabetic retinopathy is a leading cause of vision loss. Treatment options for early retinopathy are sparse. Exercise protects dying photoreceptors in models of retinal degeneration, thereby preserving vision. We tested the protective effects of exercise on retinal and cognitive deficits in a type 1 diabetes model and determined whether the TrkB pathway mediates this effect. Hyperglycaemia was induced in Long Evans rats via streptozotocin injection (STZ; 100 mg/kg). Following confirmed hyperglycaemia, both control and diabetic rats underwent treadmill exercise for 30 min, 5 days/week at 0 m/min (inactive groups) or 15 m/min (active groups) for 8 weeks. A TrkB receptor antagonist (ANA-12), or vehicle, was injected 2.5 h before exercise training. We measured spatial frequency and contrast sensitivity using optokinetic tracking biweekly post-STZ; retinal function using electroretinography at 4 and 8 weeks; and cognitive function and exploratory behaviour using Y-maze at 8 weeks. Retinal neurotrophin-4 was measured using ELISA. Compared with non-diabetic controls, diabetic rats showed significantly reduced spatial frequency and contrast sensitivity, delayed electroretinogram oscillatory potential and flicker implicit times and reduced cognitive function and exploratory behaviour. Exercise interventions significantly delayed the appearance of all deficits, except for exploratory behaviour. Treatment with ANA-12 significantly reduced this protection, suggesting a TrkB-mediated mechanism. Despite this, no changes in retinal neurotrohin-4 were observed with diabetes or exercise. Exercise protected against early visual and cognitive dysfunction in diabetic rats, suggesting that exercise interventions started after hyperglycaemia diagnosis may be a beneficial treatment. The translational potential is high, given that exercise treatment is non-invasive, patient controlled and inexpensive. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  2. Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A)-Mediated changes in Fas Expression and Fas-Dependent Apoptosis: Role of Lyn/Syk activation

    PubMed Central

    Incrocci, Ryan; Hussain, Samira; Stone, Amanda; Bieging, Kathryn; Alt, Lauren A.C.; Fay, Michael J.; Swanson-Mungerson, Michelle

    2015-01-01

    Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies. PMID:26255694

  3. Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

    PubMed

    Qin, Sisi; Yang, Canhong; Huang, Weihua; Du, Shuhua; Mai, Hantao; Xiao, Jijie; Lü, Tianming

    2018-01-31

    Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1β, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Anti-arrhythmic activities of opioid agonists and antagonists and their stereoisomers.

    PubMed Central

    Sarne, Y.; Flitstein, A.; Oppenheimer, E.

    1991-01-01

    1. A series of opioid agonists, antagonists and their (+)-stereoisomers were tested for antiarrhythmic activity in the rat coronary artery occlusion model. 2. Naloxone (0.01-2 mg kg-1) significantly reduced the incidence and severity of cardiac arrhythmias, in accordance with previous published studies. 3. The non-opioid stereoisomer, (+)-naloxone, was equipotent with naloxone against occlusion-induced arrhythmia. 4. Similar non-stereospecific antiarrhythmic effects were induced by another opioid antagonist, Win 44,441-3 and its stereoisomer Win 44,441-2. 5. The opioid agonists, morphine and levorphanol, protected against occlusion-induced arrhythmia as did the opioid antagonists, and the (+)-stereoisomer, dextrorphan, was equipotent to levorphanol. 6. It is concluded that the antiarrhythmic effects of opioid drugs are not mediated by opioid receptors. A direct effect on ionic currents in cardiac muscle is suggested as the mechanism of opioid antiarrhythmic activity. PMID:1364840

  5. Evaluation of the alpha-1 and alpha-2 adrenoceptor-mediated effects of a series of dimethoxy-substituted tolazoline derivatives in the cardiovascular system of the pithed rat.

    PubMed

    Ruffolo, R R; Messick, K

    1985-01-01

    The alpha-1 and alpha-2 adrenoceptor-mediated effects of a series of dimethoxy-substituted tolazoline derivatives were investigated in the cardiovascular system of the pithed rat. The 2,5- and 3,5-dimethoxy-substituted tolazoline derivatives produced vasopressor responses that were inhibited by the alpha-1 adrenoceptor antagonist, prazosin (0.1 mg/kg i.v.), and were not affected by the alpha-2 adrenoceptor antagonist, yohimbine (1 mg/kg i.v.), suggesting that these derivatives selectively activate postsynaptic vascular alpha-1 adrenoceptors. The 2,5- and 3,5-dimethoxy-substituted derivatives of tolazoline did not produce an alpha-2 adrenoceptor-mediated inhibition of neurogenic tachycardia in cord-stimulated pithed rats and were therefore presumed to be devoid of alpha-2 adrenoceptor agonist activity. In contrast, 2,3-dimethoxytolazoline produced a vasopressor effect that was inhibited by yohimbine but not by prazosin, suggesting selective activation of postsynaptic vascular alpha-2 adrenoceptors. Consistent with this observation is the fact that 2,3-dimethoxytolazoline elicited a dose-dependent, alpha-2 adrenoceptor-mediated inhibition of neurogenic tachycardia in cord-stimulated pithed rat. 3,4-Dimethoxytolazoline was a weak alpha-1 adrenoceptor agonist in the vasculature of the pithed rat and was devoid of agonist activity at alpha-2 adrenoceptors. However, 3,4-dimethoxytolazoline was found to be an alpha-2 adrenoceptor antagonist of similar potency as yohimbine. The results of the present study indicate that dimethoxy-substituted derivatives of tolazoline possess different activities and selectivities at alpha-1 and alpha-2 adrenoceptors depending upon the positions of substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Further investigation of the effects of 5-hydroxytryptamine, 8-OH-DPAT and DOI to mediate contraction and relaxation responses in the intestine and emesis in Suncus murinus.

    PubMed

    Javid, Farideh A; Afshin-Javid, Saeed; Horn, Charles C

    2018-02-15

    5-HT receptors are implicated in many gastrointestinal disorders. However, the precise role of 5-HT in mediating GI responses in Suncus murnius is still unclear. Therefore in this study, the effects of 5-HT and its agonists were investigated in Suncus. The involvement of 5-HT 2C receptors in mediating emesis was also investigated. The ability of 5-HT and its agonists/antagonists at 5-HT 1A and 5-HT 2 to modify GI motility was investigated in vitro and in vivo. WAY100635 (a 5-HT 1A antagonist) inhibited the contraction response to 5-HT in the proximal segments without affecting the maximum response; whilst enhancing the contraction to 5-HT (>30.0nM) in the distal intestine. The selective 5-HT 2A and 5-HT 2B receptor antagonists MDL-100907 and RS-127445 attenuated 5-HT-induced contractions (<10.0µM) in the distal segments. RS-127445 also attenuated 5-HT-induced contractions in the central segments. The selective 5-HT 2C receptor antagonist SB-242084, attenuated the responses to 5-HT (> 3.0nM) in the proximal and central but not the distal regions. 8-OH-DPAT-induced relaxation was resistant to the antagonism by 5-HT 1A/7 antagonists. DOI in the presence of 5-HT 1A/2A/2B/2C antagonists induced greater contraction responses (>1.0µM) in most tissues, whilst RS-127445, or SB-242084, reduced the responses to DOI (< 1.0µM) in some tissues. SB-242084 also suppressed emesis-induced by motion and intragastric CuSO 4 . In conclusion, within different regions of intestine, 5-HT 2 receptors are differently involved in contraction and emetic responses and that 8-OH-DPAT induces relaxation via non-5-HT 1A/7 receptors. Suncus could provide a model to investigate these diverse actions of 5-HT. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Novel long‐acting antagonists of muscarinic ACh receptors

    PubMed Central

    Randáková, Alena; Rudajev, Vladimír; Doležal, Vladimír; Boulos, John

    2018-01-01

    Background and Purpose The aim of this study was to develop potent and long‐acting antagonists of muscarinic ACh receptors. The 4‐hexyloxy and 4‐butyloxy derivatives of 1‐[2‐(4‐oxidobenzoyloxy)ethyl]‐1,2,3,6‐tetrahydropyridin‐1‐ium were synthesized and tested for biological activity. Antagonists with long‐residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long‐acting effects allow for reduced daily doses and adverse effects. Experimental Approach The binding and antagonism of functional responses to the agonist carbachol mediated by 4‐hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4‐butyloxy analogues. Key Results The 4‐hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M1 to 4 μM at M3 receptors. Under washing conditions to reverse antagonism, the half‐life of their antagonistic action ranged from 1.7 h at M2 to 5 h at M5 receptors. Conclusions and Implications The 4‐hexyloxy derivatives were found to be potent long‐acting M1‐preferring antagonists. In view of current literature, M1‐selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits. PMID:29498041

  8. Shifting physician prescribing to a preferred histamine-2-receptor antagonist. Effects of a multifactorial intervention in a mixed-model health maintenance organization.

    PubMed

    Brufsky, J W; Ross-Degnan, D; Calabrese, D; Gao, X; Soumerai, S B

    1998-03-01

    This study was undertaken to determine whether a program of education, therapeutic reevaluation of eligible patients, and performance feedback could shift prescribing to cimetidine from other histamine-2 receptor antagonists, which commonly are used in the management of ulcers and reflux, and reduce costs without increasing rates of ulcer-related hospital admissions. This study used an interrupted monthly time series with comparison series in a large mixed-model health maintenance organization. Physicians employed in health centers (staff model) and physicians in independent medical groups contracting to provide health maintenance organization services (group model) participated. The comparative percentage prescribed of specific histamine-2 receptor antagonists (market share), total histamine-2 receptor antagonist prescribing, cost per histamine-2 receptor antagonist prescription, and the rate of hospitalization for gastrointestinal illness were assessed. In the staff model, therapeutic reevaluation resulted in a sudden increase in market share of the preferred histamine-2 receptor antagonist cimetidine (+53.8%) and a sudden decrease in ranitidine (-44.7%) and famotidine (-4.8%); subsequently, cimetidine market share grew by 1.1% per month. In the group model, therapeutic reevaluation resulted in increased cimetidine market share (+9.7%) and decreased prescribing of other histamine-2 receptor antagonists (ranitidine -11.6%; famotidine -1.2%). Performance feedback did not result in further changes in prescribing in either setting. Use of omeprazole, an expensive alternative, essentially was unchanged by the interventions, as were overall histamine-2 receptor antagonist prescribing and hospital admissions for gastrointestinal illnesses. This intervention, which cost approximately $60,000 to implement, resulted in estimated annual savings in histamine-2 receptor antagonist expenditures of $1.06 million. Annual savings in histamine-2 receptor antagonist expenditures

  9. Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

    PubMed

    Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

    2010-01-01

    Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

  10. Synaptic GluN2A and GluN2B Containing NMDA Receptors within the Superficial Dorsal Horn Activated following Primary Afferent Stimulation

    PubMed Central

    MacDermott, Amy B.

    2014-01-01

    NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers, typically composed of two GluN1 and two of four GluN2 subunits: GluN2A-2D. Mice lacking some of the GluN2 subunits show deficits in pain transmission yet functional synaptic localization of these receptor subtypes in the dorsal horn has not been fully resolved. In this study, we have investigated the composition of synaptic NMDA receptors expressed in monosynaptic and polysynaptic pathways from peripheral sensory fibers to lamina I neurons in rats. We focused on substance P receptor-expressing (NK1R+) projection neurons, critical for expression of hyperalgesia and allodynia. EAB-318 and (R)-CPP, GluN2A/B antagonists, blocked both monosynaptic and polysynaptic NMDA EPSCs initiated by primary afferent activation by ∼90%. Physiological measurements exploiting the voltage dependence of monosynaptic EPSCs similarly indicated dominant expression of GluN2A/B types of synaptic NMDA receptors. In addition, at synapses between C fibers and NK1R+ neurons, NMDA receptor activation initiated a secondary, depolarizing current. Ifenprodil, a GluN2B antagonist, caused modest suppression of monosynaptic NMDA EPSC amplitudes, but had a widely variable, sometimes powerful, effect on polysynaptic responses following primary afferent stimulation when inhibitory inputs were blocked to mimic neuropathic pain. We conclude that GluN2B subunits are moderately expressed at primary afferent synapses on lamina I NK1R+ neurons, but play more important roles for polysynaptic NMDA EPSCs driven by primary afferents following disinhibition, supporting the view that the analgesic effect of the GluN2B antagonist on neuropathic pain is at least in part, within the spinal cord. PMID:25122884

  11. The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

    PubMed Central

    Daempfling, Lea

    2013-01-01

    Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals. PMID:23716589

  12. Effects of tachykinin receptor agonists and antagonists on the guinea-pig isolated oesophagus.

    PubMed

    Kerr, K P

    2000-11-01

    1. Vagal nerve stimulation of the guinea-pig isolated oesophagus produced a triphasic tetrodotoxin (TTX)-sensitive contractile response. The third phase, which was resistant to ganglion blocking drugs, was selectively abolished by capsaicin, suggesting the involvement of one or more neuropeptides released from afferent neurons. Receptors on cholinergic neurons were subsequently activated because the response was atropine sensitive. Contractile responses resulting from exogenous substance P were abolished by atropine and TTX and enhanced by physostigmine. These findings suggest that the third phase may be mediated by the action of a substance P-like neuropeptide released from sensory nerve endings that subsequently activated cholinergic neurons. 2. The tachykinin receptors in the body of the guinea-pig oesophagus were characterized by determining the relative agonist potencies of natural tachykinins as well as tachykinin receptor-selective analogues. Antagonist affinities were also determined. The results indicated the presence of both NK2 and NK3 receptors. In addition, the effects of a cocktail of peptidase inhibitors (captopril, thiorphan and amastatin) on responses to various tachykinins and synthetic analogues were determined. The results indicate that one or more peptidases are present in this preparation. 3. Experiments using various tachykinin receptor antagonists were performed to determine whether the activation of tachykinin receptors played a role in the mediation of the third phase of the response to vagal nerve stimulation. While this response was unaffected by NK1 and NK2 receptor-selective antagonists, it was only partially inhibited (23%) by the NK3 receptor antagonist SR 142801. Thus, in the guinea-pig oesophagus, it appears that NK3 receptors play only a minor role in mediating a contractile response when afferent neurons are excited by vagal nerve stimulation.

  13. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats.

    PubMed

    Blacktop, Jordan M; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A; Mantsch, John R

    2016-03-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 h/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5" duration, average every 40 s; range 10-70 s) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 μg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The novel micro-opioid receptor antagonist, [N-allyl-Dmt(1)]endomorphin-2, attenuates the enhancement of GABAergic neurotransmission by ethanol.

    PubMed

    Li, Qiang; Okada, Yoshio; Marczak, Ewa; Wilson, Wilkie A; Lazarus, Lawrence H; Swartzwelder, H S

    2009-01-01

    We investigated the effects of [N-allyl-Dmt(1)]endomorphin-2 (TL-319), a novel and highly potent micro-opioid receptor antagonist, on ethanol (EtOH)-induced enhancement of GABA(A) receptor-mediated synaptic activity in the hippocampus. Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were isolated from CA1 pyramidal cells from brain slices of male rats using whole-cell patch-clamp techniques. TL-319 had no effect on the baseline amplitude of eIPSCs or the frequency of sIPSCs. However, it induced a dose-dependent suppression of an ethanol-induced increase of sIPSC frequency with full reversal at concentrations of 500 nM and higher. The non-specific competitive opioid receptor antagonist naltrexone also suppressed EtOH-induced increases in sIPSC frequency but only at a concentration of 60 microM. These data indicate that blockade of micro-opioid receptors by low concentrations of [N-allyl-Dmt(1)]endomorphin-2 can reverse ethanol-induced increases in GABAergic neurotransmission and possibly alter its anxiolytic or sedative effects. This suggests the possibility that high potency opioid antagonists may emerge as possible candidate compounds for the treatment of ethanol addiction.

  15. Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

    PubMed

    Pozvek, G; Hilton, J M; Quiza, M; Houssami, S; Sexton, P M

    1997-04-01

    The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

  16. Vascular endothelial cells mediate mechanical stimulation-induced enhancement of endothelin hyperalgesia via activation of P2X2/3 receptors on nociceptors.

    PubMed

    Joseph, Elizabeth K; Green, Paul G; Bogen, Oliver; Alvarez, Pedro; Levine, Jon D

    2013-02-13

    Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

  17. Runx2 mediates epigenetic silencing of the bone morphogenetic protein-3B (BMP-3B/GDF10) in lung cancer cells

    PubMed Central

    2012-01-01

    Background The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes invasive properties. The transforming growth factor β (TGF-β) family member bone morphogenetic protein-3B (BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the regulatory mechanisms leading to its silencing have not been identified. Results Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2 overexpression in H1299 cells increased wound healing response to TGFβ treatment. Conclusions Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells. PMID:22537242

  18. LncRNA-uc002mbe.2 Interacting with hnRNPA2B1 Mediates AKT Deactivation and p21 Up-Regulation Induced by Trichostatin in Liver Cancer Cells.

    PubMed

    Chen, Ting; Gu, Chengxin; Xue, Cailin; Yang, Tao; Zhong, Yun; Liu, Shiming; Nie, Yuqiang; Yang, Hui

    2017-01-01

    Long non-coding RNAs (lncRNAs) have been implicated in liver carcinogenesis. We previously showed that the induction of lncRNA-uc002mbe.2 is positively associated with the apoptotic effect of trichostatin A (TSA) in hepatocellular carcinoma (HCC) cells. The current study further analyzed the role of uc002mbe.2 in TSA-induced liver cancer cell death. The level of uc002mbe.2 was markedly increased by TSA in the cytoplasm of HCC cells. Knockdown of uc002mbe.2 prohibited TSA-induced G2/M cell cycle arrest, p21 induction, and apoptosis of Huh7 cells and reversed the TSA-mediated decrease in p-AKT. RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays revealed that TSA induced an interaction between uc002mbe.2 and heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in Huh7 cells. This interaction mediated AKT deactivation and p21 induction in liver cancer cells. In an athymic xenograft mouse model, knockdown of uc002mbe.2 significantly prohibited the TSA-mediated reduction in tumor size and weight. In addition, the ability of TSA to reduce hnRNPA2B1 and p-AKT levels and induce p21 in the xenograft tumors was prevented by uc002mbe.2 knockdown. Therefore, the interaction of uc002mbe.2 and hnRNPA2B1 in mediating AKT deactivation and p21 induction is involved in the cytostatic effect of trichostatin in liver cancer cells.

  19. Non-NMDA receptor antagonist-induced drinking in rat

    NASA Technical Reports Server (NTRS)

    Xu, Z.; Johnson, A. K.

    1998-01-01

    Glutamate has been implicated in the central control of mechanisms that maintain body fluid homeostasis. The present studies demonstrate that intracerebroventricular (i.c.v.) injections of the non-N-methyl-d-aspartate (NMDA) receptor antagonists 6, 7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) induce drinking in rats. The dipsogenic effect of i.c.v. DNQX was antagonized by the non-NMDA receptor agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). The water intake induced by DNQX was also blocked by pretreatment with a NMDA receptor antagonist, MK-801, but not by angiotensin type 1 (AT1) or acetylcholine muscarinic receptor antagonists (losartan and atropine). The results indicate that non-NMDA receptors may exert a tonic inhibitory effect within brain circuits that control dipsogenic activity and that functional integrity of NMDA receptors may be required for the non-NMDA receptor antagonists to induce water intake. Copyright 1998 Published by Elsevier Science B.V.

  20. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kan-o, Keiko; Matsumoto, Koichiro, E-mail: koichi@kokyu.med.kyushu-u.ac.jp; Asai-Tajiri, Yukari

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (polymore » IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.« less

  1. Non-specific actions of the non-peptide tachykinin receptor antagonists, CP-96,345, RP 67580 and SR 48968, on neurotransmission.

    PubMed Central

    Wang, Z. Y.; Tung, S. R.; Strichartz, G. R.; Håkanson, R.

    1994-01-01

    1. Three non-peptide tachykinin receptor antagonists, CP-96,345, RP 67580 and SR 48968, were found to inhibit the electrically-evoked, tachykinin-mediated contractile responses of the rabbit iris sphincter in a concentration-dependent fashion; the pIC50 values were 5.6 +/- 0.01, 5.4 +/- 0.07 and 4.8 +/- 0.03, respectively. 2. These antagonists also inhibited the electrically-evoked, parasympathetic response of the rabbit iris sphincter and the sympathetic response of the guinea-pig vas deferens in a concentration-dependent manner; the pIC50 values were 0.3-1.2 log units lower than those recorded for the tachykinin-mediated responses. 3. Two local anaesthetics, bupivacaine and oxybuprocaine, were also found to inhibit the tachykinin-mediated, cholinergic and sympathetic contractile responses in these tissues in a concentration-dependent manner; the concentration ranges for producing the inhibition were similar to those of the non-peptide tachykinin receptor antagonists. 4. On the sciatic nerves of frogs, the tachykinin receptor antagonists inhibited action potentials in a concentration-dependent manner; the potency of the three drugs was similar to that of bupivacaine. 5. Our results suggest that, in addition to blocking tachykinin receptors, the non-peptide tachykinin receptor antagonists, CP-96,345, RP 67580 and SR 48968, may exert non-specific inhibitory effects on neurotransmission. PMID:8012694

  2. Behavioral approach to nondyskinetic dopamine antagonists: identification of seroquel.

    PubMed

    Warawa, E J; Migler, B M; Ohnmacht, C J; Needles, A L; Gatos, G C; McLaren, F M; Nelson, C L; Kirkland, K M

    2001-02-01

    A great need exists for antipsychotic drugs which will not induce extrapyramidal symptoms (EPS) and tardive dyskinesias (TDs). These side effects are deemed to be a consequence of nonselective blockade of nigrostriatal and mesolimbic dopamine D2 receptors. Nondyskinetic clozapine (1) is a low-potency D2 dopamine receptor antagonist which appears to act selectively in the mesolimbic area. In this work dopamine antagonism was assessed in two mouse behavioral assays: antagonism of apomorphine-induced climbing and antagonism of apomorphine-induced disruption of swimming. The potential for the liability of dyskinesias was determined in haloperidol-sensitized Cebus monkeys. Initial examination of a few close cogeners of 1 enhanced confidence in the Cebus model as a predictor of dyskinetic potential. Considering dibenzazepines, 2 was not dyskinetic whereas 2a was dyskinetic. Among dibenzodiazepines, 1 did not induce dyskinesias whereas its N-2-(2-hydroxyethoxy)ethyl analogue 3 was dyskinetic. The emergence of such distinctions presented an opportunity. Thus, aromatic and N-substituted analogues of 6-(piperazin-1-yl)-11H-dibenz[b,e]azepines and 11-(piperazin-1-yl)dibenzo[b,f][1,4]thiazepines and -oxazepines were prepared and evaluated. 11-(4-[2-(2-Hydroxyethoxy)ethyl]piperazin-1-yl)dibenzo[b,f][1,4]thiazepine (23) was found to be an apomorphine antagonist comparable to clozapine. It was essentially nondyskinetic in the Cebus model. With 23 as a platform, a number of N-substituted analogues were found to be good apomorphine antagonists but all were dyskinetic.

  3. Increased AIF-1-mediated TNF-α expression during implantation phase in IVF cycles with GnRH antagonist protocol.

    PubMed

    Xu, Bufang; Zhou, Mingjuan; Wang, Jingwen; Zhang, Dan; Guo, Feng; Si, Chenchen; Leung, Peter C K; Zhang, Aijun

    2018-06-12

    . Microarray results revealed that AIF-1 was upregulated in the antagonist group compared with the control group (fold change [FC] = 3.75) and the agonist (FC = 2.20) group. The raw microarray data and complete gene expression table were uploaded to GEO under the accession number of GSE107914. Both the mRNA and protein expression levels of AIF-1 and TNF-α were the higher in the antagonist group than in the other two groups (P < 0.05) which did not differ significantly (P > 0.05). The protein levels of TNF-α in both Ishikawa cells and primary endometrial cells were significantly increased (P < 0.05) at 96 h after transfection with the AIF-1 expression vector, indicating that TNF-α was mediated by AIF-1 in endometrial cells. Overexpression of AIF-1 in Ishikawa cells inhibited adhesion of JAR cells to them. Thus, increased AIF-1 might inhibit adhesion during implantation via raised TNF-α. The sample size of the microarray was small, which might weaken the accuracy of our results; however, the sample size of RT-qPCR and the Western blotting assays were sufficient to compensate for this deficiency in our study. In addition, the aberrant AIF-1 and thus TNF-α expression is one of many factors that may contribute to limiting implantation success. Therefore, further extensive in vitro mechanistic and in vivo animal studies are needed to assess the actual functional impact of this pathway. Anti-TNF-α therapy might mitigate the adverse effects of GnRH antagonist on endometrial receptivity and improve the implantation rate in GnRH antagonist protocols in IVF. This work was supported by grants from the National Natural Science Foundation of China, Grant numbers 81771656 and 81370763; Clinical research special fund of Chinese Medical Association, Grant number 16020480664; Shanghai Jiao Tong University Medicine-Engineering Fund, Grant number YG2017ZD11 and YG2017MS57; and the Merck-Serono China Research Fund for Fertility Agreement. P.C.K.L. is supported by a Canadian Institutes of

  4. UNC93B1 mediates differential trafficking of endosomal TLRs

    PubMed Central

    Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

    2013-01-01

    UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999

  5. Metabolic benefits of 1-(3-(4-(o-tolyl)piperazin-1-yl)propyl)pyrrolidin-2-one: a non-selective α-adrenoceptor antagonist.

    PubMed

    Kotańska, Magdalena; Kulig, Katarzyna; Marcinkowska, Monika; Bednarski, Marek; Malawska, Katarzyna; Zaręba, Paula

    2018-05-01

    Previous studies have shown that several components of the metabolic syndrome, such as hypertension, obesity or imbalanced lipid and carbohydrate homeostasis, are associated with the sympathetic nervous system overactivity. Therefore, the inhibition of the adrenergic nervous system seems to be a reasonable and appropriate therapeutic approach for the treatment of metabolic disturbances. It has been suggested that non-selective adrenoceptor antagonists could be particularly beneficial, since α 1 -adrenoceptor antagonists can improve disrupted lipid and carbohydrate profiles, while the inhibition of the α 2 -adrenoceptor may contribute to body weight reduction. The aim of the present study was to investigate the metabolic benefits deriving from administration of a non-selective α-adrenoceptor antagonist from the group of pyrrolidin-2-one derivatives. The aim of the present study was to investigate the potential metabolic benefits deriving from chronic administration of a non-selective α-adrenoceptor antagonist, from the group of pyrrolidin-2-one derivatives. The α 1 - and α 2 -adrenoreceptor affinities of the tested compound-1-(3-(4-(o-tolyl)piperazin-1-yl)propyl)pyrrolidin-2-one had been investigated previously by means of the radioligand binding assay. In the present study, we extended the pharmacological profile characteristics of the selected molecule by additional intrinistic activity assays. Next, we investigated the influence of the tested compound on body weight, hyperglycemia, hypertriglyceridemia, blood pressure in the animal model of obesity induced by a high-fat diet, and additionally we measured the spontaneous activity and body temperature. The intrinistic activity studies revealed that the tested compound is a potent, non-selective antagonist of α 1B and α 2A -adrenoceptors. After the chronic administration of the tested compound, we observed reduced level of triglycerides and glucose in the rat plasma. Interestingly, the tested did not reduce

  6. Antagonism at the NR2B subunit of NMDA receptors induces increased connectivity of the prefrontal and subcortical regions regulating reward behavior.

    PubMed

    Gass, Natalia; Becker, Robert; Sack, Markus; Schwarz, Adam J; Reinwald, Jonathan; Cosa-Linan, Alejandro; Zheng, Lei; von Hohenberg, Christian Clemm; Inta, Dragos; Meyer-Lindenberg, Andreas; Weber-Fahr, Wolfgang; Gass, Peter; Sartorius, Alexander

    2018-04-01

    Evidence indicates that ketamine's rapid antidepressant efficacy likely results from its antagonism of NR2B-subunit-containing NMDA receptors (NMDAR). Since ketamine equally blocks NR2A- and NR2B-containing NMDAR, and has affinity to other receptors, NR2B-selective drugs might have improved therapeutic efficiency and side effect profile. We aimed to compare the effects of (S)-ketamine and two different types of NR2B-selective antagonists on functional brain networks in rats, in order to find common circuits, where their effects intersect, and that might explain their antidepressant action. The experimental design comprised four parallel groups of rats (N = 37), each receiving (S)-Ketamine, CP-101,606, Ro 25-6981 or saline. After compound injection, we acquired resting-state functional magnetic resonance imaging time series. We used graph theoretical approach to calculate brain network properties. Ketamine and CP-101,606 diminished the global clustering coefficient and small-worldness index. At the nodal level, all compounds induced increased connectivity of the regions mediating reward and cognitive aspects of emotional processing, such as ventromedial prefrontal cortex, septal nuclei, and nucleus accumbens. The dorsal hippocampus and regions involved in sensory processing and aversion, such as superior and inferior colliculi, exhibited an opposite effect. The effects common to ketamine and NR2B-selective compounds were localized to the same brain regions as those reported in depression, but in the opposite direction. The upregulation of the reward circuitry might partially underlie the antidepressant and anti-anhedonic effects of the antagonists and could potentially serve as a translational imaging phenotype for testing putative antidepressants, especially those targeting the NR2B receptor subtype.

  7. Discovery of a Novel Series of CRTH2 (DP2) Receptor Antagonists Devoid of Carboxylic Acids

    PubMed Central

    2011-01-01

    Antagonism of the CRTH2 receptor represents a very attractive target for a variety of allergic diseases. Most CRTH2 antagonists known to date possess a carboxylic acid moiety, which is essential for binding. However, potential acid metabolites O-acyl glucuronides might be linked to idiosynchratic toxicity in humans. In this communication, we describe a new series of compounds that lack the carboxylic acid moiety. Compounds with high affinity (Ki < 10 nM) for the receptor have been identified. Subsequent optimization succeeded in reducing the high metabolic clearance of the first compounds in human and rat liver microsomes. At the same time, inhibition of the CYP isoforms was optimized, giving rise to stable compounds with an acceptable CYP inhibition profile (IC50 CYP2C9 and 2C19 > 1 μM). Taken together, these data show that compounds devoid of carboxylic acid groups could represent an interesting alternative to current CRTH2 antagonists in development. PMID:24900284

  8. Safety, tolerability, and bioavailability of topical SAR 1118, a novel antagonist of lymphocyte function-associated antigen-1: a phase 1b study.

    PubMed

    Paskowitz, D M; Nguyen, Q D; Gehlbach, P; Handa, J T; Solomon, S; Stark, W; Shaikh, O; Semba, C; Gadek, T R; Do, D V

    2012-07-01

    A growing body of evidence points to a role for inflammation mediated by lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 in the pathogenesis of diabetic macular oedema. This phase 1b clinical trial assessed the safety, tolerability, and pharmacokinetics of topically administered SAR 1118, a novel LFA-1 antagonist, in human subjects. In this prospective, randomized, double-masked trial, 13 subjects scheduled for vitrectomy received one of three concentrations of topical SAR 1118 (0.1, 1.0, or 5.0%) twice daily for 1 week before surgery. Undiluted aqueous and vitreous samples were collected at surgery and analysed for the concentration of the medication. All subjects completed the entire course of medication. The only adverse events reported were instillation site irritation (4/13, 31%) and dysgeusia (3/13, 23%). These were mild and transient, occurring at the highest dose. Mean concentrations (ng/ml) of SAR 1118 in the aqueous humour were 0.25, 37.2, and 101.1 for the 0.1%, 1.0%, and 5.0% dose groups, respectively. SAR 1118 was below the level of detection (0.5 ng/ml) for all vitreous samples except in a single subject who had a history of prior vitrectomy and a dislocated intraocular lens. Topical SAR 1118 was safe and well tolerated, and dose-dependent levels of drug were detected in aqueous. However, vitreous levels were below the threshold of detection with the concentrations tested. Further investigation of this medication for posterior segment applications would require intravitreal delivery or chemical modification of the drug.

  9. Bicuculline, a GABAA-receptor antagonist, blocked HPA axis activation induced by ghrelin under an acute stress.

    PubMed

    Gastón, M S; Cid, M P; Salvatierra, N A

    2017-03-01

    Ghrelin is a peptide of 28 amino acids with a homology between species, which acts on the central nervous system to regulate different actions, including the control of growth hormone secretion and metabolic regulation. It has been suggested that central ghrelin is a mediator of behavior linked to stress responses and induces anxiety in rodents and birds. Previously, we observed that the anxiogenic-like behavior induced by ghrelin injected into the intermediate medial mesopallium (IMM) of the forebrain was blocked by bicuculline (a GABA A receptor competitive antagonist) but not by diazepam (a GABA A receptor allosteric agonist) in neonatal meat-type chicks (Cobb). Numerous studies have indicated that hypothalamic-pituitary-adrenal (HPA) axis activation mediates the response to stress in mammals and birds. However, it is still unclear whether this effect of ghrelin is associated with HPA activation. Therefore, we investigated whether anxiety behavior induced by intra-IMM ghrelin and mediated through GABA A receptors could be associated with HPA axis activation in the neonatal chick. In the present study, in an Open Field test, intraperitoneal bicuculline methiodide blocked anxiogenic-like behavior as well as the increase in plasma ACTH and corticosterone levels induced by ghrelin (30pmol) in neonatal chicks. Moreover, we showed for the first time that a competitive antagonist of GABA A receptor suppressed the HPA axis activation induced by an anxiogenic dose of ghrelin. These results show that the anxiogenic ghrelin action involves the activation of the HPA axis, with a complex functional interaction with the GABA A receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Role of B-type natriuretic peptide in epoxyeicosatrienoic acid-mediated improved post-ischaemic recovery of heart contractile function

    PubMed Central

    Chaudhary, Ketul R.; Batchu, Sri Nagarjun; Das, Dipankar; Suresh, Mavanur R.; Falck, John R.; Graves, Joan P.; Zeldin, Darryl C.; Seubert, John M.

    2009-01-01

    Aims This study examined the functional role of B-type natriuretic peptide (BNP) in epoxyeicosatrienoic acid (EET)-mediated cardioprotection in mice with targeted disruption of the sEH or Ephx2 gene (sEH null). Methods and results Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C ε and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3β was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts. Conclusion Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events. PMID:19401302

  11. Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists.

    PubMed

    Varano, Flavia; Catarzi, Daniela; Falsini, Matteo; Vincenzi, Fabrizio; Pasquini, Silvia; Varani, Katia; Colotta, Vittoria

    2018-07-23

    In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A 1 , A 2A , A 2B and A 3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA 1 and hA 2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A 1 /A 2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA 1 K i  = 10.2 nM; hA 2A K i  = 4.72 nM) and behaved as a potent A 1 /A 2A antagonist/inverse agonist (hA 1 IC 50  = 13.4 nM; hA 2A IC 50  = 5.34 nM). Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

    PubMed

    Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

    2016-05-01

    Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

  13. Rap1 and Rap2 Antagonistically Control Endothelial Barrier Resistance

    PubMed Central

    Pannekoek, Willem-Jan; Linnemann, Jelena R.; Brouwer, Patricia M.; Bos, Johannes L.; Rehmann, Holger

    2013-01-01

    Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2. PMID:23469100

  14. Short Communication: Inhibition of DC-SIGN-Mediated HIV-1 Infection by Complementary Actions of Dendritic Cell Receptor Antagonists and Env-Targeting Virus Inactivators.

    PubMed

    Pustylnikov, Sergey; Dave, Rajnish S; Khan, Zafar K; Porkolab, Vanessa; Rashad, Adel A; Hutchinson, Matthew; Fieschi, Frank; Chaiken, Irwin; Jain, Pooja

    2016-01-01

    The DC-SIGN receptor on human dendritic cells interacts with HIV gp120 to promote both infection of antigen-presenting cells and transinfection of T cells. We hypothesized that in DC-SIGN-expressing cells, both DC-SIGN ligands such as dextrans and gp120 antagonists such as peptide triazoles would inhibit HIV infection with potential complementary antagonist effects. To test this hypothesis, we evaluated the effects of dextran (D66), isomaltooligosaccharides (D06), and several peptide triazoles (HNG156, K13, and UM15) on HIV infection of B-THP-1/DC-SIGN cells. In surface plasmon resonance competition assays, D66 (IC50 = 35.4 μM) and D06 (IC50 = 3.4 mM) prevented binding of soluble DC-SIGN to immobilized mannosylated bovine serum albumin (BSA). An efficacious dose-dependent inhibition of DC-SIGN-mediated HIV infection in both pretreatment and posttreatment settings was observed, as indicated by inhibitory potentials (EC50) [D66 (8 μM), D06 (48 mM), HNG156 (40 μM), UM15 (100 nM), and K13 (25 nM)]. Importantly, both dextrans and peptide triazoles significantly decreased HIV gag RNA levels [D66 (7-fold), D06 (13-fold), HNG156 (7-fold), K-13 (3-fold), and UM15 (6-fold)]. Interestingly, D06 at the highest effective concentration showed a 14-fold decrease of infection, while its combination with 50 μM HNG156 showed a 26-fold decrease. Hence, these compounds can combine to inactivate the viruses and suppress DC-SIGN-mediated virus-cell interaction that as shown earlier leads to dendritic cell HIV infection and transinfection dependent on the DC-SIGN receptor.

  15. Short Communication: Inhibition of DC-SIGN-Mediated HIV-1 Infection by Complementary Actions of Dendritic Cell Receptor Antagonists and Env-Targeting Virus Inactivators

    PubMed Central

    Pustylnikov, Sergey; Dave, Rajnish S.; Khan, Zafar K.; Porkolab, Vanessa; Rashad, Adel A.; Hutchinson, Matthew; Fieschi, Frank; Chaiken, Irwin

    2016-01-01

    Abstract The DC-SIGN receptor on human dendritic cells interacts with HIV gp120 to promote both infection of antigen-presenting cells and transinfection of T cells. We hypothesized that in DC-SIGN-expressing cells, both DC-SIGN ligands such as dextrans and gp120 antagonists such as peptide triazoles would inhibit HIV infection with potential complementary antagonist effects. To test this hypothesis, we evaluated the effects of dextran (D66), isomaltooligosaccharides (D06), and several peptide triazoles (HNG156, K13, and UM15) on HIV infection of B-THP-1/DC-SIGN cells. In surface plasmon resonance competition assays, D66 (IC50 = 35.4 μM) and D06 (IC50 = 3.4 mM) prevented binding of soluble DC-SIGN to immobilized mannosylated bovine serum albumin (BSA). An efficacious dose-dependent inhibition of DC-SIGN-mediated HIV infection in both pretreatment and posttreatment settings was observed, as indicated by inhibitory potentials (EC50) [D66 (8 μM), D06 (48 mM), HNG156 (40 μM), UM15 (100 nM), and K13 (25 nM)]. Importantly, both dextrans and peptide triazoles significantly decreased HIV gag RNA levels [D66 (7-fold), D06 (13-fold), HNG156 (7-fold), K-13 (3-fold), and UM15 (6-fold)]. Interestingly, D06 at the highest effective concentration showed a 14-fold decrease of infection, while its combination with 50 μM HNG156 showed a 26-fold decrease. Hence, these compounds can combine to inactivate the viruses and suppress DC-SIGN-mediated virus–cell interaction that as shown earlier leads to dendritic cell HIV infection and transinfection dependent on the DC-SIGN receptor. PMID:26383762

  16. SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke): A Randomized Controlled Phase 2 Trial.

    PubMed

    Smith, Craig J; Hulme, Sharon; Vail, Andy; Heal, Calvin; Parry-Jones, Adrian R; Scarth, Sylvia; Hopkins, Karen; Hoadley, Margaret; Allan, Stuart M; Rothwell, Nancy J; Hopkins, Stephen J; Tyrrell, Pippa J

    2018-05-01

    The proinflammatory cytokine IL-1 (interleukin-1) has a deleterious role in cerebral ischemia, which is attenuated by IL-1 receptor antagonist (IL-1Ra). IL-1 induces peripheral inflammatory mediators, such as interleukin-6, which are associated with worse prognosis after ischemic stroke. We investigated whether subcutaneous IL-1Ra reduces the peripheral inflammatory response in acute ischemic stroke. SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke) was a single-center, double-blind, randomized, placebo-controlled phase 2 trial of subcutaneous IL-1Ra (100 mg administered twice daily for 3 days) in patients presenting within 5 hours of ischemic stroke onset. Randomization was stratified for baseline National Institutes of Health Stroke Scale score and thrombolysis. Measurement of plasma interleukin-6 and other peripheral inflammatory markers was undertaken at 5 time points. The primary outcome was difference in concentration of log(interleukin-6) as area under the curve to day 3. Secondary outcomes included exploratory effect of IL-1Ra on 3-month outcome with the modified Rankin Scale. We recruited 80 patients (mean age, 72 years; median National Institutes of Health Stroke Scale, 12) of whom 73% received intravenous thrombolysis with alteplase. IL-1Ra significantly reduced plasma interleukin-6 ( P <0.001) and plasma C-reactive protein ( P <0.001). IL-1Ra was well tolerated with no safety concerns. Allocation to IL-1Ra was not associated with a favorable outcome on modified Rankin Scale: odds ratio (95% confidence interval)=0.67 (0.29-1.52), P =0.34. Exploratory mediation analysis suggested that IL-1Ra improved clinical outcome by reducing inflammation, but there was a statistically significant, alternative mechanism countering this benefit. IL-1Ra reduced plasma inflammatory markers which are known to be associated with worse clinical outcome in ischemic stroke. Subcutaneous IL-1Ra is safe and well tolerated. Further experimental

  17. Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B

    PubMed Central

    Hagenow, S.; Stasiak, A.; Ramsay, R. R.; Stark, H.

    2017-01-01

    Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer’s disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands. PMID:28084411

  18. IL-1 receptor antagonist-mediated therapeutic effect in murine myasthenia gravis is associated with suppressed serum proinflammatory cytokines, C3, and anti-acetylcholine receptor IgG1.

    PubMed

    Yang, Huan; Tüzün, Erdem; Alagappan, Dhivyaa; Yu, Xiang; Scott, Benjamin G; Ischenko, Alexander; Christadoss, Premkumar

    2005-08-01

    In myasthenia gravis (MG), TNF and IL-1beta polymorphisms and high serum levels of these proinflammatory cytokines have been observed. Likewise, TNF and IL-1beta are critical for the activation of acetylcholine receptor (AChR)-specific T and B cells and for the development of experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. We tested the therapeutic effect of human recombinant IL-1 receptor antagonist (IL-1ra) in C57BL/6 mice with EAMG. Multiple daily injections of 0.01 mg of IL-1ra administered for 2 wk following two AChR immunizations decreased the incidence and severity of clinical EAMG. Furthermore, IL-1ra treatment of mice with ongoing clinical EAMG reduced the clinical symptoms of disease. The IL-1ra-mediated suppression of clinical disease was associated with suppressed serum IFN-gamma, TNF-alpha, IL-1beta, IL-2, IL-6, C3, and anti-AChR IgG1 without influencing total serum IgG. Therefore, IL-1ra could be used as a nonsteroidal drug for the treatment of MG.

  19. Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors.

    PubMed

    El-Tayeb, Ali; Griessmeier, Kerstin J; Müller, Christa E

    2005-12-15

    The selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74Ci/mmol. In preliminary saturation binding studies, [(3)H]PSB-0413 showed high affinity for platelet P2Y(12) receptors with a K(D) value of 4.57nM. Human platelets had a high density of P2Y(12) receptors exhibiting a B(max) value of 7.66pmol/mg of protein.

  20. Selectively Targeting T- and B-Cell Lymphomas: A Benzothiazole Antagonist of α4β1 Integrin

    PubMed Central

    Carpenter, Richard D.; Andrei, Mirela; Aina, Olulanu H.; Lau, Edmond Y.; Lightstone, Felice C.; Liu, Ruiwu; Lam, Kit S.; Kurth, Mark J.

    2011-01-01

    Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately cytotoxic, however achieving selective targeting of cancer malignancies would allow for improved diagnostic and chemotherapeutic tools. Integrin α4β1, a heterodimeric cell surface receptor, is believed to have a relaxed conformation in normal cells and an active conformation in cancerous cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been selectively targeted with the bisaryl urea peptidomimetic antagonist 1. However, concerns regarding its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzothiazole moiety, resulting in an analog with improved physicochemical properties, solubility and kidney:tumor ratio while maintaining potency (6; IC50 = 53 pM). The results presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical imaging using the cyanine dye Cy5.5 conjugate. PMID:19072684

  1. Antagonistic Rgg regulators mediate quorum sensing via competitive DNA binding in Streptococcus pyogenes.

    PubMed

    Lasarre, Breah; Aggarwal, Chaitanya; Federle, Michael J

    2013-01-02

    Recent studies have established the fact that multiple members of the Rgg family of transcriptional regulators serve as key components of quorum sensing (QS) pathways that utilize peptides as intercellular signaling molecules. We previously described a novel QS system in Streptococcus pyogenes which utilizes two Rgg-family regulators (Rgg2 and Rgg3) that respond to neighboring signaling peptides (SHP2 and SHP3) to control gene expression and biofilm formation. We have shown that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes, and that SHPs function to activate the QS system. The mechanisms by which Rgg proteins regulate both QS-dependent and QS-independent processes remain poorly defined; thus, we sought to further elucidate how Rgg2 and Rgg3 mediate gene regulation. Here we provide evidence that S. pyogenes employs a unique mechanism of direct competition between the antagonistic, peptide-responsive proteins Rgg2 and Rgg3 for binding at target promoters. The highly conserved, shared binding sites for Rgg2 and Rgg3 are located proximal to the -35 nucleotide in the target promoters, and the direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP. These results suggest that exclusionary binding of target promoters by Rgg3 may prevent Rgg2 binding under SHP-limiting conditions, thereby preventing premature induction of the quorum sensing circuit. Rgg-family transcriptional regulators are widespread among low-G+C Gram-positive bacteria and in many cases contribute to bacterial physiology and virulence. Only recently was it discovered that several Rgg proteins function in cell-to-cell communication (quorum sensing [QS]) via direct interaction with signaling peptides. The mechanism(s) by which Rgg proteins mediate regulation is poorly understood, and further insight into Rgg

  2. A multiple mediator analysis approach to quantify the effects of the ADH1B and ALDH2 genes on hepatocellular carcinoma risk.

    PubMed

    Shih, Stephannie; Huang, Yen-Tsung; Yang, Hwai-I

    2018-06-01

    Previous work suggested a genetic component affecting the risk of hepatocellular carcinoma (HCC) and mediation analyses have elucidated potential indirect pathways of these genetic effects. Specifically, the effects of alcohol dehydrogenase (ADH1B) and aldehyde dehydrogenase (ALDH2) genes on HCC risk vary based on alcohol consumption habits. However, alcohol consumption may not be the only mediator in the identified pathway: factors related to alcohol consumption may contribute to the same indirect pathway. Thus, we developed a multimediator model to quantify the genetic effects on HCC risk through sequential dichotomous mediators under the counterfactual framework. Our method provided a closed form formula for the mediation effects through different indirect paths, which requires no assumption for the rarity of outcome. In simulation studies of a finite sample, we presented the utility of the method with the variance of the effects estimated using the delta method and bootstrapping. We applied our method to data from participants in Taiwan (580 cases and 3,207 controls) and quantified the mediation effects of single nucleotide polymorphisms (SNPs) in the ADH1B and ALDH2 genes on HCC through alcohol consumption (yes/no) and high alanine transaminase (ALT) levels (greater than or equal to 45 U/L or below 45 U/L). Assuming a dominant risk model, we identified that the SNPs' effects through alcohol consumption is more significant than through ALT levels on HCC risk. This new method provides insight to the magnitude of various casual mechanisms as a closed form solution and can be readily applied in other genomic studies. © 2018 WILEY PERIODICALS, INC.

  3. Serotonin-induced hypophagia is mediated via α2 and β2 adrenergic receptors in neonatal layer-type chickens.

    PubMed

    Zendehdel, M; Sardari, F; Hassanpour, S; Rahnema, M; Adeli, A; Ghashghayi, E

    2017-06-01

    1. Serotoninergic and adrenergic systems play crucial roles in feed intake regulation in avians but there is no report on possible interactions among them. So, in this study, 5 experiments were designed to evaluate the interaction of central serotonergic and adrenergic systems on food intake regulation in 3 h food deprived (FD 3 ) neonatal layer-type chickens. 2. In Experiment 1, chickens received intracerebroventricular (ICV) injection of control solution, serotonin (56.74 nmol), prazosin (α 1 receptor antagonist, 10 nmol) and co-injection of serotonin plus prazosin. In Experiment 2, control solution, serotonin (56.74 nmol), yohimbine (α 2 receptor antagonist, 13 nmol) and co-injection of serotonin plus yohimbine were used. In Experiment 3, the birds received control solution, serotonin (56.74 nmol), metoprolol (β 1 receptor antagonist, 24 nmol) and co-injection of serotonin plus metoprolol. In Experiment 4, injections were control solution, serotonin (56.74 nmol), ICI 118.551 (β 2 receptor antagonist, 5 nmol) and serotonin plus ICI 118.551. In Experiment 5, control solution, serotonin (56.74 nmol), SR59230R (β 3 receptor antagonist, 20 nmol) and co-administration of serotonin and SR59230R were injected. In all experiments the cumulative food intake was measured until 120 min post injection. 3. The results showed that ICV injection of serotonin alone decreased food intake in chickens. A combined injection of serotonin plus ICI 118.551 significantly attenuated serotonin-induced hypophagia. Also, co-administration of serotonin and yohimbine significantly amplified the hypophagic effect of serotonin. However, prazosin, metoprolol and SR59230R had no effect on serotonin-induced hypophagia in chickens. 4. These results suggest that serotonin-induced feeding behaviour is probably mediated via α 2 and β 2 adrenergic receptors in neonatal layer-type chicken.

  4. GABA/sub B/ receptor activation inhibits Ca/sup 2 +/-activated potassium channels in synaptosomes: involvement of G-proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ticku, M.K.; Delgado, A.

    1989-01-01

    /sup 86/Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca/sup 2 +/-activated K/sup +/-channels. Depolarization of /sup 86/Rb-loaded synaptosomes in physiological buffer increased Ca/sup 2 +/-activated /sup 86/Rb-efflux by 400%. The /sup 86/Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La/sup 3 +/ indicating the involvement of Ca/sup 2 +/-activated K/sup +/-channels. (-)Baclofen inhibited Ca/sup 2 +/-activated /sup 86/Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but notmore » by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca/sup 2 +/-activated K/sup +/-channels. These results suggest that baclofen inhibits Ca/sup 2 +/-activated K/sup +/-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology.« less

  5. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

    PubMed Central

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

    2015-01-01

    ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1

  6. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions.

    PubMed

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul; Melikyan, Gregory B

    2015-09-01

    HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF

  7. A novel 5HT3 antagonist 4i (N-(3-chloro-2-methylphenyl)quinoxalin-2-carboxamide) prevents diabetes-induced depressive phenotypes in mice: Modulation of serotonergic system.

    PubMed

    Gupta, Deepali; Thangaraj, Devadoss; Radhakrishnan, Mahesh

    2016-01-15

    Despite the presence of a multitudinous pharmacotherapy, diabetes-induced depressive disorder remains undertreated. Evidence suggests that brain serotonergic deficits are associated with depressive-like behavior in diabetes and that 5HT3 receptor (5HT3R) antagonists have serotonergic facilitatory effects. This study examined the effects of a novel 5HT3R antagonist, 4i (N-(3-chloro-2-methylphenyl)quinoxalin-2-carboxamide), in diabetes-induced depressive phenotypes. Experimentally, (1) to evaluate the effects of 4i, mice with 8-weeks of diabetes (induced by streptozotocin, 200mg/kg, i.p.) were treated with vehicle, 4i (0.5 and 1mg/kg/day, i.p.), fluoxetine (10mg/kg/day, i.p.) for 4-weeks and subjected to neurobehavioral assays, followed by biochemical estimation of serotonin levels in midbrain, prefrontal-cortex and cerebellum. (2) To evaluate the role of 5HT3R in the postulated effect of 4i, diabetic mice were given 4i (1mg/kg/day, i.p.) after 1h of 1-(m-chlorophenyl)-biguanide (mCPBG, a 5HT3R agonist, 10mg/kg/day, i.p.) treatment and subjected to the same protocol. The results showed that diabetic mice exhibited a significant behavioral deficit, including depression-like behavior in forced swim test, anxiety-like in open field test and sociability deficits in social interaction test, along with a significant decrease in serotonin level in these brain regions. 4i (1mg/kg), similar to fluoxetine, prevented these behavioral abnormalities and normalized brain serotonin levels. 4i (0.5mg/kg) ameliorated only diabetes-induced depressive-like behavior and serotonin deficits, but not anxiety-like effects. mCPBG blunted 4i-mediated behavioral response and increase in brain serotonin levels. Altogether, this study suggests that 4i prevents diabetes-induced depressive phenotypes in mice, which may involve antagonism of 5HT3Rs and increase in serotonin levels in discrete brain regions. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Chemoinformatics Profiling of the Chromone Nucleus as a MAO-B/A2AAR Dual Binding Scaffold

    PubMed Central

    Cruz-Monteagudo, Maykel; Borges, Fernanda; Cordeiro, M. Natália D. S.; Helguera, Aliuska Morales; Tejera, Eduardo; Paz-y-Miño, Cesar; Sánchez-Rodríguez, Aminael; Perera-Sardiña, Yunier; Perez-Castillo, Yunierkis

    2017-01-01

    Background: In the context of the current drug discovery efforts to find disease modifying therapies for Parkinson´s disease (PD) the current single target strategy has proved inefficient. Consequently, the search for multi-potent agents is attracting more and more attention due to the multiple pathogenetic factors implicated in PD. Multiple evidences points to the dual inhibition of the monoamine oxidase B (MAO-B), as well as adenosine A2A receptor (A2AAR) blockade, as a promising approach to prevent the neurodegeneration involved in PD. Currently, only two chemical scaffolds has been proposed as potential dual MAO-B inhibitors/A2AAR antagonists (caffeine derivatives and benzothiazinones). Methods: In this study, we conduct a series of chemoinformatics analysis in order to evaluate and advance the potential of the chromone nucleus as a MAO-B/A2AAR dual binding scaffold. Results: The information provided by SAR data mining analysis based on network similarity graphs and molecular docking studies support the suitability of the chromone nucleus as a potential MAO-B/A2AAR dual binding scaffold. Additionally, a virtual screening tool based on a group fusion similarity search approach was developed for the prioritization of potential MAO-B/A2AAR dual binder candidates. Among several data fusion schemes evaluated, the MEAN-SIM and MIN-RANK GFSS approaches demonstrated to be efficient virtual screening tools. Then, a combinatorial library potentially enriched with MAO-B/A2AAR dual binding chromone derivatives was assembled and sorted by using the MIN-RANK and then the MEAN-SIM GFSS VS approaches. Conclusion: The information and tools provided in this work represent valuable decision making elements in the search of novel chromone derivatives with a favorable dual binding profile as MAO-B inhibitors and A2AAR antagonists with the potential to act as a disease-modifying therapeutic for Parkinson´s disease. PMID:28093976

  9. Microbial Herd Protection Mediated by Antagonistic Interaction in Polymicrobial Communities

    PubMed Central

    Wong, Megan J. Q.; Liang, Xiaoye; Smart, Matt; Tang, Le; Moore, Richard; Ingalls, Brian

    2016-01-01

    ABSTRACT In host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist, despite significant antagonistic interactions between species, are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through the delivery of toxic effectors. It is well established that intraspecies protection is conferred by immunity proteins that neutralize effector toxicities. In contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS-active antagonistic bacterial species, Aeromonas hydrophila and Vibrio cholerae, to demonstrate that interspecies protection is dependent on effectors. A. hydrophila and V. cholerae do not share conserved immunity genes but could coexist equally in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the competing wild-type strain. Time-lapse microscopic analyses showed that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interactions inside each cluster and restricting the interactions to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulated our experimental observations. These results provide mechanistic insights regarding the general role of microbial weapons in determining the structures of complex multispecies communities. IMPORTANCE Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in

  10. Microbial herd protection mediated by antagonistic interaction in polymicrobial communities.

    PubMed

    Wong, Megan; Liang, Xiaoye; Smart, Matt; Tang, Le; Moore, Richard; Ingalls, Brian; Dong, Tao G

    2016-09-16

    In the host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist - despite significant antagonistic interactions between species - are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through delivery of toxic effectors. It is well established that intra-species protection is conferred by immunity proteins that neutralize effector toxicities. By contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS active antagonistic bacteria, Aeromonas hydrophila (AH) and Vibrio cholerae (VC), to demonstrate that interspecies protection is dependent on effectors. AH and VC do not share conserved immunity genes but could equally co-exist in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the other competing wild type. Time-lapse microscopy analyses show that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interaction inside each cluster and restricting it to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulate our experimental observation. These results provide mechanistic insights for the general role of microbial weapons in determining the structures of complex multispecies communities. Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in determining community structures and exchange of genetic materials

  11. Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

    PubMed

    Yoon, SeongHun; Gariepy, Cheryl E; Yanagisawa, Masashi; Zuccarello, Mario; Rapoport, Robert M

    2016-03-01

    The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

  12. The antiplatelet activity of magnolol is mediated by PPAR-β/γ.

    PubMed

    Shih, Ching-Yu; Chou, Tz-Chong

    2012-09-15

    Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Neuroprotective Effects of Reactive Oxygen Species Mediated by BDNF-Independent Activation of TrkB

    PubMed Central

    Huang, Yang Zhong; McNamara, James O.

    2012-01-01

    Reactive oxygen species (ROS) have diverse biological consequences in the mammalian CNS, but the molecular targets mediating these pleiotropic effects are incompletely understood. Like ROS, the neurotrophin receptor, TrkB receptor tyrosine kinase, has diverse effects in the developing and mature mammalian brain. Our discovery that zinc can transactivate TrkB, together with the finding that ROS can trigger zinc release from cytosolic zinc binding proteins, led us to hypothesize that ROS can transactivate TrkB in CNS neurons by a zinc-dependent mechanism. We found that both exogenous H2O2 and endogenous ROS activate TrkB signaling by a Src family kinase-dependent but brain-derived neurotrophic factor-independent mechanism in cultured rat cortical neurons. Exogenous H2O2 enhances cytosolic zinc content in a metallothionein-3 (MT-3)-requiring manner. Both exogenous H2O2 and endogenous ROS mediated transactivation of TrkB requires intracellular zinc and MT-3. The ROS-triggered transactivation of TrkB exerts neuroprotective effects, because inhibition of TrkB kinase activity or uncoupling Shc signaling from TrkB exacerbates neuronal cell death induced by H2O2. Thus, we propose a molecular signaling event whereby ROS induce release of zinc from cytosolic MT-3, the increased cytosolic zinc transactivates TrkB, and the enhanced Shc signaling downstream from TrkB promotes prosurvival effects. We suggest that such neuroprotective effects mediated by ROS are operative in diverse acute and chronic neurological disorders. PMID:23115189

  14. Antagonizing SET augments the effects of radiotherapy in hepatocellular carcinoma through reactivating PP2A-mediated AKT downregulation.

    PubMed

    Huang, Chao-Yuan; Hung, Man-Hsin; Shih, Chi-Ting; Hsieh, Feng-Shu; Kuo, Chiung-Wen; Tsai, Ming-Hsien; Chang, Shih-Shin; Hsiao, Yung-Jen; Chen, Li-Ju; Chao, Tzu-I; Chen, Kuen-Feng

    2018-06-18

    Increasing evidence suggests that SET functions as an oncoprotein and promotes cancer survival and therapeutic resistance. However, whether SET affects radiotherapy (RT)-mediated anti-cancer effects has not yet been explored. Here, we investigated the impact of SET on RT sensitivity in hepatocellular carcinoma (HCC). Using colony and hepatosphere formation assay, we found that RT-induced proliferative inhibition was critically associated with SET expression. Next, we tested a novel SET antagonist, EMQA, in combination with RT. We showed that additive use of EMQA significantly enhanced the effects of RT against HCC in vitro and in vivo. Notably, compared to mice receiving either RT or EMQA alone, the growth of PLC5 xenografted tumor in mice receiving RT plus EMQA was significantly reduced without compromising treatment tolerability. Furthermore, we proved that antagonizing SET to restore PP2A-mediated p-AKT downregulation was responsible for the synergism between EMQA and RT. Our data demonstrate a new oncogenic property of SET, and provide preclinical evidence that combining a SET antagonist and RT may be effective for treatment of HCC. Further investigation is warranted to validate the clinical relevance of this approach. The American Society for Pharmacology and Experimental Therapeutics.

  15. Discovery and Cocrystal Structure of Benzodiazepinedione HDM2 Antagonists that Activate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grasberger,B.; Lu, T.; Schubert, C.

    2005-01-01

    HDM2 binds to an {alpha}-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their {alpha}-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

  16. Vernolide-A, a sesquiterpene lactone from Vernonia cinerea, induces apoptosis in B16F-10 melanoma cells by modulating p53 and caspase-3 gene expressions and regulating NF-κB-mediated bcl-2 activation.

    PubMed

    Pratheeshkumar, Poyil; Kuttan, Girija

    2011-07-01

    In this study, we investigated the effect of vernolide-A on the induction of apoptosis as well as its regulatory effect on the activation of transcription factors in B16F-10 melanoma cells. Treatment of B16F-10 cells with nontoxic concentrations of vernolide-A showed the presence of apoptotic bodies and induced DNA fragmentation in a dose-dependent manner. Cell-cycle analysis and TUNEL assays also confirmed the observation. The proapoptotic genes, p53, Bax, caspase-9, and caspase-3, were upregulated in vernolide-A-treated cells, whereas the antiapoptotic gene, Bcl-2, was downregulated. vernolide-A treatment also showed a downregulation of cyclin D1 expression and upregulated p21 and p27 gene expression in B16F-10 melanoma cells. The study also reveals that vernolide-A treatment could alter the production and expression of proinflammatory cytokines and could inhibit the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor-κB and other transcription factors, such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response-element-binding protein in B16F-10 melanoma cells. These results suggest that vernolide-A induces apoptosis via activation of p53-induced, caspase-3-mediated proapoptotic signaling and suppression of NF-κB-induced, bcl-2-mediated survival signaling.

  17. BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth.

    PubMed

    Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H; Galipeau, Jacques; Deng, Xingming

    2016-05-10

    Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy.

  18. Lead Optimization Studies of Cinnamic Amide EP2 Antagonists

    PubMed Central

    2015-01-01

    Prostanoid receptor EP2 can play a proinflammatory role, exacerbating disease pathology in a variety of central nervous system and peripheral diseases. A highly selective EP2 antagonist could be useful as a drug to mitigate the inflammatory consequences of EP2 activation. We recently identified a cinnamic amide class of EP2 antagonists. The lead compound in this class (5d) displays anti-inflammatory and neuroprotective actions. However, this compound exhibited moderate selectivity to EP2 over the DP1 prostanoid receptor (∼10-fold) and low aqueous solubility. We now report compounds that display up to 180-fold selectivity against DP1 and up to 9-fold higher aqueous solubility than our previous lead. The newly developed compounds also display higher selectivity against EP4 and IP receptors and a comparable plasma pharmacokinetics. Thus, these compounds are useful for proof of concept studies in a variety of models where EP2 activation is playing a deleterious role. PMID:24773616

  19. Synthesis and properties of a new water-soluble prodrug of the adenosine A 2A receptor antagonist MSX-2.

    PubMed

    Vollmann, Karl; Qurishi, Ramatullah; Hockemeyer, Jörg; Müller, Christa E

    2008-02-12

    The compound L-valine-3-{8-[(E)-2-[3-methoxyphenyl)ethenyl]-7-methyl-1-propargylxanthine-3-yl}propyl ester hydrochloride (MSX-4) was synthesized as an amino acid ester prodrug of the adenosine A2A receptor antagonist MSX-2. It was found to be stable in artificial gastric acid, but readily cleaved by pig liver esterase.

  20. Neurokinin-1 receptor antagonists CP-96,345 and L-733,060 protect mice from cytokine-mediated liver injury.

    PubMed

    Bang, Renate; Sass, Gabriele; Kiemer, Alexandra K; Vollmar, Angelika M; Neuhuber, Winfried L; Tiegs, Gisa

    2003-04-01

    Previously, we have shown that primary afferent sensory neurons are necessary for disease activity in T cell-mediated immune hepatitis in mice. In the present study, we analyzed the possible role of substance P (SP), an important proinflammatory neuropeptide of these nerve fibers, in an in vivo mouse model of liver inflammation. Liver injury was induced by bacterial lipopolysaccharide (LPS) in D-galactosamine (GalN)-sensitized mice. Depletion of primary afferent nerve fibers by neonatal capsaicin treatment down-regulated circulating levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) and protected mice from GalN/LPS-induced liver injury. Likewise, pretreatment of mice with antagonists of the SP-specific neurokinin-1 receptor (NK-1R), i.e., (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine (CP-96,345) and (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine (L-733,060), dose dependently protected mice from GalN/LPS-induced liver injury. The presence of the NK-1R in the murine liver was demonstrated by reverse transcription-polymerase chain reaction, sequence analysis, and immunocytochemistry. NK-1R blockade reduced inflammatory liver damage, i.e., edema formation, neutrophil infiltration, hepatocyte apoptosis, and necrosis. To get further insight into the mechanism by which receptor blockade attenuated GalN/LPS-induced liver damage, we analyzed plasma levels and intrahepatic expression of TNFalpha, IFNgamma, interleukin (IL)-6, and IL-10. NK-1R blockade clearly inhibited GalN/LPS-induced production of TNFalpha and IFNgamma, whereas synthesis of the hepatoprotective cytokines IL-6 and IL-10 was increased. NK-1 receptor antagonists might be potent drugs for treatment of inflammatory liver disease, most likely by inhibiting SP effects.

  1. Environmental and cortisol-mediated control of Ca(2+) uptake in tilapia (Oreochromis mossambicus).

    PubMed

    Lin, Chia-Hao; Kuan, Wei-Chun; Liao, Bo-Kai; Deng, Ang-Ni; Tseng, Deng-Yu; Hwang, Pung-Pung

    2016-04-01

    Ca(2+) is a vital element for many physiological processes in vertebrates, including teleosts, which live in aquatic environments and acquire Ca(2+) from their surroundings. Ionocytes within the adult gills or larval skin are critical sites for transcellular Ca(2+) uptake in teleosts. The ionocytes of zebrafish were found to contain transcellular Ca(2+) transporters, epithelial Ca(2+) channel (ECaC), plasma membrane Ca(2+)-ATPase 2 (PMCA2), and Na(+)/Ca(2+) exchanger 1b (NCX1b), providing information about the molecular mechanism of transcellular Ca(2+) transports mediated by ionocytes in fish. However, more evidence is required to establish whether or not a similar mechanism of transcellular Ca(2+) transport also exists in others teleosts. In the present study, ecac, pmca2, and ncx1 were found to be expressed in the branchial ionocytes of tilapia, thereby providing further support for the mechanism of transcellular Ca(2+) transport through ionocytes previously proposed for zebrafish. In addition, we also reveal that low Ca(2+) water treatment of tilapia stimulates Ca(2+) uptake and expression of ecac and cyp11b (the latter encodes a cortisol-synthesis enzyme). Treatment of tilapia with exogenous cortisol (20 mg/l) enhanced both Ca(2+) influx and ecac expression. Therefore, increased cyp11b expression is suggested to enhance Ca(2+) uptake capacity in tilapia exposed to low Ca(2+) water. Furthermore, the application of cortisol receptor antagonists revealed that cortisol may regulate Ca(2+) uptake through glucocorticoid and/or mineralocorticoid receptor (GR and/or MR) in tilapia. Taken together, the data suggest that cortisol may activate GR and/or MR to execute its hypercalcemic action by stimulating ecac expression in tilapia.

  2. PSD-93 deletion inhibits Fyn-mediated phosphorylation of NR2B and protects against focal cerebral ischemia.

    PubMed

    Zhang, Meijuan; Li, Qingjie; Chen, Ling; Li, Jie; Zhang, Xin; Chen, Xiang; Zhang, Qingxiu; Shao, Yuan; Xu, Yun

    2014-08-01

    Modification of N-methyl-d-aspartate receptor (NMDAR)-mediated excitotoxicity appears to be a potential target in the treatment of ischemic stroke. Postsynaptic density protein-93 (PSD-93) specifically binds the C-terminal tails of the NMDAR, which is critical to couple NMDAR activity to specific intracellular signaling. This study is to investigate whether PSD-93 disruption displays neuroprotection in a focal ischemic stroke model of adult mice and, if it does, to explore possible mechanisms. It was found that, following middle cerebral artery occlusion (MCAO), PSD-93 knockout (KO) mice manifested significant reductions in infarcted volume, neurological deficits and number of degenerated neurons. PSD-93 deletion also reduced cultured cortical neuronal death caused by glucose and oxygen deprivation (OGD). Ischemic long term potentiation (i-LTP) could not be induced in the PSD-93 KO group and wild type (WT) groups pretreated with either AP-5 (NMDAR inhibitor) or PP2 (Src family inhibitor). PSD-93 KO decreased the phosphorylation of the NR2B at Tyr1472 and the interaction between NR2B and Fyn after MCAO. Together, our study demonstrated that PSD-93 KO confers profound neuroprotection against ischemic brain injury, which probably links to the inhibitory effect on Fyn-mediated phosphorylation of NR2B caused by PSD-93 deletion. These findings may provide a novel avenue for the treatment of ischemic stroke. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. EphB6 promotes anoikis by modulating EphA2 signaling.

    PubMed

    Akada, Mai; Harada, Kohei; Negishi, Manabu; Katoh, Hironori

    2014-12-01

    Anoikis is a specific type of apoptosis induced by detachment of epithelial cells from extracellular matrix, and acquiring resistance to anoikis is an important step that enables cancer cells to metastasize. EphA2, which is overexpressed in a variety of human cancers, is phosphorylated by Akt on serine 897 and mediates ligand ephrin-independent promotion of anoikis resistance through the RhoG activator Ephexin4. EphB6 is frequently silenced in invasive and metastatic cancers; however, its role in cancer progression is poorly understood. Here we show that EphB6 interacts with EphA2 and suppresses EphA2-mediated promotion of anoikis resistance in MCF7 breast cancer cells. On the other hand, knockdown of EphB6 promotes anoikis resistance. We further show that expression of EphB6 decreases serine 897 phosphorylation of EphA2 and suppresses EphA2-Ephexin4 interaction and the RhoG activation. These findings implicate EphB6 as a negative regulator of EphA2 oncogenic signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3′ Immunoglobulin Heavy Chain Regulatory Region

    PubMed Central

    Salisbury, Richard L.; Sulentic, Courtney E. W.

    2015-01-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3′Igh regulatory region (3′IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3′IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3′IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3′IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3′IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3′IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3′IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels. PMID:26377645

  5. Protective effect of lafutidine, a histamine H2 receptor antagonist, against loxoprofen-induced small intestinal lesions in rats.

    PubMed

    Amagase, Kikuko; Ochi, Akimu; Sugihara, Tetsuya; Kato, Shinichi; Takeuchi, Koji

    2010-05-01

    We examined the effect of lafutidine, a histamine H(2) receptor antagonist with a mucosal protective action mediated by capsaicin-sensitive sensory neurons (CSN), on intestinal lesions produced by loxoprofen administration in rats. Animals were given loxoprofen (10-100 mg/kg p.o.) and killed 24 h later. Lafutidine (10 and 30 mg/kg), cimetidine (100 mg/kg) or famotidine (30 mg/kg) was given twice p.o. at 0.5 h before and 6 h after loxoprofen. Omeprazole (100 mg/kg) was given p.o. once 0.5 h before. Ampicillin (800 mg/kg) was given p.o. twice at 24 h and 0.5 h before loxoprofen, while 16,16-dimethyl prostaglandin E(2) (dmPGE(2); 0.01 mg/kg) was given i.v. twice at 5 min before and 6 h after. Loxoprofen dose-dependently produced hemorrhagic lesions in the small intestine, accompanied by invasion of enterobacteria and increased inducible nitric oxide synthase (iNOS) expression as well as myeloperoxidase activity in the mucosa. The ulcerogenic response to loxoprofen (60 mg/kg) was significantly prevented by lafutidine (30 mg/kg), similar to dmPGE(2) and ampicillin, and the effect of lafutidine was totally attenuated by ablation of CSN. Neither cimetidine, famotidine nor omeprazole had a significant effect against these lesions. Lafutidine alone increased mucus secretion and reverted the decreased mucus response to loxoprofen, resulting in suppression of bacterial invasion and iNOS expression. In addition, loxoprofen downregulated Muc2 expression, and this response was totally reversed by lafutidine mediated by CSN. Lafutidine protects the small intestine against loxoprofen-induced lesions, essentially mediated by the CSN, and this effect may be functionally associated with increased Muc2 expression/mucus secretion, an important factor in the suppression of bacterial invasion.

  6. Antagonist-perturbation mechanism for activation function-2 fixed motifs: active conformation and docking mode of retinoid X receptor antagonists

    NASA Astrophysics Data System (ADS)

    Tsuji, Motonori

    2017-06-01

    HX531, which contains a dibenzodiazepine skeleton, is one of the first retinoid X receptor (RXR) antagonists. Functioning via RXR-PPARγ heterodimer, this compound is receiving a lot of attention as a therapeutic drug candidate for diabetic disease controlling differentiation of adipose tissue. However, the active conformation of HX531 for RXRs is not well established. In the present study, quantum mechanics calculations and molecular mechanical docking simulations were carried out to precisely study the docking mode of HX531 with the human RXRα ligand-binding domain, as well as to provide a new approach to drug design using a structure-based perspective. It was suggested that HX531, which has the R configuration for the bent dibenzodiazepine plane together with the equatorial configuration for the N-methyl group attached to the nitrogen atom in the seven-membered diazepine ring, is a typical activation function-2 (AF-2) fixed motif perturbation type antagonist, which destabilizes the formation of AF-2 fixed motifs. On the other hand, the docking simulations supported the experimental result that LG100754 is an RXR homodimer antagonist and an RXR heterodimer agonist.

  7. Diastereoselective intramolecular Ritter reaction: generation of a cis-fused hexahydro-4aH-indeno[1,2-b]pyridine ring system with 4a,9b-diangular substituents.

    PubMed

    Van Emelen, K; De Wit, T; Hoornaert, G J; Compernolle, F

    2000-10-05

    Indanol intermediates 5, prepared via Michael addition of 1-indanone beta-ketoester and acrylonitrile followed by reduction or Grignard reaction of the ketone group, were submitted to intramolecular Ritter reaction using various acid reaction conditions to produce tricyclic lactams 4. This cis-fused hexahydro-4aH-indeno[1,2-b]pyridine ring system, substituted at both angular positions 4a and 9b, provides access to constrained analogues of non-peptide NK(1)-antagonists with monocyclic piperidine structure.

  8. Gamma-hydroxybutyric acid (GHB) and the mesoaccumbens reward circuit: evidence for GABA(B) receptor-mediated effects.

    PubMed

    Pistis, M; Muntoni, A L; Pillolla, G; Perra, S; Cignarella, G; Melis, M; Gessa, G L

    2005-01-01

    Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid naturally occurring in the mammalian brain, which recently emerged as a major recreational drug of abuse. GHB has multiple neuronal mechanisms including activation of both the GABA(B) receptor, and a distinct GHB-specific receptor. This complex GHB-GABA(B) receptor interaction is probably responsible for the multifaceted pharmacological, behavioral and toxicological profile of GHB. Drugs of abuse exert remarkably similar effects upon reward-related circuits, in particular the mesolimbic dopaminergic system and the nucleus accumbens (NAc). We used single unit recordings in vivo from urethane-anesthetized rats to characterize the effects of GHB on evoked firing in NAc "shell" neurons and on spontaneous activity of antidromically identified dopamine (DA) cells located in the ventral tegmental area. GHB was studied in comparison with the GABA(B) receptor agonist baclofen and antagonist (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH50911). Additionally, we utilized a GHB analog, gamma-(p-methoxybenzil)-gamma-hydroxybutyric acid (NCS-435), devoid of GABA(B) binding properties, but with high affinity for specific GHB binding sites. In common with other drugs of abuse, GHB depressed firing in NAc neurons evoked by the stimulation of the basolateral amygdala. On DA neurons, GHB exerted heterogeneous effects, which were correlated to the baseline firing rate of the cells but led to a moderate stimulation of the DA system. All GHB actions were mediated by GABA(B) receptors, since they were blocked by SCH50911 and were not mimicked by NCS-435. Our study indicates that the electrophysiological profile of GHB is close to typical drugs of abuse: both inhibition of NAc neurons and moderate to strong stimulation of DA transmission are distinctive features of diverse classes of abused drugs. Moreover, it is concluded that addictive and rewarding properties of GHB do not necessarily involve a putative high affinity GHB

  9. Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor

    PubMed Central

    Brim, B. L.; Haskell, R.; Awedikian, R.; Ellinwood, N.M.; Jin, L.; Kumar, A.; Foster, T.C.; Magnusson, K.

    2012-01-01

    The GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor shows age-related declines in expression across the frontal cortex and hippocampus. This decline is strongly correlated to age-related memory declines. This study was designed to determine if increasing GluN2B subunit expression in the frontal lobe or hippocampus would improve memory in aged mice. Mice were injected bilaterally with either the GluN2B vector, containing cDNA specific for the GluN2B subunit and enhanced Green Fluorescent Protein (eGFP); a control vector or vehicle. Spatial memory, cognitive flexibility, and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression, exhibited improved long-term spatial memory, comparable to young mice. However, memory was rescued on different days in the Morris water maze; early for hippocampal GluN2B subunit enrichment and later for the frontal lobe. A higher concentration of the GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B expression, as compared to aged controls, suggesting there was an increase in the number of GluN2B-containing NMDA receptors. In addition, hippocampal slices from aged mice with increased GluN2B subunit expression exhibited enhanced NMDA receptor-mediated excitatory post-synaptic potentials (EPSP). Treatment with Ro 25-6981 showed that a greater proportion of the NMDA receptor-mediated EPSP was due to the GluN2B subunit in these animals, as compared to aged controls. These results suggest that increasing the production of the GluN2B subunit in aged animals enhances memory and synaptic transmission. Therapies that enhance GluN2B subunit expression within the aged brain may be useful for ameliorating age-related memory declines. PMID:23103326

  10. A novel identification approach for discovery of 5-HydroxyTriptamine 2A antagonists: combination of 2D/3D similarity screening, molecular docking and molecular dynamics.

    PubMed

    Kumar, Rakesh; Jade, Dhananjay; Gupta, Dinesh

    2018-03-05

    5-HydroxyTriptamine 2A antagonists are potential targets for treatment of various cerebrovascular and cardiovascular disorders. In this study, we have developed and performed a unique screening pipeline for filtering ZINC database compounds on the basis of similarities to known antagonists to determine novel small molecule antagonists of 5-HydroxyTriptamine 2A. The screening pipeline is based on 2D similarity, 3D dissimilarity and a combination of 2D/3D similarity. The shortlisted compounds were docked to a 5-HydroxyTriptamine 2A homology-based model, and complexes with low binding energies (287 complexes) were selected for molecular dynamics (MD) simulations in a lipid bilayer. The MD simulations of the shortlisted compounds in complex with 5-HydroxyTriptamine 2A confirmed the stability of the complexes and revealed novel interaction insights. The receptor residues S239, N343, S242, S159, Y370 and D155 predominantly participate in hydrogen bonding. π-π stacking is observed in F339, F340, F234, W151 and W336, whereas hydrophobic interactions are observed amongst V156, F339, F234, V362, V366, F340, V235, I152 and W151. The known and potential antagonists shortlisted by us have similar overlapping molecular interaction patterns. The 287 potential 5-HydroxyTriptamine 2A antagonists may be experimentally verified.

  11. Rapid and efficient hydrophilicity tuning of p53/mdm2 antagonists*

    PubMed Central

    Srivastava, Stuti; Beck, Barbara; Wang, Wei; Czarna, Anna; Holak, Tad A.; Dömling, Alexander

    2009-01-01

    The protein-protein interaction of p53 and mdm2 is an important anticancer target. The interface, however, is very hydrophobic and naturally results in very hydrophobic antagonists. We used the Orru three component reaction (O-3CR) along with a rapid and efficient, recently discovered amidation reaction to dramatically improve the water solubility of our recently discovered low molecular weight p53/mdm2 antagonists. Arrays of amides were synthesized with improved hydrophilicity and retainment and/or improvement of p53/mdm2 inhibitory activity. PMID:19548636

  12. Development of uncoupling between D1- and D2-mediated motor behavior in rats depleted of dopamine as neonates.

    PubMed

    Byrnes, E M; Bruno, J P

    1994-09-01

    The D1- and D2-mediation of stimulated motor behavior was studied in pups (Days 10-11) and weanlings (Days 20-21) that had been depleted of dopamine (DA) on postnatal Day 3. Administration of the D1-like agonist SKF 38393 (30.0 mg/kg) or the D2-like agonist quinpirole (3.0 mg/kg) increased the incidence of sniffing and locomotion in intact and DA-depleted animals tested at either age. However, the ability of selective DA antagonists to reduce these stimulated responses interacted with both the depletion and the age at the time of testing. When tested as pups, both the D1 antagonist SCH 23390 (0.2 or 0.4 mg/kg) and the D2 antagonist clebopride (10.0 mg/kg) suppressed the behaviors induced by either class of DA agonist. When tested as weanlings, intact animals exhibited the profile of pups (i.e., either antagonist blocked each agonist). In DA-depleted weanlings, however, only the D1 antagonist blocked the D1 agonist-induced responses and only the D2 antagonist blocked the D2 agonist-induced responses. These data demonstrate that the interactions between D1 and D2 receptors in the expression of stimulated motor behaviors are altered following DA depletions in neonates. Moreover, this change in receptor function occurs sometime between 7 and 13 days after the DA depletion.

  13. Engineered Interleukin-2 Antagonists for the Inhibition of Regulatory T cells

    PubMed Central

    Liu, David V.; Maier, Lisa M.; Hafler, David A.; Wittrup, K. Dane

    2014-01-01

    The immunosuppressive effects of CD4+ CD25high regulatory T cells interfere with anti-tumor immune responses in cancer patients. Here, we present a novel class of engineered human Interleukin (IL)-2 analogues that antagonize the IL-2 receptor, for inhibiting regulatory T cell suppression. These antagonists have been engineered for high affinity to the α subunit of the IL-2 receptor and very low affinity to either the β or γ subunit, resulting in a signaling-deficient IL-2 analogue that sequesters the IL-2 receptor α subunit from wild type IL-2. Two variants, “V91R” and “Q126T” with residue substitutions that disrupt the β and γ subunit binding interfaces, respectively, have been characterized in both a T cell line and in human primary regulatory T cells. These mutants retain their high affinity binding to IL-2 receptor α subunit, but do not activate STAT5 phosphorylation or stimulate T cell growth. The two mutants competitively antagonize wild-type IL-2 signaling through the IL-2 receptor with similar efficacy, with inhibition constants of 183 pM for V91R and 216 pM for Q126T. Here, we present a novel approach to CD25-mediated Treg inhibition, with the use of an engineered human IL-2 analogue that antagonizes the IL-2 receptor. PMID:19816193

  14. BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors.

    PubMed

    Korfi, K; Smith, M; Swan, J; Somervaille, T C P; Dhomen, N; Marais, R

    2016-04-07

    B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential.

  15. Th2 cytokine antagonists: potential treatments for severe asthma.

    PubMed

    Hansbro, Philip M; Scott, Grace V; Essilfie, Ama-Tawiah; Kim, Richard Y; Starkey, Malcolm R; Nguyen, Duc H; Allen, Paul D; Kaiko, Gerard E; Yang, Ming; Horvat, Jay C; Foster, Paul S

    2013-01-01

    Asthma is a major disease burden worldwide. Treatment with steroids and long acting β-agonists effectively manage symptoms in many patients but do not treat the underlying cause of disease and have serious side effects when used long term and in children. Therapies targeting the underlying causes of asthma are urgently needed. T helper type 2 (Th2) cells and the cytokines they release are clinically linked to the presentation of all forms of asthma. They are the primary drivers of mild to moderate and allergic asthma. They also play a pathogenetic role in exacerbations and more severe asthma though other factors are also involved. Much effort using animal models and human studies has been dedicated to the identification of the pathogenetic roles of these cells and cytokines and whether inhibition of their activity has therapeutic benefit in asthma. We discuss the current status of Th2 cytokine antagonists for the treatment of asthma. We also discuss the potential for targeting Th2-inducing cytokines, Th2 cell receptors and signaling as well as the use of Th2 cell antagonists, small interfering oligonucleotides, microRNAs, and combination therapies. Th2 antagonists may be most effective in particular asthma subtypes/endotypes where specific cytokines are known to be active through the analysis of biomarkers. Targeting common receptors and pathways used by these cytokines may have additional benefit. Animal models have been valuable in identifying therapeutic targets in asthma, however the results from such studies need to be carefully interpreted and applied to appropriately stratified patient cohorts in well-designed clinical studies and trials.

  16. β-Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α-PP2A-Mediated Dephosphorylation.

    PubMed

    Weeks, Kate L; Ranieri, Antonella; Karaś, Agnieszka; Bernardo, Bianca C; Ashcroft, Alexandra S; Molenaar, Chris; McMullen, Julie R; Avkiran, Metin

    2017-03-25

    Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. β-Adrenoceptor (β-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β-AR agonist isoproterenol to induce β 1 -AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. β-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A-mediated

  17. BDNF-TrkB signaling through Erk1/2MAPK phosphorylation mediates the enhancement of fear memory induced by glucocorticoids

    PubMed Central

    Revest, J-M; Le Roux, A; Roullot-Lacarrière, V; Kaouane, N; Vallée, M; Kasanetz, F; Rougé-Pont, F; Tronche, F; Desmedt, A; Piazza, P V

    2014-01-01

    Activation of glucocorticoid receptors (GR) by glucocorticoid hormones (GC) enhances contextual fear memories through the activation of the Erk1/2MAPK signaling pathway. However, the molecular mechanism mediating this effect of GC remains unknown. Here we used complementary molecular and behavioral approaches in mice and rats and in genetically modified mice in which the GR was conditionally deleted (GRNesCre). We identified the tPA-BDNF-TrkB signaling pathway as the upstream molecular effectors of GR-mediated phosphorylation of Erk1/2MAPK responsible for the enhancement of contextual fear memory. These findings complete our knowledge of the molecular cascade through which GC enhance contextual fear memory and highlight the role of tPA-BDNF-TrkB-Erk1/2MAPK signaling pathways as one of the core effectors of stress-related effects of GC. PMID:24126929

  18. 2-Methylacrylamide as a bioisoster of thiourea group for 1,3-dibenzylthioureido TRPV1 receptor antagonists.

    PubMed

    Park, Seol Rin; Kim, Juhyun; Lee, Sun Young; Park, Young-Ho; Kim, Hee-Doo

    2018-06-15

    In order to replace thiourea group with the more drug-like moiety for 1,3-dibenzylthioureas having TRPV1 antagonist activity, we introduced a set of functional groups between the two aromatic rings based on bioisosteric replacement. The synthesized bioisosteres of 1,3-dibenzylthioureas were tested for their antagonist activities on TRPV1 by 45 Ca 2+ -influx assay using neonatal rat cultured spinal sensory neurons. Among the tested 14 kinds of bioisosters, 2-methylacrylamide group was the best candidate to replace thiourea group. Compound 7c, 2-methylacrylamide analog of ATC-120, showed as potent as ATC-120 in its antagonist activity. In addition, 2-methylacrylamide analog 7e having vinyl moiety showed the most potent activity with 0.022 μM of IC 50 value, indicating that thiourea group of 1,3-dibenzylthioureas could be replaced to 2-methylacrylamide without loss of their potencies. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. A Novel Mechanism of γ-Irradiation-Induced IL-6 Production Mediated by P2Y11 Receptor in Epidermal Keratinocytes.

    PubMed

    Ohsaki, Airi; Miyano, Yuki; Tanaka, Rei; Tanuma, Sei-Ichi; Kojima, Shuji; Tsukimoto, Mitsutoshi

    2018-06-01

    Skin inflammation is caused by excessive production of cytokines and chemokines in response to an external stimulus, such as radiation, but the mechanisms involved are not completely understood. Here, we report a novel mechanism of γ-irradiation-induced interleukin-6 (IL-6) production mediated by P2Y11 receptors in epidermal cells. After irradiation of HaCaT cells derived from human epidermal keratinocytes with 5 Gy of γ-rays ( 137 Cs: 0.78 Gy/min), IL-6 production was unchanged at 24 h after γ-irradiation, but was increased at 48 h. IL-6 mRNA was increased at 30 h, and IL-6 production was increased at 33 h after irradiation. The production of IL-6 was sustained at least for 4 d after irradiation. P2Y11 receptor antagonist NF157 inhibited IL-6 production in irradiated cells. Treatment with ATP, a ligand of P2Y11 receptor caused IL-6 production within 24 h. ATP-induced IL-6 production was also suppressed by NF157. Extracellular ATP level was increased after irradiation. The p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling was involved in the production of IL-6 at the downstream of P2Y11 receptor activation. In addition, the cell cycle was arrested at the G2/M phase, and DNA repair foci were not disappeared at 48 h after γ-irradiation. The protein level of histone methylation enzyme G9a, which inhibits IL-6 production, was decreased after γ-irradiation. In conclusion, we suggest that γ-irradiation induces sustained IL-6 production in HaCaT cells from 33 h after irradiation, which is mediated through P2Y11 receptor-p38 MAPK-NF-κB signaling pathway and G9a degradation. This is a novel mechanism of cytokine production in γ-irradiated cells.

  20. Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo.

    PubMed

    Shi, Ming; Zhang, Ling; Gu, Hong-Tao; Jiang, Feng-Qin; Qian, Lu; Yu, Ming; Chen, Guo-Jiang; Luo, Qun; Shen, Bei-Fen; Guo, Ning

    2007-10-01

    To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin 2 (IL-2) fusion protein (HFI) in vitro and in vivo. Fusion protein HFI was constructed. The efficacy of HFI in mediating tumor cell lysis was determined by colorimetric lactate dehydrogenase release assays. The antitumor activity of HFI was evaluated in tumor xenograft models. The fusion protein was folded as a homodimer formed by covalently linking Fc portions and it retained ErbB2 specificity and IL-2 biological activity. HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and improved the therapeutic efficacy of IL-2 in experiments in vivo. The genetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high efficiency both in mediating ADCC in vitro and significant antitumor activity in tumor xenograft models.

  1. Corticospinal control of antagonistic muscles in the cat.

    PubMed

    Ethier, Christian; Brizzi, Laurent; Giguère, Dominic; Capaday, Charles

    2007-09-01

    We recently suggested that movement-related inter-joint muscle synergies are recruited by selected excitation and selected release from inhibition of cortical points. Here we asked whether a similar cortical mechanism operates in the functional linking of antagonistic muscles. To this end experiments were done on ketamine-anesthetized cats. Intracortical microstimulation (ICMS) and intramuscular electromyographic recordings were used to find and characterize wrist, elbow and shoulder antagonistic motor cortical points. Simultaneous ICMS applied at two cortical points, each evoking activity in one of a pair of antagonistic muscles, produced co-contraction of antagonistic muscle pairs. However, we found an obvious asymmetry in the strength of reciprocal inhibition; it was always significantly stronger on physiological extensors than flexors. Following intravenous injection of a single bolus of strychnine, a cortical point at which only a physiological flexor was previously activated also elicited simultaneous activation of its antagonist. This demonstrates that antagonistic corticospinal neurons are closely grouped, or intermingled. To test whether releasing a cortical point from inhibition allows it to be functionally linked with an antagonistic cortical point, one of three GABA(A) receptor antagonists, bicuculline, gabazine or picrotoxin, was injected iontophoretically at one cortical point while stimulation was applied to an antagonistic cortical point. This coupling always resulted in co-contraction of the represented antagonistic muscles. Thus, antagonistic motor cortical points are linked by excitatory intracortical connections held in check by local GABAergic inhibition, with reciprocal inhibition occurring at the spinal level. Importantly, the asymmetry of cortically mediated reciprocal inhibition would appear significantly to bias muscle maps obtained by ICMS in favor of physiological flexors.

  2. ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion.

    PubMed

    Cater, Michael A; La Fontaine, Sharon; Shield, Kristy; Deal, Yolanda; Mercer, Julian F B

    2006-02-01

    The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.

  3. The Mediator subunit MED23 couples H2B mono-ubiquitination to transcriptional control and cell fate determination.

    PubMed

    Yao, Xiao; Tang, Zhanyun; Fu, Xing; Yin, Jingwen; Liang, Yan; Li, Chonghui; Li, Huayun; Tian, Qing; Roeder, Robert G; Wang, Gang

    2015-12-02

    The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. © 2015 The Authors.

  4. Ionotropic and metabotropic glutamate receptor mediation of glucocorticoid-induced apoptosis in hippocampal cells and the neuroprotective role of synaptic N-methyl-D-aspartate receptors.

    PubMed

    Lu, J; Goula, D; Sousa, N; Almeida, O F X

    2003-01-01

    Glutamate receptors have been proposed to mediate the apoptotic actions of glucocorticoids in hippocampal cells. To further analyze the role of glutamate receptors in this process, we pretreated primary hippocampal cells from neonatal (postnatal day 4) rats with antagonists of ionotropic glutamate receptor (iGluR) and metabotropic glutamate receptor (mGluR) antagonists before exposure to the specific glucocorticoid receptor agonist dexamethasone (DEX) at a dose of 1 microM. Dizocilpine (MK801; a general N-methyl-D-aspartic acid [NMDA] receptor antagonist, NMDAR antagonist) and ifenprodil (a specific ligand of the NMDAR 2B subunit, NR2B), were used to block iGluR; (RS)-alpha-ethyl-4-carboxyphenylglycine (E4CPG) and (RS)-alpha-cyclopropyl-4-phosphonophenyl-glycine (CPPG) were employed as I/II (E4CPG) and II/III (CPPG) mGluR antagonists. Blockade of iGluR resulted in a significant attenuation of DEX-induced cell death; the finding that ifenprodil exerted a similar potency to MK801 demonstrates the involvement of NR2B receptors in glucocorticoid-induced cell death. Apoptosis accounted for a significant amount of the cell loss observed, as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling histochemistry for the in situ labeling of DNA breaks; apoptotic cells were distinguished from necrosis on the basis of morphological criteria, including chromatin condensation, membrane blebbing and presence of apoptotic bodies. Treatment with E4CPG and CPPG completely abolished the apoptotic response to DEX, thus showing the additional contribution of mGluR to the phenomenon. Further, dose-response studies with NMDA revealed that whereas high (10 microM) doses of NMDA themselves elicit cytotoxic responses, low (1-5 microM) concentrations of NMDA can effectively oppose DEX-induced cell death. Interestingly, the neuroprotective actions of low dose NMDA stimulation were abolished when either synaptic or extrasynaptic NMDA receptors were blocked with MK801

  5. Prevention of ultraviolet-B radiation damage by resveratrol in mouse skin is mediated via modulation in survivin.

    PubMed

    Aziz, Moammir Hassan; Afaq, Farrukh; Ahmad, Nihal

    2005-01-01

    Nonmelanoma skin cancer is the most frequently diagnosed malignancy in the United States, and multiple exposures to solar ultraviolet (UV) radiation (particularly its UV-B component, 290-320 nm), is its major cause. 'Chemoprevention' by naturally occurring agents is being appreciated as a newer dimension in the management of neoplasia including skin cancer. We recently demonstrated that resveratrol (trans-3, 5, 4-trihydroxystilbene), an antioxidant found in grapes, red wines and a variety of nuts and berries, imparts protection from acute UV-B-mediated cutaneous damages in SKH-1 hairless mice. Understanding the mechanism of resveratrol-mediated protection of UV responses is important. We earlier demonstrated that resveratrol imparts chemopreventive effects against multiple UV-exposure-mediated modulations in (1) cki-cyclin-cdk network, and (2) mitogen activated protein kinase (MAPK)-pathway. This study was conducted to assess the involvement of inhibitor of apoptosis protein family Survivin during resveratrol-mediated protection from multiple exposures of UV-B (180 mJ/cm(2); on alternate days; for a total of seven exposures) radiations in the SKH-1 hairless mouse skin. Our data demonstrated that topical pre-treatment of resveratrol (10 micromol in 200 microl acetone/mouse) resulted in significant inhibition of UV-B exposure-mediated increases in (1) cellular proliferations (Ki-67 immunostaining), (2) protein levels of epidermal cyclooxygenase-2 and ornithine decarboxylase, established markers of tumor promotion, (3) protein and messenger RNA levels of Survivin, and (4) phosphorylation of survivin in the skin of SKH-1 hairless mouse. Resveratrol pretreatment also resulted in (1) reversal of UV-B-mediated decrease of Smac/DIABLO, and (2) enhancement of UV-B-mediated induction of apoptosis, in mouse skin. Taken together, our study suggested that resveratrol imparts chemopreventive effects against UV-B exposure-mediated damages in SKH-1 hairless mouse skin via

  6. Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca2+ channels

    PubMed Central

    Huang, Dongyang; Liang, Ce; Zhang, Fan; Men, Hongchao; Du, Xiaona; Gamper, Nikita; Zhang, Hailin

    2016-01-01

    T-type Ca2+ channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca2+ currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca2+ channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca2+ currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a ‘reserve pool’ of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions. PMID:26944020

  7. The dopamine D2 receptor dimer and its interaction with homobivalent antagonists: homology modeling, docking and molecular dynamics.

    PubMed

    Kaczor, Agnieszka A; Jörg, Manuela; Capuano, Ben

    2016-09-01

    In order to apply structure-based drug design techniques to G protein-coupled receptor complexes, it is essential to model their 3D structure and to identify regions that are suitable for selective drug binding. For this purpose, we have developed and tested a multi-component protocol to model the inactive conformation of the dopamine D2 receptor dimer, suitable for interaction with homobivalent antagonists. Our approach was based on protein-protein docking, applying the Rosetta software to obtain populations of dimers as present in membranes with all the main possible interfaces. Consensus scoring based on the values and frequencies of best interfaces regarding four scoring parameters, Rosetta interface score, interface area, free energy of binding and energy of hydrogen bond interactions indicated that the best scored dimer model possesses a TM4-TM5-TM7-TM1 interface, which is in agreement with experimental data. This model was used to study interactions of the previously published dopamine D2 receptor homobivalent antagonists based on clozapine,1,4-disubstituted aromatic piperidines/piperazines and arylamidoalkyl substituted phenylpiperazine pharmacophores. It was found that the homobivalent antagonists stabilize the receptor-inactive conformation by maintaining the ionic lock interaction, and change the dimer interface by disrupting a set of hydrogen bonds and maintaining water- and ligand-mediated hydrogen bonds in the extracellular and intracellular part of the interface. Graphical Abstract Structure of the final model of the dopamine D2 receptor homodimer, indicating the distancebetween Tyr37 and Tyr 5.42 in the apo form (left) and in the complex with the ligand (right).

  8. A Novel Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-canonical Wnt-5a/Ror2/Disheveled Pathway Activation

    PubMed Central

    Bernatik, Ondrej; Radaszkiewicz, Tomasz; Behal, Martin; Dave, Zankruti; Witte, Florian; Mahl, Annika; Cernohorsky, Nicole H.; Krejci, Pavel; Stricker, Sigmar; Bryja, Vitezslav

    2017-01-01

    Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions. PMID:28523267

  9. Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

    PubMed

    Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

    2015-03-01

    Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

  10. CDKL5 controls postsynaptic localization of GluN2B-containing NMDA receptors in the hippocampus and regulates seizure susceptibility.

    PubMed

    Okuda, Kosuke; Kobayashi, Shizuka; Fukaya, Masahiro; Watanabe, Aya; Murakami, Takuto; Hagiwara, Mai; Sato, Tempei; Ueno, Hiroe; Ogonuki, Narumi; Komano-Inoue, Sayaka; Manabe, Hiroyuki; Yamaguchi, Masahiro; Ogura, Atsuo; Asahara, Hiroshi; Sakagami, Hiroyuki; Mizuguchi, Masashi; Manabe, Toshiya; Tanaka, Teruyuki

    2017-10-01

    Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function. Copyright © 2017 Elsevier Inc. All rights

  11. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo

    PubMed Central

    Goody, Michelle F.; Kelly, Meghan W.; Lessard, Kevin N.; Khalil, Andre; Henry, Clarissa A.

    2010-01-01

    Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn play roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

  12. Tiam–Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion

    PubMed Central

    2016-01-01

    Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB+ cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB+ cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion. PMID:27597758

  13. Serotonin 2C receptor antagonist improves fear discrimination and subsequent safety signal recall.

    PubMed

    Foilb, Allison R; Christianson, John P

    2016-02-04

    The capacity to discriminate between safety and danger is fundamental for survival, but is disrupted in individuals with posttraumatic stress disorder (PTSD). Acute stressors cause a release of serotonin (5-HT) in the forebrain, which is one mechanism for enhanced fear and anxiety; these effects are mediated by the 5-HT2Creceptor. Using a fear discrimination paradigm where a danger signal conditioned stimulus (CS+) co-terminates with a mild footshock and a safety signal (CS-) indicates the absence of shock, we demonstrate that danger/safety discrimination and fear inhibition develop over the course of 4 daily conditioning sessions. Systemic administration of the 5-HT2Creceptor antagonist SB 242084 (0.25 or 1.0mg/kg) prior to conditioning reduced behavioral freezing during conditioning, and improved learning and subsequent inhibition of fear by the safety signal. Discrimination was apparent in the first recall test, and discrimination during training was evident after 3days of conditioning versus 5days in the vehicle treated controls. These results suggest a novel therapeutic use for 5-HT2Creceptor antagonists to improve learning under stressful circumstances. Potential anatomical loci for 5-HT2Creceptor modulation of fear discrimination learning and cognitive performance enhancement are discussed. John P. Christianson and Allison R. Foilb, the authors, verify that animal research was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) and all procedures involving animals were reviewed and approved by the Boston College Animal Care and Use Committee. All efforts were made to limit the number of animals used and their suffering. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Haloperidol Suppresses NF-kappaB to Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Response in RAW 264 Cells

    PubMed Central

    Yamamoto, Shunsuke; Ohta, Noriyuki; Matsumoto, Atsuhiro; Horiguchi, Yu; Koide, Moe; Fujino, Yuji

    2016-01-01

    Background Haloperidol, a tranquilizing agent, is administered both to treat symptoms of psychotic disorders and to sedate agitated and delirious patients. Notably, haloperidol has been suggested to inhibit the immune response through unknown mechanisms. We hypothesized that the sedative modulates the immune response via NF-κB. Material/Methods Using flow cytometry, we analyzed the effects of haloperidol on expression CD80 and CD86 in RAW 264 cells and in primary macrophages derived from bone marrow. Secretion of interleukin (IL)-1β, IL-6, and IL-12 p40 was measured by enzyme-linked immunosorbent assay. In addition, NF-κB activation was evaluated using a reporter assay based on secretory embryonic alkaline phosphatase. Finally, synthetic antagonists were used to identify the dopamine receptor that mediates the effects of haloperidol. Results Haloperidol inhibited NF-κB activation, and thereby suppressed expression of CD80, as well as secretion of IL-1β, IL-6, and IL-12 p40. CD80 and IL-6 levels were similarly attenuated by a D2-like receptor antagonist, but not by a D1-like receptor antagonist. Conclusions The data strongly suggest that haloperidol inhibits the immune response by suppressing NF-κB signaling via the dopamine D2 receptor. PMID:26842661

  15. Haloperidol Suppresses NF-kappaB to Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Response in RAW 264 Cells.

    PubMed

    Yamamoto, Shunsuke; Ohta, Noriyuki; Matsumoto, Atsuhiro; Horiguchi, Yu; Koide, Moe; Fujino, Yuji

    2016-02-04

    BACKGROUND Haloperidol, a tranquilizing agent, is administered both to treat symptoms of psychotic disorders and to sedate agitated and delirious patients. Notably, haloperidol has been suggested to inhibit the immune response through unknown mechanisms. We hypothesized that the sedative modulates the immune response via NF-κB. MATERIAL AND METHODS Using flow cytometry, we analyzed the effects of haloperidol on expression CD80 and CD86 in RAW 264 cells and in primary macrophages derived from bone marrow. Secretion of interleukin (IL)-1β, IL-6, and IL-12 p40 was measured by enzyme-linked immunosorbent assay. In addition, NF-κB activation was evaluated using a reporter assay based on secretory embryonic alkaline phosphatase. Finally, synthetic antagonists were used to identify the dopamine receptor that mediates the effects of haloperidol. RESULTS Haloperidol inhibited NF-κB activation, and thereby suppressed expression of CD80, as well as secretion of IL-1β, IL-6, and IL-12 p40. CD80 and IL-6 levels were similarly attenuated by a D2-like receptor antagonist, but not by a D1-like receptor antagonist. CONCLUSIONS The data strongly suggest that haloperidol inhibits the immune response by suppressing NF-kB signaling via the dopamine D2 receptor.

  16. Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus.

    PubMed

    Takeda, Atsushi; Itagaki, Kosuke; Ando, Masaki; Oku, Naoto

    2012-03-01

    Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus. Copyright © 2011 Wiley Periodicals, Inc.

  17. The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers*

    PubMed Central

    Wilson, Parker C.; Lee, Mi-Hye; Appleton, Kathryn M.; El-Shewy, Hesham M.; Morinelli, Thomas A.; Peterson, Yuri K.; Luttrell, Louis M.; Jaffa, Ayad A.

    2013-01-01

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar1,Ile4,Ile8]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar1,Ile4, Ile8]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar1,Ile4,Ile8]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar1,Ile4,Ile8]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar1,Ile4,Ile8]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar1,Ile4,Ile8]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions. PMID:23661707

  18. The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

    PubMed

    Wilson, Parker C; Lee, Mi-Hye; Appleton, Kathryn M; El-Shewy, Hesham M; Morinelli, Thomas A; Peterson, Yuri K; Luttrell, Louis M; Jaffa, Ayad A

    2013-06-28

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic

  19. Effects of glutamate and {alpha}2-noradrenergic receptor antagonists on the development of neurotoxicity produced by chronic rotenone in rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alam, Mesbah; Danysz, Wojciech; Schmidt, Werner Juergen

    2009-10-15

    Systemic inhibition of complex I by rotenone in rats represents a model of Parkinson's disease (PD). The aim of this study was to elucidate whether neramexane (NMDA, nicotinic {alpha}9/{alpha}10 and 5-HT{sub 3} receptor antagonist), idazoxan ({alpha}{sub 2}-adrenoceptor antagonist) or 2-methyl-6-(phenyl-ethyl)-pyrimidine (MPEP, metabotropic glutamate receptor 5 antagonist) prevents rotenone-induced parkinsonian-like behaviours and neurochemical changes in rats. Rotenone (2.5 mg/kg i.p. daily) was administered over 60 days together with saline, neramexane (5 mg/kg i.p., b.i.d.), idazoxan (2.5 mg/kg i.p., b.i.d.) or MPEP (2.5 mg/kg i.p., b.i.d.). The same doses of neramexane, idazoxan and MPEP were administered to rats treated with vehicle insteadmore » of rotenone. Treatment-related effects on parkinsonian-like behaviours, such as hypokinesia/rigidity and locomotor activity, were evaluated. Moreover, concentrations of dopamine, serotonin and their metabolites were measured in rats from each experimental group. Over the 60-day treatment period, the rotenone + saline treated animals developed hypokinesia, expressed as an increase in the bar and grid descent latencies in the catalepsy test, and a decrease in locomotor activity. Neramexane and idazoxan partially prevented the development of catalepsy in rotenone-treated rats. Co-administration of MPEP with rotenone resulted only in a decrease in descent latency in the grid test on day 60. Chronic rotenone treatment reduced concentrations of dopamine and serotonin in the anterior striatum, which was blocked by co-treatment with neramexane or idazoxan but not with MPEP. Only neramexane treatment blocked the rotenone-induced decrease in dopamine levels in the substantia nigra pars compacta. In conclusion, neramexane and idazoxan counteracted to some extent the development of parkinsonian symptoms and neurochemical alterations in the rotenone model of Parkinson's disease.« less

  20. Suppressive effects of a novel CC chemokine receptor 4 antagonist on Th2 cell trafficking in ligand- and antigen-induced mouse models.

    PubMed

    Komiya, Takaki; Sugiyama, Tetsuya; Takeda, Kazuhiko; Watanabe, Noriki; Imai, Masamichi; Kokubo, Masaya; Tokuda, Natsuko; Ochiai, Hiroshi; Habashita, Hiromu; Shibayama, Shiro

    2013-11-15

    CC chemokine receptor 4 (CCR4) has been implicated as a preferential marker for T helper type 2 (Th2) cells, and is believed to be involved in the pathology of allergic diseases by controlling Th2 cell trafficking into inflamed tissues. The objective of the study was to characterize the pharmacological properties of E0001-163, a novel CCR4 antagonist. E0001-163 was tested in both in vitro chemotaxis assays as well as in vivo mouse models of CCR4 ligand-induced air pouch and antigen-induced airway inflammation by utilizing in vitro-polarized Th2 cells. In vitro, E0001-163 inhibited migratory response of human Th2-polarized cells to CCL22, a CCR4 ligand, with an IC50 value of 11.9 nM. E0001-163 significantly suppressed CCL22-induced Th2 cell trafficking into mouse air pouch in a dose-dependent manner at doses of 3 and 10mg/kg, suggesting that E0001-163 has an inhibitory effect on CCR4-mediated T cell trafficking in vivo. In addition, E0001-163 partially decreased Th2 cell trafficking and the level of IL-4 in the lungs in Th2-tansferred and ovalbumin (OVA)-challenged mice. T cell trafficking involves multiple chemokine receptors both in acute and chronic phases, and our findings suggest that CCR4, together with other chemokine receptors, may be involved in Th2 cell trafficking under disease conditions. © 2013 Elsevier B.V. All rights reserved.

  1. Effects of the GluN2B-NMDA receptor antagonist Ro 25-6981 on two types of behavioral flexibility in rats.

    PubMed

    Clark, Emma; Antoniak, Kristen; Feniquito, Alyssandra; Dringenberg, Hans C

    2017-02-15

    Recent evidence has implicated N-methyl-d-aspartate receptors (NMDARs) in several aspects of learning and behavioral flexibility in rodents. Here, we examined the effects of treatment with Ro 25-6981, a selective antagonist of NMDARs containing GluN2B subunits, on two types of behavioral flexibility in rats, spatial reversal learning and set-shifting (spatial vs. motor strategy). To examine spatial reversal learning, rats were trained to swim to a hidden platform in a water maze over four days. On the following day, the platform was moved to a new location in the maze. Administration of Ro 25-6981 (10mg/kg) selectively impaired the early phase of reversal learning, but all rats learned to navigate to the new platform location over 12 trials. To examine set-shifting, independent groups of rats were trained to either swim to a fixed location (spatial strategy) or use a motor response (e.g., "turn left"; motor strategy) to find a hidden escape platform in a cross-shaped water maze apparatus; after task acquisition, rats were trained on the second, novel strategy (set-shift) following treatment with either Ro 25-6981 (10mg/kg) or saline. Administration of Ro 25-6981 had no effect on the ability of rats to perform the set-shift and use the new strategy to locate the escape platform. These results suggest that, in rats, spatial reversal learning, but not set-shifting, is sensitive to Ro-25-6981 treatment. Thus, NMDARs-GluN2B signaling may play a selective role in some forms of behavioral plasticity, particularly for situations involving the updating of information in the spatial domain. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth

    PubMed Central

    Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H.; Galipeau, Jacques; Deng, Xingming

    2016-01-01

    Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy. PMID:27049723

  3. Misoprostol inhibits gastric mucosal release of endogenous prostaglandin E2 and thromboxane B2 in healthy volunteers.

    PubMed Central

    Mertz-Nielsen, A; Eskerod, O; Bukhave, K; Rask-Madsen, J

    1995-01-01

    Prostaglandin analogues of the E-series theoretically offer the ideal antiulcer drugs. Peptic ulcer healing with prostaglandin analogues is, however, no better than would be predicted from their ability to inhibit gastric acid secretion and they are less effective than histamine H2 receptor antagonists in preventing ulcer relapse. It could be that prostaglandin analogues inhibit gastric mucosal synthesis or release of endogenous eicosanoids, thereby abrogating their own effects. This study, therefore, examined how a single therapeutic dose (200 micrograms) of misoprostol, a synthetic analogue of prostaglandin E1, influences gastric mucosal release of endogenous prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and chemotactic leukotriene B4 (LTB4) during basal conditions and in response to gastric luminal acidification (0.1 M HCl; 5 ml/min for 10 minutes). Nine healthy volunteers were studied in a single blind, cross over design. In each subject misoprostol or placebo was instilled in randomised order into the stomach, which was subsequently perfused with isotonic mannitol. Misoprostol significantly decreased basal as well as acid stimulated output of PGE2 and TXB2, without affecting output of LTB4. These data show that misoprostol inhibits gastric mucosal synthesis of prostanoids. Decreased concentrations, or even a changed profile, of native eicosanoids modulating the release of inflammatory mediators from immune cells might explain why prostaglandin analogues have a comparatively poor clinical performance in ulcer healing and prevention. PMID:7737555

  4. Substance P antagonists and mucociliary activity in rabbit.

    PubMed

    Lindberg, S; Mercke, U

    1985-06-01

    Substance P (SP) is known to accelerate mucociliary (m.c.) activity in the rabbit maxillary sinus in vivo. The physiological significance of this finding was investigated by testing three putative SP antagonists. [Arg5, D-Trp7,9, Nle11]SP5-11 could not be used as an antagonist because it stimulated m.c. activity. [D-Arg1, D-Trp7,9, Leu11]SP had no effect on the m.c. activity changes induced by SP. [D-Pro2, D-Trp7,9]SP was found to be an effective antagonist, 1 mg/kg of this drug reversibly inhibiting both the effects of 0.1 micrograms/kg SP and the stimulating effect of 1.0 micrograms/kg bradykinin and 30.0 micrograms/kg capsaicin; the stimulating effect of 0.5 micrograms/kg methacholine was not inhibited. It is suggested that bradykinin and capsaicin stimulate m.c. activity at least partly by releasing SP. The results of this investigation also support the view that the accelerating effect of SP on m.c. activity reflects physiological SP-mediated protective mechanisms in the airways. It is concluded that [D-Pro2,D-Trp7,9]SP is a useful pharmacological tool for studying the role of SP in the control of m.c. activity in rabbits.

  5. Effects of pharmacological and genetic disruption of CXCR4 chemokine receptor function in B-cell acute lymphoblastic leukaemia.

    PubMed

    Randhawa, Shubhchintan; Cho, Byung S; Ghosh, Dipanjan; Sivina, Mariela; Koehrer, Stefan; Müschen, Markus; Peled, Amnon; Davis, Richard E; Konopleva, Marina; Burger, Jan A

    2016-08-01

    B cell acute lymphoblastic leukaemia (B-ALL) cells express high levels of CXCR4 chemokine receptors for homing and retention within the marrow microenvironment. Bone marrow stromal cells (BMSC) secrete CXCL12, the ligand for CXCR4, and protect B-ALL cells from cytotoxic drugs. Therefore, the therapeutic use of CXCR4 antagonists has been proposed to disrupt cross talk between B-ALL cells and the protective stroma. Because CXCR4 antagonists can have activating agonistic function, we compared the genetic and pharmacological deletion of CXCR4 in B-ALL cells, using CRISPR-Cas9 gene editing and CXCR4 antagonists that are in clinical use (plerixafor, BKT140). Both genetic and pharmacological CXCR4 inhibition significantly reduced B-ALL cell migration to CXCL12 gradients and beneath BMSC, and restored drug sensitivity to dexamethasone, vincristine and cyclophosphamide. NOD/SCID/IL-2rγnull mice injected with CXCR4 gene-deleted B-ALL cells had significant delay in disease progression and superior survival when compared to control mice injected with CXCR4 wild-type B-ALL cells. These findings indicate that anti-leukaemia activity of CXCR4 antagonists is primarily due to CXCR4 inhibition, rather than agonistic activity, and corroborate that CXCR4 is an important target to overcome stroma-mediated drug resistance in B-ALL. © 2016 John Wiley & Sons Ltd.

  6. Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses

    PubMed Central

    Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D

    1999-01-01

    The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and

  7. Brain regions mediating α3β4 nicotinic antagonist effects of 18-MC on methamphetamine and sucrose self-administration

    PubMed Central

    Glick, Stanley D.; Sell, Elizabeth M.; Maisonneuve, Isabelle M.

    2008-01-01

    The novel iboga alkaloid congener 18-methoxycoronaridine (18-MC) is a putative anti-addictive agent that has been shown, in rats, to decrease the self-administration of several drugs of abuse. Previous work has established that 18-MC is a potent antagonist at α3β4 nicotinic receptors. Because high densities of α3β4 nicotinic receptors occur in the medial habenula and the interpeduncular nucleus and moderate densities occur in the dorsolateral tegmentum, ventral tegmental area, and basolateral amygdala, the present study was conducted to determine if 18-MC could act in these brain areas to modulate methamphetamine self-administration in rats. Local administration of 18-MC into either the medial habenula, the interpeduncular area or the basolateral amygdala decreased methamphetamine self-administration. Similar results were produced by local administration into the same brain areas of two other α3β4 nicotinic antagonists, mecamylamine and α-conotoxin AuIB. Local administration of 18-MC, or the other antagonists, into the dorsolateral tegmentum or the ventral tegmental area had no effect on methamphetamine self-administration. In contrast, local administration of 18-MC and the other antagonists decreased sucrose self-administration when administered into the dorsolateral tegmentum or basolateral amygdala but had no effect when infused into the medial habenula, interpeduncular nucleus, or ventral tegmental area. These data are consistent with the hypothesis that 18-MC decreases methamphetamine self-administration by indirectly modulating the dopaminergic mesolimbic pathway via blockade of α3β4 nicotinic receptors in the habenulo-interpeduncular pathway and the basolateral amygdala. The data also suggest that the basolateral amygdala along with a different pathway involving α3β4 receptors in the dorsolateral tegmentum mediate the effect of 18-MC on sucrose self-administration. PMID:18930043

  8. 2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABAA-ρ1 Receptors

    PubMed Central

    Xie, An; Yan, Jun; Yue, Lan; Feng, Feng; Mir, Fozia; Abdel-Halim, Heba; Chebib, Mary; Le Breton, Guy C.; Standaert, Robert F.; Qian, Haohua

    2011-01-01

    2-Aminoethyl methylphosphonate (2-AEMP), an analog of GABA, has been found to exhibit antagonist activity at GABAA-ρ1 (also known as ρ1 GABAC) receptors. The present study was undertaken to elucidate 2-AEMP's action and to test the activities of 2-AEMP analogs. Whole-cell patch-clamp techniques were used to record membrane currents in neuroblastoma cells stably transfected with human GABAA-ρ1 receptors. The action of 2-AEMP was compared with that of 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), a commonly used GABAA-ρ1 antagonist. With 10 μM GABA, 2-AEMP's IC50 (18 μM) differed by less than 2.5-fold from that of TPMPA (7 μM), and results obtained were consistent with a primarily competitive mode of inhibition by 2-AEMP. Terminating the presentation of 2-AEMP or TPMPA in the presence of GABA produced a release from inhibition. However, the rate of inhibition release upon the termination of 2-AEMP considerably exceeded that determined with termination of TPMPA. Moreover, when presented at concentrations near their respective IC50 values, the preincubation period associated with 2-AEMP's onset of inhibition was much shorter than that for TPMPA. Analogs of 2-AEMP possessing a benzyl or n-butyl rather than a methyl substituent at the phosphorus atom, as well as analogs bearing a C-methyl substituent on the aminoethyl side chain, exhibited reduced potency relative to 2-AEMP. Of these analogs, only (R)-2-aminopropyl methylphosphonate significantly diminished the response to 10 μM GABA. Structure-activity relationships are discussed in the context of molecular modeling of ligand binding to the antagonist binding site of the GABAA-ρ1 receptor. PMID:21810922

  9. Betulinic acid exerts anti-hepatitis C virus activity via the suppression of NF-κB- and MAPK-ERK1/2-mediated COX-2 expression.

    PubMed

    Lin, Chun-Kuang; Tseng, Chin-Kai; Chen, Kai-Hsun; Wu, Shih-Hsiung; Liaw, Chih-Chuang; Lee, Jin-Ching

    2015-06-23

    This study was designed to evaluate the effect of betulinic acid (BA), extracted from Avicennia marina, on the replication of hepatitis C virus (HCV) and to investigate the mechanism of this BA-mediated anti-HCV activity. HCV replicon and infectious systems were used to evaluate the anti-HCV activity of BA. Exogenous COX-2 or knock-down of COX-2 expression was used to investigate the role of COX-2 in the anti-HCV activity of BA. The effects of BA on the phosphorylation of NF-κB and on kinases in the MAPK signalling pathway were determined. The anti-HCV activity of BA in combination with other HCV inhibitors was also determined to assess its use as an anti-HCV supplement. BA inhibited HCV replication in both Ava5 replicon cells and in a cell culture-derived infectious HCV particle system. Treatment with a combination of BA and IFN-α, the protease inhibitor telaprevir or the NS5B polymerase inhibitor sofosbuvir resulted in the synergistic suppression of HCV RNA replication. Exogenous overexpression of COX-2 gradually attenuated the inhibitory effect of BA on HCV replication, suggesting that BA reduces HCV replication by suppressing the expression of COX-2. In particular, BA down-regulated HCV-induced COX-2 expression by reducing the phosphorylation of NF-κB and ERK1/2 of the MAPK signalling pathway. BA inhibits HCV replication by suppressing the NF-κB- and ERK1/2-mediated COX-2 pathway and may serve as a promising compound for drug development or as a potential supplement for use in the treatment of HCV-infected patients. © 2015 The British Pharmacological Society.

  10. Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

    PubMed Central

    Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

    2008-01-01

    Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

  11. Functionalized Congeners of P2Y1 Receptor Antagonists:

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    de Castro, Sonia; Maruoka, Hiroshi; Hong, Kunlun

    2010-01-01

    The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of anmore » intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM

  12. Discovery and evaluation of a series of 3-acylindole imidazopyridine platelet-activating factor antagonists.

    PubMed

    Curtin, M L; Davidsen, S K; Heyman, H R; Garland, R B; Sheppard, G S; Florjancic, A S; Xu, L; Carrera, G M; Steinman, D H; Trautmann, J A; Albert, D H; Magoc, T J; Tapang, P; Rhein, D A; Conway, R G; Luo, G; Denissen, J F; Marsh, K C; Morgan, D W; Summers, J B

    1998-01-01

    Studies conducted with the goal of discovering a second-generation platelet-activating factor (PAF) antagonist have identified a novel class of potent and orally active antagonists which have high aqueous solubility and long duration of action in animal models. The compounds arose from the combination of the lipophilic indole portion of Abbott's first-generation PAF antagonist ABT-299 (2) with the methylimidazopyridine heterocycle moiety of British Biotechnology's BB-882 (1) and possess the positive attributes of both of these clinical candidates. Structure-activity relationship (SAR) studies indicated that modification of the indole and benzoyl spacer of lead compound 7b gave analogues that were more potent, longer-lived, and bioavailable and resulted in the identification of 1-(N, N-dimethylcarbamoyl)-4-ethynyl-3-[3-fluoro-4-[(1H-2-methylimidazo[4,5-c] pyrid-1-yl)methyl]benzoyl]indole hydrochloride (ABT-491, 22 m.HCl) which has been evaluated extensively and is currently in clinical development.

  13. Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine receptors, decreases ethanol consumption in alcohol-preferring UChB rats.

    PubMed

    Quiroz, Gabriel; Guerra-Díaz, Nicolás; Iturriaga-Vásquez, Patricio; Rivera-Meza, Mario; Quintanilla, María Elena; Sotomayor-Zárate, Ramón

    2018-09-03

    Alcohol abuse is a worldwide health problem with high economic costs to health systems. Emerging evidence suggests that modulation of brain nicotinic acetylcholine receptors (nAChRs) may be a therapeutic target for alcohol dependence. In this work, we assess the effectiveness of four doses of erysodine (1.5, 2.0, 4.0 or 8.0 mg/kg/day, i.p.), a competitive antagonist of nAChRs, on voluntary ethanol consumption behavior in alcohol-preferring UChB rats, administered during three consecutive days. Results show that erysodine administration produces a dose-dependent reduction in ethanol consumption respect to saline injection (control group). The highest doses of erysodine (4 and 8 mg/kg) reduce (45 and 66%, respectively) the ethanol intake during treatment period and first day of post-treatment compared to control group. While, the lowest doses of erysodine (1.5 and 2 mg/kg) only reduce ethanol intake during one day of treatment period. These effective reductions in ethanol intake were 23 and 29% for 1.5 and 2 mg/kg erysodine, respectively. Locomotor activity induced by a high dose of erysodine (10 mg/kg) was similar to those observed with saline injection in control rats, showing that the reduction in ethanol intake was not produced by hypolocomotor effect induced by erysodine. This is the first report showing that erysodine reduces ethanol intake in UChB rats in a dose-dependent manner. Our results highlight the role of nAChRs in the reward effects of ethanol and its modulation as a potentially effective pharmacological alternative for alcohol dependence treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Deficits in cognition and synaptic plasticity in a mouse model of Down syndrome ameliorated by GABAB receptor antagonists

    PubMed Central

    Kleschevnikov, A.M.; Belichenko, P.V.; Faizi, M.; Jacobs, L.F.; Htun, K.; Shamloo, M.; Mobley, W.C.

    2012-01-01

    Cognitive impairment in Down syndrome (DS) is characterized by deficient learning and memory. Mouse genetic models of DS exhibit impaired cognition in hippocampally mediated behavioral tasks and reduced synaptic plasticity of hippocampal pathways. Enhanced efficiency of GABAergic neurotransmission was implicated in those changes. We have recently shown that signaling through postsynaptic GABAB receptors is significantly increased in the dentate gyrus (DG) of Ts65Dn mice, a genetic model of DS. Here we examined a role for GABAB receptors in cognitive deficits in DS by defining the effect of selective GABAB receptor antagonists on behavior and synaptic plasticity of adult Ts65Dn mice. Treatment with the GABAB receptor antagonist CGP55845 restored memory of Ts65Dn mice in the novel place recognition, novel object recognition and contextual fear conditioning tasks, but did not affect locomotion and performance in T-maze. The treatment increased hippocampal levels of brain-derived neurotrophic factor (BDNF), equally in 2N and Ts65Dn mice. In hippocampal slices, treatment with the GABAB receptor antagonists CGP55845 or CGP52432 enhanced long-term potentiation (LTP) in the Ts65Dn DG. The enhancement of LTP was accompanied by an increase in the NMDA receptor-mediated component of the tetanus-evoked responses. These findings are evidence for a contribution of GABAB receptors to changes in hippocampal-based cognition in the Ts65Dn mouse. The ability to rescue cognitive performance through treatment with selective GABAB receptor antagonists motivates studies to further explore the therapeutic potential of these compounds in people with DS. PMID:22764230

  15. Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

    PubMed

    Rivier, Jean E; Rivier, Catherine L

    2014-04-01

    Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. The Neurogenesis Actuator and NR2B/NMDA Receptor Antagonist Ro25-6981 Consistently Improves Spatial Memory Retraining Via Brain Region-Specific Gene Expression.

    PubMed

    Gruden, Marina A; Ratmirov, Alexander M; Storozheva, Zinaida I; Solovieva, Olga A; Sherstnev, Vladimir V; Sewell, Robert D E

    2018-05-22

    NR2B-containing NMDA (NR2B/NMDA) receptors are important in controlling neurogenesis and are involved in generating spatial memory. Ro25-6981 is a selective antagonist at these receptors and actuates neurogenesis and spatial memory. Inter-structural neuroanatomical profiles of gene expression regulating adult neurogenesis and neuroapoptosis require examination in the context of memory retrieval and reversal learning. The aim was to investigate spatial memory retrieval and reversal learning in relation to gene expression-linked neurogenetic processes following blockade of NR2B/NMDA receptors by Ro25-6981. Rats were trained in Morris water maze (MWM) platform location for 5 days. Ro25-6981 was administered (protocol days 6-7) followed by retraining (days 15-18 or 29-32). Platform location was tested (on days 19 or 33) then post-mortem brain tissue sampling (on days 20 or 34). The expression of three genes known to regulate cell proliferation (S100a6), differentiation (Ascl1), and apoptosis (Casp-3) were concomitantly evaluated in the hippocampus, prefrontal cortex, and cerebellum in relation to the MWM performance protocol. Following initial training, Ro25-6981 enhanced visuospatial memory retrieval performance during further retraining (protocol days 29-32) but did not influence visuospatial reversal learning (day 33). Hippocampal Ascl1 and Casp-3 expressions were correspondingly increased and decreased while cerebellar S100a6 and Casp-3 activities were decreased and increased respectively 27 days after Ro25-6981 treatment. Chronological analysis indicated a possible involvement of new mature neurons in the reconfiguration of memory processes. This was attended by behavioral/gene correlations which revealed direct links between spatial memory retrieval enhancement and modified gene activity induced by NR2B/NMDA receptor blockade and upregulation.

  17. Antigen-specific and nonspecific mediators of T cell/B cell cooperation. III. Characterization of the nonspecific mediator(s) from different sources.

    PubMed

    Harwell, L; Kappler, J W; Marrack, P

    1976-05-01

    T cell-containing lymphoid populations produce a nonantigen-specific mediator(s) (NSM) which can replace T cell helper function in vitro in the response of B cells to sheep red blood cells (SRBC), but not to the hapten-protein conjugate, trinitrophenyl-keyhole limpet hemocyanin, (TNP-KLH). NSM produced under three conditions: 1) stimulation of KLH-primed cells with KLH; 2) allogeneic stimulation of normal spleen cells; and 3) stimulation of normal spleen cells with Con A (but not PHA) are indistinguishable on the basis of their biologic activity and m.w., estimated as 30 to 40,000 daltons by G-200 chromatography. Production of NSM is dependent on the presence of T cells. The action of NSM on B cells responding to SRBC in the presence of 2-mercaptoethanol is unaffected by severe macrophage depletion. Extensive absorption of NSM with SRBC failed to remove its activity, confirming its nonantigen-specific nature.

  18. The anti-inflammatory effects of PGE2 on human lung macrophages are mediated by the EP4 receptor.

    PubMed

    Gill, Sharonjit K; Yao, Yiwen; Kay, Linda J; Bewley, Martin A; Marriott, Helen M; Peachell, Peter T

    2016-11-01

    PGE 2 inhibits cytokine generation from human lung macrophages. However, the EP receptor that mediates this beneficial anti-inflammatory effect of PGE 2 has not been defined. The aim of this study was to identify the EP receptor by which PGE 2 inhibits cytokine generation from human lung macrophages. This was determined by using recently developed EP receptor ligands. The effects of PGE 2 and EP-selective agonists on LPS-induced generation of TNF-α and IL-6 from macrophages were evaluated. The effects of EP 2 -selective (PF-04852946, PF-04418948) and EP 4 -selective (L-161,982, CJ-042794) receptor antagonists on PGE 2 responses were studied. The expression of EP receptor subtypes by human lung macrophages was determined by RT-PCR. PGE 2 inhibited LPS-induced and Streptococcus pneumoniae-induced cytokine generation from human lung macrophages. Analysis of mRNA levels indicated that macrophages expressed EP 2 and EP 4 receptors. L-902,688 (EP 4 receptor-selective agonist) was considerably more potent than butaprost (EP 2 receptor-selective agonist) as an inhibitor of TNF-α generation from macrophages. EP 2 receptor-selective antagonists had marginal effects on the PGE 2 inhibition of TNF-α generation, whereas EP 4 receptor-selective antagonists caused rightward shifts in the PGE 2 concentration-response curves. These studies demonstrate that the EP 4 receptor is the principal receptor that mediates the anti-inflammatory effects of PGE 2 on human lung macrophages. This suggests that EP 4 receptor agonists could be effective anti-inflammatory agents in human lung disease. © 2016 The British Pharmacological Society.

  19. A physiological role of AMP-activated protein kinase in phenobarbital-mediated constitutive androstane receptor activation and CYP2B induction

    PubMed Central

    Shindo, Sawako; Numazawa, Satoshi; Yoshida, Takemi

    2006-01-01

    CAR (constitutive androstane receptor) is a nuclear receptor that regulates the transcription of target genes, including CYP (cytochrome P450) 2B and 3A. The transactivation by CAR is regulated by its subcellular localization; however, the mechanism that governs nuclear translocation has yet to be clarified. It has been reported recently that AMPK (AMP-activated protein kinase) is involved in phenobarbital-mediated CYP2B induction in a particular culture system. We therefore investigated in vivo whether AMPK is involved in the activation of CAR-dependent gene expression. Immunoblot analysis using an antibody which recognizes Thr-172-phosphorylated AMPKα1/2 revealed phenobarbital-induced AMPK activation in rat and mouse livers as well. Phenobarbital, however, failed to increase the liver phospho-AMPK level of tumour-bearing rats in which CAR nuclear translocation had been impaired. In in vivo reporter gene assays employing PBREM (phenobarbital-responsive enhancer module) from CYP2B1, an AMPK inhibitor 8-bromo-AMP abolished phenobarbital-induced transactivation. In addition, Cyp2b10 gene expression was attenuated by 8-bromo-AMP. Forced expression of a dominant-negative mutant and the wild-type of AMPKα2 in the mouse liver suppressed and further enhanced phenobarbital-induced PBREM-reporter activity respectively. Moreover, the AMPK activator AICAR (5-amino-4-imidazolecarboxamide riboside) induced PBREM transactivation and an accumulation of CAR in the nuclear fraction of the mouse liver. However, AICAR and metformin, another AMPK activator, failed to induce hepatic CYP2B in mice and rats. These observations suggest that AMPK is at least partly involved in phenobarbital-originated signalling, but the kinase activation by itself is not sufficient for CYP2B induction in vivo. PMID:17032173

  20. Pharmacological characterization of ATPM [(-)-3-aminothiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride], a novel mixed kappa-agonist and mu-agonist/-antagonist that attenuates morphine antinociceptive tolerance and heroin self-administration behavior.

    PubMed

    Wang, Yu-Jun; Tao, Yi-Min; Li, Fu-Ying; Wang, Yu-Hua; Xu, Xue-Jun; Chen, Jie; Cao, Ying-Lin; Chi, Zhi-Qiang; Neumeyer, John L; Zhang, Ao; Liu, Jing-Gen

    2009-04-01

    ATPM [(-)-3-amino-thiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] was found to have mixed kappa- and mu-opioid activity and identified to act as a full kappa-agonist and a partial mu-agonist by in vitro binding assays. The present study was undertaken to characterize its in vivo effects on morphine antinociceptive tolerance in mice and heroin self-administration in rats. ATPM was demonstrated to yield more potent antinociceptive effects than (-)U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide). It was further found that the antinociceptive effects of ATPM were mediated by kappa- and mu-, but not delta-opioid, receptors. In addition to its agonist profile on the mu-receptor, ATPM also acted as a mu-antagonist, as measured by its inhibition of morphine-induced antinociception. It is more important that ATPM had a greater ratio of the ED(50) value of sedation to that of antinociception than (-)U50,488 (11.8 versus 3.7), indicative of a less sedative effect than (-)U50,488H. In addition, ATPM showed less potential to develop antinociceptive tolerance relative to (-)U50,488H and morphine. Moreover, it dose-dependently inhibited morphine-induced antinociceptive tolerance. Furthermore, it was found that chronic treatment of rats for 8 consecutive days with ATPM (0.5 mg/kg s.c.) produced sustained decreases in heroin self-administration. (-)U50,488H (2 mg/kg s.c.) also produced similar inhibitory effect. Taken together, our findings demonstrated that ATPM, a novel mixed kappa-agonist and mu-agonist/-antagonist, could inhibit morphine-induced antinociceptive tolerance, with less potential to develop tolerance and reduce heroin self-administration with less sedative effect. kappa-Agonists with some mu-activity appear to offer some advantages over selective kappa-agonists for the treatment of heroin abuse.

  1. Synthesis of LHRH antagonists suitable for oral administration via the vitamin B12 uptake system.

    PubMed

    Russell-Jones, G J; Westwood, S W; Farnworth, P G; Findlay, J K; Burger, H G

    1995-01-01

    Conjugates have been synthesized between vitamin B12 and two lysyl derivatives of the LHRH antagonist, ANTIDE. Lys6-ANTIDE and Lys8-ANTIDE were both found to have similar activities to the native analogue in the in vitro pituitary cell assay. The in vitro bioactivity of the VB12-ANTIDE conjugates was preserved following linkage using a number of spacers; however, the in vivo bioactivity was lost. In order to produce conjugates which had similar in vivo bioactivity to the native analogue, it was necessary to link the VB12 to the ANTIDE analogues using thiol cleavable spacers. The resultant conjugates had similar activity to ANTIDE both in vitro and in vivo and were also found to be much more water soluble than ANTIDE. These VB12-ANTIDE conjugates show potential utility as water soluble ANTIDE analogues for parenteral use and are protease resistant LHRH antagonist analogues suitable for uptake from the intestine via the VB12-transport system following oral administration.

  2. Adenosine A2A receptors modulate the dopamine D2 receptor-mediated inhibition of synaptic transmission in the mouse prefrontal cortex.

    PubMed

    Real, Joana I; Simões, Ana Patrícia; Cunha, Rodrigo A; Ferreira, Samira G; Rial, Daniel

    2018-05-01

    Prefrontal cortex (PFC) circuits are modulated by dopamine acting on D 1 - and D 2 -like receptors, which are pharmacologically exploited to manage neuropsychiatric conditions. Adenosine A 2A receptors (A 2 A R) also control PFC-related responses and A 2 A R antagonists are potential anti-psychotic drugs. As tight antagonistic A 2 A R-D 2 R and synergistic A 2 A R-D 1 R interactions occur in other brain regions, we now investigated the crosstalk between A 2 A R and D 1 /D 2 R controlling synaptic transmission between layers II/III and V in mouse PFC coronal slices. Dopamine decreased synaptic transmission, a presynaptic effect based on the parallel increase in paired-pulse responses. Dopamine inhibition was prevented by the D 2 R-like antagonist sulpiride but not by the D 1 R antagonist SCH23390 and was mimicked by the D 2 R agonist sumanirole, but not by the agonists of either D 4 R (A-412997) or D 3 R (PD128907). Dopamine inhibition was prevented by the A 2 A R antagonist, SCH58261, and attenuated in A 2 A R knockout mice. Accordingly, triple-labelling immunocytochemistry experiments revealed the co-localization of A 2 A R and D 2 R immunoreactivity in glutamatergic (vGluT1-positive) nerve terminals of the PFC. This reported positive A 2 A R-D 2 R interaction controlling PFC synaptic transmission provides a mechanistic justification for the anti-psychotic potential of A 2 A R antagonists. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  3. Dual Endothelin-A/Endothelin-B Receptor Blockade and Cardiac Remodeling in Heart Failure With Preserved Ejection Fraction

    PubMed Central

    Valero-Munoz, Maria; Li, Shanpeng; Wilson, Richard M.; Boldbaatar, Batbold; Iglarz, Marc; Sam, Flora

    2017-01-01

    Background Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there remains no evidence-based therapies for HFpEF. Endothelin-1 (ET-1) antagonists are a possibility because elevated ET-1 levels are associated with adverse cardiovascular effects, such as arterial and pulmonary vasoconstriction, impaired left ventricular (LV) relaxation, and stimulation of LV hypertrophy. LV hypertrophy is a common phenotype in HFpEF, particularly when associated with hypertension. Methods and Results In the present study, we found that ET-1 levels were significantly elevated in patients with chronic stable HFpEF. We then sought to investigate the effects of chronic macitentan, a dual ET-A/ET-B receptor antagonist, on cardiac structure and function in a murine model of HFpEF induced by chronic aldosterone infusion. Macitentan caused LV hypertrophy regression independent of blood pressure changes in HFpEF. Although macitentan did not modulate diastolic dysfunction in HFpEF, it significantly reduced wall thickness and relative wall thickness after 2 weeks of therapy. In vitro studies showed that macitentan decreased the aldosterone-induced cardiomyocyte hypertrophy. These changes were mediated by a reduction in the expression of cardiac myocyte enhancer factor 2a. Moreover, macitentan improved adverse cardiac remodeling, by reducing the stiffer cardiac collagen I and titin n2b expression in the left ventricle of mice with HFpEF. Conclusions These findings indicate that dual ET-A/ET-B receptor inhibition improves HFpEF by abrogating adverse cardiac remodeling via antihypertrophic mechanisms and by reducing stiffness. Additional studies are needed to explore the role of dual ET-1 receptor antagonists in patients with HFpEF. PMID:27810862

  4. Dual Endothelin-A/Endothelin-B Receptor Blockade and Cardiac Remodeling in Heart Failure With Preserved Ejection Fraction.

    PubMed

    Valero-Munoz, Maria; Li, Shanpeng; Wilson, Richard M; Boldbaatar, Batbold; Iglarz, Marc; Sam, Flora

    2016-11-01

    Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there remains no evidence-based therapies for HFpEF. Endothelin-1 (ET-1) antagonists are a possibility because elevated ET-1 levels are associated with adverse cardiovascular effects, such as arterial and pulmonary vasoconstriction, impaired left ventricular (LV) relaxation, and stimulation of LV hypertrophy. LV hypertrophy is a common phenotype in HFpEF, particularly when associated with hypertension. In the present study, we found that ET-1 levels were significantly elevated in patients with chronic stable HFpEF. We then sought to investigate the effects of chronic macitentan, a dual ET-A/ET-B receptor antagonist, on cardiac structure and function in a murine model of HFpEF induced by chronic aldosterone infusion. Macitentan caused LV hypertrophy regression independent of blood pressure changes in HFpEF. Although macitentan did not modulate diastolic dysfunction in HFpEF, it significantly reduced wall thickness and relative wall thickness after 2 weeks of therapy. In vitro studies showed that macitentan decreased the aldosterone-induced cardiomyocyte hypertrophy. These changes were mediated by a reduction in the expression of cardiac myocyte enhancer factor 2a. Moreover, macitentan improved adverse cardiac remodeling, by reducing the stiffer cardiac collagen I and titin n2b expression in the left ventricle of mice with HFpEF. These findings indicate that dual ET-A/ET-B receptor inhibition improves HFpEF by abrogating adverse cardiac remodeling via antihypertrophic mechanisms and by reducing stiffness. Additional studies are needed to explore the role of dual ET-1 receptor antagonists in patients with HFpEF. © 2016 American Heart Association, Inc.

  5. Discovery of novel S1P2 antagonists. Part 2: Improving the profile of a series of 1,3-bis(aryloxy)benzene derivatives.

    PubMed

    Kusumi, Kensuke; Shinozaki, Koji; Yamaura, Yoshiyuki; Hashimoto, Ai; Kurata, Haruto; Naganawa, Atsushi; Ueda, Hideyuki; Otsuki, Kazuhiro; Matsushita, Takeshi; Sekiguchi, Tetsuya; Kakuuchi, Akito; Seko, Takuya

    2015-10-15

    Our initial lead compound 2 was modified to improve its metabolic stability. The resulting compound 5 showed excellent metabolic stability in rat and human liver microsomes. We subsequently designed and synthesized a hybrid compound of 5 and the 1,3-bis(aryloxy) benzene derivative 1, which was previously reported by our group to be an S1P2 antagonist. This hybridization reaction gave compound 9, which showed improved S1P2 antagonist activity and good metabolic stability. The subsequent introduction of a carboxylic acid moiety into 9 resulted in 14, which showed potent antagonist activity towards S1P2 with a much smaller species difference between human S1P2 and rat S1P2. Compound 14 also showed good metabolic stability and an improved safety profile compared with compound 9. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Angiotensin-(1-7) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stimulating STAT3 and STAT5a/b phosphorylation and inhibiting angiotensin II-stimulated ERK1/2 and Rho kinase activity.

    PubMed

    Giani, Jorge F; Gironacci, Mariela M; Muñoz, Marina C; Turyn, Daniel; Dominici, Fernando P

    2008-05-01

    Angiotensin (ANG) II contributes to cardiac remodelling by inducing the activation of several signalling molecules, including ERK1/2, Rho kinase and members of the STAT family of proteins. Angiotensin-(1-7) is produced in the heart and inhibits the proliferative actions of ANG II, although the mechanisms of this inhibition are poorly understood. Accordingly, in the present study we examined whether ANG-(1-7) affects the ANG II-mediated activation of ERK1/2 and Rho kinase, STAT3 and STAT5a/b in rat heart in vivo. We hypothesized that ANG-(1-7) inhibits these growth-promoting pathways, counterbalancing the trophic action of ANG II. Solutions of normal saline (0.9% NaCl) containing ANG II (8 pmol kg(-1)) plus ANG-(1-7) in increasing doses (from 0.08 to 800 pmol kg(-1)) were administered via the inferior vena cava to anaesthetized male Sprague-Dawley rats. After 5 min, hearts were removed and ERK1/2, Rho kinase, STAT3 and STAT5a/b phosphorylation was determined by Western blotting using phosphospecific antibodies. Angiotensin II stimulated ERK1/2 and Rho kinase phosphorylation (2.3 +/- 0.2- and 2.1 +/- 0.2-fold increase over basal values, respectively), while ANG-(1-7) was without effect. The ANG II-mediated phosphorylation of ERK1/2 and Rho kinase was prevented in a dose-dependent manner by ANG-(1-7) and disappeared in the presence of the Mas receptor antagonist d-Ala7-ANG-(1-7). Both ANG II and ANG-(1-7) increased STAT3 and STAT5a/b phosphorylation to a similar extent (130-140% increase). The ANG-(1-7)-stimulated STAT phosphorylation was blocked by the AT(1) receptor antagonist losartan and not by d-Ala7-ANG-(1-7). Our results show a dual action of ANG-(1-7), that is, a stimulatory effect on STAT3 and 5a/b phosphorylation through AT(1) receptors and a blocking action on ANG II-stimulated ERK1/2 and Rho kinase phosphorylation through Mas receptor activation. The latter effect could be representative of a mechanism for a protective role of ANG-(1-7) in the heart by

  7. Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma*

    PubMed Central

    Johnson, Jeff J.; Miller, Daniel L.; Jiang, Rong; Liu, Yueying; Shi, Zonggao; Tarwater, Laura; Williams, Russell; Balsara, Rashna; Sauter, Edward R.; Stack, M. Sharon

    2016-01-01

    Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors. PMID:26839311

  8. Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma.

    PubMed

    Johnson, Jeff J; Miller, Daniel L; Jiang, Rong; Liu, Yueying; Shi, Zonggao; Tarwater, Laura; Williams, Russell; Balsara, Rashna; Sauter, Edward R; Stack, M Sharon

    2016-03-25

    Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Novel angiotensin receptor blocker, azilsartan induces oxidative stress and NFkB-mediated apoptosis in hepatocellular carcinoma cell line HepG2.

    PubMed

    Ahmadian, Elham; Khosroushahi, Ahmad Yari; Eftekhari, Aziz; Farajnia, Safar; Babaei, Hossein; Eghbal, Mohammad Ali

    2018-03-01

    Overexpression of renin angiotensin system (RAS) components and nuclear factor-kappa B (NF-kB) has a key role in various cancers. Blockade of RAS and NF-kB pathway has been suggested to reduce cancer cell proliferation. This study aimed to investigate the role of angiotensin II and NF-kB pathway in liver hepatocellular carcinoma cell line (HepG2) proliferation by using azilsartan (as a novel Ag II antagonist) and Bay 11-7082 (as NF-kB inhibitor). HepG2 cells were treated with different concentrations of azilsartan and Bay 11-7082. Cytotoxicity was determined after 24, 48, and 72?h by MTT assay. Reactive oxygen spices (ROS) generation and cytochrome c release were measured following azilsartan and Bay11- 7082 treatment. Apoptosis was analyzed qualitatively by DAPI staining and quantitatively through flow cytometry methodologies and Bax and Bcl-2 mRNA and protein levels were assessed by real time PCR and ELISA methods, respectively. The cytotoxic effects of different concentration of azilsartan and Bay11- 7082 on HepG2 cells were observed as a reduction in cell viability, increased ROS formation, cytochrome c release and apoptosis induction. These effects were found to correlate with a shift in Bax level and a downward trend in the expression of Bcl-2. These findings suggest that azilsartan and Bay11- 7082 in combination or alone have strong potential as an agent for prevention or treatment of liver cancer after further studies. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  10. Okadaic acid induces epileptic seizures and hyperphosphorylation of the NR2B subunit of the NMDA receptor in rat hippocampus in vivo.

    PubMed

    Arias, Clorinda; Montiel, Teresa; Peña, Fernando; Ferrera, Patricia; Tapia, Ricardo

    2002-09-01

    Overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors is closely related to epilepsy and excitotoxicity, and the phosphorylation of these receptors may facilitate glutamate-mediated synaptic transmission. Here we show that in awake rats the microinjection into the hippocampus of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, induces in about 20 min intense electroencephalographic and behavioral limbic-type seizures, which are suppressed by the systemic administration of the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate and by the intrahippocampal administration of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinases. Two hours after okadaic acid, when the EEG seizures were intense, an increased serine phosphorylation of some hippocampal proteins, including an enhancement of the serine phosphorylation of the NMDA receptor subunit NR2B, was detected by immunoblotting. Twenty-four hours after okadaic acid a marked destruction of hippocampal CA1 region was observed, which was not prevented by the receptor antagonists. These findings suggest that hyperphosphorylation of glutamate receptors in vivo may result in an increased sensitivity to the endogenous transmitter and therefore induce neuronal hyperexcitability and epilepsy.

  11. Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III. Agonist and antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes.

    PubMed

    Newman-Tancredi, Adrian; Cussac, Didier; Quentric, Yann; Touzard, Manuelle; Verrièle, Laurence; Carpentier, Nathalie; Millan, Mark J

    2002-11-01

    Although certain antiparkinson agents interact with serotonin (5-HT) receptors, little information is available concerning functional actions. Herein, we characterized efficacies of apomorphine, bromocriptine, cabergoline, lisuride, piribedil, pergolide, roxindole, and terguride at human (h)5-HT(1A), h5-HT(1B), and h5-HT(1D) receptors [guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding], and at h5-HT(2A), h5-HT(2B), and h5-HT(2C) receptors (depletion of membrane-bound [(3)H]phosphatydilinositol). All drugs stimulated h5-HT(1A) receptors with efficacies (compared with 5-HT, 100%) ranging from modest (apomorphine, 35%) to high (cabergoline, 93%). At h5-HT(1B) receptors, efficacies varied from mild (terguride, 37%) to marked (cabergoline, 102%) and potencies were modest (pEC(50) values of 5.8-7.6): h5-HT(1D) sites were activated with a similar range of efficacies and greater potency (7.1-8.5). Piribedil and apomorphine were inactive at h5-HT(1B) and h5-HT(1D) receptors. At h5-HT(2A) receptors, terguride, lisuride, bromocriptine, cabergoline, and pergolide displayed potent (7.6-8.8) agonist properties (49-103%), whereas apomorphine and roxindole were antagonists and piribedil was inactive. Only pergolide (113%/8.2) and cabergoline (123%/8.6) displayed pronounced agonist properties at h5-HT(2B) receptors. At 5-HT(2C) receptors, lisuride, bromocriptine, pergolide, and cabergoline were efficacious (75-96%) agonists, apomorphine and terguride were antagonists, and piribedil was inactive. MDL100,907 and SB242,084, selective antagonists at 5-HT(2A) and 5-HT(2C) receptors, respectively, abolished these actions of pergolide, cabergoline, and bromocriptine. In conclusion, antiparkinson agents display markedly different patterns of agonist and antagonist properties at multiple 5-HT receptor subtypes. Although all show modest (agonist) activity at 5-HT(1A) sites, their contrasting actions at 5-HT(2A) and 5-HT(2C) sites may be of particular significance to their

  12. Genetic variations in UGT2B28, UGT2B17, UGT2B15 genes and the risk of prostate cancer: A case-control study.

    PubMed

    Habibi, Mohsen; Mirfakhraie, Reza; Khani, Maryam; Rakhshan, Azadeh; Azargashb, Eznollah; Pouresmaeili, Farkhondeh

    2017-11-15

    Glucuronidation is a major pathway for elimination of exogenous and endogenous compounds such as environmental carcinogens and androgens from the body. This biochemical pathway is mediated by enzymes called uridine diphosphoglucuronosyltransferases (UGTs). Null (del/del) genes polymorphisms in UGT2B17, and UGT2B28 and D85Y single-nucleotide polymorphism (SNP) of UGT2B15 have been reported to increase the risk of prostate cancer. The goal of this study was to determine the association of mentioned genetic variants with the risk of prostate cancer. We investigated the copy number variations (CNVs) of UGT2B17 and UGT2B28 loci and the association between rs1902023 polymorphism of UGT2B15 gene in 360 subjects consisted of 120 healthy controls, 120 prostate cancer (PC) patients and 120 benign prostatic hyperplasia (BPH) patients. No association was detected for the mentioned polymorphisms and the risk of PC. However, a significant association was detected between UGT2B17 copy number variation and BPH risk (OR=2.189; 95% CI, 1.303-3.675; p=0.003). Furthermore, we observed that the D85Y polymorphism increases the risk of BPH when analyzed in combination with the copy number variation of UGT2B17 gene (OR=0.135; 95% CI, 0.036-0.512; p=0.003). Our findings suggest that the D85Y polymorphism of UGT2B15 and CNVs in UGT2B28 and UGT2B17 genes is not associated with prostate cancer risk in Iranian patients. To our knowledge, this is the first report that implicates the role of CNV of UGT2B17 gene in BPH. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Small Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy

    PubMed Central

    Han, Bingshe; Park, Dongkyoo; Li, Rui; Xie, Maohua; Owonikoko, Taofeek K.; Zhang, Guojing; Sica, Gabriel L.; Ding, Chunyong; Zhou, Jia; Magis, Andrew T.; Chen, Zhuo G.; Shin, Dong M.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Curran, Walter J.; Deng, Xingming

    2015-01-01

    SUMMARY The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a small molecule Bcl2-BH4 domain-antagonist (BDA-366) that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival to a cell death inducer. BDA-366 suppresses growth of lung cancer xenografts derived from cell lines and patient without significant normal tissue toxicity at effective doses. mTOR inhibition up-regulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a strategy to improve lung cancer outcome. PMID:26004684

  14. ST 1535: a preferential A2A adenosine receptor antagonist.

    PubMed

    Stasi, Maria Antonietta; Borsini, Franco; Varani, Katia; Vincenzi, Fabrizio; Di Cesare, Maria Assunta; Minetti, Patrizia; Ghirardi, Orlando; Carminati, Paolo

    2006-10-01

    Antagonism of the A2A adenosine function has proved beneficial in the treatment of Parkinson's disease, in that it increases L-dopa therapeutical effects without concomitant worsening of its side-effects. In this paper we describe a preferential A2A adenosine antagonist, ST 1535, with long-lasting pharmacodynamic effects. It competitively antagonizes the effects of the A2A adenosine agonist NECA on cAMP in cells cloned with the human A2A adenosine receptor (IC50=353+/-30 nM), and the effects of the A1 adenosine agonist CHA on cAMP in cells cloned with the human A1 adenosine receptor (IC50=510+/-38 nM). ST 1535, at oral doses of 5 and 10 mg/kg, antagonizes catalepsy induced by intracerebroventricular administration of the A2A adenosine agonist CGS 21680 (10 microg/5 microl) in mice. At oral doses ranging between 5 and 20 mg/kg, ST 1535 induces hypermotility and antagonizes haloperidol-induced catalepsy in mice up to 7 h. Oral ST 1535, at 1.25 and 2.5 mg/kg, potentiates L-dopa effects in reducing haloperidol-induced catalepsy. ST 1535 represents a potential new compound, with long-lasting activity, for the treatment of Parkinson's disease.

  15. Comparative Analysis of Virtual Screening Approaches in the Search for Novel EphA2 Receptor Antagonists.

    PubMed

    Callegari, Donatella; Pala, Daniele; Scalvini, Laura; Tognolini, Massimiliano; Incerti, Matteo; Rivara, Silvia; Mor, Marco; Lodola, Alessio

    2015-09-17

    The EphA2 receptor and its ephrin-A1 ligand form a key cell communication system, which has been found overexpressed in many cancer types and involved in tumor growth. Recent medicinal chemistry efforts have identified bile acid derivatives as low micromolar binders of the EphA2 receptor. However, these compounds suffer from poor physicochemical properties, hampering their use in vivo. The identification of compounds able to disrupt the EphA2-ephrin-A1 complex lacking the bile acid scaffold may lead to new pharmacological tools suitable for in vivo studies. To identify the most promising virtual screening (VS) protocol aimed at finding novel EphA2 antagonists, we investigated the ability of both ligand-based and structure-based approaches to retrieve known EphA2 antagonists from libraries of decoys with similar molecular properties. While ligand-based VSs were conducted using UniPR129 and ephrin-A1 ligand as reference structures, structure-based VSs were performed with Glide, using the X-ray structure of the EphA2 receptor/ephrin-A1 complex. A comparison of enrichment factors showed that ligand-based approaches outperformed the structure-based ones, suggesting ligand-based methods using the G-H loop of ephrin-A1 ligand as template as the most promising protocols to search for novel EphA2 antagonists.

  16. A New Small-Molecule Antagonist Inhibits Graves' Disease Antibody Activation of the TSH Receptor

    PubMed Central

    Eliseeva, Elena; McCoy, Joshua G.; Napolitano, Giorgio; Giuliani, Cesidio; Monaco, Fabrizio; Huang, Wenwei; Gershengorn, Marvin C.

    2011-01-01

    Context: Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. Objective: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. Design: We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. Results: We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. Conclusion: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera. PMID:21123444

  17. A new small-molecule antagonist inhibits Graves' disease antibody activation of the TSH receptor.

    PubMed

    Neumann, Susanne; Eliseeva, Elena; McCoy, Joshua G; Napolitano, Giorgio; Giuliani, Cesidio; Monaco, Fabrizio; Huang, Wenwei; Gershengorn, Marvin C

    2011-02-01

    Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

  18. Dopamine D3 and D2 Receptor Mechanisms in the Abuse-Related Behavioral Effects of Cocaine: Studies with Preferential Antagonists in Squirrel Monkeys

    PubMed Central

    Grundt, Peter; Cao, Jianjing; Platt, Donna M.; Newman, Amy Hauck; Spealman, Roger D.

    2010-01-01

    Dopamine (DA) D3 and D2 receptor mechanisms are implicated in cocaine's abuse-related behavioral effects, but the relative contribution of the two receptor subtypes is only partially characterized. This study investigated the role of D3 and D2 subtype mechanisms by determining the degree to which the D3-preferring antagonist PG01037 [N-{4-[4-(2,3-dichlorophenyl)-piperazin- 1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl] and the D2-preferring antagonist L-741626 [3-[4-(4-chlorophenyl)-4- hydroxypiperidin-1-yl]methyl-1H-indole] attenuated several behavioral effects of cocaine in squirrel monkeys. Quantitative observational studies established doses of each antagonist that did not produce untoward effects, which were used in subsequent comparisons. In addition, the ability of the D3-preferring agonist PD128907 [(R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol)] and the D2-preferring agonist sumanirole [(R)-5,6-dihydro-5-(methylamino)-4H- imidazo[4,5,1-ij]quinolin-2(1H)-one(Z)-2-butenedioate] to reproduce cocaine's discriminative stimulus (DS) and priming effects were compared. In monkeys trained to discriminate cocaine from vehicle, both DA antagonists attenuated and both DA agonists partially reproduced cocaine's DS effects. PG01037 also selectively attenuated the cocaine-like DS effects of PD128907, whereas L-741626 attenuated the cocaine-like DS effects of both agonists. In self-administration studies, L-741626 nonselectively reduced cocaine- and food-maintained responding, whereas PG01037 was ineffective against either reinforcer. In studies involving reinstatement of extinguished cocaine seeking, both antagonists attenuated cocaine-induced reinstatement of responding, and both agonists induced at least partial reinstatement of cocaine seeking. L-741626 also attenuated sumanirole-induced, but not PD128907-induced, reinstatement of responding, whereas PG01037 was ineffective against either DA agonist. The results are

  19. Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

    PubMed

    Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

    2015-07-01

    In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

  20. Adenosine-diphosphate (ADP) receptor antagonists for the prevention of cardiovascular disease in type 2 diabetes mellitus.

    PubMed

    Valentine, Nyoli; Van de Laar, Floris A; van Driel, Mieke L

    2012-11-14

    Cardiovascular disease (CVD) is the most prevalent complication of type 2 diabetes with an estimated 65% of people with type 2 diabetes dying from a cause related to atherosclerosis. Adenosine-diphosphate (ADP) receptor antagonists like clopidogrel, ticlopidine, prasugrel and ticagrelor impair platelet aggregation and fibrinogen-mediated platelet cross-linking and may be effective in preventing CVD. To assess the effects of adenosine-diphosphate (ADP) receptor antagonists for the prevention of cardiovascular disease in type 2 diabetes mellitus. We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library (issue 2, 2011), MEDLINE (until April 2011) and EMBASE (until May 2011). We also performed a manual search, checking references of original articles and pertinent reviews to identify additional studies. Randomised controlled trials comparing an ADP receptor antagonist with another antiplatelet agent or placebo for a minimum of 12 months in patients with diabetes. In particular, we looked for trials assessing clinical cardiovascular outcomes. Two review authors extracted data for studies which fulfilled the inclusion criteria, using standard data extraction templates. We sought additional unpublished information and data from the principal investigators of all included studies. Eight studies with a total of 21,379 patients with diabetes were included. Three included studies investigated ticlopidine compared to aspirin or placebo. Five included studies investigated clopidogrel compared to aspirin or a combination of aspirin and dipyridamole, or compared clopidogrel in combination with aspirin to aspirin alone. All trials included patients with previous CVD except the CHARISMA trial which included patients with multiple risk factors for coronary artery disease. Overall the risk of bias of the trials was low. The mean duration of follow-up ranged from 365 days to 913 days.Data for diabetes patients on all-cause mortality, vascular

  1. The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

    PubMed Central

    Lin, Olivia A.; Karim, Zubair A.; Vemana, Hari Priya; Espinosa, Enma V. P.; Khasawneh, Fadi T.

    2014-01-01

    There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their

  2. Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy.

    PubMed

    Sykes, David A; Bradley, Michelle E; Riddy, Darren M; Willard, Elizabeth; Reilly, John; Miah, Asadh; Bauer, Carsten; Watson, Simon J; Sandham, David A; Dubois, Gerald; Charlton, Steven J

    2016-05-01

    Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [(3)H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2(PGD2). The binding kinetics of QAW039 determined directly using [(3)H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 10(7)M(-1)min(-1)and 0.048 minute(-1), respectively. Importantly, thekoffof QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [(35)S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2concentrations, which may be clinically relevant. Copyright © 2016 by The American Society for Pharmacology and Experimental

  3. A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins.

    PubMed

    Bergamo, Elisa; Diani, Erica; Bertazzoni, Umberto; Romanelli, Maria Grazia

    2017-01-01

    HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.

  4. Estrogen Receptor α L543A,L544A Mutation Changes Antagonists to Agonists, Correlating with the Ligand Binding Domain Dimerization Associated with DNA Binding Activity*

    PubMed Central

    Arao, Yukitomo; Hamilton, Katherine J.; Coons, Laurel A.; Korach, Kenneth S.

    2013-01-01

    A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα. PMID:23733188

  5. Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB.

    PubMed

    Prasad, Rajapaksha Gedara; Choi, Yung Hyun; Kim, Gi-Young

    2015-03-01

    According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway.

  6. Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB

    PubMed Central

    Prasad, Rajapaksha Gedara; Choi, Yung Hyun; Kim, Gi-Young

    2015-01-01

    According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE2, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway. PMID:25767678

  7. Penguin-mediated exclusive hadronic weak B decays

    NASA Astrophysics Data System (ADS)

    Deshpande, N. G.; Trampetic, J.

    1990-02-01

    We estimate a number of exclusive two-body charmless decays of B+ and B- mesons. Some of these are mediated predominantly through one-loop gluon exchange, while others have a comparable or larger contribution from the doubly Cabibbo-suppressed tree diagrams. The rates for several decays are in an observable range and should test the standard model.

  8. MicroRNA-29b regulates TGF-β1-mediated epithelial–mesenchymal transition of retinal pigment epithelial cells by targeting AKT2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Min; Li, Hui; Liu, Xiaoqiang

    2016-07-15

    The role of microRNA (miRNA) in proliferative vitreoretinopathy (PVR) progression has not been studied extensively, especially in retinal pigment epithelial–mesenchymal transition (EMT) which is the main reason for formation of PVR. In this study, we first investigated the miRNA expression profile in transforming growth factor beta 1 (TGF-β1) mediated EMT of ARPE-19 cells. Among the five changed miRNAs, miR-29b showed the most significant downregulation. Enhanced expression of miR-29b could reverse TGF-β1 induced EMT through targeting Akt2. Akt2 downregulation could inhibit TGF-β1-induced EMT. Furthermore, inhibition of miR-29b in ARPE-19 cells directly triggered EMT process, which characterized by the phenotypic transition andmore » the upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin and zona occludin-1 (ZO-1) with increased cell migration. Akt2-shRNA also inhibited miR-29 inhibitor-induced EMT process. These data indicate that miR-29b plays an important role in TGF-β1-mediated EMT in ARPE-19 cells by targeting Akt2. - Highlights: • MiR-29b expression is decreased in TGF-β1-induced EMT of ARPE-19 cells. • MiR-29b inhibits TGF-β1-induced EMT in ARPE-19 cells. • MiR-29b inhibitor induces EMT in ARPE-19 cells. • Akt2 is the target for miR-29b. • Downregulation of Akt2 prevents TGF-β1-induced EMT of ARPE-19 cells.« less

  9. E2F1 and NF-κB: Key Mediators of Inflammation-associated Cancers and Potential Therapeutic Targets.

    PubMed

    Huang, Yulin; Chen, Rui; Zhou, Jianwei

    2016-01-01

    Inflammation is the fundamental protective response; however disordered immuno-response can cause chronic human disease, including cancer. Inflammatory cells and mediators are essential to the tumor microenvironment and dissection of this complex molecular and cellular milieu may elucidate a connection between cancer and inflammation and help to identify potential novel therapeutic targets. Thus, focusing on transcription factor NF-κB and E2F1 in inflammation-associated cancer is urgent. NF-κB activation is prevalent in carcinomas, mainly driven by inflammatory cytokines in the tumor microenvironment. E2F1 is also involved in regulating immune responses. Understanding the crosstalk between the two pathways may contribute to the development of novel anti-cancer drugs.

  10. A new and specific non-NMDA receptor antagonist, FG 9065, blocks L-AP4-evoked depolarization in rat cerebral cortex.

    PubMed

    Sheardown, M J

    1988-04-13

    L(+)-AP4 (2-amino-4-phosphonobutyrate) depolarized slices of rat cerebral cortex, when applied following a 2 min priming application of quisqualate. This response diminishes with time and is not seen after NMDA application. A new selective non-N-methyl-D-aspartate (NMDA) antagonist, 6-cyano-7-nitro-2,3-dihydroxyquinoxaline (FG 9065), inhibits the L(+)-AP4 depolarization. It is argued that the response is mediated indirectly by postsynaptic quisqualate receptors.

  11. Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids.

    PubMed

    Blanc, Landry; Gilleron, Martine; Prandi, Jacques; Song, Ok-Ryul; Jang, Mi-Seon; Gicquel, Brigitte; Drocourt, Daniel; Neyrolles, Olivier; Brodin, Priscille; Tiraby, Gérard; Vercellone, Alain; Nigou, Jérôme

    2017-10-17

    Mycobacterium tuberculosis is a major human pathogen that is able to survive inside host cells and resist immune clearance. Most particularly, it inhibits several arms of the innate immune response, including phagosome maturation or cytokine production. To better understand the molecular mechanisms by which M. tuberculosis circumvents host immune defenses, we used a transposon mutant library generated in a virulent clinical isolate of M. tuberculosis of the W/Beijing family to infect human macrophages, utilizing a cell line derivative of THP-1 cells expressing a reporter system for activation of the transcription factor NF-κB, a key regulator of innate immunity. We identified several M. tuberculosis mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be deficient for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latter can interfere with innate immune responses. Using natural and synthetic molecular variants, we determined that sulfoglycolipids inhibit NF-κB activation and subsequent cytokine production or costimulatory molecule expression by acting as competitive antagonists of Toll-like receptor 2, thereby inhibiting the recognition of M. tuberculosis by this receptor. Our study reveals that producing glycolipid antagonists of pattern recognition receptors is a strategy used by M. tuberculosis to undermine innate immune defense. Sulfoglycolipids are major and specific lipids of M. tuberculosis , considered for decades as virulence factors of the bacilli. Our study uncovers a mechanism by which they may contribute to M. tuberculosis virulence.

  12. The Novel μ-Opioid Receptor Antagonist, [N-Allyl-Dmt1]Endomorphin-2, Attenuates the Enhancement of GABAergic Neurotransmission by Ethanol

    PubMed Central

    Li, Qiang; Okada, Yoshio; Marczak, Ewa; Wilson, Wilkie A.; Lazarus, Lawrence H.; Swartzwelder, H. S.

    2009-01-01

    Aims: We investigated the effects of [N-allyl-Dmt1]endomorphin-2 (TL-319), a novel and highly potent μ-opioid receptor antagonist, on ethanol (EtOH)-induced enhancement of GABAA receptor-mediated synaptic activity in the hippocampus. Methods: Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were isolated from CA1 pyramidal cells from brain slices of male rats using whole-cell patch-clamp techniques. Results: TL-319 had no effect on the baseline amplitude of eIPSCs or the frequency of sIPSCs. However, it induced a dose-dependent suppression of an ethanol-induced increase of sIPSC frequency with full reversal at concentrations of 500 nM and higher. The non-specific competitive opioid receptor antagonist naltrexone also suppressed EtOH-induced increases in sIPSC frequency but only at a concentration of 60 μM. Conclusion: These data indicate that blockade of μ-opioid receptors by low concentrations of [N-allyl-Dmt1]endomorphin-2 can reverse ethanol-induced increases in GABAergic neurotransmission and possibly alter its anxiolytic or sedative effects. This suggests the possibility that high potency opioid antagonists may emerge as possible candidate compounds for the treatment of ethanol addiction. PMID:18971291

  13. Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells.

    PubMed

    Park, Sun Joo; Kim, Yong Tae; Jeon, You Jin

    2012-04-01

    Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H(2)O(2) treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H(2)O(2)-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47(phox). Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.

  14. Antioxidant Dieckol Downregulates the Rac1/ROS Signaling Pathway and Inhibits Wiskott-Aldrich Syndrome Protein (WASP)-Family Verprolin-Homologous Protein 2 (WAVE2)-Mediated Invasive Migration of B16 Mouse Melanoma Cells

    PubMed Central

    Park, Sun Joo; Kim, Yong Tae; Jeon, You Jin

    2012-01-01

    Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H2O2 treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H2O2-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol. PMID:22441674

  15. The adenosine A2A antagonist MSX-3 reverses the effects of the dopamine antagonist haloperidol on effort-related decision making in a T-maze cost/benefit procedure

    PubMed Central

    Mott, Allison M.; Nunes, Eric J.; Collins, Lyndsey E.; Port, Russell G.; Sink, Kelly S.; Hockemeyer, Jörg; Müller, Christa E.

    2010-01-01

    Rationale Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Research involving choice tasks has shown that rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements and instead select less effortful food-seeking behaviors. Objective Previous work showed that adenosine A2A antagonism can reverse the effects of the DA antagonist haloperidol in an operant task that assesses effort-related choice. The present work used a T-maze choice procedure to assess the effects of adenosine A2A and A1 antagonism. Materials and methods With this task, the two arms of the maze have different reinforcement densities (four vs. two food pellets), and a vertical 44 cm barrier is positioned in the arm with the higher density, presenting the animal with an effort-related challenge. Untreated rats strongly prefer the arm with the high density of food reward and climb the barrier in order to obtain the food. Results Haloperidol produced a dose-related (0.05–0.15 mg/kg i.p.) reduction in the number of trials in which the rats chose the high-barrier arm. Co-administration of the adenosine A2A receptor antagonist MSX-3 (0.75, 1.5, and 3.0 mg/kg i.p.), but not the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.75, 1.5, and 3.0 mg/kg i.p.), reversed the effects of haloperidol on effort-related choice and latency. Conclusions Adenosine A2A and D2 receptors interact to regulate effort-related decision making, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing or anergia that can be observed in depression, parkinsonism, and other disorders. PMID:19132351

  16. The adenosine A2A antagonist MSX-3 reverses the effects of the dopamine antagonist haloperidol on effort-related decision making in a T-maze cost/benefit procedure.

    PubMed

    Mott, Allison M; Nunes, Eric J; Collins, Lyndsey E; Port, Russell G; Sink, Kelly S; Hockemeyer, Jörg; Müller, Christa E; Salamone, John D

    2009-05-01

    Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Research involving choice tasks has shown that rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A(2A) antagonism can reverse the effects of the DA antagonist haloperidol in an operant task that assesses effort-related choice. The present work used a T-maze choice procedure to assess the effects of adenosine A(2A) and A(1) antagonism. With this task, the two arms of the maze have different reinforcement densities (four vs. two food pellets), and a vertical 44 cm barrier is positioned in the arm with the higher density, presenting the animal with an effort-related challenge. Untreated rats strongly prefer the arm with the high density of food reward and climb the barrier in order to obtain the food. Haloperidol produced a dose-related (0.05-0.15 mg/kg i.p.) reduction in the number of trials in which the rats chose the high-barrier arm. Co-administration of the adenosine A(2A) receptor antagonist MSX-3 (0.75, 1.5, and 3.0 mg/kg i.p.), but not the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.75, 1.5, and 3.0 mg/kg i.p.), reversed the effects of haloperidol on effort-related choice and latency. Adenosine A(2A) and D2 receptors interact to regulate effort-related decision making, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing or anergia that can be observed in depression, parkinsonism, and other disorders.

  17. Association between Kinin B1 Receptor Expression and Leukocyte Trafficking across Mouse Mesenteric Postcapillary Venules

    PubMed Central

    McLean, Peter G.; Ahluwalia, Amrita; Perretti, Mauro

    2000-01-01

    Using intravital microscopy, we examined the role played by B1 receptors in leukocyte trafficking across mouse mesenteric postcapillary venules in vivo. B1 receptor blockade attenuated interleukin (IL)-1β–induced (5 ng intraperitoneally, 2 h) leukocyte–endothelial cell interactions and leukocyte emigration (∼50% reduction). The B1 receptor agonist des-Arg9bradykinin (DABK), although inactive in saline- or IL-8–treated mice, caused marked neutrophil rolling, adhesion, and emigration 24 h after challenge with IL-1β (when the cellular response to IL-1β had subsided). Reverse transcriptase polymerase chain reaction and Western blot revealed a temporal association between the DABK-induced response and upregulation of mesenteric B1 receptor mRNA and de novo protein expression after IL-1β treatment. DABK-induced leukocyte trafficking was antagonized by the B1 receptor antagonist des-arg10HOE 140 but not by the B2 receptor antagonist HOE 140. Similarly, DABK effects were maintained in B2 receptor knockout mice. The DABK-induced responses involved the release of neuropeptides from C fibers, as capsaicin treatment inhibited the responses. Treatment with the neurokinin (NK)1 and NK3 receptor antagonists attenuated the responses, whereas NK2, calcitonin gene-related peptide, or platelet-activating factor receptor antagonists had no effect. Substance P caused leukocyte recruitment that, similar to DABK, was inhibited by NK1 and NK3 receptor blockade. Mast cell depletion using compound 48/80 reduced DABK-induced leukocyte trafficking, and DABK treatment was shown histologically to induce mast cell degranulation. DABK-induced trafficking was inhibited by histamine H1 receptor blockade. Our findings provide clear evidence that B1 receptors play an important role in the mediation of leukocyte–endothelial cell interactions in postcapillary venules, leading to leukocyte recruitment during an inflammatory response. This involves activation of C fibers and mast cells

  18. Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268.

    PubMed

    Sutton, Laurie P; Rushlow, Walter J

    2011-06-01

    Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and β-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs). © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  19. 3D-QSAR studies on 1,2,4-triazolyl 5-azaspiro [2.4]-heptanes as D3R antagonists

    NASA Astrophysics Data System (ADS)

    Zhang, Xin; Zhang, Hui

    2018-07-01

    Dopamine D3 receptor has become an attractive target in the treatment of abused drugs. 3D-QSAR studies were performed on a novel series of D3 receptor antagonists, 1,2,4-triazolyl 5-azaspiro [2.4]-heptanes, using CoMFA and CoMSIA methods. Two predictive 3D-QSAR models have been generated for the modified design of D3R antagonists. Based on the steric, electrostatic, hydrophobic and hydrogen-bond acceptor information of contour maps, key structural factors affecting the bioactivity were explored. This work gives helpful suggestions on the design of novel D3R antagonists with increased activities.

  20. NMDARs Mediate the Role of Monoamine Oxidase A in Pathological Aggression

    PubMed Central

    Bortolato, Marco; Godar, Sean C.; Melis, Miriam; Soggiu, Alessio; Roncada, Paola; Casu, Angelo; Flore, Giovanna; Chen, Kevin; Frau, Roberto; Urbani, Andrea; Castelli, M. Paola; Devoto, Paola; Shih, Jean C.

    2012-01-01

    Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25–6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality. PMID:22723698

  1. NMDARs mediate the role of monoamine oxidase A in pathological aggression.

    PubMed

    Bortolato, Marco; Godar, Sean C; Melis, Miriam; Soggiu, Alessio; Roncada, Paola; Casu, Angelo; Flore, Giovanna; Chen, Kevin; Frau, Roberto; Urbani, Andrea; Castelli, M Paola; Devoto, Paola; Shih, Jean C

    2012-06-20

    Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25-6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality.

  2. Rational Design, Pharmacomodulation, and Synthesis of Dual 5-Hydroxytryptamine 7 (5-HT7)/5-Hydroxytryptamine 2A (5-HT2A) Receptor Antagonists and Evaluation by [(18)F]-PET Imaging in a Primate Brain.

    PubMed

    Deau, Emmanuel; Robin, Elodie; Voinea, Raluca; Percina, Nathalie; Satała, Grzegorz; Finaru, Adriana-Luminita; Chartier, Agnès; Tamagnan, Gilles; Alagille, David; Bojarski, Andrzej J; Morisset-Lopez, Séverine; Suzenet, Franck; Guillaumet, Gérald

    2015-10-22

    We report the synthesis of 46 tertiary amine-bearing N-alkylated benzo[d]imidazol-2(3H)-ones, imidazo[4,5-b]pyridin-2(3H)-ones, imidazo[4,5-c]pyridin-2(3H)-ones, benzo[d]oxazol-2(3H)-ones, oxazolo[4,5-b]pyridin-2(3H)-ones and N,N'-dialkylated benzo[d]imidazol-2(3H)-ones. These compounds were evaluated against 5-HT7R, 5-HT2AR, 5-HT1AR, and 5-HT6R as potent dual 5-HT7/5-HT2A serotonin receptors ligands. A thorough study of the structure-activity relationship of the aromatic rings and their substituents, the alkyl chain length and the tertiary amine was conducted. 1-(4-(4-(4-Fluorobenzoyl)piperidin-1-yl)butyl)-1H-benzo[d]imidazol-2(3H)-one (79) and 1-(6-(4-(4-fluorobenzoyl)piperidin-1-yl)hexyl)-1H-benzo[d]imidazol-2(3H)-one (81) were identified as full antagonist ligands on cyclic adenosine monophosphate (cAMP, KB = 4.9 and 5.9 nM, respectively) and inositol monophosphate (IP1, KB = 0.6 and 16 nM, respectively) signaling pathways of 5-HT7R and 5-HT2AR. Both antagonists crossed the blood-brain barrier as evaluated with [(18)F] radiolabeled compounds [(18)F]79 and [(18)F]81 in a primate's central nervous system using positron emission tomography. Both radioligands showed standard uptake values ranging from 0.8 to 1.1, a good plasmatic stability, and a distribution consistent with 5-HT7R and 5-HT2AR in the CNS.

  3. MicroRNA-10b downregulation mediates acute rejection of renal allografts by derepressing BCL2L11

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Xiaoyou; Dong, Changgui; Jiang, Zhengyao

    Kidney transplantation is the major therapeutic option for end-stage kidney diseases. However, acute rejection could cause allograft loss in some of these patients. Emerging evidence supports that microRNA (miRNA) dysregulation is implicated in acute allograft rejection. In this study, we used next-generation sequencing to profile miRNA expression in normal and acutely rejected kidney allografts. Among 75 identified dysregulated miRNAs, miR-10b was the most significantly downregulated miRNAs in rejected allografts. Transfecting miR-10b inhibitor into human renal glomerular endothelial cells recapitulated key features of acute allograft rejection, including endothelial cell apoptosis, release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor α, interferon-γ, andmore » chemokine (C–C motif) ligand 2) and chemotaxis of macrophages whereas transfection of miR-10b mimics had opposite effects. Downregulation of miR-10b directly derepressed the expression of BCL2L11 (an apoptosis inducer) as revealed by luciferase reporter assay. Taken together, miR-10b downregulation mediates many aspects of disease pathogenicity of acute kidney allograft rejection. Restoring miR-10b expression in glomerular endothelial cells could be a novel therapeutic approach to reduce acute renal allograft loss. - Highlights: • miR-10b was the most downregulated microRNAs in acutely rejected renal allografts. • miR-10b downregulation triggered glomerular endothelial cell apoptosis. • miR-10b downregulation induced release of pro-inflammatory cytokines. • miR-10b downregulation derepressed its pro-apoptotic target BCL2L11.« less

  4. Inhibition of the superantigenic activities of Staphylococcal enterotoxin A by an aptamer antagonist.

    PubMed

    Wang, Kaiyu; Wu, Dong; Chen, Zhuang; Zhang, Xianhui; Yang, Xiangyue; Yang, Chaoyong James; Lan, Xiaopeng

    2016-09-01

    Staphylococcal enterotoxin A (SEA) is an important component of Staphylococcus aureus pathogenesis. SEA induces T lymphocytes activation and proliferation, resulting in the release of a large number of inflammatory cytokines. Blocking the toxic cascade triggered by SEA may be an effective strategy for the treatment of SEA-induced diseases. Through a systematic evolution of ligands by exponential enrichment process, we obtained an aptamer (S3) that could bind SEA with both high affinity and specificity, with a Kd value 36.93 ± 7.29 nM (n = 3). This aptamer antagonist effectively inhibited SEA-mediated human peripheral blood mononuclear cells proliferation and inflammatory cytokines (IFN-γ, TNF-α, IL-2 and IL-6) secretion. Moreover, PEGylated S3 significantly reduced mortality in murine lethal toxic shock models established by lipopolysaccharide-potentiated SEA. Therefore, this novel aptamer antagonist has the potential to become a new strategy for treating S. aureus infections and SEA-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Decavanadate, a P2X receptor antagonist, and its use to study ligand interactions with P2X7 receptors.

    PubMed

    Michel, Anton D; Xing, Mengle; Thompson, Kyla M; Jones, Clare A; Humphrey, Patrick P A

    2006-03-18

    In this study we have studied decavanadate effects at P2X receptors. Decavanadate competitively blocked 2'- and 3'-O-(4benzoylbenzoyl) ATP (BzATP) stimulated ethidium accumulation in HEK293 cells expressing human recombinant P2X7 receptors (pK(B) 7.5). The effects of decavanadate were rapid (minutes) in both onset and offset and contrasted with the much slower kinetics of pyridoxal 5-phosphate (P5P), Coomassie brilliant blue (CBB) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62). Decavanadate competitively blocked the slowly reversible, or irreversible, blockade of the P2X7 receptor produced by P5P and oxidised ATP suggesting competition for a common binding site. However, the interaction between decavanadate and KN62 was non-competitive. Decavanadate also blocked P2X2 and P2X4 receptors but with slightly lower potency. These data demonstrate that decavanadate is the first reversible and competitive antagonist of the P2X7 receptor and is a useful tool for studying the mechanism of interaction of ligands with the P2X7 receptor.

  6. Lycopene inhibits NF-κB activation and adhesion molecule expression through Nrf2-mediated heme oxygenase-1 in endothelial cells.

    PubMed

    Yang, Po-Min; Chen, Huang-Zhi; Huang, Yu-Ting; Hsieh, Chia-Wen; Wung, Being-Sun

    2017-06-01

    The endothelial expression of cell adhesion molecules plays a leading role in atherosclerosis. Lycopene, a carotenoid with 11 conjugated double bonds, has been shown to have anti-inflammatory properties. In the present study, we demonstrate a putative mechanism for the anti-inflammatory effects of lycopene. We demonstrate that lycopene inhibits the adhesion of tumor necrosis factor α (TNFα)-stimulated monocytes to endothelial cells and suppresses the expression of intercellular cell adhesion molecule-1 (ICAM-1) at the transcriptional level. Moreover, lycopene was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein, IκBα, following 6 h of pre-treatment. In TNFα-stimulated endothelial cells, nuclear factor-κB (NF-κB) nuclear translocation and transcriptional activity were abolished by up to 12 h of lycopene pre-treatment. We also found that lycopene increased the intracellular glutathione (GSH) level and glutamate-cysteine ligase expression. Subsequently, lycopene induced nuclear factor-erythroid 2 related factor 2 (Nrf2) activation, leading to the increased expression of downstream of heme oxygenase-1 (HO-1). The use of siRNA targeting HO-1 blocked the inhibitory effects of lycopene on IκB degradation and ICAM-1 expression. The inhibitory effects of lycopene thus appear to be mediated through its induction of Nrf2-mediated HO-1 expression. Therefore, the findings of the present study indicate that lycopene suppresses the activation of TNFα-induced signaling pathways through the upregulation of Nrf2-mediated HO-1 expression.

  7. beta(2)-adrenoceptor antagonist ICI 118,551 decreases pulmonary vascular tone in mice via a G(i/o) protein/nitric oxide-coupled pathway.

    PubMed

    Wenzel, Daniela; Knies, Ralf; Matthey, Michaela; Klein, Alexandra M; Welschoff, Julia; Stolle, Vanessa; Sasse, Philipp; Röll, Wilhelm; Breuer, Johannes; Fleischmann, Bernd K

    2009-07-01

    beta(2)-adrenoceptors are important modulators of vascular tone, particularly in the pulmonary circulation. Because neurohormonal activation occurs in pulmonary arterial hypertension, we have investigated the effect of different adrenergic vasoactive substances on tone regulation in large and small pulmonary arteries, as well as in systemic vessels of mice. We found that the beta(2)-adrenoceptor antagonist ICI 118,551 (ICI) evoked a decrease of vascular tone in large pulmonary arteries and reduced the sensitivity of pulmonary arteries toward different contracting agents, eg, norepinephrine, serotonin, or endothelin. ICI proved to act specifically on pulmonary vessels, because it shifted the dose-response curve of norepinephrine to the right in pulmonary arteries, whereas there was no effect in the aorta. Pharmacological experiments proved that the right shift of the norepinephrine dose-response curve by ICI was mediated via a beta(2)-adrenoceptor/G(i/o) protein-dependent pathway enhancing NO production in the endothelium; these results were corroborated in beta-adrenoceptor and endothelial NO synthase knockout mice where ICI had no effect. ICI increased vascular lumen diameter in lung sections and reduced pulmonary arterial pressure under normoxia and under hypoxia in the isolated perfused lung model. These effects were found to be physiologically relevant, because ICI specifically decreased pulmonary but not systemic blood pressure in vivo. Thus, the beta(2)-adrenoceptor antagonist ICI is a pulmonary arterial-specific vasorelaxant and, therefore, a potentially interesting novel therapeutic agent for the treatment of pulmonary arterial hypertension.

  8. E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

    PubMed Central

    Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

    2001-01-01

    The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027

  9. Gene Expression Profile of NF-κB, Nrf2, Glycolytic, and p53 Pathways During the SH-SY5Y Neuronal Differentiation Mediated by Retinoic Acid.

    PubMed

    de Bittencourt Pasquali, Matheus Augusto; de Ramos, Vitor Miranda; Albanus, Ricardo D Oliveira; Kunzler, Alice; de Souza, Luis Henrinque Trentin; Dalmolin, Rodrigo Juliani Siqueira; Gelain, Daniel Pens; Ribeiro, Leila; Carro, Luigi; Moreira, José Cláudio Fonseca

    2016-01-01

    SH-SY5Y cells, a neuroblastoma cell line that is a well-established model system to study the initial phases of neuronal differentiation, have been used in studies to elucidate the mechanisms of neuronal differentiation. In the present study, we investigated alterations of gene expression in SH-SY5Y cells during neuronal differentiation mediated by retinoic acid (RA) treatment. We evaluated important pathways involving nuclear factor kappa B (NF-κB), nuclear E2-related factor 2 (Nrf2), glycolytic, and p53 during neuronal differentiation. We also investigated the involvement of reactive oxygen species (ROS) in modulating the gene expression profile of those pathways by antioxidant co-treatment with Trolox®, a hydrophilic analogue of α-tocopherol. We found that RA treatment increases levels of gene expression of NF-κB, glycolytic, and antioxidant pathway genes during neuronal differentiation of SH-SY5Y cells. We also found that ROS production induced by RA treatment in SH-SY5Y cells is involved in gene expression profile alterations, chiefly in NF-κB, and glycolytic pathways. Antioxidant co-treatment with Trolox® reversed the effects mediated by RA NF-κB, and glycolytic pathways gene expression. Interestingly, co-treatment with Trolox® did not reverse the effects in antioxidant gene expression mediated by RA in SH-SY5Y. To confirm neuronal differentiation, we quantified endogenous levels of tyrosine hydroxylase, a recognized marker of neuronal differentiation. Our data suggest that during neuronal differentiation mediated by RA, changes in profile gene expression of important pathways occur. These alterations are in part mediated by ROS production. Therefore, our results reinforce the importance in understanding the mechanism by which RA induces neuronal differentiation in SH-SY5Y cells, principally due this model being commonly used as a neuronal cell model in studies of neuronal pathologies.

  10. Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

    PubMed

    Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun

    2016-01-01

    Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Neuronally mediated non-cholinergic contraction of guinea-pig bronchial smooth muscle is inhibited by a substance P antagonist.

    PubMed

    Leander, S; Grundström, N; Andersson, R G; Håkanson, R

    1984-04-01

    The isolated main bronchi of the guinea-pig respond to electrical field stimulation with a twitch followed by a slow contraction. Atropine blocked the slow contraction. The substance P antagonist, (D-Pro2, D- Trp7 ,9)-SP, greatly reduced the atropine-resistant contraction. The results suggest the involvement of substance P in non-cholinergic neurotransmission in the guinea-pig airways.

  12. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains

    PubMed Central

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains. PMID:26105137

  13. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

    NASA Technical Reports Server (NTRS)

    Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

  14. The newly developed CRF1-receptor antagonists, NGD 98-2 and NGD 9002, suppress acute stress-induced stimulation of colonic motor function and visceral hypersensitivity in rats.

    PubMed

    Million, Mulugeta; Zhao, Jing-Fang; Luckey, Andrew; Czimmer, József; Maynard, George D; Kehne, John; Hoffman, Diane C; Taché, Yvette

    2013-01-01

    Corticotropin releasing factor receptor 1 (CRF1) is the key receptor that mediates stress-related body responses. However to date there are no CRF1 antagonists that have shown clinical efficacy in stress-related diseases. We investigated the inhibitory effects of a new generation, topology 2 selective CRF1 antagonists, NGD 98-2 and NGD 9002 on exogenous and endogenous CRF-induced stimulation of colonic function and visceral hypersensitivity to colorectal distension (CRD) in conscious rats. CRF1 antagonists or vehicle were administered orogastrically (og) or subcutaneously (sc) before either intracerebroventricular (icv) or intraperitoneal (ip) injection of CRF (10 µg/kg), exposure to water avoidance stress (WAS, 60 min) or repeated CRD (60 mmHg twice, 10 min on/off at a 30 min interval). Fecal pellet output (FPO), diarrhea and visceromotor responses were monitored. In vehicle (og)-pretreated rats, icv CRF stimulated FPO and induced diarrhea in >50% of rats. NGD 98-2 or NGD 9002 (3, 10 and 30 mg/kg, og) reduced the CRF-induced FPO response with an inhibitory IC50 of 15.7 and 4.3 mg/kg respectively. At the highest dose, og NGD 98-2 or NGD 9002 blocked icv CRF-induced FPO by 67-87% and decreased WAS-induced-FPO by 23-53%. When administered sc, NGD 98-2 or NGD 9002 (30 mg/kg) inhibited icv and ip CRF-induced-FPO. The antagonists also prevented the development of nociceptive hyper-responsivity to repeated CRD. These data demonstrate that topology 2 CRF1 antagonists, NGD 98-2 and NGD 9002, administered orally, prevented icv CRF-induced colonic secretomotor stimulation, reduced acute WAS-induced defecation and blocked the induction of visceral sensitization to repeated CRD.

  15. Cloning and pharmacological characterization of the rabbit bradykinin B2 receptor.

    PubMed

    Bachvarov, D R; Saint-Jacques, E; Larrivée, J F; Levesque, L; Rioux, F; Drapeau, G; Marceau, F

    1995-12-01

    Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecutive characterization of G protein-coupled receptors present in cultured rabbit aortic smooth muscle cells. One of the isolated resulting fragments was highly homologous to the corresponding region of the bradykinin (BK) B2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which allowed the identification of an intronless 1101-nucleotide open reading frame which codes for a 367-amino acid receptor protein. The rabbit B2 receptor sequence is more than 80% identical to the ones determined in three other species and retain putative glycosylation, palmitoylation and phosphorylation sites. In the rabbit genomic sequence, an acceptor splice sequence was found 8 base pairs upstream of the start codon. Northern blot analysis showed a high expression of a major transcript (4.2 kilobases) in the rabbit kidney and duodenum, and a less abundant expression in other tissues. Southern blot experiments suggest that a single copy of this gene exists in the rabbit genome. The cloned rabbit B2 receptor expressed in COS-1 cells binds [3H]BK in a saturable manner (KD 2.1 nM) and this ligand competes with a series of kinin agonists and antagonist with a rank order consistent with the B2 receptor identity. The insurmountable character of the antagonism exerted by Hoe 140 against BK on the rabbit B2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments with the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [3H]BK in a surmountable manner for the human B2 receptor expressed in COS-1 cells. The cloning of the rabbit B2 receptor will be useful notably for the study of the structural basis of antagonist binding and for studies on receptor

  16. Microbiome-Derived Tryptophan Metabolites and Their Aryl Hydrocarbon Receptor-Dependent Agonist and Antagonist Activities

    PubMed Central

    Jin, Un-Ho; Lee, Syng-Ook; Sridharan, Gautham; Lee, Kyongbum; Davidson, Laurie A.; Jayaraman, Arul; Chapkin, Robert S.; Alaniz, Robert

    2014-01-01

    The tryptophan metabolites indole, indole-3-acetate, and tryptamine were identified in mouse cecal extracts and fecal pellets by mass spectrometry. The aryl hydrocarbon receptor (AHR) agonist and antagonist activities of these microbiota-derived compounds were investigated in CaCo-2 intestinal cells as a model for understanding their interactions with colonic tissue, which is highly aryl hydrocarbon (Ah)–responsive. Activation of Ah-responsive genes demonstrated that tryptamine and indole 3-acetate were AHR agonists, whereas indole was an AHR antagonist that inhibited TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)–induced CYP1A1 expression. In contrast, the tryptophan metabolites exhibited minimal anti-inflammatory activities, whereas TCDD decreased phorbol ester-induced CXCR4 [chemokine (C-X-C motif) receptor 4] gene expression, and this response was AHR dependent. These results demonstrate that the tryptophan metabolites indole, tryptamine, and indole-3-acetate modulate AHR-mediated responses in CaCo-2 cells, and concentrations of indole that exhibit AHR antagonist activity (100–250 μM) are detected in the intestinal microbiome. PMID:24563545

  17. Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

    PubMed

    Wu, Liping; Oshima, Tadayuki; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2017-07-01

    Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  18. Hallucinogen-like effects of N,N-dipropyltryptamine (DPT): possible mediation by serotonin 5-HT1A and 5-HT2A receptors in rodents

    PubMed Central

    Fantegrossi, William E.; Reissig, Chad J.; Katz, Elyse B.; Yarosh, Haley L.; Rice, Kenner C.; Winter, Jerrold C.

    2008-01-01

    N,N-dipropyltryptamine (DPT) is a synthetic tryptamine hallucinogen which has been used psychotherapeutically in humans, but has been studied preclinically only rarely. In the present studies, DPT was tested in a drug-elicited head twitch assay in mice, and in rats trained to discriminate lysergic acid diethylamide (LSD), N,N-dimethyl-4-phosphoryloxytryptamine (psilocybin), or 3,4-methylenedioxymethamphetamine (MDMA). A separate group of rats was also trained to recognize DPT itself as a discriminative stimulus, and in all cases, the behavioral effects of DPT were challenged with the selective serotonin (5-HT)2A antagonist M100907, the 5-HT1A selective antagonist WAY-100635, or their combination. In the head twitch assay, DPT elicited dose-dependent effects, producing a biphasic dose-effect curve. WAY-100635 produced a parallel rightward shift in the dose-effect curve for head twitches, indicative of surmountable antagonism, but the antagonist effects of M100907 were functionally insurmountable. DPT produced partial to full substitution when tested in rats trained to discriminate LSD, psilocybin or MDMA, and served as a discriminative stimulus. In all cases, the antagonist effects of M100907 were more profound than were those of WAY-100635. DPT is thus active in two rodent models relevant to 5-HT2 agonist activity. The effectiveness with which M100907 antagonizes the behavioral actions of this compound strongly suggests that the 5-HT2A receptor is an important site of action for DPT, but the modulatory actions of WAY-100635 also imply a 5-HT1A-mediated component to the actions of this compound. PMID:17905422

  19. Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    PubMed Central

    Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G.; MacLeod, Roderick A. F.

    2013-01-01

    Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies. PMID:23637834

  20. Ectopic expression of homeobox gene NKX2-1 in diffuse large B-cell lymphoma is mediated by aberrant chromatin modifications.

    PubMed

    Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

    2013-01-01

    Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.

  1. Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

    PubMed Central

    Johnston, Jamie; Griffin, Sarah J; Baker, Claire; Skrzypiec, Anna; Chernova, Tatanya; Forsythe, Ian D

    2008-01-01

    The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K+ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half-activated at −9.2 ± 2.1 mV and half-inactivated at −35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 μm) and high concentrations of extracellular tetraethylammonium (TEA; IC50 = 11.8 mm), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing. PMID:18511484

  2. The effect of the leukotriene antagonist pranlukast on pediatric acute otitis media.

    PubMed

    Nakamura, Yoshihisa; Hamajima, Yuki; Suzuki, Motohiko; Esaki, Shinichi; Yokota, Makoto; Oshika, Masanori; Takagi, Ippei; Yasui, Keiko; Miyamoto, Naoya; Sugiyama, Kazuko; Nakayama, Meiho; Murakami, Shingo

    2016-08-01

    Conventional treatment for acute otitis media mainly targets bacteria with antibiotics, neglecting to control for mediators of inflammation. Mediators of inflammation, such as leukotrienes, have been identified in patients with acute otitis media (AOM) or subsequent secretory otitis media (SOM). They can cause functional eustachian tube dysfunction or increase mucous in the middle ear, causing persistent SOM following AOM. The objective of the present study was to evaluate whether or not administration of pranlukast, a widely used leukotriene C4, D4, and E4 antagonist, together with antibiotics could inhibit the progression to SOM. Children with AOM, who were from two to 12 years old, were randomly divided into two groups as follows: a control group in which 50 patients received antibiotic-based conventional treatment according to guidelines for treating AOM proposed by the Japan Otological Society (version 2006); and a pranlukast group, in which 52 patients were administered pranlukast for up to 28 days as well as given conventional treatment. Cases were regarded as persistent SOM when a tympanogram was type B or C2 four weeks after treatment was initiated. Two patients in the pranlukast group and 3 patients in the control group were excluded because they relapsed AOM within 28 days after initial treatment. Therefore, the analysis included 50 and 47 subjects in the pranlukast and control groups, respectively. The percentage of patients diagnosed with persistent SOM (22.0%) was significantly smaller in the pranlukast group compared with the control group (44.7%) (p = 0.018, chi-squared test). The results indicate that combined treatment of AOM with antibiotics and a leukotriene antagonist to control inflammation is useful for preventing progression to persistent SOM. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

    PubMed Central

    Fujita, Hiroaki; Akita, Mariko; Kato, Ryuichi; Sasaki, Yoshiteru; Wakatsuki, Soichi

    2014-01-01

    The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex. PMID:24469399

  4. Reversal of sibutramine-induced anorexia with a selective 5-HT(2C) receptor antagonist.

    PubMed

    Higgs, Suzanne; Cooper, Alison J; Barnes, Nicholas M

    2011-04-01

    The monoamine reuptake inhibitor sibutramine reduces food intake but the receptor subtypes mediating the effects of sibutramine on feeding remain to be clearly identified. The involvement of the 5-HT(2C) receptor subtype in the satiety-enhancing effects of sibutramine was investigated by examining the effects of co-administration of sibutramine with the selective 5-HT(2C) receptor antagonist SB 242084 Microstructural analyses of licking for a glucose solution by non-deprived, male rats were performed over a range of doses of sibutramine to identify a selective satiety-enhancing dose (experiment 1). Similar analyses were performed after administration of a vehicle control, two doses of SB 242084 alone or two doses of SB 242084 in combination with sibutramine (experiment 2). Sibutramine at doses of 1-3 mg/kg selectively reduced glucose consumption via a reduction in the number of bouts of licking. Non-selective effects to increase latency to lick were only observed at the higher dose of 6 mg/kg. Co-administration of sibutramine (3 mg/kg) with SB 242084 (1 or 3 mg/kg) reversed the effect of sibutramine on bout number whereas either dose of SB 242084 alone had no significant effect. We confirm behaviourally selective effects of sibutramine on feeding and provide further support for the satiety-enhancing effects of sibutramine. Our data also provide evidence for the involvement of the 5-HT(2C) receptor in the satiety-enhancing effects of sibutramine although additional targets may have an impact, and further investigation of the molecular mechanisms underlying the efficacy of sibutramine as an anorectic is warranted.

  5. CXCL4-induced migration of activated T lymphocytes is mediated by the chemokine receptor CXCR3.

    PubMed

    Mueller, Anja; Meiser, Andrea; McDonagh, Ellen M; Fox, James M; Petit, Sarah J; Xanthou, Georgina; Williams, Timothy J; Pease, James E

    2008-04-01

    The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via Galphai-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.

  6. Antibody-Mediated Complement C3b/iC3b Binding to Group B Streptococcus in Paired Mother and Baby Serum Samples in a Refugee Population on the Thailand-Myanmar Border

    PubMed Central

    Herbert, Jenny; Thomas, Stephen; Brookes, Charlotte; Turner, Claudia; Turner, Paul; Nosten, Francois; Le Doare, Kirsty; Hudson, Michael; Heath, Paul T.; Gorringe, Andrew

    2015-01-01

    Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations. PMID:25589553

  7. Kinase activation through dimerization by human SH2-B.

    PubMed

    Nishi, Masahiro; Werner, Eric D; Oh, Byung-Chul; Frantz, J Daniel; Dhe-Paganon, Sirano; Hansen, Lone; Lee, Jongsoon; Shoelson, Steven E

    2005-04-01

    The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.

  8. Vasopressin and a nonpeptide antidiuretic hormone receptor antagonist (OPC-31260).

    PubMed

    Burrell, L M; Phillips, P A; Stephenson, J M; Risvanis, J; Johnston, C I

    1994-03-01

    The development of nonpeptide orally active AVP analogues has provided a new tool with which to assess the physiological and pathophysiological role of vasopressin (AVP). We have previously characterised the nonpeptide vasopressin V1 receptor antagonist OPC-21268, and now report the in vitro characterisation of the nonpeptide V2 receptor antagonist OPC-31260 in the rat. OPC-31260 caused a concentration-dependent displacement of the selective AVP V2 receptor antagonist radioligand, [3H]desGly-NH2(9)[d(CH2)5, D-Ile2,Ile4]AVP from V2 receptors in rat kidney medulla membranes. The concentration of OPC-31260 that displaced 50% of specific AVP binding (IC50) was 20 +/- 2 nmol/l for renal V2 receptors. OPC-31260 also caused a concentration-dependent displacement of the selective AVP V1 receptor antagonist radioligand, [125I]-[d(CH2)5,sarcosine7]AVP from V1 receptors in both rat liver and kidney medulla membranes. The IC50 was 500 +/- 30 nmol/l for both renal and liver V1 receptors. After oral administration to rats, OPC-31260 was an effective inhibitor of AVP at renal V2 and liver V1 receptors in a time-dependent manner. In vitro binding kinetic studies showed that OPC-31260 was a competitive antagonist at both the renal V2 receptor and the hepatic V1 receptor. OPC-31260 is a nonpeptide, orally effective competitive inhibitor of AVP with a V2:V1 receptor selectivity ratio of 25:1 indicating relative V2 receptor selectivity.

  9. Virtual High-Throughput Screening To Identify Novel Activin Antagonists

    PubMed Central

    Zhu, Jie; Mishra, Rama K.; Schiltz, Gary E.; Makanji, Yogeshwar; Scheidt, Karl A.; Mazar, Andrew P.; Woodruff, Teresa K.

    2015-01-01

    Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex’s binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases. PMID:26098096

  10. The somatostatin receptor 2 antagonist 64Cu-NODAGA-JR11 outperforms 64Cu-DOTA-TATE in a mouse xenograft model

    PubMed Central

    Rylova, Svetlana N.; Stoykow, Christian; Del Pozzo, Luigi; Abiraj, Keelara; Tamma, Maria Luisa; Kiefer, Yvonne; Fani, Melpomeni; Maecke, Helmut R.

    2018-01-01

    Copper-64 is an attractive radionuclide for PET imaging and is frequently used in clinical applications. The aim of this study was to perform a side-by-side comparison of the in vitro and in vivo performance of 64Cu-NODAGA-JR11 (NODAGA = 1,4,7-triazacyclononane,1-glutaric acid,4,7-acetic acid, JR11 = p-Cl-Phe-cyclo(D-Cys-Aph(Hor)-D-Aph(cbm)-Lys-Thr-Cys)D-Tyr-NH2), a somatostatin receptor 2 antagonist, with the clinically used sst2 agonist 64Cu-DOTA-TATE ((TATE = D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)Thr). In vitro studies demonstrated Kd values of 5.7±0.95 nM (Bmax = 4.1±0.18 nM) for the antagonist 64/natCu-NODAGA-JR11 and 20.1±4.4. nM (Bmax = 0.48±0.18 nM) for the agonist 64/natCu-DOTA-TATE. Cell uptake studies showed the expected differences between agonists and antagonists. Whereas 64Cu-DOTA-TATE (the agonist) showed very effective internalization in the cell culture assay (with 50% internalized at 4 hours post-peptide addition under the given experimental conditions), 64Cu-NODAGA-JR11 (the antagonist) showed little internalization but strong receptor-mediated uptake at the cell membrane. Biodistribution studies of 64Cu-NODAGA-JR11 showed rapid blood clearance and tumor uptake with increasing tumor-to-relevant organ ratios within the first 4 hours and in some cases, 24 hours, respectively. The tumor washout was slow or non-existent in the first 4 hours, whereas the kidney washout was very efficient, leading to high and increasing tumor-to-kidney ratios over time. Specificity of tumor uptake was proven by co-injection of high excess of non-radiolabeled peptide, which led to >80% tumor blocking. 64Cu-DOTA-TATE showed less favorable pharmacokinetics, with the exception of lower kidney uptake. Blood clearance was distinctly slower and persistent higher blood values were found at 24 hours. Uptake in the liver and lung was relatively high and also persistent. The tumor uptake was specific and similar to that of 64Cu-NODAGA-JR11 at 1 h, but release from the tumor

  11. The somatostatin receptor 2 antagonist 64Cu-NODAGA-JR11 outperforms 64Cu-DOTA-TATE in a mouse xenograft model.

    PubMed

    Rylova, Svetlana N; Stoykow, Christian; Del Pozzo, Luigi; Abiraj, Keelara; Tamma, Maria Luisa; Kiefer, Yvonne; Fani, Melpomeni; Maecke, Helmut R

    2018-01-01

    Copper-64 is an attractive radionuclide for PET imaging and is frequently used in clinical applications. The aim of this study was to perform a side-by-side comparison of the in vitro and in vivo performance of 64Cu-NODAGA-JR11 (NODAGA = 1,4,7-triazacyclononane,1-glutaric acid,4,7-acetic acid, JR11 = p-Cl-Phe-cyclo(D-Cys-Aph(Hor)-D-Aph(cbm)-Lys-Thr-Cys)D-Tyr-NH2), a somatostatin receptor 2 antagonist, with the clinically used sst2 agonist 64Cu-DOTA-TATE ((TATE = D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)Thr). In vitro studies demonstrated Kd values of 5.7±0.95 nM (Bmax = 4.1±0.18 nM) for the antagonist 64/natCu-NODAGA-JR11 and 20.1±4.4. nM (Bmax = 0.48±0.18 nM) for the agonist 64/natCu-DOTA-TATE. Cell uptake studies showed the expected differences between agonists and antagonists. Whereas 64Cu-DOTA-TATE (the agonist) showed very effective internalization in the cell culture assay (with 50% internalized at 4 hours post-peptide addition under the given experimental conditions), 64Cu-NODAGA-JR11 (the antagonist) showed little internalization but strong receptor-mediated uptake at the cell membrane. Biodistribution studies of 64Cu-NODAGA-JR11 showed rapid blood clearance and tumor uptake with increasing tumor-to-relevant organ ratios within the first 4 hours and in some cases, 24 hours, respectively. The tumor washout was slow or non-existent in the first 4 hours, whereas the kidney washout was very efficient, leading to high and increasing tumor-to-kidney ratios over time. Specificity of tumor uptake was proven by co-injection of high excess of non-radiolabeled peptide, which led to >80% tumor blocking. 64Cu-DOTA-TATE showed less favorable pharmacokinetics, with the exception of lower kidney uptake. Blood clearance was distinctly slower and persistent higher blood values were found at 24 hours. Uptake in the liver and lung was relatively high and also persistent. The tumor uptake was specific and similar to that of 64Cu-NODAGA-JR11 at 1 h, but release from the tumor

  12. Methadone inhibits CYP2D6 and UGT2B7/2B4 in vivo: a study using codeine in methadone- and buprenorphine-maintained subjects

    PubMed Central

    Gelston, Eloise A; Coller, Janet K; Lopatko, Olga V; James, Heather M; Schmidt, Helmut; White, Jason M; Somogyi, Andrew A

    2012-01-01

    AIMS To compare the O-demethylation (CYP2D6-mediated), N-demethylation (CYP3A4-mediated) and 6-glucuronidation (UGT2B4/7-mediated) metabolism of codeine between methadone- and buprenorphine-maintained CYP2D6 extensive metabolizer subjects. METHODS Ten methadone- and eight buprenorphine-maintained subjects received a single 60 mg dose of codeine phosphate. Blood was collected at 3 h and urine over 6 h and assayed for codeine, norcodeine, morphine, morphine-3- and -6-glucuronides and codeine-6-glucuronide. RESULTS The urinary metabolic ratio for O-demethylation was significantly higher (P = 0.0044) in the subjects taking methadone (mean ± SD, 2.8 ± 3.1) compared with those taking buprenorphine (0.60 ± 0.43), likewise for 6-glucuronide formation (0.31 ± 0.24 vs. 0.053 ± 0.027; P < 0.0002), but there was no significant difference (P = 0.36) in N-demethylation. Similar changes in plasma metabolic ratios were also found. In plasma, compared with those maintained on buprenorphine, the methadone-maintained subjects had increased codeine and norcodeine concentrations (P < 0.004), similar morphine (P = 0.72) and lower morphine-3- and -6- and codeine-6-glucuronide concentrations (P < 0.008). CONCLUSION Methadone is associated with inhibition of CYP2D6 and UGTs 2B4 and 2B7 reactions in vivo, even though it is not a substrate for these enzymes. Plasma morphine was not altered, owing to the opposing effects of inhibition of both formation and elimination; however, morphine-6-glucuronide (analgesically active) concentrations were substantially reduced. Drug interactions with methadone are likely to include drugs metabolized by various UGTs and CYP2D6. PMID:22092298

  13. Discovery of potent, selective, orally active benzoxazepine-based Orexin-2 receptor antagonists.

    PubMed

    Fujimoto, Tatsuhiko; Kunitomo, Jun; Tomata, Yoshihide; Nishiyama, Keiji; Nakashima, Masato; Hirozane, Mariko; Yoshikubo, Shin-Ichi; Hirai, Keisuke; Marui, Shogo

    2011-11-01

    During our efforts to identify a series of potent, selective, orally active human Orexin-2 Receptor (OX2R) antagonists, we elucidated structure-activity relationship (SAR) on the 7-position of a benzoxazepine scaffold by utilizing Hammett σ(p) and Hansch-Fujita π value as aromatic substituent constants. The attempts led to the discovery of compound 1m, possessing good in vitro potency with over 100-fold selectivity against OX1R, good metabolic stability in human and rat liver microsome, good oral bioavailability in rats, and in vivo antagonistic activity in rats by oral administration. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Metabolism of a 5HT6 antagonist, 2-methyl-1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazole (SAM-760): impact of sulfonamide metabolism on diminution of a ketoconazole mediated clinical drug-drug interaction.

    PubMed

    Sawant-Basak, Aarti; Obach, R Scott; Doran, Angela C; Lockwood, Peter; Schildknegt, Klaas; Gao, Hongying; Mancuso, Jessica; Tse, Susanna; Comery, Tom

    2018-04-25

    SAM-760, (2-methyl-1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazole), a 5HT 6 antagonist, was investigated in humans for the treatment of Alzheimer's dementia. In liver microsomes and recombinant CYP450 isozymes, SAM-760 was predominantly metabolized by CYP3A (~85%). Based on these observations and an expectation of 5-fold magnitude of interaction with moderate to strong CYP3A inhibitors, a clinical DDI study was performed. In presence of ketoconazole, mean C max and AUC 0-inf of SAM-760 showed only a modest increase by 30% and 38%, respectively. In vitro investigation of this unexpectedly low interaction was undertaken using [ 14 C]SAM-760. Radiometric profiling in human hepatocytes, confirmed all oxidative metabolites observed previously with unlabeled SAM-760; however the pre-dominant radiometric peak was an unexpected polar metabolite which was insensitive to pan-CYP inhibitor, 1-aminobenzotriazole. In human hepatocytes, radiometric integration attributed 43% of total metabolism of SAM-760 to this non-CYP pathway. Using an authentic standard, this predominant metabolite was confirmed as benzenesulfinic acid. Additional investigation revealed that the benzenesulfinic acid metabolite may be a novel, non-enzymatic, thiol mediated reductive cleavage of aryl sulfonamide group of SAM-760. We also determined the relative contribution of P450 to metabolism of SAM-760 in human hepatocytes, by following the rate of formation of oxidative metabolites in presence and absence of P450 isoform specific inhibitors. P450 mediated oxidative metabolism of SAM-760 was still primarily attributed to CYP3A (33%), with minor contributions from CYP isoforms 2C19 and 2D6. Thus, disposition of [ 14 C]SAM-760 in human hepatocytes via novel sulfonamide metabolism and CYP3A verified the lower than expected clinical DDI when SAM-760 was co-administered with ketoconazole. The American Society for Pharmacology and Experimental Therapeutics.

  15. Thioredoxin-mediated denitrosylation regulates cytokine-induced nuclear factor κB (NF-κB) activation.

    PubMed

    Kelleher, Zachary T; Sha, Yonggang; Foster, Matthew W; Foster, W Michael; Forrester, Michael T; Marshall, Harvey E

    2014-01-31

    S-nitrosylation of nuclear factor κB (NF-κB) on the p65 subunit of the p50/p65 heterodimer inhibits NF-κB DNA binding activity. We have recently shown that p65 is constitutively S-nitrosylated in the lung and that LPS-induced injury elicits a decrease in SNO-p65 levels concomitant with NF-κB activation in the respiratory epithelium and initiation of the inflammatory response. Here, we demonstrate that TNFα-mediated activation of NF-κB in the respiratory epithelium similarly induces p65 denitrosylation. This process is mediated by the denitrosylase thioredoxin (Trx), which becomes activated upon cytokine-induced degradation of thioredoxin-interacting protein (Txnip). Similarly, inhibition of Trx activity in the lung attenuates LPS-induced SNO-p65 denitrosylation, NF-κB activation, and airway inflammation, supporting a pathophysiological role for this mechanism in lung injury. These data thus link stimulus-coupled activation of NF-κB to a specific, protein-targeted denitrosylation mechanism and further highlight the importance of S-nitrosylation in the regulation of the immune response.

  16. Degradation of Mcl-1 by granzyme B: implications for Bim-mediated mitochondrial apoptotic events.

    PubMed

    Han, Jie; Goldstein, Leslie A; Gastman, Brian R; Froelich, Christopher J; Yin, Xiao-Ming; Rabinowich, Hannah

    2004-05-21

    Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L.

  17. Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

    PubMed Central

    Novoselova, Tatiana V.; Zahira, Kiran; Rose, Ruth-Sarah

    2012-01-01

    Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination. PMID:22307975

  18. Highly Increased 125I-JR11 Antagonist Binding In Vitro Reveals Novel Indications for sst2 Targeting in Human Cancers.

    PubMed

    Reubi, Jean Claude; Waser, Beatrice; Mäcke, Helmut; Rivier, Jean

    2017-02-01

    There is recent in vitro and in vivo evidence that somatostatin receptor subtype 2 (sst 2 ) antagonists are better tools to target neuroendocrine tumors (NETs) than sst 2 agonists. Indeed, antagonists bind to a greater number of sst 2 sites than agonists. Whether sst 2 antagonists could be used successfully to target non-NETs, expressing low sst 2 density, is unknown. Here, we compare quantitatively 125 I-JR11 sst 2 antagonist binding in vitro with that of the sst 2 agonist 125 I-Tyr 3 -octreotide in large varieties of non-NET and NET. In vitro receptor autoradiography was performed with 125 I-JR11 and 125 I-Tyr 3 -octreotide in cancers from prostate, breast, colon, kidney, thyroid, and lymphoid tissues as well as NETs as reference. In general, 125 I-JR11 binds to many more sst 2 sites than 125 I-Tyr 3 -octreotide. In 13 breast cancers, 8 had a low binding (mean density, 844 ± 168 dpm/mg of tissue) with the agonist whereas 12 had a high binding (mean density, 4,447 ± 1,128 dpm/mg of tissue) with the antagonist. All 12 renal cell cancers showed a low binding of sst 2 with the agonist (mean density, 348 ± 49 dpm/mg of tissue) whereas all cases had a high sst 2 binding with the antagonist (mean density, 3,777 ± 582 dpm/mg of tissue). One of 5 medullary thyroid cancers was positive with the agonist, whereas 5 of 5 were positive with the antagonist. In 15 non-Hodgkin lymphomas, many more sst 2 sites were labeled with the antagonist than with the agonist. In 14 prostate cancers, none had sst 2 binding with the agonist and only 4 had a weak binding with the antagonist. None of 17 colon cancers showed sst 2 sites with the agonist, and only 3 cases were weakly positive with the antagonist. In the various tumor types, adjacent sst 2 -expressing tissues such as vessels, lymphocytes, nerves, mucosa, or stroma were more strongly labeled with the antagonist than with the agonist. The reference NET cases, incubated with a smaller amount of tracer, were also found to have many

  19. Analysis of hydrophobic interactions of antagonists with the beta2-adrenergic receptor.

    PubMed

    Novoseletsky, V N; Pyrkov, T V; Efremov, R G

    2010-01-01

    The adrenergic receptors mediate a wide variety of physiological responses, including vasodilatation and vasoconstriction, heart rate modulation, and others. Beta-adrenergic antagonists ('beta-blockers') thus constitute a widely used class of drugs in cardiovascular medicine as well as in management of anxiety, migraine, and glaucoma. The importance of the hydrophobic effect has been evidenced for a wide range of beta-blocker properties. To better understand the role of the hydrophobic effect in recognition of beta-blockers by their receptor, we carried out a molecular docking study combined with an original approach to estimate receptor-ligand hydrophobic interactions. The proposed method is based on automatic detection of molecular fragments in ligands and the analysis of their interactions with receptors separately. A series of beta-blockers, based on phenylethanolamines and phenoxypropanolamines, were docked to the beta2-adrenoceptor binding site in the crystal structure. Hydrophobic complementarity between the ligand and the receptor was calculated using the PLATINUM web-server (http://model.nmr.ru/platinum). Based on the analysis of the hydrophobic match for molecular fragments of beta-blockers, we have developed a new scoring function which efficiently predicts dissociation constant (pKd) with strong correlations (r(2) approximately 0.8) with experimental data.

  20. cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

    PubMed Central

    Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2013-01-01

    Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

  1. Spinal serum-inducible and glucocorticoid-inducible kinase 1 mediates neuropathic pain via kalirin and downstream PSD-95-dependent NR2B phosphorylation in rats.

    PubMed

    Peng, Hsien-Yu; Chen, Gin-Den; Lai, Cheng-Yuan; Hsieh, Ming-Chun; Lin, Tzer-Bin

    2013-03-20

    The coupling of the spinal postsynaptic density-95 (PSD-95) with the glutamatergic N-methyl-d-aspartate receptor NR2B subunit and the subsequent NR2B phosphorylation contribute to pain-related plasticity. Increasing evidence reveals that kalirin, a Rho-guanine nucleotide exchange factor, modulates PSD-95-NR2B-dependent neuroplasticity. Our laboratory recently demonstrated that serum-inducible and glucocorticoid-inducible kinase 1 (SGK1) participates in inflammation-associated pain hypersensitivity by modulating spinal glutamatergic neurotransmission. Because kalirin is one of the proteins in PSD that is highly phosphorylated by various kinases, we tested whether kalirin could be a downstream target of spinal SGK1 that participates in neuropathic pain development via regulation of the PSD-95-NR2B coupling-dependent phosphorylation of NR2B. We observed that spinal nerve ligation (SNL, L5) in male Sprague Dawley rats resulted in behavioral allodynia, which was associated with phosphorylated SGK1 (pSGK1), kalirin, and phosphorylated NR2B (pNR2B) expression and an increase in pSGK1-kalirin-PSD-95-pNR2B coprecipitation in the ipsilateral dorsal horn (L4-L5). SNL-enhanced kalirin immunofluorescence was coincident with pSGK1, PSD-95, and pNR2B immunoreactivity. Small-interfering RNA (siRNA) that targeted spinal kalirin mRNA expression (10 μg, 10 μl; i.t.) reduced SNL-induced allodynia, kalirin and pNR2B expression, as well as kalirin-PSD-95 and PSD-95-pNR2B coupling and costaining without affecting SGK1 phosphorylation. Daily administration of GSK-650394 (an SGK1 antagonist; 100 nm, 10 μl, i.t.) not only exhibited effects similar to the kalirin mRNA-targeting siRNA but also attenuated pSGK1-kalirin costaining and SGK1-kalirin coupling. We suggest that nerve injury could induce spinal SGK1 phosphorylation that subsequently interacts with and upregulates kalirin to participate in neuropathic pain development via PSD-95-NR2B coupling-dependent NR2B phosphorylation.

  2. Propionibacterium acnes induces an adjuvant effect in B-1 cells and affects their phagocyte differentiation via a TLR2-mediated mechanism.

    PubMed

    Gambero, Monica; Teixeira, Daniela; Butin, Liane; Ishimura, Mayari Eika; Mariano, Mario; Popi, Ana Flavia; Longo-Maugéri, Ieda Maria

    2016-09-01

    B-1 lymphocytes are present in large numbers in the mouse peritoneal cavity, as are macrophages, and are responsible for natural IgM production. These lymphocytes migrate to inflammatory foci and are also involved in innate immunity. It was also demonstrated that B-1 cells are able to differentiated into phagocytes (B-1CDP), which is characterized by expression of F4/80 and increased phagocytic activity. B-1 cell responses to antigens and adjuvants are poorly characterized. It has been shown that Propionibacterium acnes suspensions induce immunomodulatory effects in both macrophages and B-2 lymphocytes. We recently demonstrated that this bacterium has the ability to increase B-1 cell populations both in vitro and in vivo. P. acnes induces B-1CDP differentiation, increases the expression of TLR2, TLR4 and TLR9 and augments the expression of CD80, CD86 and CD40 in B-1 and B-1CDP cells. Because P. acnes has been shown to modulate TLR expression, in this study, we investigated the role of TLR2 and TLR4 in B-1 cell population, including B-1CDP differentiation and phagocytic activity in vitro and in vivo. Interestingly, we have demonstrated that TLR2 signaling could be involved in the increase in the B-1 cell population induced by P. acnes. Furthermore, the early differentiation of B-1CDP is also dependent of TLR2. It was also observed that TLR signals also interfere in the phagocytic ability of B-1 cells and their phagocytes. According to these data, it is clear that P. acnes promotes an important adjuvant effect in B-1 cells by inducing them to differentiate into B-1CDP cells and modulates their phagocytic functions both in vivo and in vitro. Moreover, most of these effects are mediated primarily via TLR2. These data reinforce the findings that such bacterial suspensions have powerful adjuvant properties. The responses of B-1 cells to exogenous stimulation indicate that these cells are important to the innate immune response. Copyright © 2016 Elsevier GmbH. All rights

  3. Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus.

    PubMed

    Lee, Nam Keun; Yeo, In-Cheol; Park, Joung Whan; Kang, Byung-Sun; Hahm, Young Tae

    2010-09-01

    In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 degrees C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5mug/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254nm. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.

    PubMed

    Bajusz, S; Kovacs, M; Gazdag, M; Bokser, L; Karashima, T; Csernus, V J; Janaky, T; Guoth, J; Schally, A V

    1988-03-01

    To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically.

  5. Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.

    PubMed Central

    Bajusz, S; Kovacs, M; Gazdag, M; Bokser, L; Karashima, T; Csernus, V J; Janaky, T; Guoth, J; Schally, A V

    1988-01-01

    To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically. PMID:3278323

  6. UV-B photoreceptor-mediated signalling in plants.

    PubMed

    Heijde, Marc; Ulm, Roman

    2012-04-01

    Ultraviolet-B radiation (UV-B) is a key environmental signal that is specifically perceived by plants to promote UV acclimation and survival in sunlight. Whereas the plant photoreceptors for visible light are rather well characterised, the UV-B photoreceptor UVR8 was only recently described at the molecular level. Here, we review the current understanding of the UVR8 photoreceptor-mediated pathway in the context of UV-B perception mechanism, early signalling components and physiological responses. We further outline the commonalities in UV-B and visible light signalling as well as highlight differences between these pathways. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Yuan, E-mail: yuan.xu@ki.se; Cardell, Lars-Olaf

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin-more » (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance

  8. P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.

    PubMed

    Swartz, Talia H; Esposito, Anthony M; Durham, Natasha D; Hartmann, Boris M; Chen, Benjamin K

    2014-10-01

    Human immunodeficiency virus type 1 (HIV-1) infection is chronic and presently still incurable. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 infection. Purinergic receptors are distributed throughout a wide variety of tissue types and detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in the transmission of HIV-1 from cell to cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection did not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, we observed CXCR4 colocalization with the newly internalized virus particles within target lymphocytes and found that the purinergic antagonists did not impair the recruitment of the coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y-selective receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target and that purinergic antagonists may have potent activity against viral infection of CD4+ T lymphocytes by both cell-free and cell-to-cell transmission. This study identifies purinergic antagonists to be potent inhibitors of HIV-1 cell-free and cell-to-cell-mediated infection and provides a

  9. The design, synthesis and pharmacological characterization of novel β2-adrenoceptor antagonists

    PubMed Central

    Hothersall, J Daniel; Black, James; Caddick, Stephen; Vinter, Jeremy G; Tinker, Andrew; Baker, James R

    2011-01-01

    BACKGROUND AND PURPOSE Selective and potent antagonists for the β2-adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology. EXPERIMENTAL APPROACH A range of pharmacological assays was used to assess potency, affinity, selectivity (β2-adrenoceptor vs. β1-adrenoceptor) and efficacy. KEY RESULTS Ten novel compounds were identified but none had as high affinity as the prototypical β2-adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for β2-adrenoceptors. Most of the ligands were inverse agonists for β2-adrenoceptor-cAMP signalling, although one (5217377) was a partial agonist and another a neutral antagonist (7929193). None of the ligands were efficacious with regard to β2-adrenoceptor-β-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the β2-adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour. CONCLUSIONS AND IMPLICATIONS In terms of improving on the pharmacology of the ligand ICI-118,551, one of the compounds was more selective (racemic JB-175), while one was a neutral antagonist (7929193), although none had as high an affinity. The results substantiate the notion that β-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology. PMID:21323900

  10. Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist.

    PubMed

    Yoshida, Yu; Naoe, Yoshimitsu; Terauchi, Taro; Ozaki, Fumihiro; Doko, Takashi; Takemura, Ayumi; Tanaka, Toshiaki; Sorimachi, Keiichi; Beuckmann, Carsten T; Suzuki, Michiyuki; Ueno, Takashi; Ozaki, Shunsuke; Yonaga, Masahiro

    2015-06-11

    The orexin/hypocretin receptors are a family of G protein-coupled receptors and consist of orexin-1 (OX1) and orexin-2 (OX2) receptor subtypes. Orexin receptors are expressed throughout the central nervous system and are involved in the regulation of the sleep/wake cycle. Because modulation of these receptors constitutes a promising target for novel treatments of disorders associated with the control of sleep and wakefulness, such as insomnia, the development of orexin receptor antagonists has emerged as an important focus in drug discovery research. Here, we report the design, synthesis, characterization, and structure-activity relationships (SARs) of novel orexin receptor antagonists. Various modifications made to the core structure of a previously developed compound (-)-5, the lead molecule, resulted in compounds with improved chemical and pharmacological profiles. The investigation afforded a potential therapeutic agent, (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006), an orally active, potent orexin antagonist. The efficacy was demonstrated in mice in an in vivo study by using sleep parameter measurements.

  11. Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

    PubMed

    Liu, Hong; Zhang, Yujin; Wu, Hongyu; D'Alessandro, Angelo; Yegutkin, Gennady G; Song, Anren; Sun, Kaiqi; Li, Jessica; Cheng, Ning-Yuan; Huang, Aji; Edward Wen, Yuan; Weng, Ting Ting; Luo, Fayong; Nemkov, Travis; Sun, Hong; Kellems, Rodney E; Karmouty-Quintana, Harry; Hansen, Kirk C; Zhao, Bihong; Subudhi, Andrew W; Jameson-Van Houten, Sonja; Julian, Colleen G; Lovering, Andrew T; Eltzschig, Holger K; Blackburn, Michael R; Roach, Robert C; Xia, Yang

    2016-08-02

    High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. Together, human and mouse studies reveal novel

  12. Radioligand binding characterization of the bradykinin B(2) receptor in the rabbit and pig ileal smooth muscle.

    PubMed

    Meini, Stefania; Cucchi, Paola; Catalani, Claudio; Bellucci, Francesca; Santicioli, Paolo; Giuliani, Sandro; Maggi, Carlo Alberto

    2010-06-10

    Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and B(2) receptor. An attempt to highlight differences which can undertake ligands selectivity across the species is presented. In conclusion, the present study indicates MEN16132 as the only nonpeptidic compound which displays an even subnanomolar affinity for the rabbit and pig B(2) receptor.

  13. Dynorphin up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR is associated with increased CREB (Ser 133) phosphorylation, but is independent of BDNF/TrkB signaling pathways.

    PubMed

    Rittase, W Bradley; Dong, Yu; Barksdale, DaRel; Galdzicki, Zygmunt; Bausch, Suzanne B

    2014-05-01

    Emerging evidence suggests that neuronal responses to N-methyl-d-aspartate (NMDAR) activation/inactivation are influenced by subunit composition. For example, activation of synaptic NMDAR (comprised of GluN2A>GluN2B) phosphorylates cAMP-response-element-binding protein (CREB) at Ser 133, induces BDNF expression and promotes neuronal survival. Activation of extrasynaptic NMDAR (comprised of GluN2B>GluN2) dephosphorylates CREB (Ser 133), reduces BDNF expression and triggers neuronal death. These results led us to hypothesize that chronic inhibition of GluN2B-containing NMDAR would increase CREB (Ser 133) phosphorylation, increase BDNF levels and subsequently alter downstream dynorphin (DYN) and neuropeptide Y (NPY) expression. We focused on DYN and NPY because these neuropeptides can decrease excitatory neurotransmission and seizure occurrence and we reported previously that seizure-like events are reduced following chronic treatment with GluN2B antagonists. Consistent with our hypothesis, chronic treatment (17-21days) of hippocampal slice cultures with the GluN2B-selective antagonists ifenprodil or Ro25,6981 increased both CREB (Ser 133) phosphorylation and granule cell mossy fiber pathway DYN expression. Similar treatment with the non-subtype-selective NMDAR antagonists d-APV or memantine had no significant effect on either CREB (Ser 133) phosphorylation or DYN expression. In contrast to our hypothesis, BDNF levels were decreased following chronic treatment with Ro25,6981, but not ifenprodil, d-APV or memantine. Blockade of BDNF actions and TrkB activation did not significantly augment hilar DYN expression in vehicle-treated cultures and had no effect in Ro25,6981 treated cultures. These findings suggest that chronic exposure to GluN2B-selective NMDAR antagonists increased DYN expression through a putatively pCREB-dependent, but BDNF/TrkB-independent mechanism. Published by Elsevier Inc.

  14. Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

    PubMed Central

    Xia, Wenle; Gooden, David; Liu, Leihua; Zhao, Sumin; Soderblom, Erik J.; Toone, Eric J.; Beyer, Wayne F.; Walder, Harold; Spector, Neil L.

    2014-01-01

    Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85ErbB2) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85ErbB2. Here we show that PUVA reduced p85ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies. PMID:24551203

  15. The Design and Delivery of a Thermally Responsive Peptide to Inhibit S100B Mediated Neurodegeneration

    PubMed Central

    Hearst, Scoty M; Walker, Leslie R; Shao, Qingmei; Lopez, Mariper; Raucher, Drazen; Vig, Parminder J S

    2011-01-01

    S100B, a glial secreted protein is believed to play a major role in neurodegeneration in Alzheimer's disease, Down syndrome, traumatic brain injury and spinocerebellar ataxia type 1 (SCA1). SCA1 is a trinucleotide repeat disorder in which the expanded polyglutamine mutation in the protein ataxin-1 primarily targets Purkinje cells (PCs) of the cerebellum. Currently, the exact mechanism of S100B mediated PC damage in SCA1 is not clear. However, here we show that S100B may act via the activation of the RAGE signaling pathway resulting in oxidative stress mediated injury to mutant ataxin-1 expressing neurons. To combat S100B mediated neurodegeneration, we have designed a selective thermally responsive S100B inhibitory peptide, Synb1-ELP-TRTK. Our therapeutic polypeptide was developed using three key elements: (1) the elastin-like polypeptide (ELP), a thermally responsive polypeptide, (2) the TRTK12 peptide, a known S100B inhibitory peptide and (3) a cell penetrating peptide, Synb1, to enhance intracellular delivery. Binding studies revealed that our peptide, Synb1-ELP-TRTK, interacts with its molecular target S100B and maintains a high S100B binding affinity as comparable with the TRTK12 peptide alone. In addition, in vitro studies revealed that Synb1-ELP-TRTK treatment reduces S100B uptake in SHSY5Y cells. Furthermore, the Synb1-ELP-TRTK peptide decreased S100B induced oxidative damage to mutant ataxin-1 expressing neurons. To test the delivery capabilities of ELP based therapeutic peptides to the cerebellum; we treated mice with fluorescently labeled Synb1-ELP and observed that thermal targeting enhanced peptide delivery to the cerebellum. Here, we have laid the framework for thermal based therapeutic targeting to regions of the brain, particularly the cerebellum. Overall, our data suggests that thermal targeting of ELP based therapeutic peptides to the cerebellum is a novel treatment strategy for cerebellar neurodegenerative disorders. PMID:21958864

  16. Quantification of the Contribution of GLP-1 to Mediating Insulinotropic Effects of DPP-4 Inhibition With Vildagliptin in Healthy Subjects and Patients With Type 2 Diabetes Using Exendin [9-39] as a GLP-1 Receptor Antagonist.

    PubMed

    Nauck, Michael A; Kind, Joachim; Köthe, Lars D; Holst, Jens J; Deacon, Carolyn F; Broschag, Matthias; He, Yan Ling; Kjems, Lise; Foley, James

    2016-08-01

    We quantified the contribution of GLP-1 as a mediator of the therapeutic effects of dipeptidyl peptidase 4 (DPP-4) inhibition (vildagliptin) by using the GLP-1 receptor antagonist exendin [9-39] in patients with type 2 diabetes and in healthy subjects. Thirty-two patients with type 2 diabetes and 29 age- and weight-matched healthy control subjects were treated in randomized order with 100 mg once daily vildagliptin or placebo for 10 days. Meal tests were performed (days 9 and 10) without and with a high-dose intravenous infusion of exendin [9-39]. The main end point was the ratio of the areas under the curve (AUCs) of integrated insulin secretion rates (total AUCISR) and glucose (total AUCglucose) over 4 h after the meal. Vildagliptin treatment more than doubled responses of intact GLP-1 and glucose-dependent insulinotropic polypeptide and lowered glucose responses without changing AUCISR/AUCglucose in healthy subjects. Vildagliptin significantly increased this ratio by 10.5% in patients with type 2 diabetes, and exendin [9-39] reduced it (both P < 0.0001). The percentage reduction in the AUCISR/AUCglucose ratio achieved with exendin [9-39] was significantly smaller after vildagliptin treatment than after placebo treatment (P = 0.026) and was equivalent to 47 ± 5% of the increments due to vildagliptin. Thus, other mediators appear to contribute significantly to the therapeutic effects of DPP-4 inhibition. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  17. Purinoceptor modulation of noradrenaline release in rat tail artery: tonic modulation mediated by inhibitory P2Y- and facilitatory A2A-purinoceptors.

    PubMed Central

    Gonçalves, J.; Queiroz, G.

    1996-01-01

    1. The effects of analogues of adenosine and ATP on noradrenaline release elicited by electrical stimulation (5 Hz, 2700 pulses) were studied in superfused preparations of rat tail artery. The effects of purinoceptor antagonists, of adenosine deaminase and of adenosine uptake blockade were also examined. Noradrenaline was measured by h.p.l.c. electrochemical detection. 2. The A1-adenosine receptor agonist, N6-cyclopentyladenosine (CPA; 0.1-100 nM) reduced, whereas the A2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3-30 nM) increased evoked noradrenaline overflow. These effects were antagonized by the A1-adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 nM) and the A2-adenosine receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), respectively. The P2Y-purinoceptor agonist, 2-methylthio-ATP (1-100 microM) reduced noradrenaline overflow, an effect prevented by the P2-purinoceptor antagonist, cibacron blue 3GA (100 microM) and suramin (100 microM). 3. Adenosine deaminase (2 u ml-1), DMPX (100 nM) and inhibition of adenosine uptake with S-(p-nitrobenzyl)-6-thioinosine (NBTI; 50 nM) decreased evoked noradrenaline overflow. DPCPX alone did not change noradrenaline overflow but prevented the inhibition caused by NBTI. The P2Y-purinoceptor antagonist, cibacron blue 3GA (100 microM) increased evoked noradrenaline overflow as did suramin, a non-selective P2-antagonist. 4. It is concluded that, in rat tail artery, inhibitory (A1 and P2Y) and facilitatory (A2A) purinoceptors are present and modulate noradrenaline release evoked by electrical stimulation. Endogenous purines tonically modulate noradrenaline release through activation of inhibitory P2Y and facilitatory A2A purinoceptors, whereas a tonic activation of inhibitory A1 purinoceptors seems to be prevented by adenosine uptake. PMID:8825357

  18. Induction of nuclear factor kappaB by the CD30 receptor is mediated by TRAF1 and TRAF2.

    PubMed Central

    Duckett, C S; Gedrich, R W; Gilfillan, M C; Thompson, C B

    1997-01-01

    CD30 is a lymphoid cell-specific surface receptor which was originally identified as an antigen expressed on Hodgkin's lymphoma cells. Activation of CD30 induces the nuclear factor kappaB (NF-kappaB) transcription factor. In this study, we define the domains in CD30 which are required for NF-kappaB activation. Two separate elements of the cytoplasmic domain which were capable of inducing NF-kappaB independently of one another were identified. The first domain (domain 1) mapped to a approximately 120-amino-acid sequence in the membrane-proximal region of the CD30 cytoplasmic tail, between residues 410 and 531. A second, more carboxy-terminal region (domain 2) was identified between residues 553 and 595. Domain 2 contains two 5- to 10-amino-acid elements which can mediate the binding of CD30 to members of the tumor necrosis factor receptor-associated factor (TRAF) family of signal transducing proteins. Coexpression of CD30 with TRAF1 or TRAF2 but not TRAF3 augmented NF-kappaB activation through domain 2 but not domain 1. NF-kappaB induction through domain 2 was inhibited by coexpression of either full-length TRAF3 or dominant negative forms of TRAF1 or TRAF2. In contrast, NF-kappaB induction by domain 1 was not affected by alterations in TRAF protein levels. Together, these data support a model in which CD30 can induce NF-kappaB by both TRAF-dependent and -independent mechanisms. TRAF-dependent induction of NF-kappaB appears to be regulated by the relative levels of individual TRAF proteins in the cell. PMID:9032281

  19. A Phase 2, Double-Blind, Placebo-Controlled Randomized Trial Assessing the Efficacy of ABT-436, a Novel V1b Receptor Antagonist, for Alcohol Dependence

    PubMed Central

    Ryan, Megan L; Falk, Daniel E; Fertig, Joanne B; Rendenbach-Mueller, Beatrice; Katz, David A; Tracy, Katherine A; Strain, Eric C; Dunn, Kelly E; Kampman, Kyle; Mahoney, Elizabeth; Ciraulo, Domenic A; Sickles-Colaneri, Laurie; Ait-Daoud, Nassima; Johnson, Bankole A; Ransom, Janet; Scott, Charles; Koob, George F; Litten, Raye Z

    2017-01-01

    Alcohol use disorder has been linked to dysregulation of the brain stress systems, producing a negative emotional state leading to chronic relapsing behavior. Vasopressin receptors appear to have a regulatory role in stress, anxiety, and alcohol. This study evaluated the novel compound, ABT-436, a V1b receptor antagonist, in alcohol-dependent participants in a 12-week clinical trial. Men and women (n=150) who met criteria for DSM–IV alcohol dependence were recruited across four sites. Participants received double-blind ABT-436 or placebo, and a computerized behavioral intervention. ABT-436 was titrated to 800 mg/day during weeks 2–12. Although the primary outcome, percentage of heavy drinking days, was lower in participants receiving ABT-436 compared with placebo, this difference was not statistically significant (31.3 vs 37.6, respectively; p=0.172; d=0.20). However, participants receiving ABT-436 had significantly greater percentage of days abstinent than those receiving placebo (51.2 vs 41.6, respectively; p=0.037; d=0.31). No significant differences were found between treatment groups on any other measures of drinking, alcohol craving, or alcohol-related consequences. Smokers receiving ABT-436 smoked significantly fewer cigarettes per week than those receiving placebo (p=0.046). ABT-436 was well tolerated, with diarrhea (mild-to-moderate severity) being the most common side effect. In subgroup analyses, participants with relatively higher baseline levels of stress responded better to ABT-436 than placebo on select drinking outcomes, suggesting there may be value in testing medications targeting the vasopressin receptor in high stress, alcohol-dependent patients. PMID:27658483

  20. The PPARγ2 A/B-Domain Plays a Gene-Specific Role in Transactivation and Cofactor Recruitment

    PubMed Central

    Bugge, Anne; Grøntved, Lars; Aagaard, Mads M.; Borup, Rehannah; Mandrup, Susanne

    2009-01-01

    We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5–8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARγ target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARγ-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARγ2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARγ lacking the A/B-domain (PPARγCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARγCDE compared with cells expressing full-length PPARγ2. Using chromatin immunoprecipitation, we demonstrate that PPARγ binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARγ-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARγ2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes. PMID:19282365

  1. N-methyl-D-aspartate receptor antagonist MK-801 prevents apoptosis in rats that have undergone fetal spinal cord transplantation following spinal hemisection.

    PubMed

    Zhang, Qiang; Shao, Yang; Zhao, Changsong; Cai, Juan; Sun, Sheng

    2014-12-01

    Spinal cord injury is the main cause of paraplegia, but effective therapies for it are lacking. Embryonic spinal cord transplantation is able to repair spinal cord injury, albeit with a large amount of neuronal apoptosis remaining in the spinal cord. MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, is able to reduce cell death by decreasing the concentration of excitatory amino acids and preventing extracellular calcium ion influx. In this study, the effect of MK-801 on the apoptosis of spinal cord neurons in rats that have received a fetal spinal cord (FSC) transplant following spinal hemisection was investigated. Wistar rats were divided into three groups: Spinal cord hemisection injury with a combination of FSC transplantation and MK-801 treatment (group A); spinal cord hemisection injury with FSC transplantation (group B); and spinal cord injury with insertion of a Gelfoam pledget (group C). The rats were sacrificed 1, 3, 7 and 14 days after the surgery. Apoptosis in spinal slices from the injured spinal cord was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling reaction, and the expression of B-cell lymphoma-2 (Bcl-2) was measured by immunohistochemistry. The positive cells were quantitatively analyzed using a computer image analysis system. The rate of apoptosis and the positive expression of Bcl-2 protein in the spinal cord neurons in the three groups decreased in the following order: C>B>A (P<0.05) and A>B>C (P<0.05), respectively. This indicates that treatment with the NMDA receptor antagonist MK-801 prevents apoptosis in the spinal cord neurons of rats that have undergone FSC transplantation following spinal hemisection.

  2. N-methyl-D-aspartate receptor antagonist MK-801 prevents apoptosis in rats that have undergone fetal spinal cord transplantation following spinal hemisection

    PubMed Central

    ZHANG, QIANG; SHAO, YANG; ZHAO, CHANGSONG; CAI, JUAN; SUN, SHENG

    2014-01-01

    Spinal cord injury is the main cause of paraplegia, but effective therapies for it are lacking. Embryonic spinal cord transplantation is able to repair spinal cord injury, albeit with a large amount of neuronal apoptosis remaining in the spinal cord. MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, is able to reduce cell death by decreasing the concentration of excitatory amino acids and preventing extracellular calcium ion influx. In this study, the effect of MK-801 on the apoptosis of spinal cord neurons in rats that have received a fetal spinal cord (FSC) transplant following spinal hemisection was investigated. Wistar rats were divided into three groups: Spinal cord hemisection injury with a combination of FSC transplantation and MK-801 treatment (group A); spinal cord hemisection injury with FSC transplantation (group B); and spinal cord injury with insertion of a Gelfoam pledget (group C). The rats were sacrificed 1, 3, 7 and 14 days after the surgery. Apoptosis in spinal slices from the injured spinal cord was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling reaction, and the expression of B-cell lymphoma-2 (Bcl-2) was measured by immunohistochemistry. The positive cells were quantitatively analyzed using a computer image analysis system. The rate of apoptosis and the positive expression of Bcl-2 protein in the spinal cord neurons in the three groups decreased in the following order: C>B>A (P<0.05) and A>B>C (P<0.05), respectively. This indicates that treatment with the NMDA receptor antagonist MK-801 prevents apoptosis in the spinal cord neurons of rats that have undergone FSC transplantation following spinal hemisection. PMID:25371724

  3. Bacterial antagonists of fungal pathogens also control root-knot nematodes by induced systemic resistance of tomato plants.

    PubMed

    Adam, Mohamed; Heuer, Holger; Hallmann, Johannes

    2014-01-01

    The potential of bacterial antagonists of fungal pathogens to control the root-knot nematode Meloidogyne incognita was investigated under greenhouse conditions. Treatment of tomato seeds with several strains significantly reduced the numbers of galls and egg masses compared with the untreated control. Best performed Bacillus subtilis isolates Sb4-23, Mc5-Re2, and Mc2-Re2, which were further studied for their mode of action with regard to direct effects by bacterial metabolites or repellents, and plant mediated effects. Drenching of soil with culture supernatants significantly reduced the number of egg masses produced by M. incognita on tomato by up to 62% compared to the control without culture supernatant. Repellence of juveniles by the antagonists was shown in a linked twin-pot set-up, where a majority of juveniles penetrated roots on the side without inoculated antagonists. All tested biocontrol strains induced systemic resistance against M. incognita in tomato, as revealed in a split-root system where the bacteria and the nematodes were inoculated at spatially separated roots of the same plant. This reduced the production of egg masses by up to 51%, while inoculation of bacteria and nematodes in the same pot had only a minor additive effect on suppression of M. incognita compared to induced systemic resistance alone. Therefore, the plant mediated effect was the major reason for antagonism rather than direct mechanisms. In conclusion, the bacteria known for their antagonistic potential against fungal pathogens also suppressed M. incognita. Such "multi-purpose" bacteria might provide new options for control strategies, especially with respect to nematode-fungus disease complexes that cause synergistic yield losses.

  4. Discovery of highly selective brain-penetrant vasopressin 1a antagonists for the potential treatment of autism via a chemogenomic and scaffold hopping approach.

    PubMed

    Ratni, Hasane; Rogers-Evans, Mark; Bissantz, Caterina; Grundschober, Christophe; Moreau, Jean-Luc; Schuler, Franz; Fischer, Holger; Alvarez Sanchez, Ruben; Schnider, Patrick

    2015-03-12

    From a micromolar high throughput screening hit 7, the successful complementary application of a chemogenomic approach and of a scaffold hopping exercise rapidly led to a low single digit nanomolar human vasopressin 1a (hV1a) receptor antagonist 38. Initial optimization of the mouse V1a activities delivered suitable tool compounds which demonstrated a V1a mediated central in vivo effect. This novel series was further optimized through parallel synthesis with a focus on balancing lipophilicity to achieve robust aqueous solubility while avoiding P-gp mediated efflux. These efforts led to the discovery of the highly potent and selective brain-penetrant hV1a antagonist RO5028442 (8) suitable for human clinical studies in people with autism.

  5. Angiotensin II attenuates NMDA receptor-mediated neuronal cell death and prevents the associated reduction in Bcl-2 expression.

    PubMed

    Schelman, William R; Andres, Robert; Ferguson, Paul; Orr, Brent; Kang, Evan; Weyhenmeyer, James A

    2004-09-10

    While angiotensin II (Ang II) plays a major role in the regulation of blood pressure, fluid homeostasis and neuroendocrine function, recent studies have also implicated the peptide hormone in cell growth, differentiation and apoptosis. In support of this, we have previously demonstrated that Ang II attenuates N-methyl-D-aspartate (NMDA) receptor signaling [Molec. Brain Res. 48 (1997) 197]. To further examine the modulatory role of Ang II on NMDA receptor function, we investigated the effect of angiotensin receptor (AT) activation on NMDA-mediated cell death and the accompanying decrease in Bcl-2 expression. The viability of differentiated N1E-115 and NG108-15 neuronal cell lines was reduced following exposure to NMDA in a dose-dependent manner. MTT analysis (mitochondrial integrity) revealed a decrease in cell survival of 49.4+/-12.3% in NG108 cells and 79.9+/-6.8% in N1E cells following treatment with 10 mM NMDA for 20 h. Cytotoxicity in N1E cells was inhibited by the noncompetitive NMDA receptor antagonist, MK-801. Further, NMDA receptor-mediated cell death in NG108 cells was attenuated by treatment with Ang II. The Ang II effect was inhibited by both AT1 and AT2 receptor antagonists, losartan and PD123319, respectively, suggesting that both receptor subtypes may play a role in the survival effect of Ang II. Since it has been shown that activation of NMDA receptors alters the expression of Bcl-2 family proteins, Western blot analysis was performed in N1E cells to determine whether Ang II alters the NMDA-induced changes in Bcl-2 expression. A concentration-dependent decrease of intracellular Bcl-2 protein levels was observed following treatment with NMDA, and this reduction was inhibited by MK801. Addition of Ang II suppressed the NMDA receptor-mediated reduction in Bcl-2. The Ang II effect on NMDA-mediated changes in Bcl-2 levels was blocked by PD123319, but was not significantly changed by losartan, suggesting AT2 receptor specificity. Taken together, these

  6. Balancing selected medication costs with total number of daily injections: a preference analysis of GnRH-agonist and antagonist protocols by IVF patients.

    PubMed

    Sills, E Scott; Collins, Gary S; Salem, Shala A; Jones, Christopher A; Peck, Alison C; Salem, Rifaat D

    2012-08-30

    During in vitro fertilization (IVF), fertility patients are expected to self-administer many injections as part of this treatment. While newer medications have been developed to substantially reduce the number of these injections, such agents are typically much more expensive. Considering these differences in both cost and number of injections, this study compared patient preferences between GnRH-agonist and GnRH-antagonist based protocols in IVF. Data were collected by voluntary, anonymous questionnaire at first consultation appointment. Patient opinion concerning total number of s.c. injections as a function of non-reimbursed patient cost associated with GnRH-agonist [A] and GnRH-antagonist [B] protocols in IVF was studied. Completed questionnaires (n = 71) revealed a mean +/- SD patient age of 34 +/- 4.1 yrs. Most (83.1%) had no prior IVF experience; 2.8% reported another medical condition requiring self-administration of subcutaneous medication(s). When out-of-pocket cost for [A] and [B] were identical, preference for [B] was registered by 50.7% patients. The tendency to favor protocol [B] was weaker among patients with a health occupation. Estimated patient costs for [A] and [B] were $259.82 +/- 11.75 and $654.55 +/- 106.34, respectively (p < 0.005). Measured patient preference for [B] diminished as the cost difference increased. This investigation found consistently higher non-reimbursed direct medication costs for GnRH-antagonist IVF vs. GnRH-agonist IVF protocols. A conditional preference to minimize downregulation (using GnRH-antagonist) was noted among some, but not all, IVF patient sub-groups. Compared to IVF patients with a health occupation, the preference for GnRH-antagonist was weaker than for other patients. While reducing total number of injections by using GnRH-antagonist is a desirable goal, it appears this advantage is not perceived equally by all IVF patients and its utility is likely discounted heavily by patients when nonreimbursed

  7. Balancing selected medication costs with total number of daily injections: a preference analysis of GnRH-agonist and antagonist protocols by IVF patients

    PubMed Central

    2012-01-01

    Background During in vitro fertilization (IVF), fertility patients are expected to self-administer many injections as part of this treatment. While newer medications have been developed to substantially reduce the number of these injections, such agents are typically much more expensive. Considering these differences in both cost and number of injections, this study compared patient preferences between GnRH-agonist and GnRH-antagonist based protocols in IVF. Methods Data were collected by voluntary, anonymous questionnaire at first consultation appointment. Patient opinion concerning total number of s.c. injections as a function of non-reimbursed patient cost associated with GnRH-agonist [A] and GnRH-antagonist [B] protocols in IVF was studied. Results Completed questionnaires (n = 71) revealed a mean +/− SD patient age of 34 +/− 4.1 yrs. Most (83.1%) had no prior IVF experience; 2.8% reported another medical condition requiring self-administration of subcutaneous medication(s). When out-of-pocket cost for [A] and [B] were identical, preference for [B] was registered by 50.7% patients. The tendency to favor protocol [B] was weaker among patients with a health occupation. Estimated patient costs for [A] and [B] were $259.82 +/− 11.75 and $654.55 +/− 106.34, respectively (p < 0.005). Measured patient preference for [B] diminished as the cost difference increased. Conclusions This investigation found consistently higher non-reimbursed direct medication costs for GnRH-antagonist IVF vs. GnRH-agonist IVF protocols. A conditional preference to minimize downregulation (using GnRH-antagonist) was noted among some, but not all, IVF patient sub-groups. Compared to IVF patients with a health occupation, the preference for GnRH-antagonist was weaker than for other patients. While reducing total number of injections by using GnRH-antagonist is a desirable goal, it appears this advantage is not perceived equally by all IVF patients and its utility is likely

  8. Nonpeptide vasopressin antagonists: a new group of hormone blockers entering the scene.

    PubMed

    Mayinger, B; Hensen, J

    1999-01-01

    After the story of success of hormone blockers for catecholamines, aldosterone and angiotensin II and their successful implementation into clinical practice another endocrine cardiovascular system has come into focus. It has long been known, that the hormone vasopressin plays an important role in peripheral vasoconstriction, hypertension and in several disease conditions with dilutional hyponatremia in edematous disorders, like congestive heart failure, liver cirrhosis, SIADH and nephrotic syndrome. A series of orally active nonpeptide antagonists against the vasopressin receptor subtypes has recently been synthesized and is now under intensive examination. Nonpeptide V1a-receptor specific antagonists, OPC 21268 and SR 49059, nonpeptide V2-receptor specific antagonists, SR 121463 A and VPA 985, and combined V1a-/V2-receptor antagonists, OPC 31260 and YM 087, have become available for clinical research. AVP-V2-receptor antagonists lead to a dose-dependent diabetes insipidus in animals and man. The term aquaretic drugs (aquaretics) has been coined for these drugs to highlight their different mechanism compared to the saluretic diuretic furosemide. V1a-receptor antagonists might offer new therapeutic advantages in the treatment of vasoconstriction and hypertension. Combined V1a-/V2-receptor antagonists might be beneficial in the treatment of congestive heart failure. Early results are promising and now need to be confirmed in large clinical studies.

  9. Plexin-B2 promotes invasive growth of malignant glioma

    PubMed Central

    Pingle, Sandeep C.; Kesari, Santosh; Wang, Huaien; Yong, Raymund L.; Zou, Hongyan; Friedel, Roland H.

    2015-01-01

    Invasive growth is a major determinant of the high lethality of malignant gliomas. Plexin-B2, an axon guidance receptor important for mediating neural progenitor cell migration during development, is upregulated in gliomas, but its function therein remains poorly understood. Combining bioinformatic analyses, immunoblotting and immunohistochemistry of patient samples, we demonstrate that Plexin-B2 is consistently upregulated in all types of human gliomas and that its expression levels correlate with glioma grade and poor survival. Activation of Plexin-B2 by Sema4C ligand in glioblastoma cells induced actin-based cytoskeletal dynamics and invasive migration in vitro. This proinvasive effect was associated with activation of the cell motility mediators RhoA and Rac1. Furthermore, costimulation of Plexin-B2 and the receptor tyrosine kinase Met led to synergistic Met phosphorylation. In intracranial glioblastoma transplants, Plexin-B2 knockdown hindered invasive growth and perivascular spreading, and resulted in decreased tumor vascularity. Our results demonstrate that Plexin-B2 promotes glioma invasion and vascularization, and they identify Plexin-B2 as a potential novel prognostic marker for glioma malignancy. Targeting the Plexin-B2 pathway may represent a novel therapeutic approach to curtail invasive growth of glioblastoma. PMID:25762646

  10. Atrial Fibrillation, Type 2 Diabetes, and Non-Vitamin K Antagonist Oral Anticoagulants: A Review.

    PubMed

    Plitt, Anna; McGuire, Darren K; Giugliano, Robert P

    2017-04-01

    Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with a 5-fold increase in the risk for stroke. Type 2 diabetes is an independent risk factor for both stroke and atrial fibrillation, and in the setting of AF, type 2 diabetes is independently associated with a 2% to 3.5% increase in absolute stroke rate per year. The overlap in the pathophysiologies of AF and type 2 diabetes are not well understood, and current practice guidelines provide few recommendations regarding patients with both conditions. In this article, we review the epidemiology and pathophysiology of the nexus of AF and type 2 diabetes. Furthermore, we analyze the subgroup of patients with type 2 diabetes enrolled in phase 3 clinical trials of non-vitamin K antagonist oral anticoagulants in prevention of arterial thromboembolism in AF, highlighting the greater absolute benefit of non-vitamin K oral anticoagulants in patients with type 2 diabetes. Finally, we offer recommendations on risk stratification and therapy for patients with concomitant AF and type 2 diabetes. We highlight the increased thromboembolic risk with coexisting AF and type 2 diabetes. We recommend that further studies be done to evaluate the potential benefits of anticoagulation for all patients who have both and the potential for non-vitamin K oral anticoagulants to have greater benefits than risks over vitamin K antagonists.

  11. Quantification of indirect pathway inhibition by the adenosine A2a antagonist SYN115 in Parkinson disease.

    PubMed

    Black, Kevin J; Koller, Jonathan M; Campbell, Meghan C; Gusnard, Debra A; Bandak, Stephen I

    2010-12-01

    Adenosine A(2a) receptor antagonists reduce symptom severity in Parkinson disease (PD) and animal models. Rodent studies support the hypothesis that A(2a) antagonists produce this benefit by reducing the inhibitory output of the basal ganglia indirect pathway. One way to test this hypothesis in humans is to quantify regional pharmacodynamic responses with cerebral blood flow (CBF) imaging. That approach has also been proposed as a tool to accelerate pharmaceutical dose finding, but has not yet been applied in humans to drugs in development. We successfully addressed both these aims with a perfusion magnetic resonance imaging (MRI) study of the novel adenosine A(2a) antagonist SYN115. During a randomized, double-blind, placebo-controlled, crossover study in 21 PD patients on levodopa but no agonists, we acquired pulsed arterial spin labeling MRI at the end of each treatment period. SYN115 produced a highly significant decrease in thalamic CBF, consistent with reduced pallidothalamic inhibition via the indirect pathway. Similar decreases occurred in cortical regions whose activity decreases with increased alertness and externally focused attention, consistent with decreased self-reported sleepiness on SYN115. Remarkably, we also derived quantitative pharmacodynamic parameters from the CBF responses to SYN115. These results suggested that the doses tested were on the low end of the effective dose range, consistent with clinical data reported separately. We conclude that (1) SYN115 enters the brain and exerts dose-dependent regional effects, (2) the most prominent of these effects is consistent with deactivation of the indirect pathway as predicted by preclinical studies; and (3) perfusion MRI can provide rapid, quantitative, clinically relevant dose-finding information for pharmaceutical development.

  12. Quantification of indirect pathway inhibition by the adenosine A2a antagonist SYN115 in Parkinson disease

    PubMed Central

    Black, Kevin J.; Koller, Jonathan M.; Campbell, Meghan C.; Gusnard, Debra A.; Bandak, Stephen I.

    2010-01-01

    Adenosine A2a receptor antagonists reduce symptom severity in Parkinson disease (PD) and animal models. Rodent studies support the hypothesis that A2a antagonists produce this benefit by reducing the inhibitory output of the basal ganglia indirect pathway. One way to test this hypothesis in humans is to quantify regional pharmacodynamic responses with cerebral blood flow (CBF) imaging. That approach has also been proposed as a tool to accelerate pharmaceutical dose-finding, but has not yet been applied in humans to drugs in development. We successfully addressed both these aims with a perfusion MRI study of the novel adenosine A2a antagonist SYN115. During a randomized, double-blind, placebo-controlled, crossover study in 21 PD patients on levodopa but no agonists, we acquired pulsed arterial spin labeling MRI at the end of each treatment period. SYN115 produced a highly significant decrease in thalamic CBF, consistent with reduced pallidothalamic inhibition via the indirect pathway. Similar decreases occurred in cortical regions whose activity decreases with increased alertness and externally-focused attention, consistent with decreased self-reported sleepiness on SYN115. Remarkably, we also derived quantitative pharmacodynamic parameters from the CBF responses to SYN115. These results suggested that the doses tested were on the low end of the effective dose range, consistent with clinical data reported separately. We conclude that (1) SYN115 enters the brain and exerts dose-dependent regional effects, (2) the most prominent of these effects is consistent with deactivation of the indirect pathway as predicted by preclinical studies; and (3) perfusion MRI can provide rapid, quantitative, clinically relevant dose-finding information for pharmaceutical development. PMID:21123574

  13. Cost-effectiveness of histamine receptor-2 antagonist versus proton pump inhibitor for stress ulcer prophylaxis in critically ill patients*.

    PubMed

    MacLaren, Robert; Campbell, Jon

    2014-04-01

    To examine the cost-effectiveness of using histamine receptor-2 antagonist or proton pump inhibitor for stress ulcer prophylaxis. Decision analysis model examining costs and effectiveness of using histamine receptor-2 antagonist or proton pump inhibitor for stress ulcer prophylaxis. Costs were expressed in 2012 U.S. dollars from the perspective of the institution and included drug regimens and the following outcomes: clinically significant stress-related mucosal bleed, ventilator-associated pneumonia, and Clostridium difficile infection. Effectiveness was the mortality risk associated with these outcomes and represented by survival. Costs, occurrence rates, and mortality probabilities were extracted from published data. A simulation model. A mixed adult ICU population. Histamine receptor-2 antagonist or proton pump inhibitor for 9 days of stress ulcer prophylaxis therapy. Output variables were expected costs, expected survival rates, incremental cost, and incremental survival rate. Univariate sensitivity analyses were conducted to determine the drivers of incremental cost and incremental survival. Probabilistic sensitivity analysis was conducted using second-order Monte Carlo simulation. For the base case analysis, the expected cost of providing stress ulcer prophylaxis was $6,707 with histamine receptor-2 antagonist and $7,802 with proton pump inhibitor, resulting in a cost saving of $1,095 with histamine receptor-2 antagonist. The associated mortality probabilities were 3.819% and 3.825%, respectively, resulting in an absolute survival benefit of 0.006% with histamine receptor-2 antagonist. The primary drivers of incremental cost and survival were the assumptions surrounding ventilator-associated pneumonia and bleed. The probabilities that histamine receptor-2 antagonist was less costly and provided favorable survival were 89.4% and 55.7%, respectively. A secondary analysis assuming equal rates of C. difficile infection showed a cost saving of $908 with histamine

  14. Discovery of an Orally Bioavailable Gonadotropin-Releasing Hormone Receptor Antagonist.

    PubMed

    Kim, Seon-Mi; Lee, Minhee; Lee, So Young; Park, Euisun; Lee, Soo-Min; Kim, Eun Jeong; Han, Min Young; Yoo, Taekyung; Ann, Jihyae; Yoon, Suyoung; Lee, Jiyoun; Lee, Jeewoo

    2016-10-13

    We developed a compound library for orally available gonadotropin-releasing hormone (GnRH) receptor antagonists that were based on a uracil scaffold. On the basis of in vitro activity and CYP inhibition profile, we selected 18a (SKI2496) for further in vivo studies. Compound 18a exhibited more selective antagonistic activity toward the human GnRH receptors over the GnRHRs in monkeys and rats, and this compound also showed inhibitory effects on GnRH-mediated signaling pathways. Pharmacokinetic and pharmacodynamic evaluations of 18a revealed improved bioavailability and superior gonadotropic suppression activity compared with Elagolix, the most clinically advanced compound. Considering that 18a exhibited highly potent and selective antagonistic activity toward the hGnRHRs along with favorable pharmacokinetic profiles, we believe that 18a may represent a promising candidate for an orally available hormonal therapy.

  15. Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

    PubMed

    Abdi, Muna H; Beswick, Paul J; Billinton, Andy; Chambers, Laura J; Charlton, Andrew; Collins, Sue D; Collis, Katharine L; Dean, David K; Fonfria, Elena; Gleave, Robert J; Lejeune, Clarisse L; Livermore, David G; Medhurst, Stephen J; Michel, Anton D; Moses, Andrew P; Page, Lee; Patel, Sadhana; Roman, Shilina A; Senger, Stefan; Slingsby, Brian; Steadman, Jon G A; Stevens, Alexander J; Walter, Daryl S

    2010-09-01

    A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies. Copyright 2010 Elsevier Ltd. All rights reserved.

  16. Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

    PubMed

    Queiroz, Glória; Quintas, Clara; Talaia, Carlos; Gonçalves, Jorge

    2004-08-01

    In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

  17. In vitro histamine H/sub 2/-antagonist activity of the novel compound HUK 978

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Coombes, J.D.; Norris, D.B.; Rising, T.J.

    1985-11-04

    Histamine stimulated adenylate cyclase from guinea-pig fundic mucosa and /sup 3/H-tiotidine binding in guinea-pig cerebral cortex were used to assess the in-vitro histamine H/sub 2/-activity of the novel H/sub 2/-antagonist HUK 978. The results showed that HUK 978 was a more potent H/sub 2/-antagonist than either cimetidine or ranitidine. HUK 978 was also shown to be devoid of activity at the histamine H-/sub 1/-receptor, the muscarinic receptor and the ..cap alpha.. and ..beta..-adrenergic receptors.

  18. Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate.

    PubMed

    Pestel, Sabine; Nath, Annegret; Jungermann, Kurt; Schieferdecker, Henrike L

    2003-08-15

    Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.

  19. Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

    PubMed Central

    2012-01-01

    Background Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway. Results The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1. Conclusions Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway

  20. A dual CCR2/CCR5 chemokine antagonist, BMS-813160? Evaluation of WO2011046916.

    PubMed

    Norman, Peter

    2011-12-01

    This application claims the dual CCR2/CCR5 antagonist (S)-1-[(1S,2R,4R)-4-isopropyl(methyl)amino)-2-propylcyclohexyl]-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-2-one, crystalline form N-1, a process for its preparation and therapeutic uses of it. The compound is shown to be a potent, well-absorbed, drug with a good duration of action. It shows better properties than similar compounds previously claimed by Bristol-Myers Squibb and appears to be a clinical development candidate.