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.

Dephosphorylation of GluN2B C-Terminal Tyrosine Residues Does Not Contribute to Acute Ethanol Inhibition of Recombinant NMDA Receptors

PubMed Central

Hughes, Benjamin A.; Smothers, Corigan T.; Woodward, John J.

2013-01-01

N-methyl-D-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10–600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors. PMID:23357553

Enhancement of bradykinin and resensitization of its B2 receptor.

PubMed

Marcic, B; Deddish, P A; Jackman, H L; Erdös, E G

1999-03-01

We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

PubMed Central

Ciaccio, Mark F.; Chuu, Chih-pin; Jones, Richard B.

2012-01-01

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This

Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster

PubMed Central

Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

2017-01-01

Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor’s pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B’s pharmacology, we evaluated the receptor’s response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo. PMID:28553207

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

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

PubMed

Loebrich, Sven; Benoit, Marc Robert; Konopka, Jaclyn Aleksandra; Cottrell, Jeffrey Richard; Gibson, Joanne; Nedivi, Elly

2016-02-08

Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors. Copyright © 2016 Elsevier Ltd. All rights reserved.

TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

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

St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.

2008-10-24

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitromore » and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.« less

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.

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

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

Expression of the ephrin receptor B2 in the embryonic chicken bursa of Fabricius

USDA-ARS?s Scientific Manuscript database

Chicken B-cells develop in a specific organ, the bursa of Fabricius. To understand the bursal microenvironment guiding B-cell development, previous studies identified ephrin (Eph) receptor B2 (EphB2) gene transcripts in the embryonic bursa. We hypothesize that the EphB2 receptors and their ligands r...

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

PubMed

Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep

2014-07-01

To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.

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.

SULT2B1b Sulfotransferase: Induction by Vitamin D Receptor and Reduced Expression in Prostate Cancer

PubMed Central

Seo, Young-Kyo; Mirkheshti, Nooshin; Song, Chung S.; Kim, Soyoung; Dodds, Sherry; Ahn, Soon C.; Christy, Barbara; Mendez-Meza, Rosario; Ittmann, Michael M.; Abboud-Werner, Sherry

2013-01-01

An elevated tumor tissue androgen level, which reactivates androgen receptor in recurrent prostate cancer, arises from the intratumor synthesis of 5α-dihydrotestosterone through use of the precursor steroid dehydroepiandrosterone (DHEA) and is fueled by the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD1), aldoketoreductase (AKR1C3), and steroid 5-alpha reductase, type 1 (SRD5A1) present in cancer tissue. Sulfotransferase 2B1b (SULT2B1b) (in short, SULT2B) is a prostate-expressed hydroxysteroid SULT that converts cholesterol, oxysterols, and DHEA to 3β-sulfates. DHEA metabolism involving sulfonation by SULT2B can potentially interfere with intraprostate androgen synthesis due to reduction of free DHEA pool and, thus, conversion of DHEA to androstenedione. Here we report that in prostatectomy specimens from treatment-naive patients, SULT2B expression is markedly reduced in malignant tissue (P < .001, Mann-Whitney U test) compared with robust expression in adjacent nonmalignant glands. SULT2B was detected in formalin-fixed specimens by immunohistochemistry on individual sections and tissue array. Immunoblotting of protein lysates of frozen cancer and matched benign tissue confirmed immunohistochemistry results. An in-house–developed rabbit polyclonal antibody against full-length human SULT2B was validated for specificity and used in the analyses. Ligand-activated vitamin D receptor induced the SULT2B1 promoter in vivo in mouse prostate and increased SULT2B mRNA and protein levels in vitro in prostate cancer cells. A vitamin D receptor/retinoid X receptor-α–bound DNA element (with a DR7 motif) mediated induction of the transfected SULT2B1 promoter in calcitriol-treated cells. SULT2B knockdown caused an increased proliferation rate of prostate cancer cells upon stimulation by DHEA. These results suggest that the tumor tissue SULT2B level may partly control prostate cancer growth, and its induction in a therapeutic setting may inhibit disease

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.

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy.

PubMed

Fonfara, Sonja; Hetzel, Udo; Oyama, Mark A; Kipar, Anja

2014-03-01

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B. The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular interactions between general anesthetics and the 5HT2B receptor.

PubMed

Matsunaga, Felipe; Gao, Lu; Huang, Xi-Ping; Saven, Jeffery G; Roth, Bryan L; Liu, Renyu

2015-01-01

Serotonin modulates many processes through a family of seven serotonin receptors. However, no studies have screened for interactions between general anesthetics currently in clinical use and serotonergic G-protein-coupled receptors (GPCRs). Given that both intravenous and inhalational anesthetics have been shown to target other classes of GPCRs, we hypothesized that general anesthetics might interact directly with some serotonin receptors and thus modify their function. Radioligand binding assays were performed to screen serotonin receptors for interactions with propofol and isoflurane as well as for affinity determinations. Docking calculations using the crystal structure of 5-HT2B were performed to computationally confirm the binding assay results and locate anesthetic binding sites. The 5-HT2B class of receptors interacted significantly with both propofol and isoflurane in the primary screen. The affinities for isoflurane and propofol were determined to be 7.78 and .95 μM, respectively, which were at or below the clinical concentrations for both anesthetics. The estimated free energy derived from docking calculations for propofol (-6.70 kcal/mol) and isoflurane (-5.10 kcal/mol) correlated with affinities from the binding assay. The anesthetics were predicted to dock at a pharmacologically relevant binding site of 5HT2B. The molecular interactions between propofol and isoflurane with the 5-HT2B class of receptors were discovered and characterized. This finding implicates the serotonergic GPCRs as potential anesthetic targets.

Colonic inflammation increases the contribution of muscarinic M2 receptors to carbachol-induced contraction of the rat colon.

PubMed

Jragh, Dina M; Khan, Islam; Oriowo, Mabayoje A

2011-01-01

Carbachol-induced contraction of the rat colon is impaired in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. The main objective of this study was to examine the effect of colitis on the expression and function of muscarinic (M) receptor subtypes in the rat colon. Rats (n = 80) were treated with TNBS and used 5 days later for measurement of contractility, myeloperoxidase activity, histology and expression of muscarinic receptor isoforms using Western blot analysis. Carbachol produced concentration-dependent contractions of colonic segments from control (n = 40) and TNBS-treated (n = 40) rats with no significant difference in potency. However, the maximum response to carbachol was significantly reduced in colon segments of TNBS-treated rats. The selective muscarinic receptor antagonists 4-diphenylacetoxy-N-methyl piperidine (4-DAMP, M(3)), pirenzepine (M(1)) and methoctramine (M(2)) antagonized carbachol-induced contraction in control (9.1 ± 0.1, 6.7 ± 0.3 and 6.0 ± 0.1, respectively) and TNBS-treated rats (9.2 ± 0.2, 6.9 ± 0.2, 6.7 ± 0.2). The -logK(B) values in control rats are consistent with an action of carbachol on muscarinic M(3) receptors. There was no significant difference in -logK(B) values for 4-DAMP and pirenzepine in control and TNBS-treated rats, but methoctramine was fivefold more potent in TNBS-treated rats, possibly indicating an increased contribution of muscarinic M(2) receptors to carbachol-induced contraction in the inflamed colon. The expression of M(2) receptors was also significantly increased in colon segments from TNBS-treated rats, confirming the increased role of muscarinic M(2) receptors in the inflamed colon. The data show that while only M(3) receptors appeared to mediate carbachol-induced contraction in control segments, expression of both M(2) and M(3) receptors was increased in the inflamed rat colon. Copyright © 2011 S. Karger AG, Basel.

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.

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

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

Furukawa, Kiyoshi; Sato, Toru; Katsuno, Tatsuro, E-mail: katsuno@faculty.chiba-u.jp

2011-02-25

Research highlights: {yields} Smad3{sup -/-} mice showed an increased number of proliferating epithelial cells in colonic crypts. {yields} Proliferating epithelial cells showed activated Wnt/{beta}-catenin pathway. {yields} Smad3{sup -/-} mice also showed intermingling of proliferating cells with differentiated cells. {yields} Loss of EphB receptor expression was observed in the colonic crypts of Smad3{sup -/-} mice. {yields} Loss of EphB receptor expression is likely responsible for cell intermingling. -- Abstract: Deficiency of Smad3, an intracellular mediator of TGF-{beta}, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonicmore » epithelial cell proliferation and crypt formation. Smad3{sup ex8/ex8} C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3{sup -/-} mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3{sup -/-} mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear {beta}-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3{sup -/-} mice in accordance with nuclear {beta}-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3{sup -/-} mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system

Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability.

PubMed

Nakagawa, Satoe H; Hua, Qing-xin; Hu, Shi-Quan; Jia, Wenhua; Wang, Shuhua; Katsoyannis, Panayotis G; Weiss, Michael A

2006-08-04

Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Cilleros, Victor; Tomàs, Josep

2017-01-01

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M 1 -, M 2 - and M 4 -subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo . Our previous results show that M 1 , M 2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M 1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M 2 receptor is largely independent of both M 1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development

PubMed Central

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A.; Cilleros, Victor; Tomàs, Josep

2017-01-01

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M1-, M2- and M4-subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo. Our previous results show that M1, M2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M2 receptor is largely independent of both M1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination. PMID:28228723

Amygdala Infusions of an NR2B-Selective or an NR2A-Preferring NMDA Receptor Antagonist Differentially Influence Fear Conditioning and Expression in the Fear-Potentiated Startle Test

ERIC Educational Resources Information Center

Walker, David L.; Davis, Michael

2008-01-01

Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the…

Tumor Suppressor Activity of the EphB2 Receptor in Prostate Cancer

DTIC Science & Technology

2006-11-01

should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of...information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM...identified in the EphB2 receptor gene in prostate cancer suggesting that EphB2, a member of the large Eph receptor tyrosine kinase family, is a tumor

Mechanisms of ErbB receptor negative regulation and relevance in cancer

PubMed Central

Fry, William H.D.; Kotelawala, Lakmal; Sweeney, Colleen; Carraway, Kermit L.

2009-01-01

The ErbB family of receptor tyrosine kinases engages a wide variety of signaling pathways that collectively direct transcriptional programs controlling organogenesis during development and tissue maintenance in the adult. These receptors are also frequently found overexpressed or aberrantly activated in various cancers, suggesting that ErbB receptor signaling activity must be very tightly regulated. Sufficient levels of ErbB signaling are necessary to mediate tissue homeostasis, for example, but over-signaling can trigger cellular processes that contribute to cancer initiation or progression. Efforts over the last quarter century have led to a thorough understanding of the signaling pathways that are activated by these receptors and the mechanisms by which ErbB receptors engage these pathways. However, the compensatory negative regulatory mechanisms responsible for attenuating receptor activation have only more recently begun to be explored. Here we review the different known mechanisms of ErbB negative regulation, with particular emphasis on those proteins that exhibit some specificity for the ErbB family. We also describe how loss or suppression of ErbB negative regulators may contribute to tumor development, and discuss how restoration or augmentation of these pathways may represent a novel avenue for the development of ErbB-targeted therapies. PMID:18706412

Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus

USDA-ARS?s Scientific Manuscript database

The cDNA of the receptor for CAP2b/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP2b-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP2b/PVK receptor, as concluded from analyses of the predicted protein sequence, ph...

GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.

PubMed

Price, Christopher J; Scott, Ricardo; Rusakov, Dmitri A; Capogna, Marco

2008-07-02

Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

Adenosine A2B receptor: from cell biology to human diseases

NASA Astrophysics Data System (ADS)

Sun, Ying; Huang, Pingbo

2016-08-01

Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR’s functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases.

Cooperation of neurotrophin receptor TrkB and Her2 in breast cancer cells facilitates brain metastases.

PubMed

Choy, Cecilia; Ansari, Khairul I; Neman, Josh; Hsu, Sarah; Duenas, Matthew J; Li, Hubert; Vaidehi, Nagarajan; Jandial, Rahul

2017-04-26

Patients with primary breast cancer that is positive for human epidermal growth factor receptor 2 (Her2+) have a high risk of developing metastases in the brain. Despite gains with systemic control of Her2+ disease using molecular therapies, brain metastases remain recalcitrant to therapeutic discovery. The clinical predilection of Her2+ breast cancer cells to colonize the brain likely relies on paracrine mechanisms. The neural niche poses unique selection pressures, and neoplastic cells that utilize the brain microenvironment may have a survival advantage. Tropomyosin-related kinase B (TrkB), Her2, and downstream targets were analyzed in primary breast cancer, breast-to-brain metastasis (BBM) tissues, and tumor-derived cell lines using quantitative real-time PCR, western blot, and immunohistochemical assessment. TrkB function on BBM was confirmed with intracranial, intracardiac, or mammary fat pad xenografts in non-obese diabetic/severe combined immunodeficiency mice. The function of brain-derived neurotrophic factor (BDNF) on cell proliferation and TrkB/Her2 signaling and interactions were confirmed using selective shRNA knockdown and selective inhibitors. The physical interaction of Her2-TrkB was analyzed using electron microscopy, co-immunoprecipitation, and in silico analysis. Dual targeting of Her2 and TrkB was analyzed using clinically utilized treatments. We observed that patient tissues and cell lines derived from Her2+ human BBM displayed increased activation of TrkB, a neurotrophin receptor. BDNF, an extracellular neurotrophin, with roles in neuronal maturation and homeostasis, specifically binds to TrkB. TrkB knockdown in breast cancer cells led to decreased frequency and growth of brain metastasis in animal models, suggesting that circulating breast cancer cells entering the brain may take advantage of paracrine BDNF-TrkB signaling for colonization. In addition, we investigated a possible interaction between TrkB and Her2 receptors on brain metastatic

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

PubMed

Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Role of kinin B1 and B2 receptors in memory consolidation during the aging process of mice.

PubMed

Lemos, Mayra Tolentino Resk; Amaral, Fabio Agostini; Dong, Karis Ester; Bittencourt, Maria Fernanda Queiroz Prado; Caetano, Ariadiny Lima; Pesquero, João Bosco; Viel, Tania Araujo; Buck, Hudson Sousa

2010-04-01

Under physiological conditions, elderly people present memory deficit associated with neuronal loss. This pattern is also associated with Alzheimer's disease but, in this case, in a dramatically intensified level. Kinin receptors have been involved in neurodegeneration and increase of amyloid-beta concentration, associated with Alzheimer's disease (AD). Considering these findings, this work evaluated the role of kinin receptors in memory consolidation during the aging process. Male C57Bl/6 (wt), knock-out B1 (koB1) or B2 (koB2) mice (3, 6, 12 and 18-month-old - mo; n=10 per group) were submitted to an acquisition session, reinforcement to learning (24h later: test 1) and final test (7days later: test 2), in an active avoidance apparatus, to evaluate memory. Conditioned avoidance responses (CAR, % of 50 trials) were registered. In acquisition sessions, similar CAR were obtained among age matched animals from all strains. However, a significant decrease in CAR was observed throughout the aging process (3mo: 8.8+/-2.3%; 6mo: 4.1+/-0.6%; 12mo: 2.2+/-0.6%, 18mo: 3.6+/-0.6%, P<0.01), indicating a reduction in the learning process. In test 1, as expected, memory retention increased significantly (P<0.05) in all 3- and 6-month-old animals as well as in 12-month-old-wt and 12-month-old-koB1 (P<0.01), compared to the training session. However, 12-month-old-koB2 and all 18-month-old animals did not show an increase in memory retention. In test 2, 3- and 6-month-old wt and koB1 mice of all ages showed a significant improvement in memory (P<0.05) compared to test 1. However, 12-month-old wt and koB2 mice of all ages showed no difference in memory retention. We suggest that, during the aging process, the B1 receptor could be involved in neurodegeneration and memory loss. Nevertheless, the B2 receptor is apparently acting as a neuroprotective factor. Copyright 2009 Elsevier Ltd. All rights reserved.

Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kγ pathway.

PubMed

Sgnaolin, V; Pereira, T C B; Bogo, M R; Zanin, R; Battastini, A M O; Morrone, F B; Campos, M M

2013-08-01

Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.

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.

Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors.

PubMed

Zemkova, Hana; He, Mu-Lan; Koshimizu, Taka-aki; Stojilkovic, Stanko S

2004-08-04

The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X2aR, P2X2bR, and P2X3R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences of P2X2aR and P2X2bR. Chimeric P2X2a/V60-F301X3R and P2X2b/V60-F301X3R inherited the P2X3R ligand-selective profile, whereas the potency of agonists for P2X2a/V60-R180X3R was in between those observed at parental receptors. Furthermore, P2X2a/V60-F301X3R and P2X2a/V60-R180X3R desensitized in a P2X2aR-specific manner, and P2X2b/V60-F301X3R desensitized with rates comparable with those of P2X2bR. In striking contrast to parental receptors, the rates of decay in P2X2a/V60-F301X3R and P2X2b/V60-F301X3R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X2a/V60-R180X3R-expressing cells. However, the recovery from desensitization of P2X2a/V60-F301X3R and P2X2a/V60-R180X3R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.

Structure and functional interaction of the extracellular domain of human GABA[subscript B] receptor GBR2

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

Geng, Yong; Xiong, Dazhi; Mosyak, Lidia

2012-10-24

Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA{sub B} receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA{sub B} receptor has been implicated in several neurological disorders. GABA{sub B} receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimericmore » interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA{sub B} receptor that is unique to the GABAergic system.« less

The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

PubMed

Gurd, J W; Bissoon, N

1997-08-01

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

Ephrin-B3 regulates glutamate receptor signaling at hippocampal synapses

PubMed Central

Antion, Marcia D.; Christie, Louisa A.; Bond, Allison M.; Dalva, Matthew B.; Contractor, Anis

2010-01-01

B-ephrin - EphB receptor signaling modulates NMDA receptors by inducing tyrosine phosphorylation of NR2 subunits. Ephrins and EphB RTKs are localized to postsynaptic compartments in the CA1, and therefore potentially interact in a non-canonical cis-configuration. However, it is not known whether cis- configured receptor-ligand signaling is utilized by this class of RTKs, and whether this might influence excitatory synapses. We found that ablation of ephrin-B3 results in an enhancement of the NMDA receptor component of synaptic transmission relative to the AMPA receptor component in CA1 synapses. Synaptic AMPA receptor expression is reduced in ephrin-B3 knockout mice, and there is a marked enhancement of tyrosine phosphorylation of the NR2B receptor subunit. In a reduced system co-expression of ephrin-B3 attenuated EphB2-mediated NR2B tyrosine phosphorylation. Moreover, phosphorylation of EphB2 was elevated in the hippocampus of ephrin-B3 knockout mice, suggesting that regulation of EphB2 activity is lost in these mice. Direct activation of EphB RTKs resulted in phosphorylation of NR2B and a potential signaling partner, the non-receptor tyrosine kinase Pyk2. Our data suggests that ephrin-B3 limits EphB RTK-mediated phosphorylation of the NR2B subunit through an inhibitory cis- interaction which is required for the correct function of glutamatergic CA1 synapses. PMID:20678574

Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

PubMed

Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

2016-01-15

After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. Copyright © 2015 Elsevier B.V. All rights reserved.

RNA Aptamer-Based Functional Ligands of the Neurotrophin Receptor, TrkB

PubMed Central

Huang, Yang Zhong; Hernandez, Frank J.; Gu, Bin; Stockdale, Katie R.; Nanapaneni, Kishore; Scheetz, Todd E.; Behlke, Mark A.; Peek, Andrew S.; Bair, Thomas; Giangrande, Paloma H.

2012-01-01

Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (KD ∼2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors. PMID:22752556

Vestibular receptors contribute to cortical auditory evoked potentials.

PubMed

Todd, Neil P M; Paillard, Aurore C; Kluk, Karolina; Whittle, Elizabeth; Colebatch, James G

2014-03-01

Acoustic sensitivity of the vestibular apparatus is well-established, but the contribution of vestibular receptors to the late auditory evoked potentials of cortical origin is unknown. Evoked potentials from 500 Hz tone pips were recorded using 70 channel EEG at several intensities below and above the vestibular acoustic threshold, as determined by vestibular evoked myogenic potentials (VEMPs). In healthy subjects both auditory mid- and long-latency auditory evoked potentials (AEPs), consisting of Na, Pa, N1 and P2 waves, were observed in the sub-threshold conditions. However, in passing through the vestibular threshold, systematic changes were observed in the morphology of the potentials and in the intensity dependence of their amplitude and latency. These changes were absent in a patient without functioning vestibular receptors. In particular, for the healthy subjects there was a fronto-central negativity, which appeared at about 42 ms, referred to as an N42, prior to the AEP N1. Source analysis of both the N42 and N1 indicated involvement of cingulate cortex, as well as bilateral superior temporal cortex. Our findings are best explained by vestibular receptors contributing to what were hitherto considered as purely auditory evoked potentials and in addition tentatively identify a new component that appears to be primarily of vestibular origin. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Clofibric acid induces hepatic CYP 2B1/2 via constitutive androstane receptor not via peroxisome proliferator activated receptor alpha in rat.

PubMed

Ibrahim, Zein Shaban; Ahmed, Mohamed Mohamed; El-Shazly, Samir Ahmed; Ishizuka, Mayumi; Fujita, Shoichi

2014-01-01

Peroxisome proliferator activated receptor α (PPARα) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPARα were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPARα transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPARα.

Human umbilical vein: involvement of cyclooxygenase-2 pathway in bradykinin B1 receptor-sensitized responses.

PubMed

Errasti, A E; Rey-Ares, V; Daray, F M; Rogines-Velo, M P; Sardi, S P; Paz, C; Podestá, E J; Rothlin, R P

2001-08-01

In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.

Tyrosine Phosphorylation of NR2B Contributes to Chronic Migraines via Increased Expression of CGRP in Rats

PubMed Central

Liang, Xiping; Wang, Sha; Qin, Guangcheng; Xie, Jingmei; Tan, Ge; Zhou, Jiying; McBride, Devin W.

2017-01-01

Tyrosine phosphorylation of NR2B (NR2B-pTyr), a subunit of the N-methyl-D-aspartate (NMDA) receptor, has been reported to develop central sensitization and persistent pain in the spine, but its effect in chronic migraines has not been examined. We hypothesized that tyrosine phosphorylation of NR2B contributes to chronic migraines (CM) through calcitonin gene-related peptide (CGRP) in rats. Ninety-four male Sprague-Dawley rats were subjected to seven inflammatory soup (IS) injections. In a subset of animals, the time course and location of NR2B tyrosine phosphorylation were detected by western blot and immunofluorescence double staining. Another set of animals were given either genistein, vehicle, or genistein and recombinant CGRP. The mechanical threshold was measured, the expressions of NR2B-pTyr, NR2B, and CGRP were quantified using western blot, and nitric oxide (NO) was measured with the nitric acid reductase method. NR2B-pTyr expression, in neurons, peaked at 24 hours after CM. Genistein improved the mechanical threshold and reduced migraine attacks 24 and 72 hours after CM. Tyrosine phosphorylation of NR2B decreased the mechanical threshold and increased migraine attacks via upregulated CGRP expression in the rat model of CM. Thus, tyrosine phosphorylation of NR2B may be a potential therapeutic target for treatment of CM. PMID:28393079

Tyrosine Phosphorylation of NR2B Contributes to Chronic Migraines via Increased Expression of CGRP in Rats.

PubMed

Liang, Xiping; Wang, Sha; Qin, Guangcheng; Xie, Jingmei; Tan, Ge; Zhou, Jiying; McBride, Devin W; Chen, Lixue

2017-01-01

Tyrosine phosphorylation of NR2B (NR2B-pTyr), a subunit of the N-methyl-D-aspartate (NMDA) receptor, has been reported to develop central sensitization and persistent pain in the spine, but its effect in chronic migraines has not been examined. We hypothesized that tyrosine phosphorylation of NR2B contributes to chronic migraines (CM) through calcitonin gene-related peptide (CGRP) in rats. Ninety-four male Sprague-Dawley rats were subjected to seven inflammatory soup (IS) injections. In a subset of animals, the time course and location of NR2B tyrosine phosphorylation were detected by western blot and immunofluorescence double staining. Another set of animals were given either genistein, vehicle, or genistein and recombinant CGRP. The mechanical threshold was measured, the expressions of NR2B-pTyr, NR2B, and CGRP were quantified using western blot, and nitric oxide (NO) was measured with the nitric acid reductase method. NR2B-pTyr expression, in neurons, peaked at 24 hours after CM. Genistein improved the mechanical threshold and reduced migraine attacks 24 and 72 hours after CM. Tyrosine phosphorylation of NR2B decreased the mechanical threshold and increased migraine attacks via upregulated CGRP expression in the rat model of CM. Thus, tyrosine phosphorylation of NR2B may be a potential therapeutic target for treatment of CM.

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

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension.

PubMed

Hood, Katie Y; Mair, Kirsty M; Harvey, Adam P; Montezano, Augusto C; Touyz, Rhian M; MacLean, Margaret R

2017-07-01

Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1 -/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT 1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT 1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT 1B receptor signaling and Nox1, confirmed in PASMCs from Nox1 -/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT 1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT 1B receptors contribute to

Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

PubMed

Yang, Yunlong; Bajracharya, Prati; Castillo, Paula; Nachman, Ronald J; Pietrantonio, Patricia V

2013-12-01

The cDNA of the receptor for CAP(2b)/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP(2b)-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP(2b)/PVK receptor, as concluded from analyses of the predicted protein sequence, phylogenetics and functional expression. Expression analyses of synganglion, salivary gland, Malpighian tubule, and ovary revealed Rhimi-CAP(2b)-R transcripts. The expression in mammalian cells of the open reading frame of Rhimi-CAP(2b)-R cDNA fused with a hemagglutinin tag at the receptor N-terminus was confirmed by immunocytochemistry. In a calcium bioluminescence assay the recombinant receptor was activated by the tick Ixodes scapularis CAP(2b)/PVK and a PVK analog with EC₅₀s of 64 nM and 249 nM, respectively. Tick pyrokinins were not active. This is the first report on the functional characterization of the CAP(2b)/PVK receptor from any tick species which will now permit the discovery of the physiological roles of these neuropeptides in ticks, as neurohormones, neuromodulators and/or neurotransmitters. Copyright © 2013 Elsevier Inc. All rights reserved.

Pathophysiological roles of P2 receptors in glial cells.

PubMed

Abbracchio, Maria P; Verderio, Claudia

2006-01-01

Extracellular nucleotides act through specific receptors on target cells: the seven ionotropic P2X and the eight G protein-coupled P2Y receptors. All these receptors are expressed by brain astroglia and microglia. In astrocytes, P2 receptors have been implicated in short-term calcium-dependent cell-cell communication. Upon mechanical stimulation or activation by other transmitters, astrocytes release ATP and respond to ATP with a propagating wave of intracellular calcium increases, allowing a homotypic astrocyte-astrocyte communication, as well as an heterotypic signalling which also involves neurons, oligodendrocytes and microglia. Astrocytic P2 receptors also mediate reactive astrogliosis, a reaction contributing to neuronal death in neurodegenerative diseases. Signalling leading to inflammatory astrogliosis involves induction of cyclo-oxygenase 2 through stimulation of ERK1,2 and of the transcriptional factors AP-1 and NF-kappaB. Microglia also express several P2 receptors linked to intracellular calcium increases. P2 receptor subtypes are differentially regulated by typical proinflammatory signals for these cells (e.g. lipopolysaccharide), suggesting specific roles in brain immune responses. Globally, these findings highlight the roles of P2 receptors in glial cell pathophysiology suggesting a contribution to neurodegenerative diseases characterized by excessive gliosis and neuro-inflammation. They also open up the possibility of modulating brain damage by ligands selectively targeting the specific P2 receptor subtypes involved in the gliotic response.

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

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.

Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB.

PubMed

Zeng, Lei; Kuti, Miklos; Mujtaba, Shiraz; Zhou, Ming-Ming

2014-07-01

The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine-binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α-PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. © 2014 Wiley Periodicals, Inc.

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

An ErbB2-Muc4 complex in rat ocular surface epithelia.

PubMed

Swan, Jeremy S; Arango, Maria E; Carothers Carraway, Coralie A; Carraway, Kermit L

2002-05-01

To show the presence and localization of type 1 growth factor receptors (ErbB2, ErbB3 and ErbB4) in rat corneal and conjunctival epithelia and investigate the association of ErbB2 with its intramembrane ligand Muc4. Methacarn-fixed, paraffin-embedded sections of corneas and eyelids from female adult rats were immunocytochemically stained using antibodies against the ErbB receptors and Muc4. Sequential immunoprecipitation and immunoblot analyses were performed on epithelial lysates to investigate the presence of a complex of Muc4 and ErbB2 in corneal and conjunctival epithelia. Immunocytochemical staining demonstrated the presence of ErbB2, ErbB3 and ErbB4 growth factor receptors throughout the rat corneal and conjunctival epithelia. Co-immunoprecipitation of the epithelial lysates demonstrated that Muc4 and ErbB2 are present as a complex. The three type 1 growth factor receptors (ErbB2, ErbB3 and ErbB4) are present in the rat corneal and conjunctival epithelia, and ErbB2 is at least partly associated with Muc4. This demonstration of the presence and localization of these three type 1 growth factor receptors may help in understanding how these receptors contribute to ocular epithelial behavior and functions.

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

PubMed

Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

2018-06-09

Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody

PubMed Central

Wacker, Daniel; Kapoor, Mili; Zhang, Ai; Han, Gye Won; Basu, Shibom; Patel, Nilkanth; Messerschmidt, Marc; Weierstall, Uwe; Liu, Wei; Katritch, Vsevolod; Roth, Bryan L.; Stevens, Raymond C.

2017-01-01

Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. PMID:28716900

Low dopamine striatal D2 receptors are associated with prefrontal metabolism in obese subjects: Possible contributing factors

PubMed Central

Volkow, Nora D.; Wang, Gene-Jack; Telang, Frank; Fowler, Joanna S.; Thanos, Panayotis K.; Logan, Jean; Alexoff, David; Ding, Yu-Shin; Wong, Christopher; Ma, Yeming; Pradhan, Kith

2009-01-01

Dopamine's role in inhibitory control is well recognized and its disruption may contribute to behavioral disorders of discontrol such as obesity. However, the mechanism by which impaired dopamine neurotransmission interferes with inhibitory control is poorly understood. We had previously documented a reduction in dopamine D2 receptors in morbidly obese subjects. To assess if the reductions in dopamine D2 receptors were associated with activity in prefrontal brain regions implicated in inhibitory control we assessed the relationship between dopamine D2 receptor availability in striatum with brain glucose metabolism (marker of brain function) in ten morbidly obese subjects (BMI>40 kg/m2) and compared it to that in twelve non-obese controls. PET was used with [11C]raclopride to assess D2 receptors and with [18F] FDG to assess regional brain glucose metabolism. In obese subjects striatal D2 receptor availability was lower than controls and was positively correlated with metabolism in dorsolateral prefrontal, medial orbitofrontal, anterior cingulate gyrus and somatosensory cortices. In controls correlations with prefrontal metabolism were not significant but comparisons with those in obese subjects were not significant, which does not permit to ascribe the associations as unique to obesity. The associations between striatal D2 receptors and prefrontal metabolism in obese subjects suggest that decreases in striatal D2 receptors could contribute to overeating via their modulation of striatal prefrontal pathways, which participate in inhibitory control and salience attribution. The association between striatal D2 receptors and metabolism in somatosensory cortices (regions that process palatability) could underlie one of the mechanisms through which dopamine regulates the reinforcing properties of food. PMID:18598772

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

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

Gentiopicroside attenuates morphine rewarding effect through downregulation of GluN2B receptors in nucleus accumbens.

PubMed

Liu, Shui-Bing; Ma, Lan; Guo, Hong-Ju; Feng, Bin; Guo, Yan-Yan; Li, Xiao-Qiang; Sun, Wen-Ji; Zheng, Lian-He; Zhao, Ming-Gao

2012-08-01

Gentiopicroside (Gent) is one of the secoiridoid compound isolated from Gentiana lutea. This compound exhibits analgesic activities and inhibits the expression of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the anterior cingulate cortex in mice. Nucleus accumbens (NAc) is a forebrain structure known for its role in drug addiction. However, little is known about the role of Gent on morphine dependence and synaptic transmission changes in the NAc. Conditioned place preference (CPP) test and behavioral sensitization of locomotor activity were used to investigate drug-seeking related behaviors. Brain slices containing NAc were prepared, and whole-cell patch-clamp recordings were performed to record the excitatory postsynaptic currents (EPSCs). Expression of proteins was detected by Western blot analysis. Systemic administration of Gent attenuated the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization. Gent significantly reversed overexpression of GluN2B-containing NMDA receptors and dopamine D2 receptors in NAc during the first week of morphine withdrawal. However, the compound did not affect the overexpression of GluN2A-containing NMDA receptors, GluA1, and dopamine D1 receptors. Lastly, Gent significantly reduced NMDA receptors-mediated EPSCs in the NAc. Our study provides strong evidence that Gent inhibits morphine dependence through downregulation of GluN2B-containing NMDA receptors in the NAc. © 2012 Blackwell Publishing Ltd.

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

Kinin-B2 receptor expression and activity during differentiation of embryonic rat neurospheres.

PubMed

Martins, Antonio H; Alves, Janaína M; Trujillo, Cleber A; Schwindt, Telma T; Barnabé, Gabriela F; Motta, Fabiana L T; Guimaraes, Alessander O; Casarini, Dulce E; Mello, Luiz E; Pesquero, João B; Ulrich, Henning

2008-04-01

Neural progenitor cells were isolated from rat fetal telencephalon and proliferate as neurospheres in the presence of EGF, FGF-2, and heparin. In the absence of these growth factors, neurospheres differentiate into neurons, astrocytes, and oligodendrocytes. Using an embryonal carcinoma cell line as in vitro differentiation model, we have already demonstrated the presence of an autocrine loop system between kinin-B2 receptor activity and secretion of its ligand bradykinin (BK) as prerequisites for final neuronal differentiation (Martins et al., J Biol Chem 2005; 280: 19576-19586). The aim of this study was to verify the activity of the kallikrein-kinin system (KKS) during neural progenitor cell differentiation. Immunofluorescence studies and flow cytometry analysis revealed increases in glial fibrillary acidic protein and beta-3 tubulin expression and decrease in the number of nestin-positive cells along neurospheres differentiation, indicating the transition of neural progenitor cells to astrocytes and neurons. Kinin-B2 receptor expression and activity, secretion of BK into the medium, and presence of high-molecular weight kininogen suggest the participation of the KKS in neurosphere differentiation. Functional kinin-B2 receptors and BK secretion indicate an autocrine loop during neurosphere differentiation to neurons, astrocytes, and oligodendrocytes, reflecting events occurring during early brain development. (c) 2008 International Society for Analytical Cytology.

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension

PubMed Central

Hood, Katie Y.; Mair, Kirsty M.; Harvey, Adam P.; Montezano, Augusto C.; Touyz, Rhian M.

2017-01-01

Objective— Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase–derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. Approach and Results— HPASMCs from controls and PAH patients, and PASMCs from Nox1−/− mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT1B receptor signaling and Nox1, confirmed in PASMCs from Nox1−/− mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Conclusions— Serotonin can induce cellular Src-related kinase–regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with

Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma messenger ribonucleic acid levels.

PubMed

Yoshiga, Daigo; Sato, Naoichi; Torisu, Takehiro; Mori, Hiroyuki; Yoshida, Ryoko; Nakamura, Seiji; Takaesu, Giichi; Kobayashi, Takashi; Yoshimura, Akihiko

2007-05-01

Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.

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.

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.

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.

Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice1, 2, 3

PubMed Central

Bonami, Rachel H.; Thomas, James W.

2015-01-01

Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or if this process is dysregulated in related autoimmunity. To resolve these issues, an editing-competent model was developed in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a non-autoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, as selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen-targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab’)2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy. PMID:26432895

The role of GluN2B-containing NMDA receptors in short- and long-term fear recall.

PubMed

Mikics, Eva; Toth, Mate; Biro, Laszlo; Bruzsik, Biborka; Nagy, Boglarka; Haller, Jozsef

2017-08-01

N-methyl-d-aspartate (NMDA) receptors are crucial synaptic elements in long-term memory formation, including the associative learning of fearful events. Although NMDA blockers were consistently shown to inhibit fear memory acquisition and recall, the clinical use of general NMDA blockers is hampered by their side effects. Recent studies revealed significant heterogeneity in the distribution and neurophysiological characteristics of NMDA receptors with different GluN2 (NR2) subunit composition, which may have differential role in fear learning and recall. To investigate the specific role of NMDA receptor subpopulations with different GluN2 subunit compositions in the formation of lasting traumatic memories, we contrasted the effects of general NMDA receptor blockade with GluN2A-, GluN2B-, and GluN2C/D subunit selective antagonists (MK-801, PEAQX, Ro25-6981, PPDA, respectively). To investigate acute and lasting consequences, behavioral responses were investigated 1 and 28days after fear conditioning. We found that MK-801 (0.05 and 0.1mg/kg) decreased fear recall at both time points. GluN2B receptor subunit blockade produced highly similar effects, albeit efficacy was somewhat smaller 28days after fear conditioning. Unlike MK-801, Ro25-6981 (3 and 10mg/kg) did not affect locomotor activity in the open-field. In contrast, GluN2A and GluN2C/D blockers (6 and 20mg/kg PEAQX; 3 and 10mg/kg PPDA, respectively) had no effect on conditioned fear recall at any time point and dose. This sharp contrast between GluN2B- and other subunit-containing NMDA receptor function indicates that GluN2B receptor subunits are intimately involved in fear memory formation, and may provide a novel pharmacological target in post-traumatic stress disorder or other fear-related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Adenosine enhances sweet taste through A2B receptors in the taste bud

PubMed Central

Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D.

2012-01-01

Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (Type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+ mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (Type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell RT-PCR on isolated vallate taste cells, we show that many Receptor cells express adenosine receptors, Adora2b, while Presynaptic (Type III) and Glial-like (Type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase (ACPP). Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. PMID:22219293

Adenosine enhances sweet taste through A2B receptors in the taste bud.

PubMed

Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

2012-01-04

Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

[Immunoenzymatic assays of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer: correlation with clinical and biological parameters].

PubMed

Métayé, T; Bareille Saint-Gaudens, A; Millet, C; Ingrand, P; Daban, A; Bégon, F

1996-01-01

Two new immunoenzymatic assays for c-erbB-2 oncoprotein and epidermal growth factor receptor (EGF-R) (Oncogene Science) in human breast cancer were validated. Correlations between these assays and some clinical and biological parameters were also studied. The repeatability and reproducibility of standard curves for the two methods gave a coefficient of variation (CV) of less than 4% and about 10% respectively. The accuracy of c-erbB-2 oncoprotein and EGF-R assays was examined by using dilution and recovery tests throughout the standard curves. The linear relations between theoretical and measured values, for these tests, had slopes close to 1 and an intercept near 0. The median value for EGF-R, measured on solubilized membranes of 290 primary tumors, was 0.12 fmol/micrograms protein, the mean value was 0.37 (range 0 to 35.7). For c-erbB-2 oncoprotein, the median value, measured using the same population, was 2.75 human neu unit/micrograms protein, the mean value was 7.85 (range 1 to 125). There was an inverse relationship between EGF-R values and those for the estrogen receptor (ER), progesterone receptor and pS2 protein as well as menopausal status. C-erbB-2 oncoprotein concentrations were positively correlated with ER, pS2 protein and cathepsin D. Furthermore, a significant positive correlation was observed between EGF-R levels and c-erbB-2 oncoprotein levels. In conclusion, immunoenzymatic assays of EGF-R and c-erbB-2 oncoprotein are easy to use, sensitive and reliable. The accurate standardisation of immunoenzymatic assays could contribute to the clinical use of EGF-R and c-erbB-2 oncoprotein as prognostic factors in breast cancer.

Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

PubMed Central

Ferreira, Joana S; Papouin, Thomas; Ladépêche, Laurent; Yao, Andrea; Langlais, Valentin C; Bouchet, Delphine; Dulong, Jérôme; Mothet, Jean-Pierre; Sacchi, Silvia; Pollegioni, Loredano; Paoletti, Pierre; Oliet, Stéphane Henri Richard; Groc, Laurent

2017-01-01

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling. DOI: http://dx.doi.org/10.7554/eLife.25492.001 PMID:28598327

Endogenous bradykinin activates ischaemically sensitive cardiac visceral afferents through kinin B2 receptors in cats

PubMed Central

Tjen-A-Looi, Stephanie C; Pan, Hui-Lin; Longhurst, John C

1998-01-01

responses of seven afferents to ischaemia (0.35 ± 0.13 to 1.89 ± 0.48 vs. 0.40 ± 0.10 to 0.76 ± 0.24 impulses s−1). Indomethacin also significantly reduced the responses of six ischaemically sensitive cardiac afferents to BK (2.65 ± 1.23 to 1.2 ± 0.51 impulses s−1). In six cats pretreated with indomethacin, NPC-17731 attenuated the impulse activity of six ischaemically sensitive cardiac afferents (0.39 ± 0.12 to 1.0 ± 0.3 vs. 0.26 ± 0.14 to 0.48 ± 0.20 impulses s−1). This study demonstrates that BK produced during ischaemia contributes to stimulation of ischaemically sensitive cardiac visceral afferents through activation of kinin B2 receptors. Furthermore, BK stimulates ischaemically sensitive cardiac visceral afferents through a mechanism that is, at least in part, independent of cyclo-oxygenase activation. PMID:9706010

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

PubMed

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L; Hille, Bertil; Koh, Duk-Su

2016-03-01

Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

PubMed Central

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

2016-01-01

Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to

Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

PubMed

Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

2016-04-01

Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

CXCL13 regulates the trafficking of GluN2B-containing NMDA receptor via IL-17 in the development of remifentanil-induced hyperalgesia in rats.

PubMed

Zhu, M; Yuan, S T; Yu, W L; Jia, L L; Sun, Y

2017-05-01

This study aimed to investigate whether CXCL13 modulated the trafficking of NMDA receptor via interleukin (IL)-17 in a rat model of remifentanil-induced hyperalgesia (RIH).Although chemokines are crucial regulators of neuroinflammation, spinal N-methyl-d-aspartate (NMDA) receptor activation, and development of hypernociceptive process, little is known about specific pathogenesis and effective treatment. Inflammatory mediators are required for excitatory synaptic transmission in pathologic pain. A neutralizing antibody against CXCL13 (anti-CXCL13), antiserum against IL-17 (anti-IL-17), and recombinant CXCL13 and IL-17 were administered intrathecally to explore the roles of CXCL13, IL-17, and NMDA receptor, as well as the prevention of hyperalgesia. Paw withdrawal threshold and paw withdrawal latency were employed to record mechanical allodynia and thermal hyperalgesia. Reverse transcriptase quantitative polymerase chain reaction was used to evaluate the levels of CXCL13/CXCR5 and IL-17/IL-17RA in the spinal dorsal horn. The trafficking of spinal GluN2B-containing NMDA receptor was assessed by Western blot after nociceptive testing. This study found mechanical allodynia and thermal hyperalgesia with a remarkable increase in the expression of spinal CXCL13/CXCR5 and IL-17/IL-17RA and trafficking of GluN2B-containing NMDA receptor after remifentanil exposure. Behavioral RIH and elevated GluN2B trafficking were dampened by intrathecal anti-CXCL13 and anti-IL-17, respectively. The delivery of exogenous CXCL13 dose-dependently generated a rapid nociceptive hypersensitivity in naïve rats, which was prevented by coadministering anti-IL-17. CXCL13-induced IL-17RA overproduction and GluN2B trafficking were reversed by anti-IL-17 treatment. GluN2B antagonist also blocked CXCL13, and IL-17 directly induced hyperalgesia. This study highlighted the contribution of IL-17 pathway in the trafficking of CXCL13-induced GluN2B-containing NMDA receptor in the pathogenesis of RIH

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

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

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.

[Polymorphic Variants of Glutamate Receptor (GRIK5, GRIN2B) and Serotonin Receptor (HTR2A) Genes Are Associated with Chronic Obstructive Pulmonary Disease].

PubMed

Korytina, G F; Akhmadishina, L Z; Kochetova, O V; Aznabaeva, Y G; Zagidullin, Sh Z; Victorova, T V

2017-01-01

Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease of the respiratory system that affects primarily distal respiratory pathways and lung parenchyma. Smoking tobacco is a major risk factor for COPD. The relationship of HTR4 (rs3995090), HTR2A (rs6313), GRIK5 (rs8099939), GRIN2B (rs2268132), and CHRNB4 (rs1948) gene polymorphisms and COPD, as well as the contribution of these polymorphisms to the variations in quantitative characteristics that describe respiratory function, smoking behavior, and nicotine dependence was assessed in an ethnically homogeneous Tatar population. The polymorphisms of HTR2A (rs6313) (P = 0.026, OR = 1.42 for the CC genotype) and GRIN2B (rs2268132) (P = 0.0001, OR = 2.39 for the TT genotype) were significantly associated with increased risk of COPD. The AA genotype of GRIK5 (rs8099939) had a protective effect (P = 0.02, OR = 0.61). Importantly, the HTR2A (rs6313), GRIN2B (rs2268132), and GRIK5 (rs8099939) polymorphisms were only associated with COPD in smokers. Smoking index (pack-years) was significantly higher in carriers of the GRIK5 genotype AC (rs8099939) (P = 0.0027). The TT genotype of GRIN2B (rs2268132) was associated with COPD in subjects with high nicotine dependence according to the Fagerstrõm test (P = 0.002, OR = 2.98). The TT genotype of HTR2A (rs6313) was associated with a reduced risk of the disease in the group with moderate nicotine dependence (P = 0.02, OR = 0.22). The CC genotype of HTR2A (rs6313) and the TT genotype of GRIN2B (rs2268132) were associated with higher levels of nicotine dependence according to the Fagerstrõm test (P = 0.0011 and P = 0.037). Our results may provide insight into potential molecular mechanisms that involve the glutamate (GRIK5, GRIN2B) and serotonin (HTR2A) receptor genes in the pathogenesis of COPD.

Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish.

PubMed

Funkenstein, Bruria; Krol, Ekaterina; Esterin, Elena; Kim, Yong-Soo

2012-12-01

Myostatin (MSTN), a negative regulator of muscle growth and a member of the transforming growth factor-β superfamily, can bind the two activin type 2 receptors (ACVR2). It has been previously shown that WT mice injected with ACVR2B extracellular domain (ACVR2B-ECD) had higher muscle mass. Likewise, fish larvae immersed in Pichia pastoris culture supernatant, containing goldfish Acvr2b-ECD, showed enhanced larval growth. However, it is not clear whether fish Mstn1 and Mstn2 signal through the same receptor and whether fish express more than one acvr2b gene. In the current study, three cDNAs encoding acvr2b (saacvr2b-1, saacvr2b-2a, and saacvr2b-2b) were cloned from gilthead sea bream. All three contain the short extracellular binding domain, a short transmembrane region, and a conserved catalytic domain of serine/threonine protein kinase. Bioinformatics analysis provided evidence for the existence of two acvr2b genes (acvr2b-1 and acvr2b-2) in several other fish species as well, probably as a result of gene or genome duplication. The two isoforms differ in their amino acid sequences. The direct inhibitory effect of Acvr2b-ECD on Mstn activity was tested in vitro. The saAcvr2b-1-ECD was expressed in the yeast P. pastoris. Evidence is provided for N-glycosylation of Acvr2b-1-ECD. The affinity-purified Acvr2b-1-ECD inhibited recombinant mouse/rat/human mature MSTN activity when determined in vitro using the CAGA-luciferase assay in A204 cells. A lower inhibitory activity was obtained when unprocessed purified, furin-digested, and activated saMstn1 was used. Results of this study demonstrate for the first time the existence of two acvr2b genes in fish. In addition, the study shows that bioactive fish Acvr2b-ECD can be produced from P. pastoris.

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

PubMed Central

Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M.; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E.; Morales, Miguel A.; Cifuentes, Fredy

2017-01-01

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity. PMID:28744222

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion.

PubMed

Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E; Morales, Miguel A; Cifuentes, Fredy

2017-01-01

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

PubMed

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

B cell receptor editing in tolerance and autoimmunity

PubMed Central

Luning Prak, Eline T.; Monestier, Marc; Eisenberg, Robert A.

2010-01-01

Receptor editing is the process of ongoing antibody gene rearrangement in a lymphocyte that already has a functional antigen receptor. The expression of a functional antigen receptor will normally terminate further rearrangement (allelic exclusion). However, lymphocytes with autoreactive receptors have a chance at escaping negative regulation by “editing” the specificities of their receptors with additional antibody gene rearrangements. Nemazee points out, “receptor editing separates receptor selection from cellular selection.”1 As such, editing complicates the Clonal Selection Hypothesis, because edited cells are not simply endowed for life with a single, invariant antigen receptor.2 For example, an edited B cell changes the specificity of its B cell receptor (BCR), and if the initial immunoglobulin gene is not inactivated during the editing process, allelic exclusion is violated, and the B cell can exhibit two specificities. Here we will describe the discovery of editing, the pathways of receptor editing at the heavy (H) and light (L) chain loci, and current evidence regarding how and where editing happens and what effects it has on the antibody repertoire. PMID:21251012

Nogo-B Receptor stabilizes Niemann-Pick Type C2 protein and regulates intracellular cholesterol trafficking

PubMed Central

Harrison, Kenneth D.; Miao, Robert Qing; Fernandez-Hernándo, Carlos; Suárez, Yajaira; Dávalos, Alberto; Sessa, William C.

2009-01-01

Summary The Nogo-B Receptor (NgBR) is a recently identified receptor for the N-terminus of Reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast-2-hybrid screen for interacting proteins using the C-terminal domain as bait and identified Niemann-Pick Type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER), and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR leads to a decrease in NPC2 levels, increased intracellular cholesterol accumulation and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking. PMID:19723497

AMP and adenosine are both ligands for adenosine 2B receptor signaling.

PubMed

Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

2018-01-15

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

Hepatitis B virus-triggered autophagy targets TNFRSF10B/death receptor 5 for degradation to limit TNFSF10/TRAIL response.

PubMed

Shin, Gu-Choul; Kang, Hong Seok; Lee, Ah Ram; Kim, Kyun-Hwan

2016-12-01

Death receptors of TNFSF10/TRAIL (tumor necrosis factor superfamily member 10) contribute to immune surveillance against virus-infected or transformed cells by promoting apoptosis. Many viruses evade antiviral immunity by modulating TNFSF10 receptor signaling, leading to persistent infection. Here, we report that hepatitis B virus (HBV) X protein (HBx) restricts TNFSF10 receptor signaling via macroautophagy/autophagy-mediated degradation of TNFRSF10B/DR5, a TNFSF10 death receptor, and thus permits survival of virus-infected cells. We demonstrate that the expression of the TNFRSF10B protein is dramatically reduced both in liver tissues of chronic hepatitis B patients and in cell lines transfected with HBV or HBx. HBx-mediated downregulation of TNFRSF10B is caused by the lysosomal, but not proteasomal, degradation pathway. Immunoblotting analysis of LC3B and SQSTM1, and microscopy analysis of tandem-fluorescence-tagged LC3B revealed that HBx promotes complete autophagy. Inhibition of autophagy with a pharmacological inhibitor and LC3B knockdown revealed that HBx-induced autophagy is crucial for TNFRSF10B degradation. Immunoprecipitation and GST affinity isolation assays showed that HBx directly interacts with TNFRSF10B and recruits it to phagophores, the precursors to autophagosomes. We confirmed that autophagy activation is related to the downregulation of the TNFRSF10B protein in liver tissues of chronic hepatitis B patients. Inhibition of autophagy enhanced the susceptibility of HBx-infected hepatocytes to TNFSF10. These results identify the dual function of HBx in TNFRSF10B degradation: HBx plays a role as an autophagy receptor-like molecule, which promotes the association of TNFRSF10B with LC3B; HBx is also an autophagy inducer. Our data suggest a molecular mechanism for HBV evasion from TNFSF10-mediated antiviral immunity, which may contribute to chronic HBV infection.

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase.

PubMed

Bornet, Olivier; Nouailler, Matthieu; Feracci, Michaël; Sebban-Kreuzer, Corinne; Byrne, Deborah; Halimi, Hubert; Morelli, Xavier; Badache, Ali; Guerlesquin, Françoise

2014-06-05

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor.

PubMed

Quitterer, Ursula; Pohl, Armin; Langer, Andreas; Koller, Samuel; Abdalla, Said

2011-06-10

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

PubMed Central

Ding, Yan; Liu, Zixing; Desai, Shruti; Zhao, Yuhua; Liu, Hao; Pannell, Lewis K; Yi, Hong; Wright, Elizabeth R; Owen, Laurie B; Dean-Colomb, Windy; Fodstad, Oystein; Lu, Jianrong; LeDoux, Susan P; Wilson, Glenn L; Tan, Ming

2012-01-01

It is well known that ErbB2, a receptor tyrosine kinase, localizes on the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through the association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to ErbB2 targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. PMID:23232401

Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.

PubMed

García-Nafría, Javier; Nehmé, Rony; Edwards, Patricia C; Tate, Christopher G

2018-06-20

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of G s to four different GPCRs have been elucidated 1-4 , but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT 1B receptor (5-HT 1B R) bound to the agonist donitriptan and coupled to an engineered G o heterotrimer. In this complex, 5-HT 1B R is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the β 2 -adrenoceptor (β 2 AR) 3 or the adenosine A 2A receptor (A 2A R) 1 in complex with G s . In contrast to the complexes with G s , the gap between the receptor and the Gβ-subunit in the G o -5-HT 1B R complex precludes molecular contacts, and the interface between the Gα-subunit of G o and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the G o α-subunit. The molecular variations between the interfaces of G o and G s in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

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.

The bradykinin B2 receptor in the early immune response against Listeria infection.

PubMed

Kaman, Wendy E; Wolterink, Arthur F W M; Bader, Michael; Boele, Linda C L; van der Kleij, Desiree

2009-02-01

The endogenous danger signal bradykinin was recently found implicated in the development of immunity against parasites via dendritic cells. We here report an essential role of the B(2) (B(2)R) bradykinin receptor in the early immune response against Listeria infection. Mice deficient in B(2)R (B(2)R(-/-) mice) were shown to suffer from increased hepatic bacterial burden and concomitant dramatic weight loss during infection with Listeria monocytogenes. Levels of cytokines known to play a pivotal role in the early phase immune response against L. monocytogenes, IL-12p70 and IFN-gamma, were reduced in B(2)R(-/-) mice. To extend these findings to the human system, we show that bradykinin potentiates the production of IL-12p70 in human monocyte-derived dendritic cells. Thus, we show that bradykinin and the B(2)R play a role in early innate immune functions during bacterial infection.

Interaction between AT1 receptor and NF-κB in hypothalamic paraventricular nucleus contributes to oxidative stress and sympathoexcitation by modulating neurotransmitters in heart failure.

PubMed

Yu, Xiao-Jing; Suo, Yu-Ping; Qi, Jie; Yang, Qing; Li, Hui-Hua; Zhang, Dong-Mei; Yi, Qiu-Yue; Zhang, Jian; Zhu, Guo-Qing; Zhu, Zhiming; Kang, Yu-Ming

2013-12-01

Angiotensin II type 1 receptor (AT1-R) and nuclear factor-kappaB (NF-κB) in the paraventricular nucleus (PVN) play important roles in heart failure (HF); however, the central mechanisms by which AT1-R and NF-κB contribute to sympathoexcitation in HF are yet unclear. In this study, we determined whether interaction between AT1-R and NF-κB in the PVN modulates neurotransmitters and contributes to NAD(P)H oxidase-dependent oxidative stress and sympathoexcitation in HF. Rats were implanted with bilateral PVN cannulae and subjected to coronary artery ligation or sham surgery (SHAM). Subsequently, animals were treated for 4 weeks through bilateral PVN infusion with either vehicle or losartan (LOS, 10 μg/h), an AT1-R antagonist; or pyrrolidine dithiocarbamate (PDTC, 5 μg/h), a NF-κB inhibitor via osmotic minipump. Myocardial infarction (MI) rats had higher levels of glutamate (Glu), norepinephrine (NE) and NF-κB p65 activity, lower levels of gamma-aminobutyric acid (GABA), and more positive neurons for phosphorylated IKKβ and gp91(phox) (a subunit of NAD(P)H oxidase) in the PVN when compared to SHAM rats. MI rats also had higher levels of renal sympathetic nerve activity (RSNA) and plasma proinflammatory cytokines (PICs), NE and epinephrine. PVN infusions of LOS or PDTC attenuated the decreases in GABA and the increases in gp91(phox), NF-κB activity, Glu and NE, in the PVN of HF rats. PVN infusions of LOS or PDTC also attenuated the increases in RSNA and plasma PICs, NE and epinephrine in MI rats. These findings suggest that interaction between AT1 receptor and NF-κB in the PVN contributes to oxidative stress and sympathoexcitation by modulating neurotransmitters in heart failure.

Uncoupling of the ITIM receptor G6b-B from the tyrosine phosphatases Shp1 and Shp2 disrupts platelet homeostasis in mice.

PubMed

Geer, Mitchell J; van Geffen, Johanna P; Gopalasingam, Piraveen; Vögtle, Timo; Smith, Christopher W; Heising, Silke; Kuijpers, Marijke J E; Tullemans, Bibian M E; Jarvis, Gavin E; Eble, Johannes A; Jeeves, Mark; Overduin, Michael; Heemskerk, Johan W M; Mazharian, Alexandra; Senis, Yotis A

2018-06-11

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of the ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine (Y) residues 212 and 238 within the ITIM and ITSM were mutated to phenylalanine (F), respectively. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, due to a reduction in platelet production, had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to that observed in G6b knockout (G6b KO) mice. Platelets from G6b-B diY/F mice were hypo-responsive to collagen, due to a significant reduction in expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, and thrombin, that could be partially rescued by co-stimulating the platelets with ADP. In contrast, platelets from G6b-B diY/F, G6b KO and megakaryocyte-specific Shp2 KO mice were hyper-responsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 in order to mediate its regulatory effects on platelet homeostasis. Copyright © 2018 American Society of Hematology.

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

Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes.

PubMed

Rivera, A; Rotter, M A; Brugnara, C

1999-10-01

Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.

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

Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

PubMed Central

Aherne, CM; Saeedi, B; Collins, CB; Masterson, JC; McNamee, EN; Perrenoud, L; Rapp, CR; Curtis, VF; Bayless, A; Fletcher, A; Glover, LE; Evans, CM; Jedlicka, P; Furuta, GT; de Zoeten, EF; Colgan, SP; Eltzschig, HK

2015-01-01

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses. PMID:25850656

Evaluation of 11C-Me-NB1 as a Potential PET Radioligand for Measuring GluN2B-Containing NMDA Receptors, Drug Occupancy, and Receptor Cross Talk.

PubMed

Krämer, Stefanie D; Betzel, Thomas; Mu, Linjing; Haider, Ahmed; Herde, Adrienne Müller; Boninsegni, Anna K; Keller, Claudia; Szermerski, Marina; Schibli, Roger; Wünsch, Bernhard; Ametamey, Simon M

2018-04-01

Clinical and preclinical research with modulators at the N -methyl-d-aspartate (NMDA) receptor GluN2B N-terminal domain (NTD) aims for the treatment of various neurologic diseases. The interpretation of the results is hampered by the lack of a suitable NMDA PET tracer for assessing the receptor occupancy of potential drugs. We have developed 11 C-Me-NB1 as a PET tracer for imaging GluN1/GluN2B-containing NMDA receptors and used it to investigate in rats the dose-dependent receptor occupancy of eliprodil, a GluN2B NTD modulator. Methods: 11 C-Me-NB1 was synthesized and characterized by in vitro displacement binding experiments with rat brain membranes, in vitro autoradiography, and blocking and displacement experiments by PET and PET kinetic modeling. Receptor occupancy by eliprodil was studied by PET with 11 C-Me-NB1. Results: 11 C-Me-NB1 was synthesized at 290 ± 90 GBq/μmol molar activity, 7.4 ± 1.9 GBq total activity at the end of synthesis ( n = 17), and more than 99% radiochemical purity. 11 C-Me-NB1 binding in rat brain was blocked in vitro and in vivo by the NTD modulators Ro-25-6981 and eliprodil. Half-maximal receptor occupancy by eliprodil occurred at 1.5 μg/kg. At 1 mg/kg of eliprodil, a dose with reported neuroprotective effects, more than 99.5% of binding sites were occupied. In vitro, 11 C-Me-NB1 binding was independent of the σ-1 receptor (Sigma1R), and the Sigma1R agonist (+)-pentazocine did not compete for high-affinity binding. In vivo, a 2.5 mg/kg dose of (+)-pentazocine abolished 11 C-Me-NB1-specific binding, indicating an indirect effect of Sigma1R on 11 C-Me-NB1 binding. Conclusion: 11 C-Me-NB1 is suitable for the in vivo imaging of NMDA GluN1/GluN2B receptors and the assessment of receptor occupancy by NTD modulators. GluN1/GluN2B NMDA receptors are fully occupied at neuroprotective doses of eliprodil. Furthermore, 11 C-Me-NB1 enables imaging of GluN1/GluN2B NMDA receptor cross talk. © 2018 by the Society of Nuclear Medicine and

Improved metabolic phenotype of hypothalamic PTP1B-deficiency is dependent upon the leptin receptor.

PubMed

Tsou, Ryan C; Rak, Kimberly S; Zimmer, Derek J; Bence, Kendra K

2014-06-01

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of central metabolic signaling, and mice with whole brain-, leptin receptor (LepRb) expressing cell-, or proopiomelanocortin neuron-specific PTP1B-deficiency are lean, leptin hypersensitive, and display improved glucose homeostasis. However, whether the metabolic effects of central PTP1B-deficiency are due to action within the hypothalamus remains unclear. Moreover, whether or not these effects are exclusively due to enhanced leptin signaling is unknown. Here we report that mice with hypothalamic PTP1B-deficiency (Nkx2.1-PTP1B(-/-)) display decreased body weight and adiposity on high-fat diet with no associated improvements in glucose tolerance. Consistent with previous reports, we find that hypothalamic deletion of the LepRb in mice (Nkx2.1-LepRb(-/-)) results in extreme hyperphagia and obesity. Interestingly, deletion of hypothalamic PTP1B and LepRb (Nkx2.1-PTP1B(-/-):LepRb(-/-)) does not rescue the hyperphagia or obesity of Nkx2.1-LepRb(-/-) mice, suggesting that hypothalamic PTP1B contributes to the central control of energy balance through a leptin receptor-dependent pathway.

The short-chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis.

PubMed

Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M; Angueira, Anthony R; Brodsky, Michael; Hayes, M Geoffrey; Kovatcheva-Datchary, Petia; Bäckhed, Fredrik; Gilbert, Jack A; Lowe, William L; Layden, Brian T

2015-11-15

The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Together, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

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

Ibrutinib (ImbruvicaTM) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells.

PubMed

Grabinski, Nicole; Ewald, Florian

2014-12-01

Ibrutinib (formerly PCI-32765) is a specific, irreversible, and potent inhibitor of Burton's tyrosine kinase (BTK) developed for the treatment of several forms of blood cancer. It is now an FDA-approved drug marketed under the name Imbruvica(TM) (Pharmacyclics, Inc.) and successfully used as an orally administered second-line drug in the treatment of mantle cell lymphoma. Since BTK is predominantly expressed in hematopoietic cells, the sensitivity of solid tumor cells to Ibrutinib has not been analyzed. In this study, we determined the effect of Ibrutinib on breast cancer cells. We demonstrate that Ibrutinib efficiently reduces the phosphorylation of the receptor tyrosine kinases ErbB1, ErbB2 and ErbB3, thereby suppressing AKT and MAPK signaling in ErbB2-positive (ErbB2+) breast cancer cell lines. Treatment with Ibrutinib significantly reduced the viability of ErbB2+ cell lines with IC50 values at nanomolar concentrations, suggesting therapeutic potential of Ibrutinib in breast cancer. Combined treatment with Ibrutinib and the dual PI3K/mTOR inhibitor BEZ235 synergistically reduces cell viability of ErbB2+ breast cancer cells. Combination indices below 0.25 at 50% inhibition of cell viability were determined by the Chou-Talalay method. Therefore, the combination of Ibrutinib and canonical PI3K pathway inhibitors could be a new and effective approach in the treatment of breast cancer with activated ErbB receptors. Ibrutinib could thus become a valuable component of targeted therapy in aggressive ErbB2+ breast cancer.

Lung injury pathways: Adenosine receptor 2B signaling limits development of ischemic bronchiolitis obliterans organizing pneumonia.

PubMed

Densmore, John C; Schaid, Terry R; Jeziorczak, Paul M; Medhora, Meetha; Audi, Said; Nayak, Shraddha; Auchampach, John; Dwinell, Melinda R; Geurts, Aron M; Jacobs, Elizabeth R

2017-02-01

Purpose/Aim of the Study: Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague-Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strains containing a mutation in the A 2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A 2A (A 2A AR) and A 2B adenosine receptor (A 2B AR) mRNA and protein were quantified. Twenty-four hours after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A 2B AR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A 2A AR mRNA and protein concentrations remained unchanged following ischemia. A 2B AR protein was increased in PA ligated lungs of SS rats after 7 days, and 4 h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. Increased A 2B AR and adenosine following unilateral lung ischemia as well as more BOOP in A 2B AR mutant rats implicate a protective role for A 2B AR signaling in countering ischemic lung injury.

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.

Transactivation of ErbB receptors by leptin in the cardiovascular system: mechanisms, consequences and target for therapy.

PubMed

Bełtowski, Jerzy; Jazmroz-Wiśniewska, Anna

2014-01-01

Many experimental and clinical studies have demonstrated that elevated leptin concentration in patients with obesity/metabolic syndrome contributes to the pathogenesis of cardiovascular disorders including arterial hypertension, atherosclerosis, restenosis after coronary angioplasty and myocardial hypertrophy. Receptor tyrosine kinases belonging to the ErbB family, especially ErbB1 (epidermal growth factor receptor) and ErbB2 are abundantly expressed in the blood vessels and the heart. EGFR is activated not only by its multiple peptide ligands but also by many other factors including angiotensin II, endothelin-1, norepinephrine, thrombin and prorenin; the phenomenon referred to as "transactivation". Augmented EGFR signaling contributes to abnormalities of vascular tone and renal sodium handling as well as vascular remodeling and myocardial hypertrophy through various intracellular mechanisms, in particular extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinase (PI3K). Recent experimental studies indicate that chronically elevated leptin transactivates the EGFR through the mechanisms requiring reactive oxygen species and cytosolic tyrosine kinase, c-Src. In addition, hyperleptinemia increases ErbB2 activity in the arterial wall. Stimulation of EGFR and ErbB2 downstream signaling pathways such as ERK and PI3K in the vascular wall and the kidney may contribute to the increase in vascular tone, enhanced tubular sodium reabsorption as well as vascular and renal lesions in hyperleptinemic obese subjects.

Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle.

PubMed

Formicola, Luigi; Pannérec, Alice; Correra, Rosa Maria; Gayraud-Morel, Barbara; Ollitrault, David; Besson, Vanessa; Tajbakhsh, Shahragim; Lachey, Jennifer; Seehra, Jasbir S; Marazzi, Giovanna; Sassoon, David A

2018-01-01

Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70-80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration.

Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle

PubMed Central

Formicola, Luigi; Pannérec, Alice; Correra, Rosa Maria; Gayraud-Morel, Barbara; Ollitrault, David; Besson, Vanessa; Tajbakhsh, Shahragim; Lachey, Jennifer; Seehra, Jasbir S.; Marazzi, Giovanna; Sassoon, David A.

2018-01-01

Degenerative myopathies typically display a decline in satellite cells coupled with a replacement of muscle fibers by fat and fibrosis. During this pathological remodeling, satellite cells are present at lower numbers and do not display a proper regenerative function. Whether a decline in satellite cells directly contributes to disease progression or is a secondary result is unknown. In order to dissect these processes, we used a genetic model to reduce the satellite cell population by ~70–80% which leads to a nearly complete loss of regenerative potential. We observe that while no overt tissue damage is observed following satellite cell depletion, muscle fibers atrophy accompanied by changes in the stem cell niche cellular composition. Treatment of these mice with an Activin receptor type-2B (AcvR2B) pathway blocker reverses muscle fiber atrophy as expected, but also restores regenerative potential of the remaining satellite cells. These findings demonstrate that in addition to controlling fiber size, the AcvR2B pathway acts to regulate the muscle stem cell niche providing a more favorable environment for muscle regeneration. PMID:29881353

The relevance of kalikrein-kinin system via activation of B2 receptor in LPS-induced fever in rats.

PubMed

Soares, Denis de Melo; Santos, Danielle R; Rummel, Christoph; Ott, Daniela; Melo, Míriam C C; Roth, Joachim; Calixto, João B; Souza, Glória E P

2017-11-01

This study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B 1 and B 2 receptors on LPS- induced fever and the POA cells involved in this response. Male Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B 2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B 1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca ++ signaling in POA cells was performed in rat pup brain tissue microcultures. Icatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE 2 concentration. Effect abolished by indomethacin (i.p.). LPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B 2 -receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B 2 -receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury

PubMed Central

Eckle, Tobias; Hughes, Kelly; Ehrentraut, Heidi; Brodsky, Kelley S.; Rosenberger, Peter; Choi, Doo-Sup; Ravid, Katya; Weng, Tingting; Xia, Yang; Blackburn, Michael R.; Eltzschig, Holger K.

2013-01-01

The signaling molecule adenosine has been implicated in attenuating acute lung injury (ALI). Adenosine signaling is terminated by its uptake through equilibrative nucleoside transporters (ENTs). We hypothesized that ENT-dependent adenosine uptake could be targeted to enhance adenosine-mediated lung protection. To address this hypothesis, we exposed mice to high-pressure mechanical ventilation to induce ALI. Initial studies demonstrated time-dependent repression of ENT1 and ENT2 transcript and protein levels during ALI. To examine the contention that ENT repression represents an endogenous adaptive response, we performed functional studies with the ENT inhibitor dipyridamole. Dipyridamole treatment (1 mg/kg; EC50=10 μM) was associated with significant increases in ALI survival time (277 vs. 395 min; P<0.05). Subsequent studies in gene-targeted mice for Ent1 or Ent2 revealed a selective phenotype in Ent2−/− mice, including attenuated pulmonary edema and improved gas exchange during ALI in conjunction with elevated adenosine levels in the bronchoalveolar fluid. Furthermore, studies in genetic models for adenosine receptors implicated the A2B adenosine receptor (Adora2b) in mediating ENT-dependent lung protection. Notably, dipyridamole-dependent attenuation of lung inflammation was abolished in mice with alveolar epithelial Adora2b gene deletion. Our newly identified crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to this protein set.—Eckle, T., Hughes, K., Ehrentraut, H., Brodsky, K. S., Rosenberger, P., Choi, D.-S., Ravid, K., Weng, T., Xia, Y., Blackburn, M. R., Eltzschig, H. K. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury. PMID:23603835

CD22 ligation inhibits downstream B cell receptor signaling and Ca(2+) flux upon activation.

PubMed

Sieger, N; Fleischer, S J; Mei, H E; Reiter, K; Shock, A; Burmester, G R; Daridon, C; Dörner, T

2013-03-01

CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation. Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1). Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling. These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases. Copyright © 2013 by the American College of Rheumatology.

Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension.

PubMed

Bauer, Michael; Wilkens, Heinrike; Langer, Frank; Schneider, Sven O; Lausberg, Henning; Schäfers, Hans-Joachim

2002-03-05

The pulmonary circulation is an important site for the production and clearance of endothelin (ET)-1, a potent vasoactive and mitogenic peptide. Increased plasma ET-1 levels are observed in pulmonary arterial hypertension (PHT) and may contribute to the regulation of pulmonary vascular resistance, as well as to proliferative changes in the pulmonary vascular bed. We prospectively assessed changes in plasma big ET-1 levels and changes in ET(A) and ET(B) receptor gene expression in 14 consecutive patients undergoing pulmonary thromboendarterectomy for thromboembolic PHT. Plasma big ET-1 levels were higher in patients with PHT (median, 2.2 pg/mL; 25th to 75th percentile, 1.5 to 3.0 pg/mL) compared with age-matched controls (median, 1.2 pg/mL; 25th to 75th percentile, 1.0 to 1.4 pg/mL; P=0.002). In addition to increased plasma big ET-1 levels, selective upregulation of ET(B) receptor mRNA transcripts and immunoreactive protein in the pulmonary artery was observed in the patients; however, ET(A) receptor gene expression was unaffected. These data suggest that changes in the ET signaling system in PHT caused by thromboembolic disease are not limited to an increased production of ET-1: they also affect ET receptor gene expression.

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.

Association between the NMDA glutamate receptor GRIN2B gene and obsessive–compulsive disorder

PubMed Central

Alonso, Pino; Gratacós, Mónica; Segalàs, Cinto; Escaramís, Georgia; Real, Eva; Bayés, Mónica; Labad, Javier; López-Solà, Clara; Estivill, Xavier; Menchón, José M.

2012-01-01

Background Recent data from neuroimaging, genetic and clinical trials and animal models suggest a role for altered glutamatergic neurotransmission in the pathogenesis of obsessive–compulsive disorder (OCD). The aim of this study was to investigate whether variants in the GRIN2B gene, the gene encoding the NR2 subunit of the N-methyl-d-aspartate (NMDA) glutamate receptor, may contribute to genetic susceptibility to OCD or to different OCD subphenotypes. Methods Between 2003 and 2008, we performed a case–control association study in which we genotyped 10 tag single-nucleotide polymorphisms (SNPs) in the 3′ untranslated region (3′ UTR) of GRIN2B. We performed SNP association and haplotype analysis considering the OCD diagnosis and different OCD subphenotypes: early-onset OCD, comorbid tic disorders and OCD clinical symptom dimensions. Results We enrolled 225 patients with OCD and 279 controls recruited from the OCD Clinic at Bellvitge Hospital (Barcelona, Spain). No significant difference in the distribution of alleles or genotypes was detected between patients with OCD and controls. Nonetheless, on analyzing OCD subphenotypes, the rs1805476 SNP in male patients (95% confidence interval [CI] 1.37–4.22, p = 0.002) and a 4-SNP haplotype in the whole sample (rs1805476, rs1805501, rs1805502 and rs1805477; odds ratio 1.92, 95% CI 1.22–3.01; permutation p = 0.023) were significantly associated with the presence of contamination obsessions and cleaning compulsions. Limitations Study limitations included the risk of population stratification associated with the case–control design, use of psychiatrically unscreened blood donors as the control group, reduced sample size of participants with certain OCD subphenotypes and tested polymorphisms limited to 3′ UTR and exon 13 of GRIN2B. Conclusion Our results converge with recent data suggesting a possible contribution of glutamatergic variants to the genetic vulnerability to OCD or at least to certain OCD

Differential effects of BDNF and neurotrophin 4 (NT4) on endocytic sorting of TrkB receptors.

PubMed

Proenca, Catia C; Song, Minseok; Lee, Francis S

2016-08-01

Neurotrophins are a family of growth factors playing key roles in the survival, development, and function of neurons. The neurotrophins brain-derived neurotrophic factor (BDNF) and NT4 both bind to and activate TrkB receptors, however, they mediate distinct neuronal functions. The molecular mechanism of how TrkB activation by BDNF and NT4 leads to diverse outcomes is unknown. Here, we report that BDNF and NT4 lead to differential endocytic sorting of TrkB receptors resulting in diverse biological functions in cultured cortical neurons. Fluorescent microscopy and surface biotinylation experiments showed that both neurotrophins stimulate internalization of TrkB with similar kinetics. Exposure to BDNF for 2-3 h reduced the surface pool of TrkB receptors to half, whereas a longer treatment (4-5 h) with NT4 was necessary to achieve a similar level of down-regulation. Although BDNF and NT4 induced TrkB phosphorylation with similar intensities, BDNF induced more rapid ubiquitination and degradation of TrkB than NT4. Interestingly, TrkB receptor ubiquitination by these ligands have substantially different pH sensitivities, resulting in varying degrees of receptor ubiquitination at lower pH levels. Consequently, NT4 was capable of maintaining longer sustained downstream signaling activation that correlated with reduced TrkB ubiquitination at endosomal pH. Thus, by leading to altered endocytic trafficking itineraries for TrkB receptors, BDNF and NT4 elicit differential TrkB signaling in terms of duration, intensity, and specificity, which may contribute to their functional differences in vivo. The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), both bind to and activate TrkB receptors, however, they mediate distinct neuronal functions. Here, we propose that BDNF and NT4 lead to differential endocytic sorting of TrkB receptors resulting in diverse biological functions. BDNF induces more rapid ubiquitination and degradation of TrkB than NT4

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

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.

The short chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis

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

Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M.

The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene ( Ffar2) expressionmore » is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Altogether, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.« less

The short chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis

DOE PAGES

Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M.; ...

2015-09-22

The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene ( Ffar2) expressionmore » is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Altogether, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.« less

Diversity in peptide recognition by the SH2 domain of SH2B1.

PubMed

McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

2018-02-01

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

Excess of Aminopeptidase A in the Brain Elevates Blood Pressure via the Angiotensin II Type 1 and Bradykinin B2 Receptors without Dipsogenic Effect

PubMed Central

Ishida, Akio; Ohya, Yusuke

2017-01-01

Aminopeptidase A (APA) cleaves angiotensin (Ang) II, kallidin, and other related peptides. In the brain, it activates the renin angiotensin system and causes hypertension. Limited data are available on the dipsogenic effect of APA and pressor effect of degraded peptides of APA such as bradykinin. Wistar-Kyoto rats received intracerebroventricular (icv) APA in a conscious, unrestrained state after pretreatment with (i) vehicle, (ii) 80 μg of telmisartan, an Ang II type-1 (AT1) receptor blocker, (iii) 800 nmol of amastatin, an aminopeptidase inhibitor, and (iv) 1 nmol of HOE-140, a bradykinin B2 receptor blocker. Icv administration of 400 and 800 ng of APA increased blood pressure by 12.6 ± 3.0 and 19.0 ± 3.1 mmHg, respectively. APA did not evoke drinking behavior. Pressor response to APA was attenuated on pretreatment with telmisartan (vehicle: 22.1 ± 2.2 mmHg versus telmisartan: 10.4 ± 3.2 mmHg). Pressor response to APA was also attenuated with amastatin and HOE-140 (vehicle: 26.5 ± 1.1 mmHg, amastatin: 14.4 ± 4.2 mmHg, HOE-140: 16.4 ± 2.2 mmHg). In conclusion, APA increase in the brain evokes a pressor response via enzymatic activity without dipsogenic effect. AT1 receptors and B2 receptors in the brain may contribute to the APA-induced pressor response. PMID:28421141

Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

PubMed

Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

2000-05-01

CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

Pharmacological endothelin receptor interaction does not occur in veins from ET(B) receptor deficient rats.

PubMed

Thakali, Keshari; Galligan, James J; Fink, Gregory D; Gariepy, Cheryl E; Watts, Stephanie W

2008-07-01

Heterodimerization of G-protein coupled receptors can alter receptor pharmacology. ET A and ET B receptors heterodimerize when co-expressed in heterologous expression lines. We hypothesized that ET A and ET B receptors heterodimerize and pharmacologically interact in vena cava from wild-type (WT) but not ET B receptor deficient (sl/sl) rats. Pharmacological endothelin receptor interaction was assessed by comparing ET-1-induced contraction in rings of rat thoracic aorta and thoracic vena cava from male Sprague Dawley rats under control conditions, ET A receptor blockade (atrasentan, 10 nM), ET B receptor blockade (BQ-788, 100 nM) or ET B receptor desensitization (Sarafotoxin 6c, 100 nM) and ET A plus ET B receptor blockade or ET A receptor blockade plus ET B receptor desensitization. In addition, similar pharmacological ET receptor antagonism experiments were performed in rat thoracic aorta and vena cava from WT and sl/sl rats. ET A but not ET B receptor blockade or ET B receptor desensitization inhibited aortic and venous ET-1-induced contraction. In vena cava but not aorta, when ET B receptors were blocked (BQ-788, 100 nM) or desensitized (S6c, 100 nM), atrasentan caused a greater inhibition of ET-1-induced contraction. Vena cava from WT but not sl/sl rats exhibited similar pharmacological ET receptor interaction. Immunocytochemistry was performed on freshly dissociated aortic and venous vascular smooth muscle cells to determine localization of ET A and ET B receptors. ET A and ET B receptors qualitatively co-localized more strongly to the plasma membrane of aortic compared to venous vascular smooth muscle cells. Our data suggest that pharmacological ET A and ET B receptor interaction may be dependent on the presence of functional ET B receptors and independent of receptor location.

Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABA(B) receptors, but not α2 adrenergic receptors.

PubMed

Zhang, Guohua; Chen, Wenling; Marvizón, Juan Carlos G

2010-09-01

GABA(B) , μ-opioid and adrenergic α(2) receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABA(B) receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high-frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABA(B) agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low-frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α(2) adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low-frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABA(B) receptors, but not by α(2) receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd. No claim to original US government works.

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

Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

NASA Astrophysics Data System (ADS)

Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

2015-07-01

Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects.

PubMed

Jackson, James G; St Clair, Patricia; Sliwkowski, Mark X; Brattain, Michael G

2004-04-01

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.

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

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

PubMed Central

Kabir, Farruk M. Lutful; DeInnocentes, Patricia; Agarwal, Payal; Mill, Christopher P.; Riese, David J.

2017-01-01

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer. PMID:27515268

Dopamine Receptor-Specific Contributions to the Computation of Value.

PubMed

Burke, Christopher J; Soutschek, Alexander; Weber, Susanna; Raja Beharelle, Anjali; Fehr, Ernst; Haker, Helene; Tobler, Philippe N

2018-05-01

Dopamine is thought to play a crucial role in value-based decision making. However, the specific contributions of different dopamine receptor subtypes to the computation of subjective value remain unknown. Here we demonstrate how the balance between D1 and D2 dopamine receptor subtypes shapes subjective value computation during risky decision making. We administered the D2 receptor antagonist amisulpride or placebo before participants made choices between risky options. Compared with placebo, D2 receptor blockade resulted in more frequent choice of higher risk and higher expected value options. Using a novel model fitting procedure, we concurrently estimated the three parameters that define individual risk attitude according to an influential theoretical account of risky decision making (prospect theory). This analysis revealed that the observed reduction in risk aversion under amisulpride was driven by increased sensitivity to reward magnitude and decreased distortion of outcome probability, resulting in more linear value coding. Our data suggest that different components that govern individual risk attitude are under dopaminergic control, such that D2 receptor blockade facilitates risk taking and expected value processing.

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

Gender-related associations of genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase and bradykinin B2 receptor with treadmill exercise test responses.

PubMed

Nunes, Rafael Amorim Belo; Barroso, Lúcia Pereira; Pereira, Alexandre da Costa; Krieger, José Eduardo; Mansur, Alfredo José

2014-01-01

Treadmill exercise test responses have been associated with cardiovascular prognosis in individuals without overt heart disease. Neurohumoral and nitric oxide responses may influence cardiovascular performance during exercise testing. Therefore, we evaluated associations between functional genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase, bradykinin receptor B2 and treadmill exercise test responses in men and women without overt heart disease. We enrolled 766 (417 women; 349 men) individuals without established heart disease from a check-up programme at the Heart Institute, University of São Paulo Medical School. Exercise capacity, chronotropic reserve, maximum heart-rate achieved, heart-rate recovery, exercise systolic blood pressure (SBP), exercise diastolic blood pressure (DBP) and SBP recovery were assessed during exercise testing. Genotypes for the α-adrenergic receptors ADRA1A Arg347Cys (rs1048101), ADRA2A 1780 C>T (rs553668), ADRA2B Del 301-303 (rs28365031), endothelial nitric synthase (eNOS) 786 T>C (rs2070744), eNOS Glu298Asp (rs1799983) and BK2R (rs5810761) polymorphisms were assessed by PCR and high-resolution melting analysis. Maximum SBP was associated with ADRA1A rs1048101 (p=0.008) and BK2R rs5810761 (p=0.008) polymorphisms in men and ADRA2A rs553668 (p=0.008) and ADRA2B rs28365031 (p=0.022) in women. Maximum DBP pressure was associated with ADRA2A rs553668 (p=0.002) and eNOS rs1799983 (p=0.015) polymorphisms in women. Exercise capacity was associated with eNOS rs2070744 polymorphisms in women (p=0.01) and with eNOS rs1799983 in men and women (p=0.038 and p=0.024). The findings suggest that genetic variants of α-adrenergic receptors and bradykinin B2 receptor may be involved with blood pressure responses during exercise tests. Genetic variants of endothelial nitric oxide synthase may be involved with exercise capacity and blood pressure responses during exercise tests. These responses may be gender-related.

Gender-related associations of genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase and bradykinin B2 receptor with treadmill exercise test responses

PubMed Central

Nunes, Rafael Amorim Belo; Barroso, Lúcia Pereira; Pereira, Alexandre da Costa; Krieger, José Eduardo; Mansur, Alfredo José

2014-01-01

Background Treadmill exercise test responses have been associated with cardiovascular prognosis in individuals without overt heart disease. Neurohumoral and nitric oxide responses may influence cardiovascular performance during exercise testing. Therefore, we evaluated associations between functional genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase, bradykinin receptor B2 and treadmill exercise test responses in men and women without overt heart disease. Methods We enrolled 766 (417 women; 349 men) individuals without established heart disease from a check-up programme at the Heart Institute, University of São Paulo Medical School. Exercise capacity, chronotropic reserve, maximum heart-rate achieved, heart-rate recovery, exercise systolic blood pressure (SBP), exercise diastolic blood pressure (DBP) and SBP recovery were assessed during exercise testing. Genotypes for the α-adrenergic receptors ADRA1A Arg347Cys (rs1048101), ADRA2A 1780 C>T (rs553668), ADRA2B Del 301–303 (rs28365031), endothelial nitric synthase (eNOS) 786 T>C (rs2070744), eNOS Glu298Asp (rs1799983) and BK2R (rs5810761) polymorphisms were assessed by PCR and high-resolution melting analysis. Results Maximum SBP was associated with ADRA1A rs1048101 (p=0.008) and BK2R rs5810761 (p=0.008) polymorphisms in men and ADRA2A rs553668 (p=0.008) and ADRA2B rs28365031 (p=0.022) in women. Maximum DBP pressure was associated with ADRA2A rs553668 (p=0.002) and eNOS rs1799983 (p=0.015) polymorphisms in women. Exercise capacity was associated with eNOS rs2070744 polymorphisms in women (p=0.01) and with eNOS rs1799983 in men and women (p=0.038 and p=0.024). Conclusions The findings suggest that genetic variants of α-adrenergic receptors and bradykinin B2 receptor may be involved with blood pressure responses during exercise tests. Genetic variants of endothelial nitric oxide synthase may be involved with exercise capacity and blood pressure responses during exercise tests

β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

PubMed Central

Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

2014-01-01

Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

PubMed

Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

2007-02-01

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor.

PubMed

Charest-Morin, Xavier; Lodge, Robert; Marceau, François

2018-05-01

To support bradykinin (BK) B 2 receptor (B 2 R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B 2 Rs (immunoblots, epifluorescence microscopy). APEX2-(NG) 15 -MK is a bona fide agonist of the rat, but not of the human B 2 R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric B 2 R detection, cell well plate format). APEX2-(NG) 15 -MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B 2 R in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B 2 R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B 2 R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

Multiple receptors contribute to the behavioral effects of indoleamine hallucinogens

PubMed Central

Halberstadt, Adam L.; Geyer, Mark A.

2011-01-01

Serotonergic hallucinogens produce profound changes in perception, mood, and cognition. These drugs include phenylalkylamines such as mescaline and 2,5-dimethoxy-4-methylamphetamine (DOM), and indoleamines such as (+)-lysergic acid diethylamide (LSD) and psilocybin. Despite their differences in chemical structure, the two classes of hallucinogens produce remarkably similar subjective effects in humans, and induce cross-tolerance. The phenylalkylamine hallucinogens are selective 5-HT2 receptor agonists, whereas the indoleamines are relatively non-selective for serotonin (5-HT) receptors. There is extensive evidence, from both animal and human studies, that the characteristic effects of hallucinogens are mediated by interactions with the 5-HT2A receptor. Nevertheless, there is also evidence that interactions with other receptor sites contribute to the psychopharmacological and behavioral effects of the indoleamine hallucinogens. This article reviews the evidence demonstrating that the effects of indoleamine hallucinogens in a variety of animal behavioral paradigms are mediated by both 5-HT2 and non-5-HT2 receptors. PMID:21256140

Multiple receptors contribute to the behavioral effects of indoleamine hallucinogens.

PubMed

Halberstadt, Adam L; Geyer, Mark A

2011-09-01

Serotonergic hallucinogens produce profound changes in perception, mood, and cognition. These drugs include phenylalkylamines such as mescaline and 2,5-dimethoxy-4-methylamphetamine (DOM), and indoleamines such as (+)-lysergic acid diethylamide (LSD) and psilocybin. Despite their differences in chemical structure, the two classes of hallucinogens produce remarkably similar subjective effects in humans, and induce cross-tolerance. The phenylalkylamine hallucinogens are selective 5-HT(2) receptor agonists, whereas the indoleamines are relatively non-selective for serotonin (5-HT) receptors. There is extensive evidence, from both animal and human studies, that the characteristic effects of hallucinogens are mediated by interactions with the 5-HT(2A) receptor. Nevertheless, there is also evidence that interactions with other receptor sites contribute to the psychopharmacological and behavioral effects of the indoleamine hallucinogens. This article reviews the evidence demonstrating that the effects of indoleamine hallucinogens in a variety of animal behavioral paradigms are mediated by both 5-HT(2) and non-5-HT(2) receptors. Copyright © 2011 Elsevier Ltd. All rights reserved.

Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response.

PubMed

Che, Pulin; Chen, Yunjia; Lu, Roujian; Peng, Ning; Gannon, Mary; Wyss, J Michael; Jiao, Kai; Wang, Qin

2015-01-01

The α2 adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α2A subtype elicits a hypotensive response whereas the α2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α2A subtype. We have previously shown that spinophilin attenuates the α2AAR-dependent hypotensive response; in spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with spinophilin over arrestin. Furthermore, Del301-303 α2BAR-induced ERK signaling is quickly desensitized in cells without spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of spinophilin in sustaining α2BAR signaling. Consistent with this notion, our in vivo study reveals that the α2BAR-elicited hypertensive response is diminished in spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with spinophilin is indispensable for the α2BAR to elicit the hypertensive response. This is opposite of the negative role of spinophilin in regulating α2AAR-mediated hypotensive response, suggesting that spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, spinophilin may represent a useful therapeutic target for treatment of hypertension.

A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density.

PubMed

Liu, Shuxi; Zhou, Liang; Yuan, Hongjie; Vieira, Marta; Sanz-Clemente, Antonio; Badger, John D; Lu, Wei; Traynelis, Stephen F; Roche, Katherine W

2017-04-12

NMDA receptors (NMDARs) are ionotropic glutamate receptors that are crucial for neuronal development and higher cognitive processes. NMDAR dysfunction is involved in a variety of neurological and psychiatric diseases; however, the mechanistic link between the human pathology and NMDAR dysfunction is poorly understood. Rare missense variants within NMDAR subunits have been identified in numerous patients with mental or neurological disorders. We specifically focused on the GluN2B NMDAR subunit, which is highly expressed in the hippocampus and cortex throughout development. We analyzed several variants located in the GluN2B C terminus and found that three variants in patients with autism (S1415L) or schizophrenia (L1424F and S1452F) (S1413L, L1422F, and S1450F in rodents, respectively) displayed impaired binding to membrane-associated guanylate kinase (MAGUK) proteins. In addition, we observed a deficit in surface expression for GluN2B S1413L. Furthermore, there were fewer dendritic spines in GluN2B S1413L-expressing neurons. Importantly, synaptic NMDAR currents in neurons transfected with GluN2B S1413L in GluN2A/B-deficient mouse brain slices revealed only partial rescue of synaptic current amplitude. Functional properties of GluN2B S1413L in recombinant systems revealed no change in receptor properties, consistent with synaptic defects being the result of reduced trafficking and targeting of GluN2B S1413L to the synapse. Therefore, we find that GluN2B S1413L displays deficits in NMDAR trafficking, synaptic currents, and spine density, raising the possibility that this mutation may contribute to the phenotype in this autism patient. More broadly, our research demonstrates that the targeted study of certain residues in NMDARs based on rare variants identified in patients is a powerful approach to studying receptor function. SIGNIFICANCE STATEMENT We have used a "bedside-to-bench" approach to investigate the functional regulation of NMDA receptors (NMDARs). Using

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

A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

PubMed

Wen, Jiaming; Wang, Bohan; Du, Chuanjun; Xu, Gang; Zhang, Zhewei; Li, Yi; Zhang, Nan

2015-10-01

Diabetes is an important risk factor for erectile dysfunction (ED). Recent studies have indicated that A2B adenosine receptor (ADORA2B) signaling is essential for penile erection. Thus, we hypothesize that diabetic ED may be attributed to impaired A2B adenosine signaling. To test this hypothesis, we generated diabetic rats by injecting streptozocin as animal model. After 12 weeks, immunohistochemistry staining was used to localize the expression of ADORA2B. Western Blot and quantitative PCR were employed to determine ADORA2B expression level. Intracavernosal pressure (ICP) measurement was used to evaluate erectile function. Diabetic rats received a single intravenous injection of BAY 60-6583, an ADORA2B agonist, or vehicle solution, at 60 min before the ICP measurement. The results showed that ADORA2B expressed in the nerve bundle, smooth muscle, and endothelium in penile tissue of control mice. Western Blot and quantitative PCR results indicated that the expression levels of ADORA2B protein and mRNA were significantly reduced in penile tissues of diabetic rats. Functional studies showed that the erectile response induced by electrical stimulation was remarkably decreased in diabetic rats, compared with age-matched control rats. However, at 60 min after BAY 60-6583 treatment, the erectile function was improved in diabetic rats, suggesting that enhancement of ADORA2B signaling may improve erectile function in diabetic ED. This preclinical study has revealed a previously unrecognized therapeutic possibility of BAY 60-6583 as an effective and mechanism-based drug to treat diabetic ED. In conclusion, we propose that impaired A2B adenosine signaling is one of the pathological mechanisms of diabetic ED.

Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors

PubMed Central

Kardava, Lela; Moir, Susan; Wang, Wei; Ho, Jason; Buckner, Clarisa M.; Posada, Jacqueline G.; O’Shea, Marie A.; Roby, Gregg; Chen, Jenny; Sohn, Hae Won; Chun, Tae-Wook; Pierce, Susan K.; Fauci, Anthony S.

2011-01-01

Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor–mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor–like–4 (FCRL4) and sialic acid–binding Ig-like lectin 6 (Siglec-6). Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell–associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections. PMID:21633172

Positive feedback of NR2B-containing NMDA receptor activity is the initial step toward visual imprinting: a model for juvenile learning.

PubMed

Nakamori, Tomoharu; Sato, Katsushige; Kinoshita, Masae; Kanamatsu, Tomoyuki; Sakagami, Hiroyuki; Tanaka, Kohichi; Ohki-Hamazaki, Hiroko

2015-01-01

Imprinting in chicks is a good model for elucidating the processes underlying neural plasticity changes during juvenile learning. We recently reported that neural activation of a telencephalic region, the core region of the hyperpallium densocellulare (HDCo), was critical for success of visual imprinting, and that N-Methyl-D-aspartic (NMDA) receptors containing the NR2B subunit (NR2B/NR1) in this region were essential for imprinting. Using electrophysiological and multiple-site optical imaging techniques with acute brain slices, we found that long-term potentiation (LTP) and enhancement of NR2B/NR1 currents in HDCo neurons were induced in imprinted chicks. Enhancement of NR2B/NR1 currents as well as an increase in surface NR2B expression occurred even following a brief training that was too weak to induce LTP or imprinting behavior. This means that NR2B/NR1 activation is the initial step of learning, well before the activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors which induces LTP. We also showed that knockdown of NR2B/NR1 inhibited imprinting, and inversely, increasing the surface NR2B expression by treatment with a casein kinase 2 inhibitor successfully reduced training time required for imprinting. These results suggest that imprinting stimuli activate post-synaptic NR2B/NR1 in HDCo cells, increase NR2B/NR1 signaling through up-regulation of its expression, and induce LTP and memory acquisition. The study investigated the neural mechanism underlying juvenile learning. In the initial stage of chick imprinting, NMDA receptors containing the NMDA receptor subunit 2B (NR2B) are activated, surface expression of NR2B/NR1 (NMDA receptor subunit 1) is up-regulated, and consequently long-term potentiation is induced in the telencephalic neurons. We suggest that the positive feedback in the NR2B/NR1 activation is a unique process of juvenile learning, exhibiting rapid memory acquisition. © 2014 International Society for Neurochemistry.

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

Insulin receptor isoforms A and B as well as insulin receptor substrates-1 and -2 are differentially expressed in prostate cancer.

PubMed

Heni, Martin; Hennenlotter, Jörg; Scharpf, Marcus; Lutz, Stefan Z; Schwentner, Christian; Todenhöfer, Tilman; Schilling, David; Kühs, Ursula; Gerber, Valentina; Machicao, Fausto; Staiger, Harald; Häring, Hans-Ulrich; Stenzl, Arnulf

2012-01-01

In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS) isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation. We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA- and protein expression of the cell cycle regulator p27(Kip1) was quantified by real-time RT-PCR and immunohistochemistry. Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05). IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05). These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27(Kip1) content (p<0.02). Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019). We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy.

Adenosine A2B Receptor Deficiency Promotes Host Defenses against Gram-Negative Bacterial Pneumonia

PubMed Central

Barletta, Kathryn E.; Cagnina, R. Elaine; Burdick, Marie D.; Linden, Joel

2012-01-01

Rationale: Activation of the adenosine A2B receptor (A2BR) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. Objectives: To test the hypothesis that absence of adenosine A2B receptor signaling promotes host defense against bacterial pneumonia. Methods: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A2BR. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. Measurements and Main Results: A2BR–/– mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow–derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A2BR–/– and wild-type mice, but A2BR–/– neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A2BR–/– mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. Conclusions: These data suggest that the absence of A2BR signaling enhances antimicrobial activity in gram-negative bacterial pneumonia. PMID:22997203

Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity.

PubMed

Planagumà, Jesús; Haselmann, Holger; Mannara, Francesco; Petit-Pedrol, Mar; Grünewald, Benedikt; Aguilar, Esther; Röpke, Luise; Martín-García, Elena; Titulaer, Maarten J; Jercog, Pablo; Graus, Francesc; Maldonado, Rafael; Geis, Christian; Dalmau, Josep

2016-09-01

To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016;80:388-400. © 2016 American Neurological Association.

The role of bradykinin receptor type 2 in spontaneous extravasation in mice skin: implications for non-allergic angio-oedema.

PubMed

Bisha, Marion; Dao, Vu Thao-Vi; Gholamreza-Fahimi, Ehsan; Vogt, Michael; van Zandvoort, Marc; Weber, Sarah; Bas, Murat; Khosravani, Farbod; Kojda, Georg; Suvorava, Tatsiana

2018-05-01

Non-allergic angio-oedema is a life-threatening disease mediated by activation of bradykinin type 2 receptors (B 2 receptors). The aim of this study was to investigate whether activation of B 2 receptors by endogenous bradykinin contributes to physiological extravasation. This may shed new light on the assumption that treatment with an angiotensin converting enzyme inhibitor (ACEi) results in an alteration in the vascular barrier function predisposing to non-allergic angio-oedema. We generated a new transgenic mouse model characterized by endothelium-specific overexpression of the B 2 receptor (B2 tg ) and established a non-invasive two-photon laser microscopy approach to measure the kinetics of spontaneous extravasation in vivo. The B2 tg mice showed normal morphology and litter size as compared with their transgene-negative littermates (B2 n ). Overexpression of B 2 receptors was functional in conductance vessels and resistance vessels as evidenced by B 2 receptor-mediated aortic dilation to bradykinin in presence of non-specific COX inhibitor diclofenac and by significant hypotension in B2 tg respectively. Measurement of dermal extravasation by Miles assay showed that bradykinin induced extravasation was significantly increased in B2 tg as compared with B2 n . However, neither endothelial overexpression of B 2 receptors nor treatment with the ACEi moexipril or B 2 antagonist icatibant had any effect on spontaneous extravasation measured by two-photon laser microscopy. Activation of B 2 receptors does not appear to be involved in spontaneous extravasation. Therefore, the assumption that treatment with an ACEi results in an alteration in the physiological vascular barrier function predisposing to non-allergic angio-oedema is not supported by our findings. © 2018 The British Pharmacological Society.

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

PubMed Central

Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

2015-01-01

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

Fibroblast Growth Factor 10-Fibroblast Growth Factor Receptor 2b Mediated Signaling Is Not Required for Adult Glandular Stomach Homeostasis

PubMed Central

Sala, Frederic G.; Ford, Henri R.; Bellusci, Saverio; Grikscheit, Tracy C.

2012-01-01

The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis. PMID:23133671

Structure of dual receptor binding to botulinum neurotoxin B.

PubMed

Berntsson, Ronnie P-A; Peng, Lisheng; Dong, Min; Stenmark, Pål

2013-01-01

Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3-Å structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points.

Increased hypothalamic 5-HT2A receptor gene expression and effects of pharmacologic 5-HT2A receptor inactivation in obese A{sup y} mice

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

Nonogaki, Katsunori; Nozue, Kana; Oka, Yoshitomo

2006-12-29

Serotonin (5-hydroxytryptamine; 5-HT) 2A receptors contribute to the effects of 5-HT on platelet aggregation and vascular smooth muscle cell proliferation, and are reportedly involved in decreases in plasma levels of adiponectin, an adipokine, in diabetic subjects. Here, we report that systemic administration of sarpogrelate, a 5-HT2A receptor antagonist, suppressed appetite and increased hypothalamic pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript, corticotropin releasing hormone, 5-HT2C, and 5-HT1B receptor gene expression. A{sup y} mice, which have ectopic expression of the agouti protein, significantly increased hypothalamic 5-HT2A receptor gene expression in association with obesity compared with wild-type mice matched for age. Systemic administration ofmore » sarpogrelate suppressed overfeeding, body weight gain, and hyperglycemia in obese A{sup y} mice, whereas it did not increase plasma adiponectin levels. These results suggest that obesity increases hypothalamic 5-HT2A receptor gene expression, and pharmacologic inactivation of 5-HT2A receptors inhibits overfeeding and obesity in A{sup y} mice, but did not increase plasma adiponectin levels.« less

Contribution of TyrB26 to the Function and Stability of Insulin

PubMed Central

Pandyarajan, Vijay; Phillips, Nelson B.; Rege, Nischay; Lawrence, Michael C.; Whittaker, Jonathan; Weiss, Michael A.

2016-01-01

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of TyrB26, a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the GluB26 analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although TyrB26 is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences. PMID:27129279

TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

PubMed

Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

2016-09-15

Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

PubMed

Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E

2010-02-01

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

Role of NF-κB in oxidative stress-induced defective dopamine D1 receptor signaling in the renal proximal tubules of Sprague Dawley rats

PubMed Central

Fardoun, Riham Zein; Asghar, Mohammad; Lokhandwala, Mustafa

2009-01-01

Dopamine promotes sodium excretion, in part, via activation of D1 receptors in renal proximal tubules (PT) and subsequent inhibition of Na, K-ATPase. Recently, we have reported that oxidative stress causes D1 receptors-G-protein uncoupling via mechanisms involving Protein Kinase C (PKC) and G-protein Coupled Receptor Kinase 2 (GRK2) in the primary culture of renal PT of Sprague Dawley (SD) rats. There are reports suggesting that redox-sensitive nuclear transcription factor, NF-κB, is activated in conditions associated with oxidative stress. This study was designed to identify the role of NF-κB in oxidative stress–induced defective renal D1 receptor –G-protein coupling and function. Treatment of the PT with hydrogen peroxide (H2O2, 50 μM/20 min) induced the nuclear translocation of NF-κB, increased PKC activity, and triggered the translocation of GRK2 to the proximal tubular membranes. This was accompanied by hyperphosphorylation of D1 receptors and defective D1 receptor-G-protein coupling. The functional consequence of these changes was decreased D1 receptor activation-mediated inhibition of Na, K-ATPase activity. Interestingly, pre-treatment with pyrrolidine dithiocarbamate (PDTC, 25 μM/10min), an NF-κB inhibitor, blocked the H2O2-induced nuclear translocation of NF-κB, increase in PKC activity, as well as GRK2 translocation and hyperphosphorylation of D1 receptors in the proximal tubular membranes. Furthermore, PDTC restored D1 receptor G-protein coupling and D1 receptor agonist-mediated inhibition of the Na, KATPase activity. Therefore, we suggest that oxidative stress causes nuclear translocation of NF-κB in the renal proximal tubules, which contributes to defective D1-receptor-G-protein coupling and function via mechanism involving PKC, membranous translocation of GRK 2, and subsequent phosphorylation of dopamine D1 receptors. PMID:17320758

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.

Dopamine suppresses neuronal activity of Helisoma B5 neurons via a D2-like receptor, activating PLC and K channels.

PubMed

Zhong, L R; Artinian, L; Rehder, V

2013-01-03

Dopamine (DA) plays fundamental roles as a neurotransmitter and neuromodulator in the central nervous system. How DA modulates the electrical excitability of individual neurons to elicit various behaviors is of great interest in many systems. The buccal ganglion of the freshwater pond snail Helisoma trivolvis contains the neuronal circuitry for feeding and DA is known to modulate the feeding motor program in Helisoma. The buccal neuron B5 participates in the control of gut contractile activity and is surrounded by dopaminergic processes, which are expected to release DA. In order to study whether DA modulates the electrical activity of individual B5 neurons, we performed experiments on physically isolated B5 neurons in culture and on B5 neurons within the buccal ganglion in situ. We report that DA application elicited a strong hyperpolarization in both conditions and turned the electrical activity from a spontaneously firing state to an electrically silent state. Using the cell culture system, we demonstrated that the strong hyperpolarization was inhibited by the D2 receptor antagonist sulpiride and the phospholipase C (PLC) inhibitor U73122, indicating that DA affected the membrane potential of B5 neurons through the activation of a D2-like receptor and PLC. Further studies revealed that the DA-induced hyperpolarization was inhibited by the K channel blockers 4-aminopyridine and tetraethylammonium, suggesting that K channels might serve as the ultimate target of DA signaling. Through its modulatory effect on the electrical activity of B5 neurons, the release of DA in vivo may contribute to a neuronal output that results in a variable feeding motor program. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Hypothyroidism affects D2 receptor-mediated breathing without altering D2 receptor expression.

PubMed

Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D

2014-03-01

Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. Copyright © 2014 Elsevier B.V. All rights reserved.

Structure and signalling functions of C3 receptors on human B cells.

PubMed

Frade, R

1990-03-01

CR1 (C3b receptor) and CR2 (C3d/EBV receptor) are two C3 receptors expressed on B lymphocytes. CR1 and CR2 have structural similarities and their cross-linking at the B cell surface by antibodies or specific ligands in multimeric forms induce B cell activation. However, activation of human B cells through cell surface interactions or by intracellular protein kinase C activators leads to phosphorylation of CR2 but not CR1. CR2 is phosphorylated on serine and tyrosine residues. Analysis of post-membrane events associated with CR2 revealed intracellular interactions of CR2 with p53, a plasma membrane anti-oncogene-encoded phosphoprotein, and with p120, a nuclear phosphoribonucleoprotein. These intracellular interactions probably represent important steps in the signalling functions of CR2.

GluN2B N-methyl-D-aspartate receptor and excitatory amino acid transporter 3 are upregulated in primary sensory neurons after 7 days of morphine administration in rats: implication for opiate-induced hyperalgesia.

PubMed

Gong, Kerui; Bhargava, Aditi; Jasmin, Luc

2016-01-01

The contribution of the peripheral nervous system to opiate-induced hyperalgesia (OIH) is not well understood. In this study, we determined the changes in excitability of primary sensory neurons after sustained morphine administration for 7 days. Changes in the expression of glutamate receptors and glutamate transporters after morphine administration were ascertained in dorsal root ganglions. Patch clamp recordings from intact dorsal root ganglions (ex vivo preparation) of morphine-treated rats showed increased excitability of small diameter (≤30 μm) neurons with respect to rheobase and membrane threshold, whereas the excitability of large diameter (>30 μm) neurons remained unchanged. Small diameter neurons also displayed increased responses to glutamate, which were mediated mainly by GluN2B containing N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree by the neuronal excitatory amino acid transporter 3/excitatory amino acid carrier 1. Coadministration in vivo of the GluN2B selective antagonist Ro 25-6981 with morphine for 7 days prevented the appearance of OIH and increased morphine-induced analgesia. Administration of morphine for 7 days led to an increased expression of GluN2B and excitatory amino acid transporter 3/excitatory amino acid carrier 1, but not of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate, kainate, or group I metabotropic glutamate receptors, or of the vesicular glutamate transporter 2. These results suggest that peripheral glutamatergic neurotransmission contributes to OIH and that GluN2B subunit of NMDA receptors in the periphery may be a target for therapy.

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics

PubMed Central

Robles, Eloy F.; Mena-Varas, Maria; Barrio, Laura; Merino-Cortes, Sara V.; Balogh, Péter; Du, Ming-Qing; Akasaka, Takashi; Parker, Anton; Roa, Sergio; Panizo, Carlos; Martin-Guerrero, Idoia; Siebert, Reiner; Segura, Victor; Agirre, Xabier; Macri-Pellizeri, Laura; Aldaz, Beatriz; Vilas-Zornoza, Amaia; Zhang, Shaowei; Moody, Sarah; Calasanz, Maria Jose; Tousseyn, Thomas; Broccardo, Cyril; Brousset, Pierre; Campos-Sanchez, Elena; Cobaleda, Cesar; Sanchez-Garcia, Isidro; Fernandez-Luna, Jose Luis; Garcia-Muñoz, Ricardo; Pena, Esther; Bellosillo, Beatriz; Salar, Antonio; Baptista, Maria Joao; Hernandez-Rivas, Jesús Maria; Gonzalez, Marcos; Terol, Maria Jose; Climent, Joan; Ferrandez, Antonio; Sagaert, Xavier; Melnick, Ari M.; Prosper, Felipe; Oscier, David G.; Carrasco, Yolanda R.; Dyer, Martin J. S.; Martinez-Climent, Jose A.

2016-01-01

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas. PMID:27297662

Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain.

PubMed

Dziedzic, Barbara; Prevot, Vincent; Lomniczi, Alejandro; Jung, Heike; Cornea, Anda; Ojeda, Sergio R

2003-02-01

Hypothalamic astroglial erbB tyrosine kinase receptors are required for the timely initiation of mammalian puberty. Ligand-dependent activation of these receptors sets in motion a glia-to-neuron signaling pathway that prompts the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development, from hypothalamic neuroendocrine neurons. The neuronal systems that may regulate this growth factor-mediated back signaling to neuroendocrine neurons have not been identified. Here we demonstrate that hypothalamic astrocytes contain metabotropic receptors of the metabotropic glutamate receptor 5 subtype and the AMPA receptor subunits glutamate receptor 2 (GluR2) and GluR3. As in excitatory synapses, these receptors are in physical association with their respective interacting/clustering proteins Homer and PICK1. In addition, they are associated with erbB-1 and erbB-4 receptors. Concomitant activation of astroglial metabotropic and AMPA receptors results in the recruitment of erbB tyrosine kinase receptors and their respective ligands to the glial cell membrane, transactivation of erbB receptors via a mechanism requiring metalloproteinase activity, and increased erbB receptor gene expression. By facilitating erbB-dependent signaling and promoting erbB receptor gene expression in astrocytes, a neuron-to-glia glutamatergic pathway may represent a basic cell-cell communication mechanism used by the neuroendocrine brain to coordinate the facilitatory transsynaptic and astroglial input to LHRH neurons during sexual development.

Celecoxib offsets the negative renal influences of cyclosporine via modulation of the TGF-β1/IL-2/COX-2/endothelin ET(B) receptor cascade.

PubMed

El-Gowelli, Hanan M; Helmy, Maged W; Ali, Rabab M; El-Mas, Mahmoud M

2014-03-01

Endothelin (ET) signaling provokes nephrotoxicity induced by the immunosuppressant drug cyclosporine A (CSA). We tested the hypotheses that (i): celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, counterbalances renal derangements caused by CSA in rats and (ii) the COX-2/endothelin ET(B) receptor signaling mediates the CSA-celecoxib interaction. Ten-day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea, creatinine), inflammation (interleukin-2, IL-2) and fibrosis (transforming growth factor-β₁, TGF-β₁). Histologically, CSA caused renal tubular atrophy along with interstitial fibrosis. These detrimental renal effects of CSA were largely reduced in rats treated concurrently with celecoxib (10 mg/kg/day). We also report that cortical glomerular and medullary tubular protein expressions of COX-2 and ET(B) receptors were reduced by CSA and restored to near-control values in rats treated simultaneously with celecoxib. The importance of ET(B) receptors in renal control and in the CSA-celecoxib interaction was further verified by the findings (i) most of the adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1 mg/kg/day, 10 days), and (ii) the BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, the data suggest that the facilitation of the interplay between the TGF-β1/IL-2/COX-2 pathway and the endothelin ET(B) receptors constitutes the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in rats. Copyright © 2014 Elsevier Inc. All rights reserved.

α1b-Adrenergic Receptor Localization and Relationship to the D1-Dopamine Receptor in the Rat Nucleus Accumbens.

PubMed

Mitrano, Darlene A; Jackson, Kelsey; Finley, Samantha; Seeley, Allison

2018-02-10

The α1-adrenergic receptors (α1ARs) have been implicated in numerous actions of the brain, including attention and wakefulness. Additionally, they have been identified as contributing to disorders of the brain, such as drug addiction, and recent work has shown a role of these receptors in relapse to psychostimulants. While some functionality is known, the actual subcellular localization of the subtypes of the α1ARs remains to be elucidated. Further, their anatomical relationship to receptors for other neurotransmitters, such as dopamine (DA), remains unclear. Therefore, using immunohistochemistry and electron microscopy techniques, this study describes the subcellular localization of the α1b-adrenergic receptor (α1bAR), the subtype most tied to relapse behaviors, as well as its relationship to the D1-dopamine receptor (D1R) in both the shell and core of the rat nucleus accumbens (NAc). Overall, α1bARs were found in unmyelinated axons and axon terminals with some labeling in dendrites. In accordance with other studies of the striatum, the D1R was found mainly in dendrites and spines; therefore, colocalization of the D1R with the α1bAR was rare postsynaptically. However, in the NAc shell, when the receptors were co-expressed in the same neuronal elements there was a trend for both receptors to be found on the plasma membrane, as opposed to the intracellular compartment. This study provides valuable anatomical information about the α1bAR and its relationship to the D1R and the regulation of DA and norepinephrine (NE) neurotransmission in the brain which have been examined previously. Published by Elsevier Ltd.

Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling

PubMed Central

Mi, Tiejuan; Abbasi, Shahrzad; Zhang, Hong; Uray, Karen; Chunn, Janci L.; Xia, Ling Wei; Molina, Jose G.; Weisbrodt, Norman W.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

2008-01-01

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor–mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine–induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada–/– and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder. PMID:18340377

ErbB receptors in the biology and pathology of the aerodigestive tract

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

Morgan, Sarah; Suite 500, Pittsburgh, PA 15213; Grandis, Jennifer R.

2009-02-15

The most common sites of malignancies in the aerodigestive tract include the lung, head and neck and the esophagus. Esophageal adenocarcinomas (EA), esophageal squamous cell carcinomas (ESCC), and squamous cell carcinomas of the head and neck (SCCHN) are the primary focus of this review. Traditional treatment for aerodigestive tract cancers includes primary chemoradiotherapy (CRT) or surgical resection followed by radiation (or CRT). Recent developments in treatment have focused increasingly on molecular targeting strategies including cetuximab (a monoclonal antibody against epidermal growth factor receptor (EGFR)). Cetuximab was FDA approved in 2006 for treatment of SCCHN, underscoring the importance of understanding themore » biology of these malignancies. EGFR is a member of the ErbB family of growth factor receptor tyrosine kinases. The major pathways activated by ErbB receptors include Ras/Raf/MAPK; PI3K/AKT; PLC{gamma} and STATs, all of which lead to the transcription of target genes that may contribute to aerodigestive tumor progression. This review explores the expression of ErbB receptors in EA, ESCC and SCCHN and the signaling pathways of EGFR in SCCHN.« less

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.

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

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

Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold)more » and CYP3A4 (11-fold) promoter activities over control at 10 {mu}M. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 {mu}M. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 {mu}M, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 {mu}M and 10 {mu}M, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.« less

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

PubMed

Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

2010-06-15

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. Copyright 2010 Elsevier Inc. All rights reserved.

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

Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca2+ signalling.

PubMed

Ackermann, Jochen A; Radtke, Daniel; Maurberger, Anna; Winkler, Thomas H; Nitschke, Lars

2011-04-20

Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B-cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR-induced Ca(2+) signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B-cell-specific Grb2-deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B-cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR-induced Ca(2+) signalling, alterations in mitogen-activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B-cell-specific Grb2-deficient mice showed impaired IgG and B-cell memory responses, and impaired germinal centre formation. Thus, Grb2-dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.

ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells.

PubMed

Zaoui, Kossay; Benseddik, Khedidja; Daou, Pascale; Salaün, Danièle; Badache, Ali

2010-10-26

Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.

Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

PubMed Central

Griffioen, A W; Rijkers, G T; Janssens-Korpela, P; Zegers, B J

1991-01-01

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS. PMID:1826897

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

PubMed Central

Khandelwal, Sanjay; Lee, Grace M.; Hester, C. Garren; Poncz, Mortimer; McKenzie, Steven E.; Sachais, Bruce S.; Rauova, Lubica; Kelsoe, Garnett; Cines, Douglas B.; Frank, Michael

2016-01-01

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes. PMID:27412887

Contribution of P2X4 receptors to ethanol intake in male C57BL/6 mice

PubMed Central

Wyatt, Letisha R.; Finn, Deborah A.; Khoja, Sheraz; Yardley, Megan M; Asatryan, Liana; Alkana, Ronald L.; Davies, Daryl L.

2014-01-01

P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels activated by synaptically released extracellular ATP. The P2X4 subtype is abundantly expressed in the CNS and is sensitive to low intoxicating ethanol concentrations. Genetic meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake and/or preference. The current study used mice lacking the p2rx4 gene (knockout, KO) and wildtype (WT) C57BL/6 controls to test the hypothesis that P2X4Rs contribute to ethanol intake. The early acquisition and early maintenance phases of ethanol intake were measured with three different drinking procedures. Further, we tested the effects of ivermectin (IVM), a drug previously shown to reduce ethanol’s effects on P2X4Rs and to reduce ethanol intake and preference, for its ability to differentially alter stable ethanol intake in KO and WT mice. Depending on the procedure and the concentration of the ethanol solution, ethanol intake was transiently increased in P2X4R KO versus WT mice during the acquisition of 24-hr and limited access ethanol intake. IVM significantly reduced ethanol intake in P2X4R KO and WT mice, but the degree of reduction was 50% less in the P2X4R KO mice. Western blot analysis identified significant changes in -γ aminobutyric acidA receptor (GABAAR) α1 subunit expression in brain regions associated with the regulation of ethanol behaviors in P2X4R KO mice. These findings add to evidence that P2X4Rs contribute to ethanol intake and indicate that there is a complex interaction between P2X4Rs, ethanol, and other neurotransmitter receptor systems. PMID:24671605

Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?

PubMed

Barwell, James; Gingell, Joseph J; Watkins, Harriet A; Archbold, Julia K; Poyner, David R; Hay, Debbie L

2012-05-01

The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

GluN2B-containing NMDA receptors and AMPA receptors in medial prefrontal cortex are necessary for odor span in rats.

PubMed

Davies, Don A; Greba, Quentin; Howland, John G

2013-01-01

Working memory is a type of short-term memory involved in the maintenance and manipulation of information essential for complex cognition. While memory span capacity has been extensively studied in humans as a measure of working memory, it has received considerably less attention in rodents. Our aim was to examine the role of the N-methyl-D-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors in odor span capacity using systemic injections or infusions of receptor antagonists into the medial prefrontal cortex (mPFC). Long Evans rats were trained on a well-characterized odor span task (OST). Initially, rats were trained to dig for a food reward in sand followed by training on a non-match to sample discrimination using sand scented with household spices. The rats were then required to perform a serial delayed non-match to sample procedure which was their odor span. Systemic injection of the broad spectrum NMDA receptor antagonist 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) (10 mg/kg) or the GluN2B-selective antagonist Ro 25-6981 (10 mg/kg but not 6 mg/kg) significantly reduced odor span capacity. Infusions of the GluN2B- selective antagonist Ro 25-6981 (2.5 μg/hemisphere) into mPFC reduced span capacity, an effect that was nearly significant (p = 0.069). Infusions of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (1.25 μg/hemisphere) into mPFC reduced span capacity and latency for the rats to make a choice in the task. These results demonstrate span capacity in rats depends on ionotropic glutamate receptor activation in the mPFC. Further understanding of the circuitry underlying span capacity may aid in the novel therapeutic drug development for persons with working memory impairments as a result of disorders such as schizophrenia and Alzheimer's disease.

Induced Pluripotent Stem Cell‐Derived Endothelial Cells Overexpressing Interleukin‐8 Receptors A/B and/or C‐C Chemokine Receptors 2/5 Inhibit Vascular Injury Response

PubMed Central

Giordano, Samantha; Zhao, Xiangmin; Chen, Yiu‐Fai; Litovsky, Silvio H.; Hage, Fadi G.; Townes, Tim M.; Sun, Chiao‐Wang; Wu, Li‐Chen; Oparil, Suzanne

2017-01-01

Abstract Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C‐C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat‐induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS‐ECs overexpressing IL8RA/B (RiPS‐IL8RA/B‐ECs), CCR2/5 (RiPS‐CCR2/5‐ECs), or both receptors (RiPS‐IL8RA/B+CCR2/5‐ECs) will inhibit inflammatory responses and neointima formation in balloon‐injured rat carotid artery. Twelve‐week‐old male Sprague‐Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS‐Null‐ECs (ECs transduced with empty virus), (c) RiPS‐IL8RA/B‐ECs, (d) RiPS‐CCR2/5‐ECs, or (e) RiPS‐IL8RA/B+CCR2/5‐ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS‐ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury‐induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS‐ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS‐ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168–1177 PMID:28233474

Activation-Induced Killer Cell Immunoglobulin-like Receptor 3DL2 Binding to HLA-B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis.

PubMed

Ridley, Anna; Hatano, Hiroko; Wong-Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K; Al-Mossawi, Hussein; Ladell, Kristin; Price, David A; Bowness, Paul; Kollnberger, Simon

2016-04-01

In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA-B27 and the phenotype and function of KIR-3DL2-expressing CD4+ T cells in SpA. In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27- and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription-polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor-related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. KIR-3DL2 binding to HLA-B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA-B27-KIR-3DL2 interactions for the treatment of B27+ patients with SpA. © 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

PubMed

Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

2009-10-01

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

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.

Aquaporin-4 deficiency facilitates fear memory extinction in the hippocampus through excessive activation of extrasynaptic GluN2B-containing NMDA receptors.

PubMed

Wu, Xin; Zhang, Jie-Ting; Li, Di; Zhou, Jun; Yang, Jun; Zheng, Hui-Ling; Chen, Jian-Guo; Wang, Fang

2017-01-01

Aquaporin-4 (AQP-4) is the predominant water channel in the brain and primarily expressed in astrocytes. Astrocytes have been generally believed to play important roles in regulating synaptic plasticity and information processing. A growing number of evidence shows that AQP-4 plays a potential role in the regulation of astrocyte function. However, little is known about the function of AQP-4 for synaptic plasticity in the hippocampus. Therefore, we evaluated long-term depression (LTD) in the hippocampus and the extinction of fear memory of AQP-4 knockout (KO) and wild-type (WT) mice. We found that AQP-4 deficiency facilitated fear memory extinction and NMDA receptors (NMDARs)-dependent LTD in the CA3-CA1 pathway. Furthermore, AQP-4 deficiency selectively increased GluN2B-NMDAR-mediated excitatory postsynaptic currents (EPSCs). The excessive activation of extrasynaptic GluN2B-NMDAR contributed to the facilitation of NMDAR-dependent LTD and enhancement of fear memory extinction in AQP-4 KO mice. Thus, it appears that AQP-4 may be a potential target for intervention in fear memory extinction. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

PubMed

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Lung Injury Pathways: Adenosine Receptor 2B Signaling Limits Development of Ischemic Bronchiolitis Obliterans Organizing Pneumonia

PubMed Central

Densmore, John C.; Schaid, Terry R.; Jeziorczak, Paul M.; Medhora, Meetha; Audi, Said; Nayak, Shraddha; Auchampach, John; Dwinell, Melinda R.; Geurts, Aron M.; Jacobs, Elizabeth R.

2018-01-01

Purpose/Aim of the study Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. Materials and Methods Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strain containing a mutation in the A2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A2A (A2AAR) and A2B adenosine receptor (A2BAR) mRNA and protein were quantified. Results 24h after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A2BAR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A2AAR mRNA and protein concentrations remained unchanged following ischemia. A2BAR protein was increased in PA ligated lungs of SS rats after 7d, and 4h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. Conclusions Increased A2BAR and adenosine following unilateral lung ischemia as well as more BOOP in A2BAR mutant rats implicate a protective role for A2BAR signaling in countering ischemic lung injury. PMID:28266889

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

PubMed

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

PubMed Central

Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478

SRC Inhibition Reduces NR2B Surface Expression and Synaptic Plasticity in the Amygdala

ERIC Educational Resources Information Center

Sinai, Laleh; Duffy, Steven; Roder, John C.

2010-01-01

The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src…

Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration*

PubMed Central

Ou, Young; Chan, Gordon; Zuo, Jeremy; Rattner, Jerome B.; van der Hoorn, Frans A.

2016-01-01

The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process. PMID:27226580

Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis.

PubMed

Larrivée, Bruno; Freitas, Catarina; Trombe, Marc; Lv, Xiang; Delafarge, Benjamin; Yuan, Li; Bouvrée, Karine; Bréant, Christiane; Del Toro, Raquel; Bréchot, Nicolas; Germain, Stéphane; Bono, Françoise; Dol, Frédérique; Claes, Filip; Fischer, Christian; Autiero, Monica; Thomas, Jean-Léon; Carmeliet, Peter; Tessier-Lavigne, Marc; Eichmann, Anne

2007-10-01

Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target.

Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis

PubMed Central

Larrivée, Bruno; Freitas, Catarina; Trombe, Marc; Lv, Xiang; DeLafarge, Benjamin; Yuan, Li; Bouvrée, Karine; Bréant, Christiane; Del Toro, Raquel; Bréchot, Nicolas; Germain, Stéphane; Bono, Françoise; Dol, Frédérique; Claes, Filip; Fischer, Christian; Autiero, Monica; Thomas, Jean-Léon; Carmeliet, Peter; Tessier-Lavigne, Marc; Eichmann, Anne

2007-01-01

Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target. PMID:17908930

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes.

PubMed

Allen, Bruce G; Phuong, Luu Lien; Farhat, Hala; Chevalier, Dominique

2003-02-01

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.

The A-Current Modulates Learning via NMDA Receptors Containing the NR2B Subunit

PubMed Central

Fontán-Lozano, Ángela; Suárez-Pereira, Irene; González-Forero, David; Carrión, Ángel Manuel

2011-01-01

Synaptic plasticity involves short- and long-term events, although the molecular mechanisms that underlie these processes are not fully understood. The transient A-type K+ current (IA) controls the excitability of the dendrites from CA1 pyramidal neurons by regulating the back-propagation of action potentials and shaping synaptic input. Here, we have studied how decreases in IA affect cognitive processes and synaptic plasticity. Using wild-type mice treated with 4-AP, an IA inhibitor, and mice lacking the DREAM protein, a transcriptional repressor and modulator of the IA, we demonstrate that impairment of IA decreases the stimulation threshold for learning and the induction of early-LTP. Hippocampal electrical recordings in both models revealed alterations in basal electrical oscillatory properties toward low-theta frequencies. In addition, we demonstrated that the facilitated learning induced by decreased IA requires the activation of NMDA receptors containing the NR2B subunit. Together, these findings point to a balance between the IA and the activity of NR2B-containing NMDA receptors in the regulation of learning. PMID:21966384

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

Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

PubMed Central

Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

2012-01-01

Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.

PubMed

Hadjighassem, Mahmoud R; Austin, Mark C; Szewczyk, Bernadeta; Daigle, Mireille; Stockmeier, Craig A; Albert, Paul R

2009-08-01

Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

Prostaglandin E2 regulates B cell proliferation through a candidate tumor suppressor, Ptger4.

PubMed

Murn, Jernej; Alibert, Olivier; Wu, Ning; Tendil, Simon; Gidrol, Xavier

2008-12-22

B cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies, and most B cell lymphomas depend on BCR signals for survival. Identification of genes that restrain BCR-mediated proliferation is therefore an important goal toward improving the therapy of B cell lymphoma. Here, we identify Ptger4 as a negative feedback regulator of proliferation in response to BCR signals and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). Stable knockdown of Ptger4 in B cell lymphoma markedly accelerated tumor spread in mice, whereas Ptger4 overexpression yielded significant protection. Mechanistically, we show that the intrinsic activity of Ptger4 and PGE2-EP4 signaling target a similar set of activating genes, and find Ptger4 to be significantly down-regulated in human B cell lymphoma. We postulate that Ptger4 functions in B cells as a candidate tumor suppressor whose activity is regulated by PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B cell malignancies.

Prostaglandin E2 regulates B cell proliferation through a candidate tumor suppressor, Ptger4

PubMed Central

Murn, Jernej; Alibert, Olivier; Wu, Ning; Tendil, Simon; Gidrol, Xavier

2008-01-01

B cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies, and most B cell lymphomas depend on BCR signals for survival. Identification of genes that restrain BCR-mediated proliferation is therefore an important goal toward improving the therapy of B cell lymphoma. Here, we identify Ptger4 as a negative feedback regulator of proliferation in response to BCR signals and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). Stable knockdown of Ptger4 in B cell lymphoma markedly accelerated tumor spread in mice, whereas Ptger4 overexpression yielded significant protection. Mechanistically, we show that the intrinsic activity of Ptger4 and PGE2–EP4 signaling target a similar set of activating genes, and find Ptger4 to be significantly down-regulated in human B cell lymphoma. We postulate that Ptger4 functions in B cells as a candidate tumor suppressor whose activity is regulated by PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B cell malignancies. PMID:19075289

The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B

PubMed Central

Sheng, Zhenyu; Liang, Zhong; Geiger, James H.; Prorok, Mary; Castellino, Francis J.

2009-01-01

The conantokins are short, naturally-occurring peptides that inhibit ion flow through N-methyl-D-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), specifically antagonizes NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E657-I814) were replaced by the corresponding S2 region of NR2B (E658-I815), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M739 of NR2B for the equivalent K738 of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K755 of NR2B for Y754 of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes. PMID:19427876

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Attenuation of reserpine-induced pain/depression dyad by gentiopicroside through downregulation of GluN2B receptors in the amygdala of mice.

PubMed

Liu, Shui-bing; Zhao, Rong; Li, Xu-sheng; Guo, Hong-ju; Tian, Zhen; Zhang, Nan; Gao, Guo-dong; Zhao, Ming-gao

2014-06-01

Epidemiological studies demonstrate that pain frequently occurs comorbid with depression. Gentiopicroside (Gent) is a secoiridoid compound isolated from Gentiana lutea that exhibits analgesic properties and inhibits the expression of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the anterior cingulate cortex of mice. However, the effects of Gent on the reserpine-induced pain/depression dyad and its underlying mechanisms are unclear. Reserpine administration (1 mg/kg subcutaneous daily for 3 days) caused a significant decrease in the nociceptive threshold as evidenced by the reduced paw withdrawal latency in response to a radiant heat source and mechanical allodynia. Behavioral detection indicated a significant increase in immobility time during a forced swim test, as well as decreased time in the central area and total travel distance in an open field test. Furthermore, reserpinized animals exhibited increased oxidative stress. Systemic Gent administration dose-dependently ameliorated the behavioral deficits associated with reserpine-induced pain/depression dyad. At the same time, the decrease in biogenic amine levels (norepinephrine, dopamine, and serotonin) was integrated with the increase in caspase-3 levels and GluN2B-containing NMDA receptors in the amygdala of the reserpine-injected mice. Gent significantly reversed the changes in the levels of biogenic amines, caspase-3, and GluN2B-containing NMDA receptors in amygdala. However, Gent did not affect the expression of GluN2A-containing NMDA receptors. The inhibitory effects of Gent on oxidative stress were occluded by simultaneous treatment of GluN2B receptors antagonist Ro25-6981. Our study provides strong evidence that Gent inhibits reserpine-induced pain/depression dyad by downregulating GluN2B receptors in the amygdala.

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.

Toll-Like Receptor 2 Mediates Cellular Activation by the B Subunits of Type II Heat-Labile Enterotoxins

PubMed Central

Hajishengallis, George; Tapping, Richard I.; Martin, Michael H.; Nawar, Hesham; Lyle, Elizabeth A.; Russell, Michael W.; Connell, Terry D.

2005-01-01

The type II heat-labile enterotoxins (LT-IIa and LT-IIb) of Escherichia coli have an AB5 subunit structure similar to that of cholera toxin (CT) and other type I enterotoxins, despite significant differences in the amino acid sequences of their B subunits and different ganglioside receptor specificities. LT-II holotoxins and their nontoxic B subunits display unique properties as immunological adjuvants distinct from those of CT and its B subunits. In contrast to type II holotoxins, the corresponding pentameric B subunits, LT-IIaB and LT-IIbB, stimulated cytokine release in both human and mouse cells dependent upon Toll-like receptor 2 (TLR2). Induction of interleukin-1β (IL-1β), IL-6, IL-8, or tumor necrosis factor alpha in human THP-1 cells by LT-IIaB or LT-IIbB was inhibited by anti-TLR2 but not by anti-TLR4 antibody. Furthermore, transient expression of TLR1 and TLR2 in human embryonic kidney 293 cells resulted in activation of a nuclear factor-κB-dependent luciferase gene in response to LT-IIaB or LT-IIbB. Moreover, peritoneal macrophages from TLR2-deficient mice failed to respond to LT-IIaB or LT-IIbB, in contrast to wild-type or TLR4-deficient cells. These results demonstrate that besides their established binding to gangliosides, the B subunits of type II enterotoxins also interact with TLR2. Although a ganglioside-nonbinding mutant (T34I) of LT-IIaB effectively induced cytokine release, a phenotypically similar point mutation (T13I) in LT-IIbB abrogated cytokine induction, suggesting a variable requirement for gangliosides as coreceptors in TLR2 agonist activity. TLR2-dependent activation of mononuclear cells by type II enterotoxin B subunits appears to be a novel mechanism whereby these molecules may exert their immunomodulatory and adjuvant activities. PMID:15731031

Trypsin-protease activated receptor-2 signaling contributes to pancreatic cancer pain

PubMed Central

Zhu, Jiao; Miao, Xue-Rong; Tao, Kun-Ming; Zhu, Hai; Liu, Zhi-Yun; Yu, Da-Wei; Chen, Qian-Bo; Qiu, Hai-Bo; Lu, Zhi-Jie

2017-01-01

Pain treatment is a critical aspect of pancreatic cancer patient clinical care. This study investigated the role of trypsin-protease activated receptor-2 (PAR-2) in pancreatic cancer pain. Pancreatic tissue samples were collected from pancreatic cancer (n=22) and control patients (n=22). Immunofluorescence analyses confirmed colocalization of PAR-2 and neuronal markers in pancreatic cancer tissues. Trypsin levels and protease activities were higher in pancreatic cancer tissue specimens than in the controls. Supernatants from cultured human pancreatic cancer tissues (PC supernatants) induced substance P and calcitonin gene-related peptide release in dorsal root ganglia (DRG) neurons, and FS-NH2, a selective PAR-2 antagonist, inhibited this effect. A BALB/c nude mouse orthotopic tumor model was used to confirm the role of PAR-2 signaling in pancreatic cancer visceral pain, and male Sprague-Dawley rats were used to assess ambulatory pain. FS-NH2 treatment decreased hunch scores, mechanical hyperalgesia, and visceromotor reflex responses in tumor-bearing mice. In rats, subcutaneous injection of PC supernatant induced pain behavior, which was alleviated by treatment with FS-NH2 or FUT-175, a broad-spectrum serine protease inhibitor. Our findings suggest that trypsin-PAR-2 signaling contributes to pancreatic cancer pain in vivo. Treatment strategies targeting PAR-2 or its downstream signaling molecules might effectively relieve pancreatic cancer pain. PMID:28977906

Mineralocorticoid Receptor Activation Contributes to the Supine Hypertension of Autonomic Failure.

PubMed

Arnold, Amy C; Okamoto, Luis E; Gamboa, Alfredo; Black, Bonnie K; Raj, Satish R; Elijovich, Fernando; Robertson, David; Shibao, Cyndya A; Biaggioni, Italo

2016-02-01

Primary autonomic failure is characterized by disabling orthostatic hypotension, but at least half of these patients have paradoxical supine hypertension. Renin-angiotensin mechanisms were not initially thought to contribute to this hypertension because plasma renin activity is often undetectable in autonomic failure. Plasma aldosterone levels are normal, however, and we recently showed that plasma angiotensin II is elevated and acts at AT1 (angiotensin type 1) receptors to contribute to hypertension in these patients. Because aldosterone and angiotensin II can also bind mineralocorticoid receptors to elevate blood pressure, we hypothesized that mineralocorticoid receptor activation plays a role in the hypertension of autonomic failure. To test this hypothesis, we determined the acute effects of the mineralocorticoid receptor antagonist eplerenone (50 mg, oral) versus placebo on supine blood pressure in a randomized, double-blind, crossover study. Medications were given at 8:00 pm with blood pressure recorded every 2 hours for 12 hours. Ten primary autonomic failure patients with supine hypertension completed this study (7 pure autonomic failure, 2 multiple system atrophy, 1 parkinson's disease; 7 male; 70±2 years of age). Eplerenone maximally reduced supine systolic blood pressure by 32±6 mm Hg at 8 hours after administration (versus 8±10 mm Hg placebo, P=0.016), with no effect on nocturia (12-hour urine volume: 985±134 mL placebo versus 931±94 mL eplerenone, P=0.492; nocturnal weight loss: -1.19±0.15 kg placebo versus -1.18±0.15 kg eplerenone, P=0.766). These findings suggest that inappropriate mineralocorticoid receptor activation contributes to the hypertension of autonomic failure, likely independent of canonical mineralocorticoid effects, and provides rationale for use of eplerenone in these patients. © 2015 American Heart Association, Inc.

2-Methyltetrahydro-3-benzazepin-1-ols - The missing link in SAR of GluN2B selective NMDA receptor antagonists.

PubMed

Dey, Sougata; Schepmann, Dirk; Wünsch, Bernhard

2018-01-15

The NMDA receptor containing GluN2B subunits represents a promising target for the development of drugs for the treatment of various neurological disorders including neurodegenerative diseases. In order to study the role of CH 3 and OH moieties trisubstituted tetrahydro-3-benzazepines 4 were designed as missing link between tetra- and disubstituted 3-benzazepines 2 and 5. The synthesis of 4 comprises eight reaction steps starting from alanine. The intramolecular Friedel-Crafts acylation to obtain the ketone 12 and the base-catalyzed elimination of trifluoromethanesulfinate (CF 3 SO 2 - ) followed by NaBH 4 reduction represent the key steps. The GluN2B affinity of the cis-configured 3-benzazepin-1-ol cis-4a with a 4-phenylbutyl side chain (K i  = 252 nM) is considerably lower than the GluN2B affinity of (R,R)-2 (K i  = 17 nM) indicating the importance of the phenolic OH moiety for the interaction with the receptor protein. Introduction of an additional CH 3 moiety in 2-position led to a slight decrease of GluN2B affinity as can be seen by comparing the affinity data of cis-4a and 5. The homologous phenylpentyl derivative cis-4b shows the highest GluN2B affinity (K i  = 56 nM) of this series of compounds. According to docking studies cis-4a adopts the same binding mode as the cocrystallized ligand ifenprodil-keto 1A and 5 at the interface of the GluN2B and GluN1a subunits. The same crucial H-bonds are formed between the C(O)NH 2 moiety of Gln110 within the GluN2B subunit and the protonated amino moiety and the OH moiety of (R,R)-cis-4a. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endothelin B receptor blockade attenuates pulmonary vasodilation in oxygen-ventilated fetal lambs.

PubMed

Ivy, D Dunbar; Lee, Dong-Seok; Rairigh, Robyn L; Parker, Thomas A; Abman, Steven H

2004-01-01

Endothelin-1 (ET-1) contributes to the regulation of pulmonary vascular tone in the normal ovine fetus and in models of perinatal pulmonary hypertension. In the fetal lamb lung, the effects of ET-1 depend on the balance of at least two endothelin receptor subtypes: ETA and ETB. ETA receptors are located on smooth muscle cells and mediate vasoconstriction and smooth muscle proliferation. Stimulation of endothelial ETB receptors causes vasodilation through release of nitric oxide and also functions to remove ET-1 from the circulation. However, whether activation of ETB receptors contributes to the fall in pulmonary vascular tone at birth is unknown. To determine the role of acute ETB receptor blockade in pulmonary vasodilation in response to birth-related stimuli, we studied the hemodynamic effects of selective ETB receptor blockade with BQ-788 during mechanical ventilation with low (<10%) and high FiO2 (100%) in near-term fetal sheep. Intrapulmonary infusion of BQ-788 did not change left pulmonary artery (LPA) blood flow and pulmonary vascular resistance (PVR) at baseline. In comparison with controls, BQ-788 treatment attenuated the rise in LPA flow with low and high FiO2 ventilation (p <0.001 vs. control for each FiO2 concentration). PVR progressively decreased during mechanical ventilation with low and high FiO2 in both groups, but PVR remained higher after BQ-788 treatment throughout the study period (p <0.001). We conclude that selective ETB receptor blockade attenuates pulmonary vasodilation at birth. We speculate that ETB receptor stimulation contributes to pulmonary vasodilation at birth in the ovine fetus.

Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

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

Karakas, E.; Simorowski, N; Furukawa, H

2009-01-01

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-freemore » and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.« less

Mice lacking the serotonin 5-HT2B receptor as an animal model of resistance to selective serotonin reuptake inhibitors antidepressants.

PubMed

Diaz, Silvina Laura; Narboux-Nême, Nicolas; Boutourlinsky, Katia; Doly, Stéphane; Maroteaux, Luc

2016-02-01

Depressive disorders are among the most prevalent neuropsychiatric dysfunctions worldwide, with high rates of resistance to antidepressant treatment. Genetic factors clearly contribute to the manifestation of depression as well as to the response to antidepressants. Transgenic mouse models appear as seminal tools to disentangle this complex disorder. Here, we analyzed new key aspects of the phenotype of knock-out mice for the gene encoding the serotonin 2B receptor (Htr(2B)(-/-)), including basal phenotype, ability to develop a depressive-like phenotype upon chronic isolation, and effect of chronic exposure to fluoxetine on chronically stressed Htr(2B)(-/-) mice. We find, here, that Htr(2B)(-/-) mice display an antidepressant-like phenotype, which includes reduced latency to feed in the novelty suppressed feeding test, basal increase in hippocampal BDNF levels, no change in TrkB and p75 protein levels, and an increased preference for sucrose consumption compared to wild type (Htr(2B)(+/+)) mice. Nevertheless, we show that these mice can develop depressive-like behaviors when socially isolated during four weeks. Selective serotonin reuptake inhibitors (SSRI) have been previously shown to be ineffective in non-stressed Htr(2B)(-/-) mice. We evaluated, here, the effects of the SSRI fluoxetine in chronically stressed Htr(2B)(-/-) mice and similarly no behavioral or plastic effect was induced by this antidepressant. All together, these results highlight the suitability to study resistance to SSRI antidepressants of this mouse model displaying panoply of conditions among which behavioral, neurotrophic and plastic causative factors can be analyzed. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.