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Sample records for receptor inhibits progastrin-dependent

  1. Inhibition of the acetylcholine receptor by histrionicotoxin.

    PubMed Central

    Anwyl, R.; Narahashi, T.

    1980-01-01

    1 The action of C5-decahydrohistrionicotoxin (C5-HTX) has been investigated on the extrajunctional acetylcholine (ACh) receptors of denervated rat muscle. 2 C5-HTX causes both a rapid and slow reduction in amplitude of iontophoretic ACh potentials evoked at all frequencies from the extrajunctional receptors. 3 C5-HTX also causes a time-dependent inhibition of the iontophoretic potentials evoked at frequencies greater than 0.02 Hz. This inhibition was observed either alone or superimposed upon desensitization, and may be caused by a similar mechanism to desensitization. PMID:7378635

  2. Metabotropic glutamate receptors inhibit microglial glutamate release

    PubMed Central

    McMullan, Stephen M; Phanavanh, Bounleut; Guo Li, Gary; Barger, Steven W

    2012-01-01

    Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action. PMID:22770428

  3. Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition.

    PubMed

    Hubers, Scott A; Brown, Nancy J

    2016-03-15

    Heart failure affects ≈5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, β-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the US Food and Drug Administration approved the first of a new class of drugs for the treatment of heart failure: Valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses 2 of the pathophysiological mechanisms of heart failure: activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared with enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacological properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension. PMID:26976916

  4. Aluminum fluoride inhibition of glucocorticoid receptor inactivation and transformation

    SciTech Connect

    Housley, P.R. )

    1990-04-10

    Fluoride, in the presence of aluminum ions, reversibly inhibits the temperature-mediated inactivation of unoccupied glucocorticoid receptors in cytosol preparations from mouse L cells. The effect is concentration-dependent, with virtually complete stabilization of specific glucocorticoid-binding capacity at 2 mM fluoride and 100 microM aluminum. These concentrations of aluminum and fluoride are ineffective when used separately. Aluminum fluoride also stabilizes receptors toward inactivation by gel filtration and ammonium sulfate precipitation. Aluminum fluoride prevents temperature-dependent transformation of steroid-receptor complexes to the DNA-binding state. Aluminum fluoride does not inhibit calf intestine alkaline phosphatase, and unoccupied receptors inactivated by this enzyme in the presence of aluminum fluoride can be completely reactivated by dithiothreitol. The effects of aluminum fluoride are due to stabilization of the complex between the glucocorticoid receptor and the 90-kDa mammalian heat-shock protein hsp90, which suggests that aluminum fluoride interacts directly with the receptor. Endogenous thermal inactivation of receptors in cytosol is not accompanied by receptor dephosphorylation. However, inactivation is correlated with dissociation of hsp90 from the unoccupied receptor. These results support the proposal that hsp90 is required for the receptor to bind steroid and dissociation of hsp90 is sufficient to inactivate the unoccupied receptor.

  5. Inhibition of receptor/G protein coupling by suramin analogues.

    PubMed

    Beindl, W; Mitterauer, T; Hohenegger, M; Ijzerman, A P; Nanoff, C; Freissmuth, M

    1996-08-01

    Suramin analogues act as direct antagonists of heterotrimeric G proteins because they block the rate-limiting step of G protein activation (i.e., the dissociation of GDP prebound to the G protein alpha subunit). We have used the human brain A1 adenosine receptor and the rat striatal D2 dopamine receptor, two prototypical Gi/G(o)-coupled receptors, as a model system to test whether the following analogues suppress the receptor-dependent activation of G proteins: 8-(3-nitrobenzamido)-1,3,5-naphthalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)-benzamido)-1,3,5-naphthalenetrisulfonic acid (NF018); 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalenetr isulfonic acid) (NF023); 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1- phenylene)) bis(1,3,5-naphthalenetrisulfonic acid) (NF037); and suramin. Suramin and its analogues inhibit the formation of the agonist-specific ternary complex (agonist/receptor/G protein). This inhibition is (i) quasicompetitive with respect to agonist binding in that it can be overcome by increasing receptor occupancy but (ii) does not result from an interaction of the analogues with the ligand binding pocket of the receptors because the binding of antagonists or of agonists in the absence of functional receptor/G protein interaction is not affected. In addition to suppressing the spontaneous release of GDP from defined G protein alpha subunits, suramin and its analogues reduce receptor-catalyzed guanine nucleotide exchange. The site, to which suramin analogues bind, overlaps with the docking site for the receptor on the G protein alpha subunit. The structure-activity relationships for inhibition of agonist binding to the A1 adenosine receptor (suramin > NF037 > NF023) and of agonist binding to the inhibition D2 dopamine receptor (suramin = NF037 > NF023 > NF018) differ. Thus, NF037 discriminates between the ternary complexes formed by the agonist-liganded D2 dopamine receptors and those formed by the A1 adenosine

  6. Perampanel Inhibition of AMPA Receptor Currents in Cultured Hippocampal Neurons

    PubMed Central

    Chen, Chao-Yin; Matt, Lucas; Hell, Johannes Wilhelm; Rogawski, Michael A.

    2014-01-01

    Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM), concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M−1 s−1 and 0.58 s−1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM) was similar at all kainate concentrations (3–100 µM), demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics. PMID:25229608

  7. Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

    PubMed Central

    Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-01-01

    Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

  8. Luteolin inhibits GABAA receptors in HEK cells and brain slices

    PubMed Central

    Shen, Mei-Lin; Wang, Chen-Hung; Chen, Rita Yu-Tzu; Zhou, Ning; Kao, Shung-Te; Wu, Dong Chuan

    2016-01-01

    Modulation of the A type γ-aminobutyric acid receptors (GABAAR) is one of the major drug targets for neurological and psychological diseases. The natural flavonoid compound luteolin (2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone) has been reported to have antidepressant, antinociceptive, and anxiolytic-like effects, which possibly involve the mechanisms of modulating GABA signaling. However, as yet detailed studies of the pharmacological effects of luteolin are still lacking, we investigated the effects of luteolin on recombinant and endogenous GABAAR-mediated current responses by electrophysiological approaches. Our results showed that luteolin inhibited GABA-mediated currents and slowed the activation kinetics of recombinant α1β2, α1β2γ2, α5β2, and α5β2γ2 receptors with different degrees of potency and efficacy. The modulatory effect of luteolin was likely dependent on the subunit composition of the receptor complex: the αβ receptors were more sensitive than the αβγ receptors. In hippocampal pyramidal neurons, luteolin significantly reduced the amplitude and slowed the rise time of miniature inhibitory postsynaptic currents (mIPSCs). However, GABAAR-mediated tonic currents were not significantly influenced by luteolin. These data suggested that luteolin has negative modulatory effects on both recombinant and endogenous GABAARs and inhibits phasic rather than tonic inhibition in hippocampus. PMID:27292079

  9. Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation.

    PubMed

    Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V; Liang, Jie; Arkowitz, Robert A; Stone, David E

    2016-01-01

    Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion. PMID:27072657

  10. Effects of pyridoxal 5'-phosphate on uterine estrogen receptor. II. Inhibition of estrogen . receptor transformation.

    PubMed

    Traish, A; Müller, R E; Wotiz, H H

    1980-05-10

    Previous observations suggested that pyridoxal 5'-phosphate was capable of inhibiting estrogen . receptor (R . E2) activation, or translocation to the nucleus, or both. The present study attempts to define more specifically the locus of this action. To this end we have examined the physicochemical alteration produced by interaction of pyridoxal 5'-phosphate with estrogen . receptor complex, using sucrose density gradient analysis and dissociation kinetics. Receptor transformation was inhibited when activation was performed in the presence of pyridoxal 5'-phosphate. This effect was protein- and pyridoxal 5'-phosphate concentration-dependent. When pyridoxal 5'-phosphate was introduced postactivation it did not have any effect on the activated receptor, but when similar treatment was followed by NABH4 reduction, the complex reverted to the monomeric entity. The dissociation behavior obtained with cytosol R . E2, warmed in the presence of pyridoxal 5'-phosphate, showed a biphasic curve suggesting that a significant portion of receptors remained nonactivated as demonstrated by the fast dissociating component. Due to the fact that Tris buffers cannot be used for pyridoxal 5'-phosphate experiments, we have used a borate buffer which resulted in a displacement of the sedimentation values from a 4S to 4.6 S for the unactivated receptor and 5S to 6 S for the activated form. The observations reported suggest that at least the initial effect of pyridoxal 5'-phosphate results in the inhibition of cytosolic receptor transformation from the nonactivated to the activated form. PMID:7372667

  11. Nuclear receptor SHP inhibition of Dnmt1 expression via ERRγ.

    PubMed

    Zhang, Yuxia; Wang, Li

    2011-05-01

    We describe a transcriptional mechanism regulating the expression of Dnmt1 by nuclear receptors. We show that ERRγ functions as a transcriptional activator of mouse and human Dnmt1 expression by direct binding to its response elements (ERE1/ERE2) in the dnmt1/DNMT1 promoters. The induction of Dnmt1 by ERRγ is repressed by SHP through SHP inhibition of ERRγ transactivity, diminishing ERRγ recruitment to the Dnmt1 promoter, and altering the conformation of local chromatin from an active mode by ERRγ to an inactive mode. Our study provides the first evidence for nuclear receptor mediated regulation of Dnmt1 expression through ERRγ and SHP crosstalk. PMID:21459093

  12. Nuclear receptor SHP inhibition of Dnmt1 expression via ERRγ

    PubMed Central

    Zhang, Yuxia; Wang, Li

    2011-01-01

    We describe a transcriptional mechanism regulating the expression of Dnmt1 by nuclear receptors. We show that ERRγ functions as a transcriptional activator of mouse and human Dnmt1 expression by direct binding to its response elements (ERE1/ERE2) in the dnmt1/DNMT1 promoters. The induction of Dnmt1 by ERRγ is repressed by SHP through SHP inhibition of ERRγ transactivity, diminishing ERRγ recruitment to the Dnmt1 promoter, and altering the conformation of local chromatin from an active mode by ERRγ to an inactive mode. Our study provides the first evidence for nuclear receptor mediated regulation of Dnmt1 expression through ERRγ and SHP crosstalk. PMID:21459093

  13. Inhibition of Anopheles gambiae odorant receptor function by mosquito repellents.

    PubMed

    Tsitoura, Panagiota; Koussis, Konstantinos; Iatrou, Kostas

    2015-03-20

    The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca(2+)-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET. PMID:25657000

  14. Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents*

    PubMed Central

    Tsitoura, Panagiota; Koussis, Konstantinos; Iatrou, Kostas

    2015-01-01

    The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca2+-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET. PMID:25657000

  15. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

    PubMed

    Chen, Ying-Nan P; LaMarche, Matthew J; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G; Dobson, Jason R; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J; Sellers, William R; Stams, Travis; Fortin, Pascal D

    2016-07-01

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers. PMID:27362227

  16. IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

    SciTech Connect

    Ohshima-Hosoyama, Sachiko; Hosoyama, Tohru; Nelon, Laura D.; Keller, Charles

    2010-09-03

    Research highlights: {yields} Igf1r is overexpressed and activated in a Sonic Hedgehog driven model of medulloblastoma. {yields} Picropodophyllin targets and abrogates IGF signaling in medulloblastoma. {yields} Picropodophyllin inhibits medulloblastoma tumor cell growth by induction of apoptosis. -- Abstract: The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.

  17. Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission

    PubMed Central

    Ferreira, S G; Gonçalves, F Q; Marques, J M; Tomé, Â R; Rodrigues, R J; Nunes-Correia, I; Ledent, C; Harkany, T; Venance, L; Cunha, R A; Köfalvi, A

    2015-01-01

    Background and Purpose Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1−A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. Experimental Approach Pharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices. Key Results Flow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K+-evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. Conclusions and Implications Activation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic

  18. Proton inhibition of unitary currents of vanilloid receptors

    PubMed Central

    Liu, Beiying; Yao, Jing; Wang, Yingwei; Li, Hui

    2009-01-01

    Protons, which are released during inflammation and injury, regulate many receptors and ion channels involved in pain transduction, including capsaicin channels (transient receptor potential vanilloid receptors 1). Whereas extracellular acidification both sensitizes and directly activates the channel, it also causes concomitant reduction of the unitary current amplitudes. Here, we investigate the mechanisms and molecular basis of this inhibitory effect of protons on channel conductance. Single-channel recordings showed that the unitary current amplitudes decreased with extracellular pH in a dose-dependent manner, consistent with a model in which protons bind to a site within the channel with an apparent pKa of ∼6. The inhibition was voltage dependent, ∼65% at −60 mV and 37% at +60 mV when pH was reduced from 7.4 to 5.5. The unitary current amplitudes reached saturation at [K+] ≥ 1 M, and notably the maximum amplitudes did not converge with different pHs, inconsistent with a blockade model based on surface charge screening or competitive inhibition of permeating ions. Mutagenesis experiments uncovered two acidic residues critical for proton inhibition, one located at the pore entrance and the other on the pore helix. Based on homology to the KcsA structure, the two acidic residues, along with another basic residue also on the pore helix, could form a triad interacting with each other through extensive hydrogen bonds and electrostatic contacts, suggesting that protons may mediate the interactions between the selectivity filter and pore helix, thereby altering the local structure in the filter region and consequently the conductance of the channel. PMID:19720962

  19. The apelin receptor inhibits the angiotensin II type 1 receptor via allosteric trans-inhibition

    PubMed Central

    Siddiquee, K; Hampton, J; McAnally, D; May, LT; Smith, LH

    2013-01-01

    Background and Purpose The apelin receptor (APJ) is often co-expressed with the angiotensin II type-1 receptor (AT1) and acts as an endogenous counter-regulator. Apelin antagonizes Ang II signalling, but the precise molecular mechanism has not been elucidated. Understanding this interaction may lead to new therapies for the treatment of cardiovascular disease. Experimental Approach The physical interaction of APJ and AT1 receptors was detected by co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Functional and pharmacological interactions were measured by G-protein-dependent signalling and recruitment of β-arrestin. Allosterism and cooperativity between APJ and AT1 were measured by radioligand binding assays. Key Results Apelin, but not Ang II, induced APJ : AT1 heterodimerization forced AT1 into a low-affinity state, reducing Ang II binding. Likewise, apelin mediated a concentration-dependent depression in the maximal production of inositol phosphate (IP1) and β-arrestin recruitment to AT1 in response to Ang II. The signal depression approached a limit, the magnitude of which was governed by the cooperativity indicative of a negative allosteric interaction. Fitting the data to an operational model of allosterism revealed that apelin-mediated heterodimerization significantly reduces Ang II signalling efficacy. These effects were not observed in the absence of apelin. Conclusions and Implications Apelin-dependent heterodimerization between APJ and AT1 causes negative allosteric regulation of AT1 function. As AT1 is significant in the pathogenesis of cardiovascular disease, these findings suggest that impaired apelin and APJ function may be a common underlying aetiology. Linked Article This article is commented on by Goupil et al., pp. 1101–1103 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12040 PMID:22935142

  20. Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels

    PubMed Central

    Dilger, James P.; Boguslavsky, Rebecca; Barann, Martin; Katz, Tamir; Vidal, Ana Maria

    1997-01-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds ≥10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 μM pentobarbital. PMID:9089445

  1. Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

    PubMed

    Dilger, J P; Boguslavsky, R; Barann, M; Katz, T; Vidal, A M

    1997-03-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital. PMID:9089445

  2. Alpha-2 adrenergic receptor-mediated inhibition of thermogenesis

    PubMed Central

    Madden, Christopher J.; Tupone, Domenico; Cano, Georgina; Morrison, Shaun F.

    2013-01-01

    Alpha2-adrenergic receptor (α2-AR) agonists have been use as anti-hypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since α2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat brown adipose tissue (BAT) and shivering thermogenesis by α2-AR agonists. Nanoinjection of the α2-AR agonist, clonidine (1.2 nmol), into the rostral raphe pallidus (rRPa) inhibited BAT sympathetic nerve activity (SNA) and BAT thermogenesis. Subsequent nanoinjection of the α2-AR antagonist, idazoxan (6nmol) into the rRPa reversed the clonidine-evoked inhibition of BAT SNA and BAT thermogenesis. Systemic administration of the α2-AR agonists, dexmedetomidine (25ug/kg, iv) or clonidine (100ug/kg, iv) inhibited shivering EMGs, BAT SNA and BAT thermogenesis effects that were reversed by nanoinjection of idazoxan (6nmol) into the rRPa. Dexmedetomidine (100µg/kg, ip) prevented and reversed lipopolysaccharide (10µg/kg ip)-evoked thermogenesis in free-behaving rats. Cholera toxin subunit b retrograde tracing from rRPa and pseudorabies virus transynaptic retrograde tracing from BAT combined with immunohistochemistry for catecholaminergic biosynthetic enzymes revealed the ventrolateral medulla as the source of catecholaminergic input to the rRPa and demonstrated that these catecholaminergic neurons are synaptically connected to BAT. Photostimulation of VLM neurons expressing of the PRSx8-ChR2-mCherry lentiviral vector inhibited BAT SNA via activation of α2-ARs in the rRPa. These results indicate a potent inhibition of BAT and shivering thermogenesis by α2-AR activation in the rRPa, and suggest a therapeutic potential of α2-AR agonists for reducing potentially-lethal elevations in body temperature during excessive fever. PMID:23365239

  3. Seizure control by decanoic acid through direct AMPA receptor inhibition.

    PubMed

    Chang, Pishan; Augustin, Katrin; Boddum, Kim; Williams, Sophie; Sun, Min; Terschak, John A; Hardege, Jörg D; Chen, Philip E; Walker, Matthew C; Williams, Robin S B

    2016-02-01

    The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones β-hydroxybutryate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet. PMID:26608744

  4. Seizure control by decanoic acid through direct AMPA receptor inhibition

    PubMed Central

    Chang, Pishan; Augustin, Katrin; Boddum, Kim; Williams, Sophie; Sun, Min; Terschak, John A.; Hardege, Jörg D.; Chen, Philip E.

    2016-01-01

    See Rogawski (doi:10.1093/awv369) for a scientific commentary on this article.  The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones β-hydroxybutryate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet. PMID:26608744

  5. Inhibition of Rotavirus Infectivity by a Neoglycolipid Receptor Mimetic

    PubMed Central

    Bergner, Daniel W.; Kuhlenschmidt, Theresa B.; Hanafin, William P.; Firkins, Lawrence D.; Kuhlenschmidt, Mark S.

    2011-01-01

    Group A rotaviruses are a major cause of diarrhea in the young of many mammalian species. In rotavirus infected piglets mortality can be as high as 60%. Previous research in this laboratory has identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we previously demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding and infectivity of host cells in vitro. Sialyllactose, the carbohydrate moiety of GM3, is approximately 3 orders of magnitude less effective than GM3 at inhibiting rotavirus binding to cells. Furthermore, production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic in swine is cost prohibitive. In an effort to circumvent these problems, a sialyllactose-containing neoglycolipid was synthesized and evaluated for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoylphosphatidylethanolamine (PE) by reductive amination and the product (SLPE) purified by HPLC. Characterization of the product showed a single primulin (lipid) and resorcinol (sialic acid) positive band by thin layer chromatography and quantification of phosphate and sialic acid yielded a 1:1 molar ratio. Mass spectroscopy confirmed a molecular weight coinciding with SLPE. Concentration-dependent binding of rotavirus to SLPE was demonstrated using a thin-layer overlay assay. Using concentrations comparable to GM3, SLPE was also shown to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE was shown to decrease rotavirus infection of host cells by over 90%. Finally, preliminary results of in vivo animal challenge studies using newborn piglets in their natural environment, demonstrated SLPE afforded complete protection from rotavirus disease. The efficacy of SLPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large

  6. Estrogen receptor beta signals to inhibition of cardiac fibrosis.

    PubMed

    Pedram, Ali; Razandi, Mahnaz; Narayanan, Ramesh; Levin, Ellis R

    2016-10-15

    Cardiac fibrosis evolves from the cardiac hypertrophic state. In this respect, estrogen and estrogen receptor beta (ERβ) inhibit the effects of cardiac hypertrophic peptides that also stimulate fibrosis. Here we determine details of the anti-fibrotic functions of ERβ. In acutely isolated rat cardiac fibroblasts. E2 or a specific ERβ agonist (βLGND2) blocked angiotensin II (AngII) signaling to fibrosis. This resulted from ERβ activating protein kinase A and AMP kinase, inhibiting both AngII de-phosphorylation of RhoA and the resulting stimulation of Rho kinase. Inhibition of Rho kinase from ERβ signaling resulted in marked decrease of TGFβ expression, connective tissue growth factor production and function, matrix metalloproteinases 2 and 9 expression and activity, and the conversion of fibroblasts to myofibroblasts. Production of collagens I and III were also significantly decreased. Several important aspects were corroborated in-vivo from βLGND2-treated mice that underwent AngII-induced cardiac hypertrophy. Thus, ERβ in cardiac fibroblasts prevents key aspects of cardiac fibrosis development. PMID:27321970

  7. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    SciTech Connect

    Wang, Feng; Yang, Yong

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  8. Inhibition of C5a receptor alleviates experimental CNS lupus

    PubMed Central

    Jacob, Alexander; Hack, Bradley; Bai, Tao; Brorson, James R.; Quigg, Richard J.; Alexander, Jessy J.

    2010-01-01

    To investigate the role of C5a generated on complement activation in brain, the lupus model, MRL/lpr mice were treated with C5a receptor(R) antagonist (ant). Neutrophil infiltration, ICAM, TNF-α and iNOS mRNA expression, neuronal apoptosis and the expression of p-JNK, pSTAT1 and p-Erk were reduced and p-Akt increased on C5aR inhibition in MRL/lpr brains. MRL/lpr serum caused increased apoptosis in neurons showing that lupus had a direct effect on these cells. C5aRant pretreatment prevented the lupus serum induced loss of neuronal cells. Our findings demonstrate for the first time that C5a/C5aR signaling plays an important role in the pathogenesis of CNS lupus. PMID:20207017

  9. MDM2 binds and inhibits vitamin D receptor

    PubMed Central

    Heyne, Kristina; Heil, Tessa-Carina; Bette, Birgit; Reichrath, Jörg; Roemer, Klaus

    2015-01-01

    The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Herein, we report that vitamin D receptor (VDR), another transcriptional regulator and probably, tumor suppressor, is also bound and inhibited by MDM2. This interaction was not affected by vitamin D ligand. VDR was ubiquitylated in the cell and its steady-state level was controlled by the proteasome. Strikingly, overproduced MDM2 reduced the level of VDR whereas knockdown of endogenous MDM2 increased the level of VDR. In addition to ubiquitin-marking proteins for degradation, MDM2, once recruited to promoters by DNA-binding interaction partners, can inhibit the transactivation of genes. Transient transfections with a VDR-responsive luciferase reporter revealed that low levels of MDM2 potently suppress VDR-mediated transactivation. Conversely, knockdown of MDM2 resulted in a significant increase of transcript from the CYP24A1 and p21 genes, noted cellular targets of transactivation by liganded VDR. Our findings suggest that MDM2 negatively regulates VDR in some analogy to p53. PMID:25969952

  10. Cutoff in potency implicates alcohol inhibition of N-methyl-D-aspartate receptors in alcohol intoxication.

    PubMed Central

    Peoples, R W; Weight, F F

    1995-01-01

    As the number of carbon atoms in an aliphatic n-alcohol is increased from one to five, intoxicating potency, lipid solubility, and membrane lipid disordering potency all increase in a similar exponential manner. However, the potency of aliphatic n-alcohols for producing intoxication reaches a maximum at six to eight carbon atoms and then decreases. The molecular basis of this "cutoff" effect is not understood, as it is not correlated with either the lipid solubility or the membrane disordering potency of the alcohols, which continue to increase exponentially. Since it has been suggested that inhibition of N-methyl-D-aspartate (NMDA) receptors by alcohols may play a role in alcohol intoxication, we investigated whether a series of aliphatic n-alcohols would exhibit a cutoff in potency for inhibition of NMDA receptors. We found that although potency for inhibition of NMDA receptors increased exponentially for alcohols with one to five carbon atoms, potency for inhibition of NMDA receptors reached a maximum at six to eight carbon atoms and then abruptly disappeared. This cutoff for alcohol inhibition of NMDA receptors is consistent with an interaction of the alcohols with a hydrophobic pocket on the receptor protein. In addition, the similarity of the cutoffs for alcohol inhibition of NMDA receptors and alcohol intoxication suggests that the cutoff for NMDA receptor inhibition may contribute to the cutoff for alcohol intoxication, which is consistent with an important role of NMDA receptors in alcohol intoxication. PMID:7708732

  11. Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus.

    PubMed

    Nigro, V; Medici, N; Abbondanza, C; Minucci, S; Molinari, A M; Puca, G A

    1989-11-15

    Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited. PMID:2480113

  12. Dopamine D1 receptor inhibition of NMDA receptor currents mediated by tyrosine kinase-dependent receptor trafficking in neonatal rat striatum

    PubMed Central

    Tong, Huaxia; Gibb, Alasdair J

    2008-01-01

    NMDA receptors are of particular importance in the control of synaptic strength and integration of synaptic activity. Dopamine receptor modulation of NMDA receptors in neonatal striatum may influence the efficacy of synaptic transmission in the cortico-striatal pathway and if so, this modulation will affect the behaviour of the basal ganglia network. Here, we show that in acute brain slices of neonatal (P7) rat striatum the dopamine D1 receptor agonist SKF-82958 significantly decreases NMDA receptor currents in patch-clamp whole-cell recordings. This inhibition is not abolished by application of a G protein inhibitor (GDP-β-S) or irreversible G protein activator (GTP-γ-S) suggesting a G protein-independent mechanism. In addition, intracellular application of protein tyrosine kinase inhibitors (lavendustin A or PP2) abolished D1 inhibition of NMDA currents. In contrast, in older animals (P28) D1 receptor activation produces a potentiation of the NMDA response which suggests there is a developmental switch in D1 modulation of striatal NMDA receptors. Single-channel recordings show that direct D1 receptor inhibition of NMDA receptors cannot be observed in isolated membrane patches. We hypothesize that D1 inhibition in whole-cell recordings from neonatal rats may be mediated by a change in NMDA receptor trafficking. Consistent with this hypothesis, intracellular application of a dynamin inhibitory peptide (QVPSRPNRAP) abolished D1 inhibition of NMDA receptor currents. We therefore conclude that a tyrosine kinase-dependent alteration of NMDA receptor trafficking underlies D1 dopamine receptor-mediated down-regulation of NMDA receptor currents in medium spiny neurons of neonatal rat striatum. PMID:18703578

  13. Lysophosphatidic Acid (LPA) Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response1

    PubMed Central

    Shotts, Kristin; Donovan, Erin E.; Strauch, Pamela; Pujanauski, Lindsey M.; Victorino, Francisco; Al-Shami, Amin; Fujiwara, Yuko; Tigyi, Gabor; Oravecz, Tamas; Pelanda, Roberta; Torres, Raul M.

    2014-01-01

    Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. PMID:24890721

  14. Epidermal growth factor receptor inhibition in metastatic anal cancer.

    PubMed

    Rogers, Jane E; Ohinata, Aki; Silva, Ninoska N; Mehdizadeh, Amir; Eng, Cathy

    2016-09-01

    Metastatic squamous cell carcinoma (SCCA) anal cancer is relatively rare. With limited data, cisplatin plus 5-fluorouracil has traditionally been utilized in the first-line setting. Treatment beyond front-line cisplatin progression is not well defined. Epidermal growth factor receptor (EGFR) is highly overexpressed in SCCA anal cancer and EGFR inhibition may represent a potential treatment target for this population in need. Our case series evaluated metastatic SCCA anal cancer patients who received an EGFR monoclonal antibody as second-line or third-line therapy. Data collected consisted of demographics, previous treatment, metastatic disease sites, localized therapy received, regimen received, first radiographic result, progression-free survival, and overall survival. A total of 17 patients were included, with most (76%) patients receiving an EGFR monoclonal antibody in the second-line setting. Common regimens identified combined cetuximab or panitumumab with a fluoropyrimidine plus platinum (35%), carboplatin plus paclitaxel (29%), or cisplatin plus vinorelbine (18%). Thirty-five percent of patients achieved a response and 24% had stable disease. The overall median progression-free survival and overall survival were 7.3 and 24.7 months, respectively. Compared with our large retrospective study in the front-line metastatic anal cancer setting, our study suggests that anti-EGFR therapy in combination with certain chemotherapy derived additional benefit in the refractory setting. In the metastatic setting, there is a need to discover effective therapies. We present a diverse metastatic SCCA anal cancer patient population who received cetuximab or panitumumab with chemotherapy in the second-line or third-line setting. Our case series strengthens the concept of EGFR inhibition in metastatic SCCA anal cancer. PMID:27272412

  15. Stimulation by toll-like receptors inhibits osteoclast differentiation.

    PubMed

    Takami, Masamichi; Kim, Nacksung; Rho, Jaerang; Choi, Yongwon

    2002-08-01

    Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-kappa B activation and up-regulated TNF-alpha production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone. PMID:12133979

  16. Honokiol Inhibits Androgen Receptor Activity in Prostate Cancer Cells

    PubMed Central

    Hahm, Eun-Ryeong; Karlsson, A. Isabella; Bonner, Michael Y.; Arbiser, Jack L.; Singh, Shivendra V.

    2014-01-01

    BACKGROUND We have shown previously that honokiol (HNK), a bioactive component of the medicinal plant Magnolia officinalis, inhibits growth of human prostate cancer cells in vitro and in vivo. However, the effect of HNK on androgen receptor (AR) signaling is not known. METHODS LNCaP, C4-2, and TRAMP-C1 cells were used for various assays. Trypan blue dye exclusion assay or clonogenic assay was performed for determination of cell viability. The effects of HNK and/or its analogs on protein levels of AR and its target gene product prostate specific antigen (PSA) were determined by western blotting. RNA interference of p53 was achieved by transient transfection. Reverse transcription-polymerase chain reaction was performed for mRNA expression of AR. Nuclear translocation of AR was visualized by microscopy. Apoptosis was quantified by DNA fragmentation assay or flow cytometry after Annexin V-propidium iodide staining. RESULTS HNK and its dichloroacetate analog (HDCA) were relatively more effective in suppressing cell viability and AR protein level than honokiol epoxide or biseugenol. Nuclear translocation of AR stimulated by a synthetic androgen (R1881) was markedly suppressed in the presence of HNK. Downregulation of AR protein resulting from HNK exposure was attributable to transcriptional repression as well as proteasomal degradation. HNK-mediated suppression of AR protein was maintained in LNCaP cells after knockdown of p53 protein. HNK-induced apoptosis was not affected by R1881 treatment. CONCLUSIONS The present study demonstrates, for the first time, that HNK inhibits activity of AR in prostate cancer cells regardless of the p53 status. PMID:24338950

  17. Angiotensin-(1-7) inhibits epidermal growth factor receptor transactivation via a Mas receptor-dependent pathway

    PubMed Central

    Akhtar, Saghir; Yousif, Mariam HM; Dhaunsi, Gursev S; Chandrasekhar, Bindu; Al-Farsi, Omama; Benter, Ibrahim F

    2012-01-01

    BACKGROUND AND PURPOSE The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] via its Mas receptor can prevent the development of hyperglycaemia-induced cardiovascular complications. Here, we investigated whether Ang-(1-7) can inhibit hyperglycaemia-induced EGF receptor transactivation and its classical signalling via ERK1/2 and p38 MAPK in vivo and in vitro. EXPERIMENTAL APPROACH Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG1478, a selective EGF receptor inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in primary cultures of vascular smooth muscle cells (VSMCs). KEY RESULTS Diabetes significantly enhanced phosphorylation of EGF receptor at tyrosine residues Y992, Y1068, Y1086, Y1148, as well as ERK1/2 and p38 MAPK in the mesenteric vasculature bed whereas these changes were significantly attenuated upon Ang-(1–7) or AG1478 treatment. In VSMCs grown in conditions of high glucose (25 mM), an Src-dependent elevation in EGF receptor phosphorylation was observed. Ang-(1-7) inhibited both Ang II- and glucose-induced transactivation of EGF receptor. The inhibition of high glucose-mediated Src-dependant transactivation of EGF receptor by Ang-(1-7) could be prevented by a selective Mas receptor antagonist, D-Pro7-Ang-(1-7). CONCLUSIONS AND IMPLICATIONS These results show for the first time that Ang-(1-7) inhibits EGF receptor transactivation via a Mas receptor/Src-dependent pathway and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular dysfunction. PMID:21806601

  18. Presynaptic inhibition of corticothalamic feedback by metabotropic glutamate receptors.

    PubMed

    Alexander, Georgia M; Godwin, Dwayne W

    2005-07-01

    The thalamus relays sensory information to cortex, but this information may be influenced by excitatory feedback from cortical layer VI. The full importance of this feedback has only recently been explored, but among its possible functions are influences on the processing of sensory features, synchronization of thalamic firing, and transitions in response mode of thalamic relay cells. Uncontrolled, corticothalamic feedback has also been implicated in pathological thalamic rhythms associated with certain neurological disorders. We have found a form of presynaptic inhibition of corticothalamic synaptic transmission that is mediated by a Group II metabotropic glutamate receptor (mGluR) and activated by high-frequency corticothalamic activity. We tested putative retinogeniculate and corticogeniculate synapses for Group II mGluR modulation within the dorsal lateral geniculate nucleus of the ferret thalamus. Stimulation of optic-tract fibers elicited paired-pulse depression of excitatory postsynaptic currents (EPSCs), whereas stimulation of the optic radiations elicited paired-pulse facilitation. Paired-pulse responses were subsequently used to characterize the pathway of origin of stimulated synapses. Group II mGluR agonists (LY379268 and DCG-IV) applied to thalamic neurons under voltage-clamp conditions reduced the amplitude of corticogeniculate EPSCs. Stimulation with high-frequency trains produced a facilitating response that was reduced by Group II mGluR agonists, but was enhanced by the selective antagonist LY341495, revealing a presynaptic, mGluR-mediated reduction of high-frequency corticogeniculate feedback. Agonist treatment did not affect EPSCs from stimulation of the optic tract. NAAG (reported to be selective for mGluR3) was ineffective at the corticogeniculate synapse, implicating mGluR2 in the observed effects. Our data are the first to show a synaptically elicited form of presynaptic inhibition of corticothalamic synaptic transmission that is mediated by

  19. Intestinal epithelial vitamin D receptor signaling inhibits experimental colitis

    PubMed Central

    Liu, Weicheng; Chen, Yunzi; Golan, Maya Aharoni; Annunziata, Maria L.; Du, Jie; Dougherty, Urszula; Kong, Juan; Musch, Mark; Huang, Yong; Pekow, Joel; Zheng, Changqing; Bissonnette, Marc; Hanauer, Stephen B.; Li, Yan Chun

    2013-01-01

    The inhibitory effects of vitamin D on colitis have been previously documented. Global vitamin D receptor (VDR) deletion exaggerates colitis, but the relative anticolitic contribution of epithelial and nonepithelial VDR signaling is unknown. Here, we showed that colonic epithelial VDR expression was substantially reduced in patients with Crohn’s disease or ulcerative colitis. Moreover, targeted expression of human VDR (hVDR) in intestinal epithelial cells (IECs) protected mice from developing colitis. In experimental colitis models induced by 2,4,6-trinitrobenzenesulfonic acid, dextran sulfate sodium, or CD4+CD45RBhi T cell transfer, transgenic mice expressing hVDR in IECs were highly resistant to colitis, as manifested by marked reductions in clinical colitis scores, colonic histological damage, and colonic inflammation compared with WT mice. Reconstitution of Vdr-deficient IECs with the hVDR transgene completely rescued Vdr-null mice from severe colitis and death, even though the mice still maintained a hyperresponsive Vdr-deficient immune system. Mechanistically, VDR signaling attenuated PUMA induction in IECs by blocking NF-κB activation, leading to a reduction in IEC apoptosis. Together, these results demonstrate that gut epithelial VDR signaling inhibits colitis by protecting the mucosal epithelial barrier, and this anticolitic activity is independent of nonepithelial immune VDR actions. PMID:23945234

  20. pH-dependent inhibition of native GABAA receptors by HEPES

    PubMed Central

    Hugel, S; Kadiri, N; Rodeau, JL; Gaillard, S; Schlichter, R

    2012-01-01

    BACKGROUND AND PURPOSE Artificial buffers such as HEPES are extensively used to control extracellular pH (pHe) to investigate the effect of H+ ions on GABAA receptor function. EXPERIMENTAL APPROACH In neurones cultured from spinal cord dorsal horn (DH), dorsal root ganglia (DRG) and cerebellar granule cells (GC) of neonatal rats, we studied the effect of pHe on currents induced by GABAA receptor agonists, controlling pHe with HCO3- or different concentrations of HEPES. KEY RESULTS Changing HEPES concentration from 1 to 20 mM at constant pHe strongly inhibited the currents induced by submaximal GABA applications, but not those induced by glycine or glutamate, on DH, DRG or GC neurones, increasing twofold the EC50 for GABA in DH neurones and GC. Submaximal GABAA receptor-mediated currents were also inhibited by piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino)propanesulfonic acid, tris(hydroxymethyl)aminomethane or imidazole. PIPES and HEPES, both piperazine derivatives, similarly inhibited GABAA receptors, whereas the other buffers had weaker effects and 2-(N-morpholino)ethanesulfonic acid had no effect. HEPES-induced inhibition of submaximal GABAA receptor-mediated currents was unaffected by diethylpyrocarbonate, a histidine-modifying reagent. HEPES-induced inhibition of GABAA receptors was independent of membrane potential, HCO3- and intracellular Cl- concentration and was not modified by flumazenil, which blocks the benzodiazepine binding site. However, it strongly depended on pHe. CONCLUSIONS AND IMPLICATIONS Inhibition of GABAA receptors by HEPES depended on pHe, leading to an apparent H+-induced inhibition of DH GABAA receptors, unrelated to the pH sensitivity of these receptors in both low and physiological buffering conditions, suggesting that protonated HEPES caused this inhibition. PMID:22452286

  1. A conserved structural mechanism of NMDA receptor inhibition: A comparison of ifenprodil and zinc

    PubMed Central

    Sirrieh, Rita E.; MacLean, David M.

    2015-01-01

    N-methyl-d-aspartate (NMDA) receptors, one of the three main types of ionotropic glutamate receptors (iGluRs), are involved in excitatory synaptic transmission, and their dysfunction is implicated in various neurological disorders. NMDA receptors, heterotetramers typically composed of GluN1 and GluN2 subunits, are the only members of the iGluR family that bind allosteric modulators at their amino-terminal domains (ATDs). We used luminescence resonance energy transfer to characterize the conformational changes the receptor undergoes upon binding ifenprodil, a synthetic compound that specifically inhibits activation of NMDA receptors containing GluN2B. We found that ifenprodil induced an overall closure of the GluN2B ATD without affecting conformation of the GluN1 ATD or the upper lobes of the ATDs, the same mechanism whereby zinc inhibits GluN2A. These data demonstrate that the conformational changes induced by zinc and ifenprodil represent a conserved mechanism of NMDA receptor inhibition. Additionally, we compared the structural mechanism of zinc inhibition of GluN1–GluN2A receptors to that of ifenprodil inhibition of GluN1–GluN2B. The similarities in the conformational changes induced by inhibitor binding suggest a conserved structural mechanism of inhibition independent of the binding site of the modulator. PMID:26170175

  2. Inhibition of prostaglandin E2 receptor EP3 mitigates thrombin-induced brain injury.

    PubMed

    Han, Xiaoning; Lan, Xi; Li, Qiang; Gao, Yufeng; Zhu, Wei; Cheng, Tian; Maruyama, Takayuki; Wang, Jian

    2016-06-01

    Prostaglandin E2 EP3 receptor is the only prostaglandin E2 receptor that couples to multiple G-proteins, but its role in thrombin-induced brain injury is unclear. In the present study, we exposed mouse hippocampal slice cultures to thrombin in vitro and injected mice with intrastriatal thrombin in vivo to investigate the role of EP3 receptor in thrombin-induced brain injury and explore its underlying cellular and molecular mechanisms. In vitro, EP3 receptor inhibition reduced thrombin-induced hippocampal CA1 cell death. In vivo, EP3 receptor was expressed in astrocytes and microglia in the perilesional region. EP3 receptor inhibition reduced lesion volume, neurologic deficit, cell death, matrix metalloproteinase-9 activity, neutrophil infiltration, and the number of CD68(+) microglia, but increased the number of Ym-1(+) M2 microglia. RhoA-Rho kinase levels were increased after thrombin injection and were decreased by EP3 receptor inhibition. In mice that received an intrastriatal injection of autologous arterial blood, inhibition of thrombin activity with hirudin decreased RhoA expression compared with that in vehicle-treated mice. However, EP3 receptor activation reversed this effect of hirudin. These findings show that prostaglandin E2 EP3 receptor contributes to thrombin-induced brain damage via Rho-Rho kinase-mediated cytotoxicity and proinflammatory responses. PMID:26661165

  3. Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2014-02-01

    Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8μg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension. PMID:24216055

  4. Competitive inhibition of (TH)dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    SciTech Connect

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-03-01

    The inhibitory effect of leupeptin on (TH)dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of (TH)dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of (TH)dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).

  5. Knockdown of GnT-Va expression inhibits ligand-induced downregulation of the epidermal growth factor receptor and intracellular signaling by inhibiting receptor endocytosis

    PubMed Central

    Guo, Hua-Bei; Johnson, Heather; Randolph, Matthew; Lee, Intaek; Pierce, Michael

    2009-01-01

    Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAcβ(1,6)Man branching, we utilized specific siRNA to selectively knockdown GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in the attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced downregulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor downregulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause the inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced downregulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by the expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by the treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes. PMID:19225046

  6. Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

    NASA Astrophysics Data System (ADS)

    Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  7. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  8. Inhibition of opioid release in the rat spinal cord by α2C adrenergic receptors

    PubMed Central

    Chen, Wenling; Song, Bingbing; Marvizón, Juan Carlos G.

    2008-01-01

    Neurotransmitter receptors that control the release of opioid peptides in the spinal cord may play an important role in pain modulation. Norepinephrine, released by a descending pathway originating in the brainstem, is a powerful inducer of analgesia in the spinal cord. Adrenergic α2C receptors are present in opioid-containing terminals in the dorsal horn, where they could modulate opioid release. The goal of this study was to investigate this possibility. Opioid release was evoked from rat spinal cord slices by incubating them with the sodium channel opener veratridine in the presence of peptidase inhibitors (actinonin, captopril and thiorphan), and was measured in situ through the internalization of μ-opioid receptors in dorsal horn neurons. Veratridine produced internalization in 70% of these neurons. The α2 receptor agonists clonidine, guanfacine, medetomidine and UK-14304 inhibited the evoked μ-opioid receptor internalization with IC50s of 1.7 μM, 248 nM, 0.3 nM and 22 nM, respectively. However, inhibition by medetomidine was only partial, and inhibition by UK-14304 reversed itself at concentrations higher than 50 nM. None of these agonists inhibited μ-opioid receptor internalization produced by endomorphin-2, showing that they inhibited opioid release and not the internalization itself. The inhibition produced by clonidine, guanfacine or UK-14304 was completely reversed by the selective α2C antagonist JP-1203. In contrast, inhibition by guanfacine was not prevented by the α2A antagonist BRL-44408. These results show that α2C receptors inhibit the release of opioids in the dorsal horn. This action may serve to shut down the opioid system when the adrenergic system is active. PMID:18343461

  9. Fractional vesamicol receptor occupancy and acetylcholine active transport inhibition in synaptic vesicles.

    PubMed

    Kaufman, R; Rogers, G A; Fehlmann, C; Parsons, S M

    1989-09-01

    Vesamicol [(-)-(trans)-2-(4-phenylpiperidino)cyclohexanol] receptor binding and inhibition of acetylcholine (AcCh) active transport by cholinergic synaptic vesicles that were isolated from Torpedo electric organ were studied for 23 vesamicol enantiomers, analogues, and other drugs. Use of trace [3H]vesamicol and [14C]AcCh allowed simultaneous determination of the concentrations of enantiomer, analogue, or drug required to half-saturate the vesamicol receptor (Ki) and to half-inhibit transport (IC50), respectively. Throughout a wide range of potencies for different compounds, the Ki/IC50 ratios varied from 1.5 to 24. Compounds representative of the diverse structures studied, namely deoxyvesamicol, chloroquine, and levorphanol, were competitive inhibitors of vesamicol binding. It is concluded that many drugs can bind to the vesamicol receptor and binding to only a small fraction of the receptors can result in AcCh active transport inhibition. Possible mechanisms for this effect are discussed. PMID:2550778

  10. Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

    PubMed Central

    Cho, Eunsil; Kim, Dong-Hyun; Hur, Young-Na; Whitcomb, Daniel J.; Regan, Philip; Hong, Jung-Hwa; Kim, Hanna; Ho Suh, Young; Cho, Kwangwook; Park, Mikyoung

    2015-01-01

    Cyclin Y (CCNY) is a member of the cyclin protein family, known to regulate cell division in proliferating cells. Interestingly, CCNY is expressed in neurons that do not undergo cell division. Here, we report that CCNY negatively regulates long-term potentiation (LTP) of synaptic strength through inhibition of AMPA receptor trafficking. CCNY is enriched in postsynaptic fractions from rat forebrain and is localized adjacent to postsynaptic sites in dendritic spines in rat hippocampal neurons. Using live-cell imaging of a pH-sensitive AMPA receptor, we found that during LTP-inducing stimulation, CCNY inhibits AMPA receptor exocytosis in dendritic spines. Furthermore, CCNY abolishes LTP in hippocampal slices. Taken together, our findings demonstrate that CCNY inhibits plasticity-induced AMPA receptor delivery to synapses and thereby blocks LTP, identifying a novel function for CCNY in post-mitotic cells. PMID:26220330

  11. CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

    PubMed Central

    Mamedova, Liaman; Capra, Valérie; Accomazzo, Maria Rosa; Gao, Zhan-Guo; Ferrario, Silvia; Fumagalli, Marta; Abbracchio, Maria P.; Rovati, G. Enrico; Jacobson, Kenneth A.

    2016-01-01

    Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 μM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 μM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y1,2,4,6 receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 μM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 μM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions. PMID:16280122

  12. The Melanocortin Receptor Accessory Protein 2 promotes food intake through inhibition of the Prokineticin Receptor-1

    PubMed Central

    Chaly, Anna L; Srisai, Dollada; Gardner, Ellen E; Sebag, Julien A

    2016-01-01

    The Melanocortin Receptor Accessory Protein 2 (MRAP2) is an important regulator of energy homeostasis and its loss causes severe obesity in rodents. MRAP2 mediates its action in part through the potentiation of the MC4R, however, it is clear that MRAP2 is expressed in tissues that do not express MC4R, and that the deletion of MRAP2 does not recapitulate the phenotype of Mc4r KO mice. Consequently, we hypothesized that other GPCRs involved in the control of energy homeostasis are likely to be regulated by MRAP2. In this study we identified PKR1 as the first non-melanocortin GPCR to be regulated by MRAP2. We show that MRAP2 significantly and specifically inhibits PKR1 signaling. We also demonstrate that PKR1 and MRAP2 co-localize in neurons and that Mrap2 KO mice are hypersensitive to PKR1 stimulation. This study not only identifies new partners of MRAP2 but also a new pathway through which MRAP2 regulates energy homeostasis. DOI: http://dx.doi.org/10.7554/eLife.12397.001 PMID:26829592

  13. Structure and mechanism of activity-based inhibition of the EGF-Receptor by Mig6

    PubMed Central

    Ficarro, Scott B.; Zhang, Yi; Lee, Byung Il; Cho, Ahye; Kim, Kihong; Park, Angela K.J.; Park, Woong-Yang; Murray, Bradley; Meyerson, Matthew; Beroukhim, Rameen; Marto, Jarrod A.; Cho, Jeonghee; Eck, Michael J.

    2016-01-01

    Mig6 is a feedback inhibitor that directly binds, inhibits and drives internalization of ErbB-family receptors. Mig6 selectivity targets activated receptors. Here we find that the EGF receptor phosphorylates Mig6 on Tyr394, and that this phosphorylation is primed by prior phosphorylation of an adjacent residue, Tyr395, by Src. Crystal structures of human EGFR–Mig6 complexes reveal the structural basis for enhanced phosphorylation of primed Mig6 and show how Mig6 rearranges after phosphorylation by EGFR to effectively irreversibly inhibit the same receptor that catalyzed its phosphorylation. This dual phosphorylation site allows Mig6 to inactivate EGFR in a manner that requires activation of the target receptor and can be modulated by Src. Loss of Mig6 is a driving event in human cancer; analysis of 1057 gliomas reveals frequent focal deletions of ERRFI, the gene that encodes Mig6, in EGFR-amplified glioblastomas. PMID:26280531

  14. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    PubMed

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions. PMID:26944929

  15. Pramipexole inhibits MPTP toxicity in mice by dopamine D3 receptor dependent and independent mechanisms.

    PubMed

    Ramirez, Andres D; Wong, Stephen K-F; Menniti, Frank S

    2003-08-15

    The role of dopamine D3 receptors was investigated in mediating the neuroprotective effect of the dopamine D2/D3 receptor agonist (S)-2-amino-4,5,6,7-tetrahydro-6-propylamine-benzothiazole (pramipexole) in vivo. Pramipexole retained the ability to inhibit 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion in mice in which the dopamine D3 receptor had been deleted. However, the neuroprotective efficacy was reduced in the dopamine D3 receptor-deleted mice compared to that in littermates expressing the wildtype receptor. Furthermore, the dopamine D3 receptor selective antagonist 2-(3-[4-(2-tert-butyl-6-trifluoromethyl-4-pyrimidinyl)-1-piperazinyl]propylthio)-4-pyrimidinol (A-437203) partially inhibited the neuroprotective effect of pramipexole in dopamine D3 receptor expressing mice but not in receptor-deleted mice. These results indicate that pramipexole protects dopamine neurons from MPTP-induced toxicity by mechanisms that are both dependent and independent of an interaction with dopamine D3 receptors. PMID:12954356

  16. IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

    SciTech Connect

    Taketazu, F.; Chiba, S.; Shibuya, K.; Kuwaki, T.; Tsumura, H.; Miyazono, K.; Miyagawa, K.; Takaku, F. )

    1991-02-01

    The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF binding to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.

  17. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    PubMed

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS). PMID:26031312

  18. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    PubMed

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. PMID:26976217

  19. Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.

    PubMed

    Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D

    2002-04-15

    We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

  20. GABA-A Receptor Inhibition of Local Calcium Signaling in Spines and Dendrites

    PubMed Central

    Marlin, Joseph J.

    2014-01-01

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca2+ signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca2+ signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca2+ signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca2+ signaling in pyramidal neurons, with implications for cortical function and disease. PMID:25429132

  1. Expression of heteromeric glycine receptor-channels in rat spinal cultures and inhibition by neuroactive steroids.

    PubMed

    Fodor, László; Boros, András; Dezso, Péter; Maksay, Gábor

    2006-11-01

    Ionotropic glycine receptors were studied in cultured spinal cord neurons prepared from 17-day-old rat embryos, using whole-cell patch clamp electrophysiology. Glycine receptors of 3-17 days in vitro were characterized via subtype-specific channel blockade by micromolar picrotoxin and cyanotriphenylborate, as well as nanomolar strychnine. Potentiation by nanomolar tropisetron indicated coexpression of beta with alpha subunits. The neuroactive steroids pregnenolone sulfate and dehydroepiandrosterone sulfate, as well as alphaxalone and its 3beta epimer betaxalone inhibited the chloride current with IC(50) values of 19, 46, 16 and 208 microM, respectively, with no potentiation. Reverse transcription polymerase chain reaction and immunocytochemistry demonstrated mRNAs and proteins of alpha1, alpha2, alpha3 and beta subunits in rat spinal cord cultures. In conclusion, neuroactive steroids, both positive and negative modulators of gamma-aminobutyric-acid(A) receptors, inhibited heteromeric glycine receptors at micromolar concentrations. PMID:16784797

  2. Dopamine inhibits somatolactin gene expression in tilapia pituitary cells through the dopamine D2 receptors.

    PubMed

    Jiang, Quan; Lian, Anji; He, Qi

    2016-07-01

    Dopamine (DA) is an important neurotransmitter in the central nervous system of vertebrates and possesses key hypophysiotropic functions. Early studies have shown that DA has a potent inhibitory effect on somatolactin (SL) release in fish. However, the mechanisms responsible for DA inhibition of SL gene expression are largely unknown. To this end, tilapia DA type-1 (D1) and type-2 (D2) receptor transcripts were examined in the neurointermediate lobe (NIL) of the tilapia pituitary by real-time PCR. In tilapia, DA not only was effective in inhibiting SL mRNA levels in vivo and in vitro, but also could abolish pituitary adenylate cyclase-activating polypeptide (PACAP)- and salmon gonadotropin-releasing hormone (sGnRH)-stimulated SL gene expression at the pituitary level. In parallel studies, the specific D2 receptor agonists quinpirole and bromocriptine could mimic the DA-inhibited SL gene expression. Furthermore, the D2 receptor antagonists domperidone and (-)-sulpiride could abolish the SL response to DA or the D2 agonist quinpirole, whereas D1 receptor antagonists SCH23390 and SKF83566 were not effective in this respect. In primary cultures of tilapia NIL cells, D2 agonist quinpirole-inhibited cAMP production could be blocked by co-treatment with the D2 antagonist domperidone and the ability of forskolin to increase cAMP production was also inhibited by quinpirole. Using a pharmacological approach, the AC/cAMP pathway was shown to be involved in quinpirole-inhibited SL mRNA expression. These results provide evidence that DA can directly inhibit SL gene expression at the tilapia pituitary level via D2 receptor through the AC/cAMP-dependent mechanism. PMID:26970582

  3. Quantitation of GABAA receptor inhibition required for quinolone-induced convulsions in mice.

    PubMed

    Tsutomi, Y; Matsubayashi, K; Akahane, K

    1994-11-01

    We quantified the amount of inhibition of gamma-aminobutyric acid (GABA)A receptor binding required for the onset of convulsions induced by ciprofloxacin in combination with biphenylacetic acid (BPAA) in mice. In fasting mice iv ciprofloxacin given 30 min after oral BPAA (50 mg/kg) induced convulsions at doses of 40 mg/kg or above. In contrast, ofloxacin caused no convulsions even at 100 mg/kg, the highest dose tested. When mice received 40 mg/kg of ciprofloxacin or ofloxacin, maximal brain concentrations of each quinolone at 30 min were 0.37 or 1.97 micrograms/g, respectively. These brain concentrations of ciprofloxacin and ofloxacin were not affected by combination with BPAA. In the presence of ciprofloxacin and BPAA (at brain tissue concentrations which induced convulsions), the binding of 3H-muscimol to GABAA receptor sites was inhibited by approximately 30%. Using results from a similar binding study, an impracticable iv dose of ofloxacin (500 mg/kg) was estimated to be required to inhibit GABAA receptor binding by 30%, and therefore to induce similar convulsions to those seen with ciprofloxacin at a dose of 40 mg/kg. These results may indicate that epileptic convulsions occur when ciprofloxacin and BPAA interact with each other to antagonize at least 30% of GABAA receptor binding in mice, and provide evidence for a significant role of GABAA receptor inhibition in the occurrence of quinolone-induced convulsions. PMID:7706169

  4. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

    PubMed

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. PMID:27048878

  5. Epidermal growth factor receptor inhibition in lung cancer: status 2012.

    PubMed

    Hirsch, Fred R; Jänne, Pasi A; Eberhardt, Wilfried E; Cappuzzo, Federico; Thatcher, Nick; Pirker, Robert; Choy, Hak; Kim, Edward S; Paz-Ares, Luis; Gandara, David R; Wu, Yi-Long; Ahn, Myung-Ju; Mitsudomi, Tetsuya; Shepherd, Frances A; Mok, Tony S

    2013-03-01

    Lung cancer is the most common cause of cancer deaths. Most patients present with advanced-stage disease, and the prognosis is generally poor. However, with the understanding of lung cancer biology, and development of molecular targeted agents, there have been improvements in treatment outcomes for selected subsets of patients with non-small-cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significantly improved tumor responses and progression-free survival in subsets of patients with advanced NSCLC, particularly those with tumors harboring activating EGFR mutations. Testing for EGFR mutations is a standard procedure for identification of patients who will benefit from first-line EGFR TKIs. For patients with advanced NSCLC and no activating EGFR mutations (EGFR wild-type) or no other driving oncogenes such as ALK-gene rearrangement, chemotherapy is still the standard of care. A new generation of EGFR TKIs, targeting multiple receptors and with irreversible bindings to the receptors, are in clinical trials and have shown encouraging effects. Research on primary and acquired resistant mechanisms to EGFR TKIs are ongoing. Monoclonal antibodies (e.g. cetuximab), in combination with chemotherapy, have demonstrated improved outcomes, particularly for subsets of NSCLC patients, but further validations are needed. Novel monoclonal antibodies are combined with chemotherapy, and randomized comparative studies are ongoing. This review summarizes the current status of EGFR inhibitors in NSCLC in 2012 and some of the major challenges we are facing. PMID:23370315

  6. CPAP inhibits non-nutritive swallowing through stimulation of bronchopulmonary receptors.

    PubMed

    Samson, Nathalie; Duvareille, Charles; St-Hilaire, Marie; Clapperton, Véronique; Praud, Jean-Paul

    2008-01-01

    While swallowing and respiratory problems are among the most frequent disorders encountered in neonates, the interrelationships between both functions are not completely understood. This is especially true for non-nutritive swallowing (NNS), which fulfills the important function of clearing upper airways from both local secretions and liquids refluxed from the stomach. Recently, we showed that nasal CPAP inhibits NNS during quiet sleep in the newborn lamb (Samson, St-Hilaire, Nsegbe, Reix, Moreau-Bussière and Praud 2005). The present study was aimed at testing the hypothesis that NNS inhibition is eliminated when CPAP is directly administered through a tracheostomy, thus eliminating reflexes originating from upper airway receptors. Results show that both nasal and tracheal CPAP 6cm H2O similarly inhibit total NNS during quiet sleep, thus suggesting that the inhibiting effect of nasal CPAP on NNS is mainly mediated through bronchopulmonary mechanical receptors with minimal participation of the upper airways. PMID:18085310

  7. Inhibition of mechanosensitivity in visceral primary afferents by GABAB receptors involves calcium and potassium channels.

    PubMed

    Page, A J; O'Donnell, T A; Blackshaw, L A

    2006-01-01

    GABA(B) receptors inhibit mechanosensitivity of visceral afferents. This is associated with reduced triggering of events that lead to gastro-esophageal reflux, with important therapeutic consequences. In other neuronal systems, GABA(B) receptor activation may be linked via G-proteins to reduced N-type Ca(2+) channel opening, increased inward rectifier K(+) channel opening, plus effects on a number of intracellular messengers. Here we aimed to determine the role of Ca(2+) and K(+) channels in the inhibition of vagal afferent mechanoreceptor function by the GABA(B) receptor agonist baclofen. The responses of three types of ferret gastro-esophageal vagal afferents (mucosal, tension and tension mucosal receptors) to graded mechanical stimuli were investigated in vitro. The effects of baclofen (200 microM) alone on these responses were quantified, and the effects of baclofen in the presence of the G-protein-coupled inward rectifier potassium channel blocker Rb(+) (4.7 mM) and/or the N-type calcium channel blocker omega-conotoxin GVIA (0.1 microM). Baclofen inhibition of mucosal receptor mechanosensitivity was abolished by both blockers. Its inhibitory effect on tension mucosal receptors was partly reduced by both. The inhibitory effect of baclofen on tension receptors was unaffected. The data indicate that the inhibitory action of GABA(B) receptors is mediated via different pathways in mucosal, tension and tension mucosal receptors via mechanisms involving both N-type Ca(2+) channels and inwardly rectifying K(+) channels and others. PMID:16289839

  8. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2015-07-01

    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism. PMID:25910812

  9. Disruption of insulin receptor function inhibits proliferation in endocrine resistant breast cancer cells

    PubMed Central

    Chan, Jie Ying; LaPara, Kelly; Yee, Douglas

    2015-01-01

    The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen resistant breast cancer cells. Here we examined if inhibition of InsR affected tamoxifen-resistant (TamR) breast cancer cells. InsR function was inhibited by three different mechanisms: InsR shRNA, a small InsR blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely due to the presence of IGFR/InsR hybrid receptors. Down-regulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared to single receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine sensitive and resistant breast cancer. PMID:26876199

  10. Disruption of insulin receptor function inhibits proliferation in endocrine-resistant breast cancer cells.

    PubMed

    Chan, J Y; LaPara, K; Yee, D

    2016-08-11

    The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine-resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen-resistant (TamR) breast cancer cells. Here we examined if inhibition of InsR affected TamR breast cancer cells. InsR function was inhibited by three different mechanisms: InsR short hairpin RNA, a small InsR-blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely because of the presence of IGFR /InsR hybrid receptors. Downregulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared with single-receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine-sensitive and -resistant breast cancer. PMID:26876199

  11. Inhibition of Eph receptor-ephrin ligand interaction by tea polyphenols

    PubMed Central

    Noberini, Roberta; Koolpe, Mitchell; Lamberto, Ilaria; Pasquale, Elena B.

    2013-01-01

    Tea contains a variety of bioactive chemicals, such as catechins and other polyphenols. These compounds are thought to be responsible for the health benefits of tea consumption by affecting the function of many cellular targets, not all of which have been identified. In a high-throughput screen for small molecule antagonists of the EphA4 receptor tyrosine kinase, we identified five tea polyphenols that substantially inhibit EphA4 binding to a synthetic peptide ligand. Further characterization of theaflavin monogallates from black tea and epigallocatechin-3,5-digallate from green tea revealed that these compounds at low micromolar concentrations also inhibit binding of the natural ephrin ligands to EphA4 and several other Eph receptors in in vitro assays. The compounds behave as competitive EphA4 antagonists, and their inhibitory activity is affected by amino acid mutations within the ephrin binding pocket of EphA4. In contrast, the major green tea catechin, epigallocatechin-3-gallate (EGCG), does not appear to be an effective Eph receptor antagonist. In cell culture assays, theaflavin monogallates and epigallocatechin-3,5-digallate inhibit ephrin-induced tyrosine phosphorylation (activation) of Eph receptors and endothelial capillary-like tube formation. However, the wider spectrum of Eph receptors affected by the tea derivatives in cells suggests additional mechanisms of inhibition besides interfering with ephrin binding. These results show that tea polyphenols derived from both black and green tea can suppress the biological activities of Eph receptors. Thus, the Eph receptor tyrosine kinase family represents an important class of targets for tea-derived phytochemicals. PMID:22750215

  12. Pharmacological or genetic orexin1 receptor inhibition attenuates MK-801 induced glutamate release in mouse cortex.

    PubMed

    Aluisio, Leah; Fraser, Ian; Berdyyeva, Tamara; Tryputsen, Volha; Shireman, Brock T; Shoblock, James; Lovenberg, Timothy; Dugovic, Christine; Bonaventure, Pascal

    2014-01-01

    The orexin/hypocretin neuropeptides are produced by a cluster of neurons within the lateral posterior hypothalamus and participate in neuronal regulation by activating their receptors (OX1 and OX2 receptors). The orexin system projects widely through the brain and functions as an interface between multiple regulatory systems including wakefulness, energy balance, stress, reward, and emotion. Recent studies have demonstrated that orexins and glutamate interact at the synaptic level and that orexins facilitate glutamate actions. We tested the hypothesis that orexins modulate glutamate signaling via OX1 receptors by monitoring levels of glutamate in frontal cortex of freely moving mice using enzyme coated biosensors under inhibited OX1 receptor conditions. MK-801, an NMDA receptor antagonist, was administered subcutaneously (0.178 mg/kg) to indirectly disinhibit pyramidal neurons and therefore increase cortical glutamate release. In wild-type mice, pretreatment with the OX1 receptor antagonist GSK-1059865 (10 mg/kg S.C.) which had no effect by itself, significantly attenuated the cortical glutamate release elicited by MK-801. OX1 receptor knockout mice had a blunted glutamate release response to MK-801 and exhibited about half of the glutamate release observed in wild-type mice in agreement with the data obtained with transient blockade of OX1 receptors. These results indicate that pharmacological (transient) or genetic (permanent) inhibition of the OX1 receptor similarly interfere with glutamatergic function in the cortex. Selectively targeting the OX1 receptor with an antagonist may normalize hyperglutamatergic states and thus may represent a novel therapeutic strategy for the treatment of various psychiatric disorders associated with hyperactive states. PMID:24904253

  13. Pharmacological or genetic orexin1 receptor inhibition attenuates MK-801 induced glutamate release in mouse cortex

    PubMed Central

    Aluisio, Leah; Fraser, Ian; Berdyyeva, Tamara; Tryputsen, Volha; Shireman, Brock T.; Shoblock, James; Lovenberg, Timothy; Dugovic, Christine; Bonaventure, Pascal

    2014-01-01

    The orexin/hypocretin neuropeptides are produced by a cluster of neurons within the lateral posterior hypothalamus and participate in neuronal regulation by activating their receptors (OX1 and OX2 receptors). The orexin system projects widely through the brain and functions as an interface between multiple regulatory systems including wakefulness, energy balance, stress, reward, and emotion. Recent studies have demonstrated that orexins and glutamate interact at the synaptic level and that orexins facilitate glutamate actions. We tested the hypothesis that orexins modulate glutamate signaling via OX1 receptors by monitoring levels of glutamate in frontal cortex of freely moving mice using enzyme coated biosensors under inhibited OX1 receptor conditions. MK-801, an NMDA receptor antagonist, was administered subcutaneously (0.178 mg/kg) to indirectly disinhibit pyramidal neurons and therefore increase cortical glutamate release. In wild-type mice, pretreatment with the OX1 receptor antagonist GSK-1059865 (10 mg/kg S.C.) which had no effect by itself, significantly attenuated the cortical glutamate release elicited by MK-801. OX1 receptor knockout mice had a blunted glutamate release response to MK-801 and exhibited about half of the glutamate release observed in wild-type mice in agreement with the data obtained with transient blockade of OX1 receptors. These results indicate that pharmacological (transient) or genetic (permanent) inhibition of the OX1 receptor similarly interfere with glutamatergic function in the cortex. Selectively targeting the OX1 receptor with an antagonist may normalize hyperglutamatergic states and thus may represent a novel therapeutic strategy for the treatment of various psychiatric disorders associated with hyperactive states. PMID:24904253

  14. Garlic (Allium sativum) Extracts Inhibits Lipopolysaccharide-Induced Toll-Like Receptor 4 Dimerization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Garlic has been used as a folk medicine for a long history. Numerous studies demonstrated that garlic extracts and its sulfur-containing compounds inhibit nuclear factor-kappa B (NF-kB) activation induced by various receptor agonist including lipopolysaccharide (LPS). These effects suggest that garl...

  15. Effects of P2Y12 receptor antagonists beyond platelet inhibition - comparison of ticagrelor with thienopyridines.

    PubMed

    Nylander, Sven; Schulz, Rainer

    2016-04-01

    The effect and clinical benefit of P2Y12 receptor antagonists may not be limited to platelet inhibition and the prevention of arterial thrombus formation. Potential additional effects include reduction of the pro-inflammatory role of activated platelets and effects related to P2Y12 receptor inhibition on other cells apart from platelets. P2Y12 receptor antagonists, thienopyridines and ticagrelor, differ in their mode of action being prodrugs instead of direct acting and irreversibly instead of reversibly binding to P2Y12 . These key differences may provide different potential when it comes to additional effects. In addition to P2Y12 receptor blockade, ticagrelor is unique in having the only well-documented additional target of inhibition, the equilibrative nucleoside transporter 1. The current review will address the effects of P2Y12 receptor antagonists beyond platelets and the protection against arterial thrombosis. The discussion will include the potential for thienopyridines and ticagrelor to mediate anti-inflammatory effects, to conserve vascular function, to affect atherosclerosis, to provide cardioprotection and to induce dyspnea. PMID:26758983

  16. Highly effective poly(ethylene glycol) architectures for specific inhibition of immune receptor activation.

    PubMed

    Baird, Emily J; Holowka, David; Coates, Geoffrey W; Baird, Barbara

    2003-11-11

    Architectural features of synthetic ligands were systematically varied to optimize inhibition of mast cell degranulation initiated by multivalent crossing of IgE-receptor complexes. A series of ligands were generated by end-capping poly(ethylene glycol) (PEG) polymers and amine-based dendrimers with the hapten 2,4-dinitrophenyl (DNP). These were used to explore the influence of polymeric backbone length, valency, and hapten presentation on binding to anti-DNP IgE and inhibition of stimulated activation of RBL cells. Monovalent MPEG(5000)-DNP (IC(50) = 50 nM), bivalent DNP-PEG(3350)-DNP (IC(50) = 8 nM), bismonovalent MPEG(5000)-DNP(2) (IC(50) = 20 nM), bisbivalent DNP(2)-PEG(3350)-DNP(2) (IC(50) = 3nM) and DNP(4)-dendrimer ligands (IC(50) = 50 nM) all effectively inhibit cellular activation caused by multivalent antigen, DNP-bovine serum albumin. For different DNP ligands, we provide evidence for more effective inhibition due to (i) preferential formation of intra-IgE cross-links by bivalent ligands of sufficient length, (ii) self-association of monovalent ligands with longer tails, and (iii) higher probability of binding for bisvalent ligands. We also show that larger DNP(16)-dendrimers of higher valency trigger degranulation by cross-linking IgE-receptor complexes, whereas smaller DNP-dendrimers are inhibitory. Thus, features of synthetic ligands can be manipulated to control receptor occupation, aggregation, and inhibition of the cellular response. PMID:14596588

  17. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    PubMed Central

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  18. Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.

    PubMed

    Tajima, Nami; Karakas, Erkan; Grant, Timothy; Simorowski, Noriko; Diaz-Avalos, Ruben; Grigorieff, Nikolaus; Furukawa, Hiro

    2016-06-01

    The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1-GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel. PMID:27135925

  19. Adenosine A1 receptor inhibits postnatal neurogenesis and sustains astrogliogenesis from the subventricular zone.

    PubMed

    Benito-Muñoz, Monica; Matute, Carlos; Cavaliere, Fabio

    2016-09-01

    We previously demonstrated that activation of ATP P2X receptors during oxygen and glucose deprivation inhibits neuroblast migration and in vitro neurogenesis from the subventricular zone (SVZ). Here, we have studied the effects of adenosine, the natural end-product of ATP hydrolysis, in modulating neurogenesis and gliogenesis from the SVZ. We provide immunochemical, molecular and pharmacological evidence that adenosine via A1 receptors reduces neuronal differentiation of neurosphere cultures generated from postnatal SVZ. Furthermore, activation of A1 receptors induces downregulation of genes related to neurogenesis as demonstrated by gene expression analysis. Specifically, we found that A1 receptors trigger a signaling cascade that, through the release of IL10, turns on the Bmp2/SMAD pathway. Furthermore, activating A1 receptors in SVZ-neural progenitor cells inhibits neurogenesis and stimulates astrogliogenesis as assayed in vitro in neurosphere cultures and in vivo in the olfactory bulb. Together, these data indicate that adenosine acting at A1 receptors negatively regulates adult neurogenesis while promoting astrogliogenesis, and that this feature may be relevant to pathological conditions whereby purines are profusely released. GLIA 2016;64:1465-1478. PMID:27301342

  20. Inhibition of clathrin-mediated endocytosis selectively attenuates specific insulin receptor signal transduction pathways.

    PubMed

    Ceresa, B P; Kao, A W; Santeler, S R; Pessin, J E

    1998-07-01

    To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor. PMID:9632770

  1. Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

    PubMed Central

    Ceresa, Brian P.; Kao, Aimee W.; Santeler, Scott R.; Pessin, Jeffrey E.

    1998-01-01

    To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor. PMID:9632770

  2. Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase.

    PubMed Central

    Estrada, C; Gómez, C; Martín-Nieto, J; De Frutos, T; Jiménez, A; Villalobo, A

    1997-01-01

    Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-¿4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl¿propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor. PMID:9291107

  3. A non-peptide NK1-receptor antagonist, RP 67580, inhibits neurogenic inflammation postsynaptically.

    PubMed

    Moussaoui, S M; Montier, F; Carruette, A; Blanchard, J C; Laduron, P M; Garret, C

    1993-05-01

    1. The non-peptide neurokinin NK1-receptor antagonist, RP 67580 (3aR, 7aR), a perhydroisoindolone derivative, powerfully reduced plasma extravasation in rat hind paw skin induced by local application of xylene (ID50 = 0.03 mg kg-1, i.v.) or capsaicin (ID50 = 0.06 mg kg-1, i.v.), or by i.v. injection of exogenous substance P (SP) or septide ([pGlu6,Pro9]SP(6-11)) (ID50 = 0.04-0.05 mg kg-1, i.v.). RP 67580 (1 mg kg-1, i.v.) also abolished capsaicin-induced nasal fluid hypersecretion (by 82 +/- 5%). These effects were found to be stereospecific, the enantiomer, RP 68651 (3aS, 7aS), being inactive at 1 mg kg-1, i.v. 2. In rats neonatally treated with capsaicin (50 mg kg-1, s.c.), plasma extravasation induced by SP was significantly increased (by 43 +/- 7%). RP 67580 (1 mg kg-1, i.v.) completely inhibited the SP-induced plasma extravasation in capsaicin neonatally treated-animals, as it did in control animals. This result suggests that RP 67580 acts at the postsynaptic level for the inhibition of plasma extravasation. 3. Opioid receptor agonists, mu-(morphine) and kappa-(PD-117302) at 10 mg kg-1, s.c., in contrast to NK1-receptor antagonists, did not inhibit plasma extravasation induced by exogenous SP. They were, however, partially effective against plasma extravasation induced by electrical nerve stimulation (74 +/- 4% and 48 +/- 9% inhibition at 10 mg kg-1, s.c. of morphine and PD-117302, respectively, compared to 90 +/- 3% inhibition obtained with RP 67580, 3 mg kg-1, s.c.). These results indicate the presynaptic action of opioid receptor agonists, in contrast to the postsynaptic action of NK1-receptor antagonists for the inhibition of plasma extravasation.4. Ligature of the saphenous nerve distal to the point of electrical stimulation, local application of lignocaine to the saphenous nerve, neonatal capsaicin pretreatment, and colchicine at very low doses(120 microg kg-1 day-1 given for 3 days) were found to prevent plasma extravasation elicited by electrical nerve

  4. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

    PubMed Central

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

    2015-01-01

    ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1

  5. Potentiation of tonic GABAergic inhibition by activation of postsynaptic kainate receptors.

    PubMed

    Jiang, L; Kang, D; Kang, J

    2015-07-01

    Presynaptic kainate-type glutamate ionotropic receptors (KARs) that mediate either the depression or the facilitation of GABA release have been intensively studied. Little attention has been given to the modulation of GABAA receptors (GABAARs) by postsynaptic KARs. Recent studies suggest that two GABAAR populations, synaptic (sGABAAR) and extrasynaptic (eGABAAR) GABAARs, mediate phasic and tonic forms of inhibition, respectively. Tonic inhibition plays an important role in the excitability of neuronal circuits and the occurrence of epileptic seizures. For this study, we are the first to report that the activation of postsynaptic KARs by the KAR agonist, Kainic acid (KA, 5 μM), enhanced tonic inhibition by potentiating eGABAARs. KA enhanced THIP-induced eGABAAR currents and prolonged the rise and decay time of muscimol-induced sGABAAR/eGABAAR currents, but also depressed the amplitude of evoked inhibitory postsynaptic currents (IPSCs), unitary IPSCs (uIPSCs), and muscimol-induced sGABAAR/eGABAAR currents. The PKC inhibitor, staurosporine (1 μM), in the patch pipette solution fully blocked the KA-induced potentiation of tonic inhibition, suggesting the involvement of an intracellular PKC pathway. Our study suggests that the activation of postsynaptic KARs potentiates eGABAARs but depresses sGABAARs. By activating postsynaptic KARs, synaptically released glutamate depresses phasic inhibition to facilitate neuronal plasticity, but potentiates tonic inhibition to protect neurons from over-excitation. PMID:25934031

  6. Telmisartan Induced Inhibition of Vascular Cell Proliferation beyond Angiotensin Receptor Blockade and PPARγ Activation

    PubMed Central

    Yamamoto, Koichi; Ohishi, Mitsuru; Ho, Christopher; Kurtz, Theodore W; Rakugi, Hiromi

    2010-01-01

    We investigated the ability of ARBs with PPARγ agonist activity (telmisartan and irbesartan), and ARBs devoid of PPARγ agonist activity (eprosartan and valsartan), to inhibit vascular cell proliferation studied in the absence of angiotensin II stimulation. Telmisartan and to a lesser extent irbesartan, inhibited proliferation of human aortic vascular smooth muscle cells in a dose dependent fashion whereas eprosartan and valsartan did not. To investigate the role of PPARγ in the antiproliferative effects of telmisartan, we studied genetically engineered NIH3T3 cells that express PPARγ. Pioglitazone inhibited proliferation of NIH3T3 cells expressing PPARγ, but had little effect on control NIH3T3 cells that lack PPARγ. In contrast, telmisartan inhibited proliferation equally in NIH3T3 with and without PPARγ. Valsartan failed to inhibit proliferation of either cell line. In addition, telmisartan inhibited proliferation equally in aortic smooth muscle cells derived from mice with targeted knockout of PPARγ in smooth muscle and from control mice whereas valsartan had no effect on cell proliferation. Telmisartan but not valsartan, reduced phosphorylation of AKT but not ERK otherwise induced by exposure to serum of either quiescent human smooth muscle cells, quiescent mice smooth muscle cells lacking PPARγ or quiescent CHO-K1 cells lacking AT1 receptor. In summary, the antiproliferative effect of telmisartan in the absence of exogenously supplemented angiotensin II involve more than just AT1 receptor blockade and do not require activation of PPARγ. It might be postulated that inhibition of AKT activation is a mechanism mediating the antiproliferative effects of telmisartan including in cells lacking AT1 receptors or PPARγ. PMID:19822796

  7. Effects of NMDA receptor inhibition by phencyclidine on the neuronal differentiation of PC12 cells.

    PubMed

    Lee, Eunsook; Williams, Zakia; Goodman, Carl B; Oriaku, Ebenezer T; Harris, Cynthia; Thomas, Mathews; Soliman, Karam F A

    2006-07-01

    Phencyclidine (PCP) is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist and exposing the developing brain to PCP has been shown to cause deficits in neurobehavioral functions. In the present study we tested the effects of PCP, as an NMDA receptor inhibitor, on the neuronal differentiation and biogenic amines levels including norepinephrine (NE), epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rat pheochromocytoma (PC12) cells. After PC12 cells were differentiated with nerve growth factor (NGF) in the presence of PCP, NMDA binding kinetics, biogenic amines analysis and NMDA receptor protein expression assay were conducted. The results showed that NMDA receptor binding activities were significantly increased after differentiated with NGF in PC12 cells. B(max) values were increased in differentiated cells by four-folds, whereas K(d) values were not changed. All of biogenic amines were significantly increased in differentiated cells. On the other hand, PCP at 50 and 100 microM inhibited neuronal differentiation in a dose-dependent manner in NGF-stimulated PC12 cells without affecting cell viability. PCP treatment during differentiation significantly reduced NMDA binding activity and biogenic amine levels. Western blotting analysis revealed that NMDA receptor protein expression was significantly higher in NGF-differentiated cells and PCP treatment decreased the expression of NMDA receptor proteins. These results indicate that NMDA receptor functions and monoaminergic nervous systems are significantly stimulated during NGF-induced differentiation. PCP suppresses neuronal outgrowth and hampers neuronal functions possibly by inhibiting NMDA receptor functions and biogenic amine production, implying the suppressive effects of PCP exposure on neuronal developments. PMID:16580729

  8. Inhibition of cortical acetylcholine release and cognitive performance by histamine H3 receptor activation in rats.

    PubMed Central

    Blandina, P.; Giorgetti, M.; Bartolini, L.; Cecchi, M.; Timmerman, H.; Leurs, R.; Pepeu, G.; Giovannini, M. G.

    1996-01-01

    1. The effects of histamine and agents at histamine receptors on spontaneous and 100 mM K(+)-evoked release of acetylcholine, measured by microdialysis from the cortex of freely moving, rats, and on cognitive tests are described. 2. Local administration of histamine (0.1-100 microM) failed to affect spontaneous but inhibited 100 mM K(+)-stimulated release of acetylcholine up to about 50%. The H3 receptor agonists (R)-alpha-methylhistamine (RAMH) (0.1-10 microM), imetit (0.01-10 microM) and immepip (0.01-10 microM) mimicked the effect of histamine. 3. Neither 2-thiazolylethylamine (TEA), an agonist showing some selectivity for H1 receptors, nor the H2 receptor agonist, dimaprit, modified 100 mM K(+)-evoked release of acetylcholine. 4. The inhibitory effect of 100 microM histamine was completely prevented by the highly selective histamine H3 receptor antagonist, clobenpropit but was resistant to antagonism by triprolidine and cimetidine, antagonists at histamine H1 and H2 but not H3 receptors. 5. The H3 receptor-induced inhibition of K(+)-evoked release of acetylcholine was fully sensitive to tetrodotoxin (TTX). 6. The effects of intraperitoneal (i.p.) injection of imetit (5 mg kg-1) and RAMH (5 mg kg-1) were tested on acetylcholine release and short term memory paradigms. Both drugs reduced 100 mM K(+)-evoked release of cortical acetylcholine, and impaired object recognition and a passive avoidance response. 7. These observations provide the first evidence of a regulatory role of histamine H3 receptors on cortical acetylcholine release in vivo. Moreover, they suggest a role for histamine in learning and memory and may have implications for the treatment of degenerative disorders associated with impaired cholinergic function. PMID:8982515

  9. Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

    SciTech Connect

    Jang, Eun Hyang; Jang, Soon Young; Cho, In-Hye; Hong, Darong; Jung, Bom; Park, Min-Ju; Kim, Jong-Ho

    2015-08-07

    Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy. - Highlights: • Hispolon decreased ERα expression at both mRNA and protein levels. • Hispolon decreased ERα transcriptional activity. • Hispolon treatment inhibited expression of ERα target gene pS2. • Shikonin is a candidate chemotherapeutic target in the treatment of human breast cancer.

  10. Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

    PubMed

    Aoshima, H; Inoue, Y; Hori, K

    1992-10-01

    Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1337082

  11. Inhibition of Receptor Signaling and of Glioblastoma-derived Tumor Growth by a Novel PDGFRβ Aptamer

    PubMed Central

    Camorani, Simona; Esposito, Carla L; Rienzo, Anna; Catuogno, Silvia; Iaboni, Margherita; Condorelli, Gerolama; de Franciscis, Vittorio; Cerchia, Laura

    2014-01-01

    Platelet-derived growth factor receptor β (PDGFRβ) is a cell-surface tyrosine kinase receptor implicated in several cellular processes including proliferation, migration, and angiogenesis. It represents a compelling therapeutic target in many human tumors, including glioma. A number of tyrosine kinase inhibitors under development as antitumor agents have been found to inhibit PDGFRβ. However, they are not selective as they present multiple tyrosine kinase targets. Here, we report a novel PDGFRβ-specific antagonist represented by a nuclease-resistant RNA-aptamer, named Gint4.T. This aptamer is able to specifically bind to the human PDGFRβ ectodomain (Kd: 9.6 nmol/l) causing a strong inhibition of ligand-dependent receptor activation and of downstream signaling in cell lines and primary cultures of human glioblastoma cells. Moreover, Gint4.T aptamer drastically inhibits cell migration and proliferation, induces differentiation, and blocks tumor growth in vivo. In addition, Gint4.T aptamer prevents PDGFRβ heterodimerization with and resultant transactivation of epidermal growth factor receptor. As a result, the combination of Gint4.T and an epidermal growth factor receptor–targeted aptamer is better at slowing tumor growth than either single aptamer alone. These findings reveal Gint4.T as a PDGFRβ-drug candidate with translational potential. PMID:24566984

  12. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  13. The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

    PubMed

    Smith, L Cody; Ralston-Hooper, Kimberly J; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-06-01

    Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens. PMID:27026707

  14. Characterization of the PGE receptor subtype mediating inhibition of superoxide production in human neutrophils.

    PubMed Central

    Talpain, E; Armstrong, R A; Coleman, R A; Vardey, C J

    1995-01-01

    1. The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)-stimulated human neutrophils, and to test the hypothesis that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E2. 2. PGE2 (0.001-10 microM) inhibited FMLP (100 nM)-induced O2-generation from human peripheral blood neutrophils in a concentration-dependent manner, with an EC50 of 0.15 +/- 0.03 microM, and a maximum effect ranging from 36-84% (mean inhibition of 68.7 +/- 2.5%, n = 32). 3. The EP2-receptor agonists, misoprostol, 11-deoxy PGE1, AH13205 and butaprost, all at 10 microM, inhibited O2- generation, causing 95.5 +/- 2.9%, 56.8 +/- 5.2%, 37.1 +/- 6.6% and 18.9 +/- 4.4% inhibition respectively, the latter two being much less effective than PGE2. Similarly, the EP1-receptor agonist, 17-phenyl PGE2 (10 microM), and the EP3/EP1-receptor agonist, sulprostone (10 microM), also inhibited O2- generation, causing 32.2 +/- 7.0% and 15.3 +/- 3.4% inhibition respectively. 4. The non-selective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX, 0.25 mM) inhibited the FMLP response by 54.5 +/- 5.0%. In addition, IBMX shifted concentration-effect curves for PGE2, misoprostol, 11-deoxy PGE1, butaprost, and AH 13205 to the left, to give EC50s of 0.04 +/- 0.03 (n = 13), 0.07 +/- 0.03 (n = 4), 0.08 +/- 0.03 (n = 4), 0.33 +/- 0.13 (n = 4) and 0.41 +/- 0.2 microM (n = 3) respectively, allowing equieffective concentration-ratios (EECs, PGE2 = 1) of 11.5, 5.3, 50.7 and 12.7 to be calculated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7606349

  15. Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

    SciTech Connect

    Green, S.J.; Underhill, C.B. ); Tarone, G. )

    1988-10-01

    To examine the role of the hyaluronate receptor in cell to cell adhesion, the authors have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, they investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind ({sup 3})hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a M{sub r} of 99,500, which is considerably larger than the 85,000 M{sub r} for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.

  16. Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

    PubMed

    Akahane, K; Kimura, Y; Tsutomi, Y; Hayakawa, I

    1994-10-01

    The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA. PMID:7840564

  17. Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

    PubMed Central

    Akahane, K; Kimura, Y; Tsutomi, Y; Hayakawa, I

    1994-01-01

    The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA. PMID:7840564

  18. Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization.

    PubMed

    LaMontagne, Kenneth; Littlewood-Evans, Amanda; Schnell, Christian; O'Reilly, Terence; Wyder, Lorenza; Sanchez, Teresa; Probst, Beatrice; Butler, Jeannene; Wood, Alexander; Liau, Gene; Billy, Eric; Theuer, Andreas; Hla, Timothy; Wood, Jeanette

    2006-01-01

    FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis. PMID:16397235

  19. Agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action

    SciTech Connect

    Forman, S.A.; Firestone, L.L.; Miller, K.W.

    1987-05-19

    Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring /sup 86/Rb/sup +/ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: (1) integrated net ion efflux (in 10 s) from untreated vesicles, (2) integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with ..cap alpha..-bungarotoxin, and (3) initial rates of efflux (5-100 ms) from vesicles that were partially blocked with ..cap alpha..-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10/sup 8/-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum /sup 86/Rb/sup +/ efflux by 50% (K/sub B/ values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, K/sub B/ values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times K/sub B/ did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. The authors conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

  20. Cannabinoids inhibit cholinergic contraction in human airways through prejunctional CB1 receptors

    PubMed Central

    Grassin-Delyle, S; Naline, E; Buenestado, A; Faisy, C; Alvarez, J-C; Salvator, H; Abrial, C; Advenier, C; Zemoura, L; Devillier, P

    2014-01-01

    Background and Purpose Marijuana smoking is widespread in many countries, and the use of smoked synthetic cannabinoids is increasing. Smoking a marijuana joint leads to bronchodilation in both healthy subjects and asthmatics. The effects of Δ9-tetrahydrocannabinol and synthetic cannabinoids on human bronchus reactivity have not previously been investigated. Here, we sought to assess the effects of natural and synthetic cannabinoids on cholinergic bronchial contraction. Experimental Approach Human bronchi isolated from 88 patients were suspended in an organ bath and contracted by electrical field stimulation (EFS) in the presence of the phytocannabinoid Δ9-tetrahydrocannabinol, the endogenous 2-arachidonoylglycerol, the synthetic dual CB1 and CB2 receptor agonists WIN55,212-2 and CP55,940, the synthetic, CB2-receptor-selective agonist JWH-133 or the selective GPR55 agonist O-1602. The receptors involved in the response were characterized by using selective CB1 and CB2 receptor antagonists (SR141716 and SR144528 respectively). Key Results Δ9-tetrahydrocannabinol, WIN55,212-2 and CP55,940 induced concentration-dependent inhibition of cholinergic contractions, with maximum inhibitions of 39, 76 and 77% respectively. JWH-133 only had an effect at high concentrations. 2-Arachidonoylglycerol and O-1602 were devoid of any effect. Only CB1 receptors were involved in the response because the effects of cannabinoids were antagonized by SR141716, but not by SR144528. The cannabinoids did not alter basal tone or contractions induced by exogenous Ach. Conclusions and Implications Activation of prejunctional CB1 receptors mediates the inhibition of EFS-evoked cholinergic contraction in human bronchus. This mechanism may explain the acute bronchodilation produced by marijuana smoking. PMID:24467410

  1. Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation, potential implications for COX-2 inhibition

    PubMed Central

    Arehart, Eric; Stitham, Jeremiah; Asselbergs, Folkert W.; Douville, Karen; MacKenzie, Todd; Fetalvero, Kristina M.; Gleim, Scott; Kasza, Zsolt; Rao, Yamini; Martel, Laurie; Segel, Sharon; Robb, John; Kaplan, Aaron; Simons, Michael; Powell, Richard J.; Moore, Jason H.; Rimm, Eric B.; Martin, Kathleen A.

    2009-01-01

    Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 (COX-2) inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major endproduct of COX-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in three separate Caucasian cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease, but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low risk cohort (n=2263). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared to age and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent upon the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the COX-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute in part to the underlying adverse cardiovascular outcomes observed with COX-2 inhibition. PMID:18323528

  2. Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition.

    PubMed

    Arehart, Eric; Stitham, Jeremiah; Asselbergs, Folkert W; Douville, Karen; MacKenzie, Todd; Fetalvero, Kristina M; Gleim, Scott; Kasza, Zsolt; Rao, Yamini; Martel, Laurie; Segel, Sharon; Robb, John; Kaplan, Aaron; Simons, Michael; Powell, Richard J; Moore, Jason H; Rimm, Eric B; Martin, Kathleen A; Hwa, John

    2008-04-25

    Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition. PMID:18323528

  3. Inhibition of Adult Rat Retinal Ganglion Cells by D1-type Dopamine Receptor Activation

    PubMed Central

    Hayashida, Yuki; Rodríguez, Carolina Varela; Ogata, Genki; Partida, Gloria J.; Oi, Hanako; Stradleigh, Tyler W.; Lee, Sherwin C.; Colado, Anselmo Felipe; Ishida, Andrew T.

    2011-01-01

    The spike output of neural pathways can be regulated by modulating output neuron excitability and/or their synaptic inputs. Dopaminergic interneurons synapse onto cells that route signals to mammalian retinal ganglion cells, but it is unknown whether dopamine can activate receptors in these ganglion cells and, if it does, how this affects their excitability. Here, we show D1a-receptor-like immunoreactivity in ganglion cells identified in adult rats by retrogradely transported dextran, and that dopamine, D1-type receptor agonists, and cAMP analogs inhibit spiking in ganglion cells dissociated from adult rats. These ligands curtailed repetitive spiking during constant current injections, and reduced the number and rate of rise of spikes elicited by fluctuating current injections without significantly altering the timing of the remaining spikes. Consistent with mediation by D1-type receptors, SCH-23390 reversed the effects of dopamine on spikes. Contrary to a recent report, spike inhibition by dopamine was not precluded by blocking Ih. Consistent with the reduced rate of spike rise, dopamine reduced voltage-gated Na+ current (INa) amplitude and tetrodotoxin, at doses that reduced INa as moderately as dopamine, also inhibited spiking. These results provide the first direct evidence that D1-type dopamine receptor activation can alter mammalian retinal ganglion cell excitability, and demonstrate that dopamine can modulate spikes in these cells by a mechanism different from the pre- and postsynaptic means proposed by previous studies. To our knowledge, our results also provide the first evidence that dopamine receptor activation can reduce excitability without altering the temporal precision of spike firing. PMID:19940196

  4. Inhibition of the receptor-mediated virion attachment to a lipid membrane

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2012-10-01

    The forefront of the anti-viral defence is sometimes aimed at virion attachment to a host membrane. This step or, more specifically, virion contacts with cellular membrane receptors (or, e.g., glycolipids) can be inhibited by antibodies (or specially chosen or designed compounds) via their association with virions. In this case, the full-scale attachment of virions to a host membrane occurs via a subtle interplay of the formation and rupture of multiple virion-inhibitor and virion-receptor bonds. We present a kinetic model describing this interplay and illustrating general trends in the process under consideration.

  5. Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase.

    PubMed Central

    Palaparti, A; Li, Y; Anand-Srivastava, M B

    2000-01-01

    Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP(4-23) [des(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4-23)-NH(2)]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP(4-23). In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 microM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not

  6. Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: potential role of the NFkappa B pathway.

    PubMed

    Ismail, Wan Iryani W; King, Judy A; Anwar, Khawar; Pillay, Tahir S

    2013-08-01

    The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO-IR) and 3T3-L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor β-subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate-1 (IRS-1) on serine 307 (S307) in both CHO-IR cells and 3T3-L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin-stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling-1 and -3 (SOCS-1 and -3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO-IR cells were pre-treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR β-subunit and phosphorylation of IRS-1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs. PMID:23386514

  7. Inhibition of cation channel function at the nicotinic acethylcholine receptor from Torpedo: Agonist self-inhibition and anesthetic drugs

    SciTech Connect

    Forman, S.A.

    1989-01-01

    Modulation of the nicotinic acethylcholine receptor from Torpedo by cholinergic agonists, local anesthetics, and n-alkanols was studied using {sup 86}Rb{sup +} flux studies in sealed native Torpedo electroplaque membrane vesicles. Reliable concentration-response and kinetic data were obtained using manual ten sec filtration assays in vesicles partially blocked with alpha-bungarotoxin to remove spare receptors and quenched-flow assays to assess initial {sup 86}Rb{sup +} flux rates or the rate of drug-induced receptor inactivation. Concentration response relationships for the agonists acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, and (-)-nicotine are all bell-shape due to stimulation of cation channel opening at low concentrations and inhibition of channels at higher concentrations. The rate of agonist-induced fast desensitization (k{sub d}) increases with (acetylcholine) in parallel with channel activation, suggesting that desensitization proceeds from the open state and/or states in rapid equilibrium with it. At self-inhibitory acetylcholine concentrations, a new rapid inactivation (rate = k{sub f}) is observed before fast desensitization. The rate and extent of rapid inactivation is compatible with bimolecular association between acethylcholine and inhibitory site with K{sub B} = 40 mM.

  8. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    PubMed

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K

    2013-04-01

    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia. PMID:23276608

  9. Neurosteroid interactions with synaptic and extrasynaptic GABAa receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network excitability

    PubMed Central

    Chase Matthew, Carver; Doodipala Samba, Reddy

    2013-01-01

    Rationale Neurosteroids are steroids synthesized within the brain with rapid effects on neuronal excitability. Allopregnanolone, allotetrahydrodeoxycorticosterone, and androstanediol are three widely explored prototype endogenous neurosteroids. They have very different targets and functions compared to conventional steroid hormones. Neuronal GABAa receptors are one of the prime molecular targets of neurosteroids. Objective This review provides a critical appraisal of recent advances in the pharmacology of endogenous neurosteroids that interact with GABAa receptors in the brain. Neurosteroids possess distinct, characteristic effects on the membrane potential and current conductance of the neuron, mainly via potentiation of GABAa receptors at low concentrations and direct activation of receptor chloride channel at higher concentrations. The GABAa receptor mediates two types of inhibition, now characterized as synaptic (phasic) and extrasynaptic (tonic) inhibition. Synaptic release of GABA results in the activation of low-affinity γ2-containing synaptic receptors, while high-affinity δ-containing extrasynaptic receptors are persistently activated by the ambient GABA present in the extracellular fluid. Neurosteroids are potent positive allosteric modulators of synaptic and extrasynaptic GABAa receptors and therefore enhance both phasic and tonic inhibition. Tonic inhibition is specifically more sensitive to neurosteroids. The resulting tonic conductance generates a form of shunting inhibition that controls neuronal network excitability, seizure susceptibility, and behavior. Conclusion The growing understanding of the mechanisms of neurosteroid regulation of the structure and function of the synaptic and extrasynaptic GABAa receptors provide many opportunities to create improved therapies for sleep, anxiety, stress, epilepsy, and other neuropsychiatric conditions. PMID:24071826

  10. Endogenous inhibition of hippocampal LTD and depotentiation by vasoactive intestinal peptide VPAC1 receptors.

    PubMed

    Cunha-Reis, Diana; Aidil-Carvalho, Maria de Fatima; Ribeiro, Joaquim A

    2014-11-01

    Vasoactive intestinal peptide (VIP), an important modulator of hippocampal synaptic transmission, influences exploration and hippocampal-dependent learning in rodents. Homosynaptic long-term depression (LTD) and depotentiation are two plasticity phenomena implicated in learning of behavior flexibility and spatial novelty detection. In this study, we investigated the influence of endogenous VIP on LTD and depotentiation induced by low-frequency stimulation (1 Hz, 900 pulses) of the hippocampal CA1 area in vitro in juvenile and young adult rats, respectively. LTD and depotentiation were enhanced by the VIP receptor antagonist Ac-Tyr(1) , D-Phe(2) GRF (1-29), and the selective VPAC1 receptor antagonist, PG 97-269, but not the selective VPAC2 receptor antagonist, PG 99-465. This action was mimicked by an anti-VIP antibody, suggesting that VIP, and not pituitary adenylate cyclase-activating polypeptide (PACAP), is the endogenous mediator of these effects. Selective inhibition of PAC1 receptors with PACAP (6-38) enhanced depotentiation, but not LTD. VPAC1 receptor blockade also revealed LTD in young adult rats, an effect abolished by the GABAA antagonist bicuculline, evidencing an involvement of GABAergic transmission. We conclude that inhibition of LTD and depotentiation by endogenous VIP occurs through VPAC1 receptor-mediated mechanisms and suggest that disinhibition of pyramidal cell dendrites is the most likely physiological mechanism underlying this effect. As such, VPAC1 receptor ligands may be considered promising pharmacological targets for treatment of cognitive dysfunction in diseases involving altered GABAergic circuits and pathological saturation of LTP/LTD like Down's syndrome and temporal lobe epilepsy. PMID:24935659

  11. A Cannabinoid Receptor Agonist N-Arachidonoyl Dopamine Inhibits Adipocyte Differentiation in Human Mesenchymal Stem Cells

    PubMed Central

    Ahn, Seyeon; Yi, Sodam; Seo, Won Jong; Lee, Myeong Jung; Song, Young Keun; Baek, Seung Yong; Yu, Jinha; Hong, Soo Hyun; Lee, Jinyoung; Shin, Dong Wook; Jeong, Lak Shin; Noh, Minsoo

    2015-01-01

    Endocannabinoids can affect multiple cellular targets, such as cannabinoid (CB) receptors, transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and peroxisome proliferator-activated receptor γ (PPARγ). The stimuli to induce adipocyte differentiation in hBM-MSCs increase the gene transcription of the CB1 receptor, TRPV1 and PPARγ. In this study, the effects of three endocannabinoids, N-arachidonoyl ethanolamine (AEA), N-arachidonoyl dopamine (NADA) and 2-arachidonoyl glycerol (2-AG), on adipogenesis in hBM-MSCs were evaluated. The adipocyte differentiation was promoted by AEA whereas inhibited by NADA. No change was observed by the treatment of non-cytotoxic concentrations of 2-AG. The difference between AEA and NADA in the regulation of adipogenesis is associated with their effects on PPARγ transactivation. AEA can directly activate PPARγ. The effect of AEA on PPARγ in hBM-MSCs may prevail over that on the CB1 receptor mediated signal transduction, giving rise to the AEA-induced promotion of adipogenesis. In contrast, NADA had no effect on the PPARγ activity in the PPARγ transactivation assay. The inhibitory effect of NADA on adipogenesis in hBM-MSCs was reversed not by capsazepine, a TRPV1 antagonist, but by rimonabant, a CB1 antagonist/inverse agonist. Rimonabant by itself promoted adipogenesis in hBM-MSCs, which may be interpreted as the result of the inverse agonism of the CB1 receptor. This result suggests that the constantly active CB1 receptor may contribute to suppress the adipocyte differentiation of hBM-MSCs. Therefore, the selective CB1 agonists that are unable to affect cellular PPARγ activity inhibit adipogenesis in hBM-MSCs. PMID:25995819

  12. Farnesyl pyrophosphate inhibits epithelialization and wound healing through the glucocorticoid receptor.

    PubMed

    Vukelic, Sasa; Stojadinovic, Olivera; Pastar, Irena; Vouthounis, Constantinos; Krzyzanowska, Agata; Das, Sharmistha; Samuels, Herbert H; Tomic-Canic, Marjana

    2010-01-15

    Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing. PMID:19903814

  13. Farnesyl Pyrophosphate Inhibits Epithelialization and Wound Healing through the Glucocorticoid Receptor*

    PubMed Central

    Vukelic, Sasa; Stojadinovic, Olivera; Pastar, Irena; Vouthounis, Constantinos; Krzyzanowska, Agata; Das, Sharmistha; Samuels, Herbert H.; Tomic-Canic, Marjana

    2010-01-01

    Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing. PMID:19903814

  14. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR).

    PubMed Central

    Tabb, Michelle M; Kholodovych, Vladyslav; Grün, Felix; Zhou, Changcheng; Welsh, William J; Blumberg, Bruce

    2004-01-01

    Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk. PMID:14754570

  15. Improved guanide compounds which bind the CXCR4 co-receptor and inhibit HIV-1 infection

    PubMed Central

    Wilkinson, Royce A.; Pincus, Seth H.; Song, Kejing; Shepard, Joyce B.; Weaver, Alan J.; Labib, Mohamed E.; Teintze, Martin

    2013-01-01

    The G-protein coupled receptor CXCR4 is a co-receptor for HIV-1 infection and is involved in signaling cell migration and proliferation. In a previous study of non-peptide, guanide-based CXCR4-binding compounds, spermine and spermidine phenylguanides inhibited HIV-1 entry at low micromolar concentrations. Subsequently, crystal structures of CXCR4 were used to dock a series of naphthylguanide derivatives of the polyamines spermidine and spermine. Synthesis and evaluation of the naphthylguanide compounds identified our best compound, spermine tris-1-naphthylguanide, which bound CXCR4 with an IC50 of 40nM and inhibited the infection of TZM-bl cells with X4, but not R5, strains of HIV-1 with an IC50 of 50–100nM. PMID:23434419

  16. Midazolam inhibits long-term potentiation through modulation of GABAA receptors.

    PubMed

    Evans, M S; Viola-McCabe, K E

    1996-03-01

    Benzodiazepine drugs (BZ) are used for anxiety, insomnia, and seizures. They worsen memory, especially in large doses, but the mechanism of this action is uncertain. In micromolar concentrations, benzodiazepines have been shown to reduce long-term potentiation (LTP), which could be a cellular basis for their amnesic action. We have found that the LTP-inhibiting effects of BZ occur in the nanomolar concentrations attained in humans, and that this effect occurs through modulation of GABAA receptor function. We recorded extracellular synaptic input/output (I/O) curves for population spikes (PS) and EPSPs in rat hippocampal slices before and after induction of LTP. LTP increased maximal PS and EPSPs and shifted I/O curves for PS and EPSPs to the left, reflecting increased synaptic responsiveness after LTP. Curves relating EPSPs to PS were also shifted, so that after LTP larger PS were elicited for the same size EPSP (E-S potentiation). Midazolam (0.5 microM) markedly inhibited the left-shift in PS I/O curves due to E-S potentiation but did not significantly affect other parameters. 8-Phenyltheophylline (10 microM), an adenosine receptor antagonist, did not prevent midazolam inhibition of LTP. Bicuculline, a GABAA receptor antagonist, caused a dose-dependent antagonism of midazolam's LTP inhibition. Our results suggest that benzodiazepines reduce LTP primarily through reduction of E-S potentiation, and that this effect occurs through modulation of GABAA receptor function. This could in part account for the ability of benzodiazepines to disturb new memory formation. PMID:8783210

  17. Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves.

    PubMed Central

    Ishac, E. J.; Jiang, L.; Lake, K. D.; Varga, K.; Abood, M. E.; Kunos, G.

    1996-01-01

    1. Activation of CB1 receptors by plant cannabinoids or the endogenous ligand, anandamide, causes hypotension via a sympathoinhibitory action in anaesthetized rats. In mouse isolated vas deferens, activation of CB1 receptors inhibits the electrically evoked twitch response. To determine if these effects are related to presynaptic inhibition of noradrenaline (NA) release, we examined the effects of delta 9-tetrahydrocannabinol (delta 9-THC), anandamide and the CB1 antagonist, SR141716A, on exocytotic NA release in rat isolated atria and vasa deferentia. 2. In isolated atria and vasa deferentia preloaded with [3H]-NA, electrical field stimulation caused [3H]-NA release, which was abolished by tetrodotoxin 0.5 microM and concentration-dependently inhibited by delta 9-THC or anandamide, 0.3-10 microM. The inhibitory effect of delta 9-THC and anandamide was competitively antagonized by SR 141716A, 1-10 microM. 3. Tyramine, 1 microM, also induced [3H]-NA release, which was unaffected by tetrodotoxin, delta 9-THC or anandamide in either atria or vasa deferentia. 4. CB1 receptor mRNA is present in the superior cervical ganglion, as well as in whole brain, cerebellum, hypothalamus, spleen, and vas deferens and absent in medulla oblongata and atria, as demonstrated by reverse transcription-polymerase chain reaction. There was no evidence of the presence of CB1A receptor mRNA in ganglia, brain, or cerebellum. These results suggest that activation of presynaptic CB1 receptors located on peripheral sympathetic nerve terminals mediate sympathoinhibitory effects in vitro and in vivo. Images Figure 5 Figure 6 PMID:8864538

  18. Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma

    PubMed Central

    Farnebo, Lovisa; Shahangian, Arash; Lee, Yunqin; Shin, June Ho; Scheeren, Ferenc A.; Sunwoo, John B.

    2015-01-01

    Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth. PMID:25846753

  19. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    PubMed

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-01

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies. PMID:26849939

  20. The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Cheng, Max A.; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers. PMID:25221777

  1. Role of spinal GABAA receptors in pudendal inhibition of nociceptive and nonnociceptive bladder reflexes in cats.

    PubMed

    Xiao, Zhiying; Reese, Jeremy; Schwen, Zeyad; Shen, Bing; Wang, Jicheng; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2014-04-01

    Picrotoxin, an antagonist for γ-aminobutyric acid receptor subtype A (GABAA), was used to investigate the role of GABAA receptors in nociceptive and nonnociceptive reflex bladder activities and pudendal inhibition of these activities in cats under α-chloralose anesthesia. Acetic acid (AA; 0.25%) was used to irritate the bladder and induce nociceptive bladder overactivity, while saline was used to distend the bladder and induce nonnociceptive bladder activity. To modulate the bladder reflex, pudendal nerve stimulation (PNS) was applied at multiple threshold (T) intensities for inducing anal sphincter twitching. AA irritation significantly (P < 0.01) reduced bladder capacity to 34.3 ± 7.1% of the saline control capacity, while PNS at 2T and 4T significantly (P < 0.01) increased AA bladder capacity to 84.0 ± 7.8 and 93.2 ± 15.0%, respectively, of the saline control. Picrotoxin (0.4 mg it) did not change AA bladder capacity but completely removed PNS inhibition of AA-induced bladder overactivity. Picrotoxin (iv) only increased AA bladder capacity at a high dose (0.3 mg/kg) but significantly (P < 0.05) reduced 2T PNS inhibition at low doses (0.01-0.1 mg/kg). During saline cystometry, PNS significantly (P < 0.01) increased bladder capacity to 147.0 ± 7.6% at 2T and 172.7 ± 8.9% at 4T of control capacity, and picrotoxin (0.4 mg it or 0.03-0.3 mg/kg iv) also significantly (P < 0.05) increased bladder capacity. However, picrotoxin treatment did not alter PNS inhibition during saline infusion. These results indicate that spinal GABAA receptors have different roles in controlling nociceptive and nonnociceptive reflex bladder activities and in PNS inhibition of these activities. PMID:24523385

  2. Neuropeptide Y inhibits the trigeminovascular pathway through NPY Y1 receptor: implications for migraine

    PubMed Central

    Oliveira, Margarida-Martins; Akerman, Simon; Tavares, Isaura; Goadsby, Peter J.

    2016-01-01

    Abstract Migraine is a painful neurologic disorder with premonitory symptomatology that can include disturbed appetite. Migraine pathophysiology involves abnormal activation of trigeminocervical complex (TCC) neurons. Neuropeptide Y (NPY) is synthesized in the brain and is involved in pain modulation. NPY receptors are present in trigeminal ganglia and trigeminal nucleus caudalis suggesting a role in migraine pathophysiology. The present study aimed to determine the effect of systemic administration of NPY on TCC neuronal activity in response to dural nociceptive trigeminovascular activation. We performed in vivo electrophysiology in anesthetized rats, administered NPY (10, 30, and 100 µg·kg−1), and investigated the receptors involved by studying NPY Y1 (30 µg·kg−1), Y2 (30 µg·kg−1), and Y5 receptor agonists (100·µg·kg−1), and NPY Y1 receptor antagonist (30 µg·kg−1). NPY (30 and 100 µg·kg−1) significantly reduced TCC neuronal firing in response to dural-evoked trigeminovascular activation, but only NPY (30 µg·kg−1) significantly reduced spontaneous trigeminal firing. NPY Y1 receptor agonist also significantly reduced dural-evoked and spontaneous TCC neuronal firing. NPY (10 µg·kg−1), NPY Y2, and Y5 receptor agonists, and the NPY Y1 receptor antagonist had no significant effects on nociceptive dural-evoked neuronal firing in the TCC or spontaneous trigeminal firing. This study demonstrates that NPY dose dependently inhibits dural-evoked trigeminal activity, through NPY Y1 receptor activation, indicating antinociceptive actions of NPY in a migraine animal model. Based on the role of NPY in appetite regulation, it is possible that disruption of the NPY system might explain changes of appetite in migraineurs. PMID:27023421

  3. Neuropeptide Y inhibits the trigeminovascular pathway through NPY Y1 receptor: implications for migraine.

    PubMed

    Oliveira, Margarida-Martins; Akerman, Simon; Tavares, Isaura; Goadsby, Peter J

    2016-08-01

    Migraine is a painful neurologic disorder with premonitory symptomatology that can include disturbed appetite. Migraine pathophysiology involves abnormal activation of trigeminocervical complex (TCC) neurons. Neuropeptide Y (NPY) is synthesized in the brain and is involved in pain modulation. NPY receptors are present in trigeminal ganglia and trigeminal nucleus caudalis suggesting a role in migraine pathophysiology. The present study aimed to determine the effect of systemic administration of NPY on TCC neuronal activity in response to dural nociceptive trigeminovascular activation. We performed in vivo electrophysiology in anesthetized rats, administered NPY (10, 30, and 100 µg·kg), and investigated the receptors involved by studying NPY Y1 (30 µg·kg), Y2 (30 µg·kg), and Y5 receptor agonists (100·µg·kg), and NPY Y1 receptor antagonist (30 µg·kg). NPY (30 and 100 µg·kg) significantly reduced TCC neuronal firing in response to dural-evoked trigeminovascular activation, but only NPY (30 µg·kg) significantly reduced spontaneous trigeminal firing. NPY Y1 receptor agonist also significantly reduced dural-evoked and spontaneous TCC neuronal firing. NPY (10 µg·kg), NPY Y2, and Y5 receptor agonists, and the NPY Y1 receptor antagonist had no significant effects on nociceptive dural-evoked neuronal firing in the TCC or spontaneous trigeminal firing. This study demonstrates that NPY dose dependently inhibits dural-evoked trigeminal activity, through NPY Y1 receptor activation, indicating antinociceptive actions of NPY in a migraine animal model. Based on the role of NPY in appetite regulation, it is possible that disruption of the NPY system might explain changes of appetite in migraineurs. PMID:27023421

  4. Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

    PubMed

    Basiorka, Ashley A; McGraw, Kathy L; De Ceuninck, Leentje; Griner, Lori N; Zhang, Ling; Clark, Justine A; Caceres, Gisela; Sokol, Lubomir; Komrokji, Rami S; Reuther, Gary W; Wei, Sheng; Tavernier, Jan; List, Alan F

    2016-06-15

    In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR. PMID:27197154

  5. Molecular Mechanisms of Cross-inhibition Between Nicotinic Acetylcholine Receptors and P2X Receptors in Myenteric Neurons and HEK-293 cells

    PubMed Central

    Decker, Dima A.; Galligan, James J.

    2010-01-01

    Background P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit-containing nAChRs. Methods The function of P2X2 and α3β4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Results In HEK-293 cells expressing α3β4 nAChRs and P2X2 receptors, co-application of ATP and ACh caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR)(P >0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μM), but not high (≥100 μM) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with α3β4 nAChRs. Conclusions The C-terminal tail of P2X2 receptors mediates cross-inhibition between α3β4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. PMID:20426799

  6. Beta2-adrenergic receptor stimulation inhibits nitric oxide generation by Mycobacterium avium infected macrophages.

    PubMed

    Boomershine, C S; Lafuse, W P; Zwilling, B S

    1999-11-01

    Catecholamine regulation of nitric oxide (NO) production by IFNgamma-primed macrophages infected with Mycobacterium avium was investigated. Epinephrine treatment of IFNgamma-primed macrophages at the time of M. avium infection inhibited the anti-mycobacterial activity of the cells. The anti-mycobacterial activity of macrophages correlated with NO production. Using specific adrenergic receptor agonists, the abrogation of mycobacterial killing and decreased NO production by catecholamines was shown to be mediated via the beta2-adrenergic receptor. Elevation of intracellular cAMP levels mimicked the catecholamine-mediated inhibition of NO in both M. avium infected and LPS stimulated macrophages. Specific inhibitors of both adenylate cyclase and protein kinase A prevented the beta2-adrenoceptor-mediated inhibition of nitric oxide production. Beta2-adrenoreceptor stimulation at the time of M. avium infection of IFNgamma-primed macrophages also inhibited expression of iNOS mRNA. These observations show that catecholamine hormones can affect the outcome of macrophage-pathogen interactions and suggest that one result of sympathetic nervous system activation is the suppression of the capacity of macrophages to produce anti-microbial effector molecules. PMID:10580815

  7. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  8. A New Transferrin Receptor Aptamer Inhibits New World Hemorrhagic Fever Mammarenavirus Entry.

    PubMed

    Maier, Keith E; Jangra, Rohit K; Shieh, Kevin R; Cureton, David K; Xiao, Hui; Snapp, Erik L; Whelan, Sean P; Chandran, Kartik; Levy, Matthew

    2016-01-01

    Pathogenic New World hemorrhagic fever mammarenaviruses (NWM) utilize Glycoprotein 1 (GP1) to target the apical domain of the human transferrin receptor (hTfR) for facilitating cell entry. However, the conservation between their GP1s is low. Considering this and the slow evolutionary progression of mammals compared to viruses, therapeutic targeting of hTfR provides an attractive avenue for cross-strain inhibition and diminishing the likelihood of escape mutants. Aptamers present unique advantages for the development of inhibitors to vial entry, including ease of synthesis, lack of immunogenicity, and potentially cold-chain breaking solutions to diseases endemic to South America. Here, recognizing that in vivo competition with the natural ligand, transferrin (Tf), likely drove the evolution of GP1 to recognize the apical domain, we performed competitive in vitro selections against hTfR-expressing cells with supplemented Tf. The resultant minimized aptamer, Waz, binds the apical domain of the receptor and inhibits infection of human cells by recombinant NWM in culture (EC50 ~400 nmol/l). Aptamer multimerization further enhanced inhibition >10-fold (EC50 ~30 nmol/l). Together, our results highlight the ability to use a competitor to bias the outcome of a selection and demonstrate how avidity effects can be leveraged to enhance both aptamer binding and the potency of viral inhibition. PMID:27219515

  9. Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection.

    PubMed

    Chan, Kuan Rong; Zhang, Summer Li-Xin; Tan, Hwee Cheng; Chan, Ying Kai; Chow, Angelia; Lim, Angeline Pei Chiew; Vasudevan, Subhash G; Hanson, Brendon J; Ooi, Eng Eong

    2011-07-26

    The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes. PMID:21746897

  10. Delayed Gelatinase Inhibition Induces Reticulon 4 Receptor Expression in the Peri-Infarct Cortex.

    PubMed

    Nardai, Sándor; Dobolyi, Arpád; Skopál, Judit; Lakatos, Kinga; Merkely, Béla; Nagy, Zoltán

    2016-04-01

    Matrix metalloproteinase (MMP) inhibition can potentially prevent hemorrhagic transformation following cerebral infarction; however, delayed-phase MMP activity is also necessary for functional recovery after experimental stroke. We sought to identify potential mechanisms responsible for the impaired recovery associated with subacute MMP inhibition in a transient middle cerebral artery occlusion model of focal ischemia in CD rats. Gelatinase inhibition was achieved by intracerebral injection of the Fn-439 MMP inhibitor 7 days after stroke. Treatment efficacy was determined on day 9 by in situ gelatin zymography. The peri-infarct cortex was identified by triphenyl tetrazolium chloride staining, and tissue samples were dissected for TaqMan array gene-expression study. Of 84 genes known to influence poststroke regeneration, we found upregulation of mRNA for the reticulon 4 receptor (Rtn4r), a major inhibitor of regenerative nerve growth in the adult CNS, and borderline expression changes for 3 additional genes (DCC, Jun, andNgfr). Western blot confirmed increased Rtn4r protein in the peri-infarct cortex of treated animals, and double immunolabeling showed colocalization primarily with the S100 astrocyte marker. These data suggest that increased Rtn4 receptor expression in the perilesional cortex may contribute to the impaired regeneration associated with MMP inhibition in the subacute phase of cerebral infarction. PMID:26945033

  11. Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways.

    PubMed

    Pandey, Manoj K; Kale, Vijay P; Song, Chunhua; Sung, Shen-shu; Sharma, Arun K; Talamo, Giampaolo; Dovat, Sinisa; Amin, Shantu G

    2014-10-01

    Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-κB) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1α-induced chemotaxis of MM cells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MM cells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MM cells. PMID:25034231

  12. PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor-positive breast cancer

    PubMed Central

    Bosch, Ana; Li, Zhiqiang; Bergamaschi, Anna; Ellis, Haley; Toska, Eneda; Prat, Aleix; Tao, Jessica J.; Spratt, Daniel E.; Viola-Villegas, Nerissa T.; Castel, Pau; Minuesa, Gerard; Morse, Natasha; Rodón, Jordi; Ibrahim, Yasir; Cortes, Javier; Perez-Garcia, Jose; Galvan, Patricia; Grueso, Judit; Guzman, Marta; Katzenellenbogen, John A.; Kharas, Michael; Lewis, Jason S.; Dickler, Maura; Serra, Violeta; Rosen, Neal; Chandarlapaty, Sarat; Scaltriti, Maurizio; Baselga, José

    2015-01-01

    Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective PI3Kα inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER binding sites and increased occupancy by the ER of promoter regions of upregulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors, and that combined PI3K and ER inhibition is a rational approach to target these tumors. PMID:25877889

  13. 5-HT2B Receptor Antagonists Inhibit Fibrosis and Protect from RV Heart Failure

    PubMed Central

    Janssen, Wiebke; Schymura, Yves; Novoyatleva, Tatyana; Luitel, Himal; Tretyn, Aleksandra; Pullamsetti, Soni Savai; Weissmann, Norbert; Seeger, Werner; Ghofrani, Hossein Ardeschir; Schermuly, Ralph Theo

    2015-01-01

    Objective. The serotonin (5-HT) pathway was shown to play a role in pulmonary hypertension (PH), but its functions in right ventricular failure (RVF) remain poorly understood. The aim of the current study was to investigate the effects of Terguride (5-HT2A and 2B receptor antagonist) or SB204741 (5-HT2B receptor antagonist) on right heart function and structure upon pulmonary artery banding (PAB) in mice. Methods. Seven days after PAB, mice were treated for 14 days with Terguride (0.2 mg/kg bid) or SB204741 (5 mg/kg day). Right heart function and remodeling were assessed by right heart catheterization, magnetic resonance imaging (MRI), and histomorphometric methods. Total secreted collagen content was determined in mouse cardiac fibroblasts isolated from RV tissues. Results. Chronic treatment with Terguride or SB204741 reduced right ventricular fibrosis and showed improved heart function in mice after PAB. Moreover, 5-HT2B receptor antagonists diminished TGF-beta1 induced collagen synthesis of RV cardiac fibroblasts in vitro. Conclusion. 5-HT2B receptor antagonists reduce collagen deposition, thereby inhibiting right ventricular fibrosis. Chronic treatment prevented the development and progression of pressure overload-induced RVF in mice. Thus, 5-HT2B receptor antagonists represent a valuable novel therapeutic approach for RVF. PMID:25667920

  14. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    PubMed

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents. PMID:21763704

  15. Frondoside A inhibits breast cancer metastasis and antagonizes prostaglandin E receptors EP4 and EP2

    PubMed Central

    Ma, Xinrong; Kundu, Namita; Collin, Peter D; Goloubeva, Olga; Fulton, Amy

    2013-01-01

    Frondoside A, derived from the sea cucumber Cucumaria frondosa has demonstrable anticancer activity in several models, however, the ability of Frondoside A to affect tumor metastasis has not been reported. Using a syngeneic murine model of metastatic breast cancer, we now show that Frondoside A has potent antimetastatic activity. Frondoside A given i.p. to mice bearing mammary gland implanted mammary tumors, inhibits spontaneous tumor metastasis to the lungs. The elevated Cyclooxygenase -2 activity in many malignancies promotes tumor growth and metastasis by producing high levels of PGE2 which acts on the prostaglandin E receptors, chiefly EP4 and EP2. We examined the ability of Frondoside A to modulate the functions of these EP receptors. We now show that Frondoside A antagonizes the prostaglandin E receptors EP2 and EP4. 3H-PGE2 binding to recombinant EP2 or EP4-expressing cells was inhibited by Frondoside A at low μM concentrations. Likewise, EP4 or EP2-linked activation of intracellular cAMP as well as EP4-mediated ERK1/2 activation were also inhibited by Frondoside A. Consistent with the antimetastatic activity observed in vivo, migration of tumor cells in vitro in response to EP4 or EP2 agonists was also inhibited by Frondoside A. These studies identify a new function for an agent with known antitumor activity, and show that the antimetastatic activity may be due in part to a novel mechanism of action. These studies add to the growing body of evidence that Frondoside A may be a promising new agent with potential to treat cancer and may also represent a potential new modality to antagonize EP4. PMID:21761157

  16. Frondoside A inhibits breast cancer metastasis and antagonizes prostaglandin E receptors EP4 and EP2.

    PubMed

    Ma, Xinrong; Kundu, Namita; Collin, Peter D; Goloubeva, Olga; Fulton, Amy M

    2012-04-01

    Frondoside A, derived from the sea cucumber Cucumaria frondosa has demonstrable anticancer activity in several models, however, the ability of Frondoside A to affect tumor metastasis has not been reported. Using a syngeneic murine model of metastatic breast cancer, we now show that Frondoside A has potent antimetastatic activity. Frondoside A given i.p. to mice bearing mammary gland-implanted mammary tumors, inhibits spontaneous tumor metastasis to the lungs. The elevated Cyclooxygenase-2 activity in many malignancies promotes tumor growth and metastasis by producing high levels of PGE(2) which acts on the prostaglandin E receptors, chiefly EP4 and EP2. We examined the ability of Frondoside A to modulate the functions of these EP receptors. We now show that Frondoside A antagonizes the prostaglandin E receptors EP2 and EP4. (3)H-PGE(2) binding to recombinant EP2 or EP4-expressing cells was inhibited by Frondoside A at low μM concentrations. Likewise, EP4 or EP2-linked activation of intracellular cAMP as well as EP4-mediated ERK1/2 activation were also inhibited by Frondoside A. Consistent with the antimetastatic activity observed in vivo, migration of tumor cells in vitro in response to EP4 or EP2 agonists was also inhibited by Frondoside A. These studies identify a new function for an agent with known antitumor activity, and show that the antimetastatic activity may be due in part to a novel mechanism of action. These studies add to the growing body of evidence that Frondoside A may be a promising new agent with potential to treat cancer and may also represent a potential new modality to antagonize EP4. PMID:21761157

  17. The Cannabinoid Delta-9-tetrahydrocannabinol Mediates Inhibition of Macrophage Chemotaxis to RANTES/CCL5 through the CB2 Receptor

    PubMed Central

    Raborn, Erinn S.; Marciano-Cabral, Francine; Buckley, Nancy E.; Martin, Billy R.; Cabral, Guy A.

    2009-01-01

    The chemotactic response of murine peritoneal macrophages to RANTES/CCL5 was inhibited significantly following pretreatment with delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana. Significant inhibition of this chemokine directed migratory response was obtained also when the full cannabinoid agonist CP55940 was used. The CB2 receptor-selective ligand O-2137 exerted a robust inhibition of chemotaxis while the CB1 receptor-selective ligand ACEA had a minimal effect. The THC-mediated inhibition was reversed by the CB2 receptor-specific antagonist SR144528 but not by the CB1 receptor-specific antagonist SR141716A. In addition, THC treatment had a minimal effect on the chemotactic response of peritoneal macrophages from CB2 knockout mice. Collectively, these results suggest that cannabinoids act through the CB2 receptor to trans-deactivate migratory responsiveness to RANTES/CCL5. Furthermore, the results suggest that the CB2 receptor may be a constituent element of a network of G protein-coupled receptor signal transductional systems, inclusive of chemokine receptors, that act coordinately to modulate macrophage migration. PMID:18247131

  18. The Interaction between the Drosophila Secreted Protein Argos and the Epidermal Growth Factor Receptor Inhibits Dimerization of the Receptor and Binding of Secreted Spitz to the Receptor

    PubMed Central

    Jin, Ming-hao; Sawamoto, Kazunobu; Ito, Mikiko; Okano, Hideyuki

    2000-01-01

    Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor α-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases. PMID:10688656

  19. Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

    SciTech Connect

    Forman, S.A.; Miller, K.W. )

    1989-02-21

    The relationship between the high-affinity procaine channel inhibition site and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid {sup 86}Rb{sup +} quenched-flux assays at 4 {degree}C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with {alpha}-bungarotoxin ({alpha}-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations alone, AChR undergoes one phase of inactivation in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting ACh concentrations rapid inactivation complete in 30-75 ms is followed by fast desensitization at the same k{sub d} observed without procaine. The dependence of k{sub r} on (procaine) is consistent with a bimolecular association between procaine and its AChR site. Inhibition of AChR function by mixtures of procaine plus self-inhibiting concentrations of ACh or suberyldicholine was studied by reducing the level of {alpha}-BTX block in vesicles. The data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and k{sub r}'s dependence on (ACh) in channel-activating range closely parallels that of {sup 86}Rb{sup +} flux response to ACh.

  20. Somatostatin receptor-1 induces cell cycle arrest and inhibits tumor growth in pancreatic cancer.

    PubMed

    Li, Min; Wang, Xiaochi; Li, Wei; Li, Fei; Yang, Hui; Wang, Hao; Brunicardi, F Charles; Chen, Changyi; Yao, Qizhi; Fisher, William E

    2008-11-01

    Functional somatostatin receptors (SSTR) are lost in human pancreatic cancer. Transfection of SSTR-1 inhibited pancreatic cancer cell proliferation in vitro. We hypothesize that stable transfection of SSTR-1 may inhibit pancreatic cancer growth in vivo possibly through cell cycle arrest. In this study, we examined the expression of SSTR-1 mRNA in human pancreatic cancer tissue specimens, and investigated the effect of SSTR-1 overexpression on cell proliferation, cell cycle, and tumor growth in a subcutaneous nude mouse model. We found that SSTR-1 mRNA was downregulated in the majority of pancreatic cancer tissue specimens. Transfection of SSTR-1 caused cell cycle arrest at the G(0)/G(1) growth phase, with a corresponding decline of cells in the S (mitotic) phase. The overexpression of SSTR-1 significantly inhibited subcutaneous tumor size by 71% and 43% (n = 5, P < 0.05, Student's t-test), and inhibited tumor weight by 69% and 47% (n = 5, P < 0.05, Student's t-test), in Panc-SSTR-1 and MIA-SSTR-1 groups, respectively, indicating the potent inhibitory effect of SSTR-1 on pancreatic cancer growth. Our data demonstrate that overexpression of SSTR-1 significantly inhibits pancreatic cancer growth possibly through cell cycle arrest. This study suggests that gene therapy with SSTR-1 may be a potential adjuvant treatment for pancreatic cancer. PMID:18823376

  1. Somatostatin Receptor-1 Induces Cell Cycle Arrest and Inhibits Tumor Growth in Pancreatic Cancer

    PubMed Central

    Li, Min; Wang, Xiaochi; Li, Wei; Li, Fei; Yang, Hui; Wang, Hao; Brunicardi, F. Charles; Chen, Changyi; Yao, Qizhi; Fisher, William E.

    2010-01-01

    Functional somatostatin receptors (SSTRs) are lost in human pancreatic cancer. Transfection of SSTR-1 inhibited pancreatic cancer cell proliferation in vitro. We hypothesize that stable transfection of SSTR-1 may inhibit pancreatic cancer growth in vivo possibly through cell cycle arrest. In this study, we examined the expression of SSTR-1 mRNA in human pancreatic cancer tissue specimens, and investigated the effect of SSTR-1 overexpression on cell proliferation, cell cycle, and tumor growth in in a subcutaneous nude mouse model. We found that SSTR-1 mRNA was downregulated in the majority of pancreatic cancer tissue specimens. Transfection of SSTR-1 caused cell cycle arrest at the G0/G1 growth phase, with a corresponding decline of cells in the S (mitotic) phase. The overexpression of SSTR-1 significantly inhibited subcutaneous tumor size by 71% and 43% (n=5, p<0.05, t-test), and inhibited tumor weight by 69% and 47%, (n=5, p<0.05, t-test), in Panc-SSTR-1 and MIA-SSTR-1 groups, respectively, indicating the potent inhibitory effect of SSTR-1 on pancreatic cancer growth. Our data demonstrate that overexpression of SSTR-1 significantly inhibits pancreatic cancer growth possibly through cell cycle arrest. This study suggests that gene therapy with SSTR-1 may be a potential adjuvant treatment for pancreatic cancer. PMID:18823376

  2. Bisphenol A inhibits cultured mouse ovarian follicle growth partially via the aryl hydrocarbon receptor signaling pathway

    PubMed Central

    Ziv-Gal, Ayelet; Craig, Zelieann R.; Wang, Wei; Flaws, Jodi A.

    2013-01-01

    Bisphenol A (BPA) is an endocrine disruptor that inhibits growth of mouse ovarian follicles and disrupts steroidogenesis at a dose of 438 μM. However, the effects of lower doses of BPA and its mechanism of action in ovarian follicles are unknown. We hypothesized that low doses of BPA inhibit follicular growth and decrease estradiol levels through the aryl hydrocarbon receptor (AHR) pathway. Antral follicles from wild-type and Ahr knock-out (AhrKO) mice were cultured for 96 hours. Follicle diameters and estradiol levels then were compared in wild-type and AhrKO follicles ± BPA (0.004 - 438 μM). BPA inhibited follicle growth (110 - 438 μM) and decreased estradiol levels (43.8 - 438 μM) in wild-type and AhrKO follicles. However, at BPA 110 μM, inhibition of growth in AhrKO follicles was attenuated compared to wild-type follicles. These data suggest that BPA may inhibit follicle growth partially via the AHR pathway, whereas its effects on estradiol synthesis likely involve other mechanisms. PMID:23928317

  3. The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

    PubMed Central

    Lehmann, Geniece M.; Xi, Xia; Kulkarni, Ajit A.; Olsen, Keith C.; Pollock, Stephen J.; Baglole, Carolyn J.; Gupta, Shikha; Casey, Ann E.; Huxlin, Krystel R.; Sime, Patricia J.; Feldon, Steven E.; Phipps, Richard P.

    2011-01-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR−/− fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. PMID:21406171

  4. Targeting Platelet-Derived Growth Factor Receptor β(+) Scaffold Formation Inhibits Choroidal Neovascularization.

    PubMed

    Strittmatter, Karin; Pomeroy, Hayley; Marneros, Alexander G

    2016-07-01

    Neovascular age-related macular degeneration is among the most common causes of irreversible blindness and manifests with choroidal neovascularization (CNV). Anti-vascular endothelial growth factor-A therapies are only partially effective and their chronic administration may impair functions of the choriocapillaris and retina. Thus, novel therapeutic targets are needed urgently. We have observed in a laser-induced model of CNV that a platelet-derived growth factor receptor β positive (PDGFRβ(+)) scaffold is formed before infiltration of neovessels into this scaffold to form CNV lesions, and that this scaffold limits the extent of neovascularization. Based on these observations we hypothesized that ablation of proliferating PDGFRβ(+) cells to prevent the formation of this scaffold might inhibit CNV growth and present a novel therapeutic approach for neovascular age-related macular degeneration. To test this hypothesis we targeted proliferating PDGFRβ(+) cells through independent distinct approaches after laser injury: i) by using an inducible genetic model to inhibit specifically proliferating PDGFRβ(+) cells, ii) by treating mice with a neutralizing anti-PDGFRβ antibody, iii) by administering an anti-PDGF-AB/BB aptamer, and iv) by using small chemical inhibitor approaches. The results show that therapeutic targeting of proliferating PDGFRβ(+) cells potently inhibits the formation of the pericyte-like scaffold, with concomitant attenuation of CNV. Moreover, we show that early inhibition of PDGFRβ(+) cell proliferation before neovessel formation is sufficient to inhibit scaffold formation and neovascularization. PMID:27338108

  5. Neurotrophins inhibit major histocompatibility class II inducibility of microglia: Involvement of the p75 neurotrophin receptor

    PubMed Central

    Neumann, Harald; Misgeld, Thomas; Matsumuro, Kenji; Wekerle, Hartmut

    1998-01-01

    Major histocompatibility complex (MHC) molecules are rare in the healthy brain tissue, but are heavily expressed on microglial cells after inflammatory or neurodegenerative processes. We studied the conditions leading to the induction of MHC class II molecules in microglia by using explant cultures of neonatal rat hippocampus, a model of interacting neuronal networks. Interferon-γ (IFN-γ)-dependent MHC class II inducibility in microglia cells was very low, but strongly increased in the hippocampal slices after the blockade of neuronal activity by neurotoxins [tetrodotoxin (TTX), ω-conotoxin] or glutamate antagonists. None of these agents acted directly on isolated microglia cells. We found that neurotrophins modulate microglial MHC class II expression. MHC class II inducibility was enhanced by neutralization of neurotrophins produced locally within the cultured tissues and was inhibited by the addition of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT3). NGF and, to a lower extent, NT3 acted directly on isolated microglia via the p75 neurotrophin receptor and inhibited MHC class II inducibility as shown by blockade of the p75 neurotrophin receptor with antibodies. Our data suggest that neurotrophins secreted by electrically active neurons control the antigen-presenting potential of microglia cells, and indicate that this effect is mediated partly via the p75 neurotrophin receptor. PMID:9576961

  6. Antisense oligonucleotides targeting the progesterone receptor inhibit hormone-independent breast cancer growth in mice

    PubMed Central

    Lamb, Caroline A; Helguero, Luisa A; Giulianelli, Sebastián; Soldati, Rocío; Vanzulli, Silvia I; Molinolo, Alfredo; Lanari, Claudia

    2005-01-01

    Introduction Previous data from our laboratory suggested that progesterone receptors (PRs) are involved in progestin-independent growth of mammary carcinomas. To investigate this possibility further, we studied the effects of PR antisense oligodeoxynucleotides (asPR) on in vivo tumor growth. Method BALB/c mice with subcutaneous 25 mm2 mammary carcinomas expressing estrogen receptor-α and PR were either injected intraperitoneally with 1 mg asPR every 24 or 12 hours for 5–10 days, or subcutaneously with RU 486 (6.5 mg/kg body weight) every 24 hours. Control mice received vehicle or scPR. Results Significant inhibition of tumor growth as well as a significant decrease in bromodeoxyuridine uptake was observed in asPR-treated mice, which correlated with histological signs of regression and increased apoptosis. Mice treated with RU 486 experienced almost complete tumor regression. No differences were detected between vehicle-treated and scPR-treated mice. Anti-progestin-treated and asPR-treated mice were in a continuous estrous/meta-estrous state. Decreased phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2 levels and estrogen receptor-α expression were observed as late events in RU 486-treated and asPR-treated mice with regressing tumors. Conclusion We demonstrate, for the first time, inhibition of tumor growth in vivo using asPR. Our results provide further evidence for a critical and hierarchical role of the PR pathway in mammary carcinomas. PMID:16457691

  7. Nanoparticle Targeting and Cholesterol Flux Through Scavenger Receptor Type B-1 Inhibits Cellular Exosome Uptake

    PubMed Central

    Plebanek, Michael P.; Mutharasan, R. Kannan; Volpert, Olga; Matov, Alexandre; Gatlin, Jesse C.; Thaxton, C. Shad

    2015-01-01

    Exosomes are nanoscale vesicles that mediate intercellular communication. Cellular exosome uptake mechanisms are not well defined partly due to the lack of specific inhibitors of this complex cellular process. Exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts, and can be blocked by non-specific depletion of plasma membrane cholesterol. Scavenger receptor type B-1 (SR-B1), found in lipid rafts, is a receptor for cholesterol-rich high-density lipoproteins (HDL). We hypothesized that a synthetic nanoparticle mimic of HDL (HDL NP) that binds SR-B1 and removes cholesterol through this receptor would inhibit cellular exosome uptake. In cell models, our data show that HDL NPs bind SR-B1, activate cholesterol efflux, and attenuate the influx of esterified cholesterol. As a result, HDL NP treatment results in decreased dynamics and clustering of SR-B1 contained in lipid rafts and potently inhibits cellular exosome uptake. Thus, SR-B1 and targeted HDL NPs provide a fundamental advance in studying cholesterol-dependent cellular uptake mechanisms. PMID:26511855

  8. A novel thromboxane receptor antagonist, nstpbp5185, inhibits platelet aggregation and thrombus formation in animal models.

    PubMed

    Huang, Shiu-Wen; Kuo, Heng-Lan; Hsu, Ming-Tsung; Tseng, Yufeng Jane; Lin, Shu-Wha; Kuo, Sheng-Chu; Peng, Hui-Chin; Lien, Jin-Cherng; Huang, Tur-Fu

    2016-08-01

    A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis. PMID:27173725

  9. Nogo Receptor Inhibition Enhances Functional Recovery following Lysolecithin-Induced Demyelination in Mouse Optic Chiasm

    PubMed Central

    Pourabdolhossein, Fereshteh; Mozafari, Sabah; Morvan-Dubois, Ghislaine; Mirnajafi-Zadeh, Javad; Lopez-Juarez, Alejandra; Pierre-Simons, Jacqueline; Demeneix, Barbara A.; Javan, Mohammad

    2014-01-01

    Background Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm. Methodology/Principal Findings A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down NgR levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time. Conclusions/Significance Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis. PMID:25184636

  10. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    SciTech Connect

    Guo, Li-Juan; Liao, Lan; Yang, Li; Li, Yu; Jiang, Tie-Jian

    2014-02-15

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis.

  11. Sigma-1 receptor activation inhibits osmotic swelling of rat retinal glial (Müller) cells by transactivation of glutamatergic and purinergic receptors.

    PubMed

    Vogler, Stefanie; Winters, Helge; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-01-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (σ) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective σ receptor agonist ditolylguanidine, and the selective σ1 receptor agonist PRE-084, inhibit the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of σ receptor agonists on the Müller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that σ receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Müller cell swelling. The swelling-inhibitory effect of 17β-estradiol was prevented by the σ1 receptor antagonist BD1047, suggesting that the effect is mediated by σ1 receptor activation. The data may suggest that the neuroprotective effect of σ receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells. PMID:26499958

  12. Methamphetamine inhibits Toll-like receptor 9-mediated anti-HIV activity in macrophages.

    PubMed

    Cen, Ping; Ye, Li; Su, Qi-Jian; Wang, Xu; Li, Jie-Liang; Lin, Xin-Qin; Liang, Hao; Ho, Wen-Zhe

    2013-08-01

    Toll-like receptor 9 (TLR9) is one of the key sensors that recognize viral infection/replication in the host cells. Studies have demonstrated that methamphetamine (METH) dysregulated host cell innate immunity and facilitated HIV infection of macrophages. In this study, we present new evidence that METH suppressed TLR9-mediated anti-HIV activity in macrophages. Activation of TLR9 by its agonist CpG-ODN 2216 inhibits HIV replication, which was demonstrated by increased expression of TLR9, interferon (IFN)-α, IFN regulatory factor-7 (IRF-7), myeloid differentiation factor 88 (MyD88), and myxovirus resistance gene A (MxA) in macrophages. However, METH treatment of macrophages greatly compromised the TLR9 signaling-mediated anti-HIV effect and inhibited the expression of TLR9 downstream signaling factors. Dopamine D1 receptor (D1R) antagonists (SCH23390) could block METH-mediated inhibition of anti-HIV activity of TLR9 signaling. Investigation of the underlying mechanisms of the METH action showed that METH treatment selectively down-regulated the expression of TLR9 on macrophages, whereas it had little effect on the expression of other TLRs. Collectively, our results provide further evidence that METH suppresses host cell innate immunity against HIV infection by down-regulating TLR9 expression and its signaling-mediated antiviral effect in macrophages. PMID:23751096

  13. Inhibition of cancer cell proliferation by midazolam by targeting transient receptor potential melastatin 7.

    PubMed

    Dou, Yunling; Li, Yuan; Chen, Jingkao; Wu, Sihan; Xiao, Xiao; Xie, Shanshan; Tang, Lipeng; Yan, Min; Wang, Youqiong; Lin, Jun; Zhu, Wenbo; Yan, Guangmei

    2013-03-01

    Transient receptor potential melastatin 7 (TRPM7), a Ca(2+)-permeable channel, has been demonstrated to be present in cancer cells and involved in their growth and proliferation. The present study used midazolam, a benzodiazepine class anesthesic, to pharmacologically intervene in the expression of TRPM7 and to inhibit cancer cell proliferation. Midazolam significantly inhibited the growth and proliferation of FaDu human hypopharyngeal squamous cell carcinoma cells, concurring with the induction of G(0)/G(1) cell cycle arrest and blockage of Rb activation. Central-type and peripheral-type benzodiazepine receptor antagonists did not abrogate proliferation inhibition by midazolam, while the specific TRPM7 agonist bradykinin reversed this effect. In addition, other benzodiazepines, diazepam and clonazepam also exhibited anti-proliferative activities. The inhibitory activity on cancer cell growth and proliferation, combined with the TRPM-dependent mechanism, reveals the anticancer potential of midazolam as a TRPM7 inhibitor and supports the suggestion that TRPM7 is a valuable target for pharmaceutical intervention. PMID:23426784

  14. Tributyltin and triphenyltin inhibit osteoclast differentiation through a retinoic acid receptor-dependent signaling pathway

    SciTech Connect

    Yonezawa, Takayuki; Hasegawa, Shin-ichi; Ahn, Jae-Yong; Cha, Byung-Yoon; Teruya, Toshiaki; Hagiwara, Hiromi; Nagai, Kazuo; Woo, Je-Tae; E-mail: jwoo@isc.chubu.ac.jp

    2007-03-30

    Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used in agriculture and industry. Although these compounds are known to have many toxic effects, including endocrine-disrupting effects, their effects on bone resorption are unknown. In this study, we investigated the effects of organotin compounds, such as monobutyltin (MBT), dibutyltin (DBT), TBT, and TPT, on osteoclast differentiation using mouse monocytic RAW264.7 cells. MBT and DBT had no effects, whereas TBT and TPT dose-dependently inhibited osteoclast differentiation at concentrations of 3-30 nM. Treatment with a retinoic acid receptor (RAR)-specific antagonist, Ro41-5253, restored the inhibition of osteoclastogenesis by TBT and TPT. TBT and TPT reduced receptor activator of nuclear factor-{kappa}B ligand (RANKL) induced nuclear factor of activated T cells (NFAT) c1 expression, and the reduction in NFATc1 expression was recovered by Ro41-5253. Our results suggest that TBT and TPT suppress osteoclastogenesis by inhibiting RANKL-induced NFATc1 expression via an RAR-dependent signaling pathway.

  15. A novel cholinergic receptor mediates inhibition of chick cochlear hair cells.

    PubMed

    Fuchs, P A; Murrow, B W

    1992-04-22

    The central nervous system provides feedback regulation at several points within the peripheral auditory apparatus. One component of that feedback is inhibition of cochlear hair cells by release of acetylcholine (ACh) from efferent brainstem neurons. The mechanism of hair cell inhibition, and the character of the presumed cholinergic receptor, however, have eluded understanding. Both nicotinic and muscarinic, as well as some non-cholinergic ligands can affect the efferent action. We have made whole-cell, tight-seal recordings from short (outer) hair cells isolated from the chick's cochlea. These are the principal targets of cochlear efferents in birds. ACh hyperpolarizes short hair cells by opening a cation channel through which Ca2+ enters the cell and subsequently activates Ca(2+)-dependent K+ current (Fuchs & Murrow 1991, 1992). Both curare and atropine are effective-antagonists of cholinergic inhibition at 3 microM, whereas trimethaphan camsylate and strychnine block at 1 microM. The normally irreversible nicotinic antagonist, alpha-bungarotoxin, reversibly blocked the hair cell response, as did kappa-bungarotoxin. The half-blocking concentration for alpha-bungarotoxin was 26 nM. It is proposed that the hair cell AChR is a ligand-gated cation channel related to the nicotinic receptor of nerve and muscle. PMID:1355909

  16. Prostaglandin D2 inhibits wound-induced hair follicle neogenesis through the receptor, Gpr44.

    PubMed

    Nelson, Amanda M; Loy, Dorothy E; Lawson, John A; Katseff, Adiya S; Fitzgerald, Garret A; Garza, Luis A

    2013-04-01

    Prostaglandins (PGs) are key inflammatory mediators involved in wound healing and regulating hair growth; however, their role in skin regeneration after injury is unknown. Using wound-induced hair follicle neogenesis (WIHN) as a marker of skin regeneration, we hypothesized that PGD2 decreases follicle neogenesis. PGE2 and PGD2 were elevated early and late, respectively, during wound healing. The levels of WIHN, lipocalin-type prostaglandin D2 synthase (Ptgds), and its product PGD2 each varied significantly among background strains of mice after wounding, and all correlated such that the highest Ptgds and PGD2 levels were associated with the lowest amount of regeneration. In addition, an alternatively spliced transcript variant of Ptgds missing exon 3 correlated with high regeneration in mice. Exogenous application of PGD2 decreased WIHN in wild-type mice, and PGD2 receptor Gpr44-null mice showed increased WIHN compared with strain-matched control mice. Furthermore, Gpr44-null mice were resistant to PGD2-induced inhibition of follicle neogenesis. In all, these findings demonstrate that PGD2 inhibits hair follicle regeneration through the Gpr44 receptor and imply that inhibition of PGD2 production or Gpr44 signaling will promote skin regeneration. PMID:23190891

  17. Prostaglandin D2 inhibits wound-induced hair follicle neogenesis through the receptor, Gpr44

    PubMed Central

    Nelson, Amanda M.; Loy, Dorothy E.; Lawson, John A.; Katseff, Adiya S.; FitzGerald, Garret A.; Garza, Luis A.

    2012-01-01

    Prostaglandins (PGs) are key inflammatory mediators involved in wound healing and regulating hair growth; however, their role in skin regeneration after injury is unknown. Using wound-induced hair follicle neogenesis (WIHN) as a marker of skin regeneration, we hypothesized that PGD2 decreases follicle neogenesis. PGE2 and PGD2 were elevated early and late respectively during wound healing. The levels of WIHN, lipocalin-type prostaglandin D2 synthase (Ptgds) and its product PGD2 each varied significantly among background strains of mice after wounding and all correlated such that the highest Ptgds and PGD2 levels were associated with the lowest amount of regeneration. Additionally, an alternatively spliced transcript variant of Ptgds missing exon 3 correlated with high regeneration in mice. Exogenous application of PGD2 decreased WIHN in wild type mice and PGD2 receptor Gpr44 null mice showed increased WIHN compared to strain-matched control mice. Furthermore, Gpr44 null mice were resistant to PGD2-induced inhibition of follicle neogenesis. In all, these findings demonstrate that PGD2 inhibits hair follicle regeneration through the Gpr44 receptor and imply that inhibition of PGD2 production or Gpr44 signaling will promote skin regeneration. PMID:23190891

  18. α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

    PubMed Central

    Lu, Ben; Kwan, Kevin; Levine, Yaakov A; Olofsson, Peder S; Yang, Huan; Li, Jianhua; Joshi, Sonia; Wang, Haichao; Andersson, Ulf; Chavan, Sangeeta S; Tracey, Kevin J

    2014-01-01

    The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release. PMID:24849809

  19. Proanthocyanidins inhibit in vitro and in vivo growth of human non-small cell lung cancer cells by inhibiting the prostaglandin E(2) and prostaglandin E(2) receptors.

    PubMed

    Sharma, Som D; Meeran, Syed M; Katiyar, Santosh K

    2010-03-01

    Overexpression of cyclooxygenase-2 (COX-2) and prostaglandins (PG) is linked to a wide variety of human cancers. Here, we assessed whether the chemotherapeutic effect of grape seed proanthocyanidins (GSP) on non-small cell lung cancer (NSCLC) cells is mediated through the inhibition of COX-2 and PGE(2)/PGE(2) receptor expression. The effects of GSPs on human NSCLC cell lines in terms of proliferation, apoptosis, and expression of COX-2, PGE(2), and PGE(2) receptors were determined using Western blotting, fluorescence-activated cell sorting analysis, and reverse transcription-PCR. In vitro treatment of NSCLC cells (A549, H1299, H460, H226, and H157) with GSPs resulted in significant growth inhibition and induction of apoptosis, which were associated with the inhibitory effects of GSPs on the overexpression of COX-2, PGE(2), and PGE(2) receptors (EP1 and EP4) in these cells. Treatment of cells with indomethacin, a pan-COX inhibitor, or transient transfection of cells with COX-2 small interfering RNA, also inhibited cell growth and induced cell death. The effects of a GSP-supplemented AIN76A control diet fed to nude mice bearing tumor xenografts on the expression of COX-2, PGE(2), and PGE(2) receptors in the xenografts were also evaluated. The growth-inhibitory effect of dietary GSPs (0.5%, w/w) on the NSCLC xenograft tumors was associated with the inhibition of COX-2, PGE(2), and PGE(2) receptors (EP1, EP3, and EP4) in tumors. This preclinical study provides evidence that the chemotherapeutic effect of GSPs on lung cancer cells in vitro and in vivo is mediated, at least in part, through the inhibition of COX-2 expression and subsequently the inhibition of PGE(2) and PGE(2) receptors. PMID:20145019

  20. Neurogenic contractions in intraocular porcine ciliary arteries are mediated by α₂-adrenoceptors and NPY₁ receptors and are inhibited by prostaglandin E₂ acting on prejunctional EP₄ receptors.

    PubMed

    Kringelholt, Sidse; Simonsen, Ulf; Bek, Toke

    2013-02-01

    Prostaglandin analogues and adrenergic drugs are used to reduce the intraocular pressure in glaucoma, which may partly be due to an effect on the tone of the intraocular arteries supplying the ciliary body. The aim of the present study was to investigate the interaction between prostaglandins and autonomic nervous activity induced by electrical stimulation of the tone in these ciliary vessels. The intraocular part of porcine ciliary arteries were isolated and mounted in a microvascular myograph for isometric tension recordings, and the effect of prostaglandin E(2) on electrically induced contractions was studied in the presence of selective EP receptor antagonists. PGE(2) induced concentration-dependent inhibition of electrically induced contractions of intraocular ciliary arteries which depended on the presence of the vascular endothelium. The effect of PGE(2) was blocked by an EP(4) receptor antagonist but not by an EP(1) receptor antagonist. The neurogenic contractions were partially inhibited by an α(2)-adrenoceptor antagonist and totally inhibited by a NPY(1) receptor antagonist. The effect of these antagonists was similar when contraction was induced by noradrenaline and NPY. Neurogenic contractions in intraocular porcine arteries are mediated by α(2)-adrenoceptors and NPY(1) receptors and can be inhibited by PGE(2) acting on prejunctional EP(4) receptors. This contributes to a further understanding of the role of the autonomic nervous system and prostaglandins for regulating blood flow to the anterior segment of the eye. PMID:23178872

  1. Zinc finger protein 131 inhibits estrogen signaling by suppressing estrogen receptor {alpha} homo-dimerization

    SciTech Connect

    Oh, Yohan; Chung, Kwang Chul

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ZNF131 directly interacts with ER{alpha}. Black-Right-Pointing-Pointer The binding affinity of ZNF131 to ER{alpha} increases upon E2 stimulation. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-dimerization and E2-induced breast cancer cell proliferation. Black-Right-Pointing-Pointer ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor. -- Abstract: Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ER{alpha} and ER{beta}. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of a DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ER{alpha} and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ER{alpha} inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ER{alpha}, which consequently inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor.

  2. Role of µ, κ, and δ opioid receptors in tibial inhibition of bladder overactivity in cats.

    PubMed

    Zhang, Zhaocun; Slater, Richard C; Ferroni, Matthew C; Kadow, Brian T; Lyon, Timothy D; Shen, Bing; Xiao, Zhiying; Wang, Jicheng; Kang, Audry; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2015-11-01

    In α-chloralose anesthetized cats, we examined the role of opioid receptor (OR) subtypes (µ, κ, and δ) in tibial nerve stimulation (TNS)-induced inhibition of bladder overactivity elicited by intravesical infusion of 0.25% acetic acid (AA). The sensitivity of TNS inhibition to cumulative i.v. doses of selective OR antagonists (cyprodime for µ, nor-binaltorphimine for κ, or naltrindole for δ ORs) was tested. Naloxone (1 mg/kg, i.v., an antagonist for µ, κ, and δ ORs) was administered at the end of each experiment. AA caused bladder overactivity and significantly (P < 0.01) reduced bladder capacity to 21.1% ± 2.6% of the saline control. TNS at 2 or 4 times threshold (T) intensity for inducing toe movement significantly (P < 0.01) restored bladder capacity to 52.9% ± 3.6% or 57.4% ± 4.6% of control, respectively. Cyprodime (0.3-1.0 mg/kg) completely removed TNS inhibition without changing AA control capacity. Nor-binaltorphimine (3-10 mg/kg) also completely reversed TNS inhibition and significantly (P < 0.05) increased AA control capacity. Naltrindole (1-10 mg/kg) reduced (P < 0.05) TNS inhibition but significantly (P < 0.05) increased AA control capacity. Naloxone (1 mg/kg) had no effect in cyprodime pretreated cats, but it reversed the nor-binaltorphimine-induced increase in bladder capacity and eliminated the TNS inhibition remaining in naltrindole pretreated cats. These results indicate a major role of µ and κ ORs in TNS inhibition, whereas δ ORs play a minor role. Meanwhile, κ and δ ORs also have an excitatory role in irritation-induced bladder overactivity. PMID:26354994

  3. Inhibition of the Dopamine D1 Receptor Signaling by PSD-95*◆

    PubMed Central

    Zhang, Jingping; Vinuela, Angel; Neely, Mark H.; Hallett, Penelope J.; Grant, Seth G. N.; Miller, Gregory M.; Isacson, Ole; Caron, Marc G.; Yao, Wei-Dong

    2008-01-01

    Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing gluta-mate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH2terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function. PMID:17369255

  4. Role of 5-HT(1A) receptors in fluoxetine-induced lordosis inhibition.

    PubMed

    Guptarak, Jutatip; Sarkar, Jhimly; Hiegel, Cindy; Uphouse, Lynda

    2010-07-01

    The selective serotonin reuptake inhibitor (SSRI), fluoxetine (Prozac(R)), is an effective antidepressant that is also prescribed for other disorders (e.g. anorexia, bulimia, and premenstrual dysphoria) that are prevalent in females. However, fluoxetine also produces sexual side effects that may lead patients to discontinue treatment. The current studies were designed to evaluate several predictions arising from the hypothesis that serotonin 1A (5-HT(1A)) receptors contribute to fluoxetine-induced sexual dysfunction. In rodent models, 5-HT(1A) receptors are potent negative modulators of female rat sexual behavior. Three distinct experiments were designed to evaluate the contribution of 5-HT(1A) receptors to the effects of fluoxetine. In the first experiment, the ability of the 5-HT(1A) receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY100635), to prevent fluoxetine-induced lordosis inhibition was examined. In the second experiment, the effects of prior treatment with fluoxetine on the lordosis inhibitory effect of the 5-HT(1A) receptor agonist, (+/-)-8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT), were studied. In the third experiment, the ability of progesterone to reduce the acute response to fluoxetine was evaluated. WAY100635 attenuated the effect of fluoxetine; prior treatment with fluoxetine decreased 8-OH-DPAT's potency in reducing lordosis behavior; and progesterone shifted fluoxetine's dose-response curve to the right. These findings are consistent with the hypothesis that 5-HT(1A) receptors contribute to fluoxetine-induced sexual side effects. PMID:20223238

  5. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of β-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  6. Endothelin inhibits renin release from juxtaglomerular cells via endothelin receptors A and B via a transient receptor potential canonical‐mediated pathway

    PubMed Central

    Ortiz‐Capisano, M. Cecilia

    2014-01-01

    Abstract Renin is the rate‐limiting step in the production of angiotensin II: a critical element in the regulation of blood pressure and in the pathogenesis of hypertension. Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by increases in calcium (Ca). Endothelins (ETs) inhibit renin release in a Ca‐dependent manner. JG cells contain multiple isoforms of canonical transient receptor potential (TRPC) Ca‐permeable channels. The proposed hypothesis is that endothelin inhibits renin release by activating TRPC store‐operated Ca channels. RT‐PCR and immunofluorescence revealed expression of both ETA and ETB receptors in mouse JG cells. Incubation of primary cultures of JG cells with ET‐1 (10 nmol/L) decreased renin release by 28%. Addition of either an ETA or an ETB receptor blocker completely prevented the ET inhibition of renin release. Incubation with the TRPC blocker (SKF 96365, 50 μmol/L) completely reversed the Ca‐mediated inhibition of renin release by ETs. These results suggest that endothelin inhibits renin release from JG cells via both ETA and ETB receptors, which leads to the activation of TRPC store‐operated Ca channels. PMID:25524278

  7. Challenges for drug discovery - a case study of urokinase receptor inhibition

    PubMed Central

    Chen, Zhuo; Lin, Lin; Huai, Qing; Huang, Mingdong

    2009-01-01

    Urokinase receptor (uPAR) is a widely recognized target for potential treatment of cancer. The development of uPAR inhibitors has been going on for over a decade. Despite the identification and validation of many highly potent hits using screening or medicinal approaches, none of them has been moved further along the drug discovery pipeline. The development of uPAR inhibitors exemplifies several challenges now faced by drug discovery. These include 1) hydrophobicity and thus poor bioavailability of the inhibitors from screening approaches; 2) specificity of the inhibitor, where a peptidyl inhibitor causes conformational change of the receptor; 3) species specificity, where some inhibitors developed based on the human receptor do not inhibit the murine receptor and thus cannot be validated in mouse models. The recently determined crystal structures of uPAR in complex with its ligand or inhibitor not only provide the structural insight to understand these challenges but also offer a potential solution for further inhibitor development and thus illustrate the importance of structural information in facilitating drug discovery. PMID:20025562

  8. Inhibition of Acetylcholinesterase Modulates NMDA Receptor Antagonist Mediated Alterations in the Developing Brain

    PubMed Central

    Bendix, Ivo; Serdar, Meray; Herz, Josephine; von Haefen, Clarissa; Nasser, Fatme; Rohrer, Benjamin; Endesfelder, Stefanie; Felderhoff-Mueser, Ursula; Spies, Claudia D.; Sifringer, Marco

    2014-01-01

    Exposure to N-methyl-d-aspartate (NMDA) receptor antagonists has been demonstrated to induce neurodegeneration in newborn rats. However, in clinical practice the use of NMDA receptor antagonists as anesthetics and sedatives cannot always be avoided. The present study investigated the effect of the indirect cholinergic agonist physostigmine on neurotrophin expression and the extracellular matrix during NMDA receptor antagonist induced injury to the immature rat brain. The aim was to investigate matrix metalloproteinase (MMP)-2 activity, as well as expression of tissue inhibitor of metalloproteinase (TIMP)-2 and brain-derived neurotrophic factor (BDNF) after co-administration of the non-competitive NMDA receptor antagonist MK801 (dizocilpine) and the acetylcholinesterase (AChE) inhibitor physostigmine. The AChE inhibitor physostigmine ameliorated the MK801-induced reduction of BDNF mRNA and protein levels, reduced MK801-triggered MMP-2 activity and prevented decreased TIMP-2 mRNA expression. Our results indicate that AChE inhibition may prevent newborn rats from MK801-mediated brain damage by enhancing neurotrophin-associated signaling pathways and by modulating the extracellular matrix. PMID:24595240

  9. Neuromedin B receptor antagonism inhibits migration, invasion, and epithelial-mesenchymal transition of breast cancer cells.

    PubMed

    Park, Hyun-Joo; Kim, Mi-Kyoung; Choi, Kyu-Sil; Jeong, Joo-Won; Bae, Soo-Kyung; Kim, Hyung Joon; Bae, Moon-Kyoung

    2016-09-01

    Neuromedin B (NMB) acts as an autocrine growth factor and a pro-angiogenic factor. Its receptor, NMB receptor (NMB-R), is overexpressed in solid tumors. In the present study, we showed that an NMB-R antagonist, PD168368, suppresses migration and invasion of the human breast cancer cell line MDA-MB-231. In addition, PD168368 reduced epithelial-mesenchymal transition (EMT) of breast cancer cells by E-cadherin upregulation and vimentin downregulation. Moreover, we found that PD168368 potently inhibits in vivo metastasis of breast cancer. Taken together, these findings suggest that NMB-R antagonism may be an alternative approach to prevent breast cancer metastasis, and targeting NMB-R may provide a novel therapeutic strategy for breast cancer treatment. PMID:27571778

  10. P2Y12 receptor inhibition and LPS-induced coagulation.

    PubMed

    Essex, David W; Rao, A Koneti

    2016-03-01

    Platelets play a major role in the complex interactions involved in blood coagulation via multiple mechanisms. As reported in this issue, Schoergenhofer et al. tested the hypothesis that platelet inhibition by prasugrel, a potent platelet P2Y12 ADP receptor antagonist, attenuates the effect of lipopolysaccharide (LPS) on the blood coagulation system in healthy human subjects. LPS, a bacterial product with potent pro-inflammatory and pro-thrombotic effects, plays a central role in sepsis. It activates monocytes and endothelial cells via Toll-like receptor (TLR) 4 and other TLRs to stimulate production of TF and other pro-coagulant molecules, chemokines and cytokines. Treatment with prasugrel did not decrease biomarkers of coagulaion. A better understanding of the relative roles of platelet and coagulation mechanisms in triggering the pro-thrombotic state may lead to more effective antithrombotic strategies. PMID:26846581

  11. Lung inflation inhibits rapidly adapting receptor relay neurons in the rat.

    PubMed

    Ezure, K; Tanaka, I

    2000-06-01

    Pulmonary slowly adapting receptors (SARs) and rapidly adapting receptors (RARs) are important components of various respiratory reflexes. In anesthetized, paralyzed and artificially ventilated rats, we found an inhibitory linkage from the former to the latter system at the level of their second-order relay neurons (P cells and RAR cells, respectively). Lung inflation which activates RARs as well as SARs suppressed RAR cell activity evoked by electrical stimulation of the vagus nerve. Intracellular recordings from RAR cells showed IPSPs locked to electrical stimulation of the ipsilateral and contralateral vagus nerves at an intensity just above the threshold for P cell activation. Activation of P cells with glutamate suppressed RAR cell firing. Since P cells project to the area of RAR cells, taken together, these results strongly suggest that P cells synaptically inhibit RAR cells. PMID:10852230

  12. Presynaptic GABAB receptors reduce transmission at parabrachial synapses in the lateral central amygdala by inhibiting N-type calcium channels

    PubMed Central

    Delaney, A.J.; Crane, J.W.

    2016-01-01

    The nocioceptive information carried by neurons of the pontine parabrachial nucleus to neurons of the lateral division of the central amydala (CeA-L) is thought to contribute to the affective components of pain and is required for the formation of conditioned-fear memories. Importantly, excitatory transmission between parabrachial axon terminals and CeA-L neurons can be inhibited by a number of presynaptic receptors linked to Gi/o-type G-proteins, including α2-adrenoceptors and GABAB receptors. While the intracellular signalling pathway responsible for α2-adrenoceptor inhibition of synaptic transmission at this synapse is known, the mechanism by which GABAB receptors inhibits transmission has not been determined. The present study demonstrates that activation of presynaptic GABAB receptors reduces excitatory transmission between parabrachial axon terminals and CeA-L neurons by inhibiting N-type calcium channels. While the involvement of Gβγ subunits in mediating the inhibitory effects of GABAB receptors on N-type calcium channels is unclear, this inhibition does not involve Gβγ-independent activation of pp60C-src tyrosine kinase. The results of this study further enhance our understanding of the modulation of the excitatory input from parabrachial axon terminals to CeA-L neurons and indicate that presynaptic GABAB receptors at this synapse could be valuable therapeutic targets for the treatment of fear- and pain-related disorders. PMID:26755335

  13. Presynaptic GABAB receptors reduce transmission at parabrachial synapses in the lateral central amygdala by inhibiting N-type calcium channels.

    PubMed

    Delaney, A J; Crane, J W

    2016-01-01

    The nocioceptive information carried by neurons of the pontine parabrachial nucleus to neurons of the lateral division of the central amydala (CeA-L) is thought to contribute to the affective components of pain and is required for the formation of conditioned-fear memories. Importantly, excitatory transmission between parabrachial axon terminals and CeA-L neurons can be inhibited by a number of presynaptic receptors linked to Gi/o-type G-proteins, including α2-adrenoceptors and GABAB receptors. While the intracellular signalling pathway responsible for α2-adrenoceptor inhibition of synaptic transmission at this synapse is known, the mechanism by which GABAB receptors inhibits transmission has not been determined. The present study demonstrates that activation of presynaptic GABAB receptors reduces excitatory transmission between parabrachial axon terminals and CeA-L neurons by inhibiting N-type calcium channels. While the involvement of Gβγ subunits in mediating the inhibitory effects of GABAB receptors on N-type calcium channels is unclear, this inhibition does not involve Gβγ-independent activation of pp60C-src tyrosine kinase. The results of this study further enhance our understanding of the modulation of the excitatory input from parabrachial axon terminals to CeA-L neurons and indicate that presynaptic GABAB receptors at this synapse could be valuable therapeutic targets for the treatment of fear- and pain-related disorders. PMID:26755335

  14. G protein-coupled estrogen receptor inhibits vascular prostanoid production and activity.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Barton, Matthias; Prossnitz, Eric R

    2015-10-01

    Complications of atherosclerotic vascular disease, such as myocardial infarction and stroke, are the most common causes of death in postmenopausal women. Endogenous estrogens inhibit vascular inflammation-driven atherogenesis, a process that involves cyclooxygenase (COX)-derived vasoconstrictor prostanoids such as thromboxane A2. Here, we studied whether the G protein-coupled estrogen receptor (GPER) mediates estrogen-dependent inhibitory effects on prostanoid production and activity under pro-inflammatory conditions. Effects of estrogen on production of thromboxane A(2) were determined in human endothelial cells stimulated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α). Moreover, Gper-deficient (Gper(-/-)) and WT mice were fed a pro-inflammatory diet and underwent ovariectomy or sham surgery to unmask the role of endogenous estrogens. Thereafter, contractions to acetylcholine-stimulated endothelial vasoconstrictor prostanoids and the thromboxane-prostanoid receptor agonist U46619 were recorded in isolated carotid arteries. In endothelial cells, TNF-α-stimulated thromboxane A2 production was inhibited by estrogen, an effect blocked by the GPER-selective antagonist G36. In ovary-intact mice, deletion of Gper increased prostanoid-dependent contractions by twofold. Ovariectomy also augmented prostanoid-dependent contractions by twofold in WT mice but had no additional effect in Gper(-/-) mice. These contractions were blocked by the COX inhibitor meclofenamate and unaffected by the nitric oxide synthase inhibitor l-N(G)-nitroarginine methyl ester. Vasoconstrictor responses to U46619 did not differ between groups, indicating intact signaling downstream of thromboxane-prostanoid receptor activation. In summary, under pro-inflammatory conditions, estrogen inhibits vasoconstrictor prostanoid production in endothelial cells and activity in intact arteries through GPER. Selective activation of GPER may therefore be considered as a novel strategy to

  15. G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

    PubMed

    Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

    2015-01-15

    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. PMID:25485460

  16. Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

    SciTech Connect

    Elangovan, Indira; Thirugnanam, Sivasakthivel; Chen, Aoshuang; Zheng, Guoxing; Bosland, Maarten C.; Kajdacsy-Balla, Andre; Gnanasekar, Munirathinam

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Targeting RAGE by RNAi induces apoptosis in prostate cancer cells. Black-Right-Pointing-Pointer Silencing RAGE expression abrogates rHMGB1 mediated cell proliferation. Black-Right-Pointing-Pointer Down regulation of RAGE by RNAi inhibits PSA secretion of prostate cancer cells. Black-Right-Pointing-Pointer Knock down of RAGE abrogates prostate tumor growth in vivo. Black-Right-Pointing-Pointer Disruption of RAGE expression in prostate tumor activates death receptors. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

  17. TP receptor activation and inhibition in atherothrombosis: the paradigm of diabetes mellitus.

    PubMed

    Santilli, Francesca; Mucci, Luciana; Davì, Giovanni

    2011-06-01

    Patients with type 2 diabetes mellitus are characterized by increased incidence of cardiovascular events and enhanced thromboxane-dependent platelet activation. Urinary enzymatic TXA(2) metabolites (such as 11-dehydro-TXB(2)), reflecting the whole TXA(2) biosynthesis by platelet and extra-platelet sources, are significantly increased in diabetes with the absolute post-aspirin values of 11-dehydro-TXB(2) in diabetics being comparable to non-aspirated controls and such residual TXA(2) biosynthesis despite low-dose aspirin treatment is predictive of vascular events in high-risk patients. Thus, elevated urinary 11-dehydro-TXB(2) levels identify patients who are partially insensitive to aspirin and who may benefit from alternative antiplatelet therapies or treatments that more effectively block in vivo TXA(2) production or activity. Potential mechanisms relatively insensitive to aspirin include extraplatelet, nucleate sources of TXA(2) biosynthesis, possibly triggered by inflammatory stimuli, or lipid peroxidation with enhanced generation of F2-isoprostane (reflecting ongoing in vivo oxidative stress) than can activate platelets via the platelet TP receptor thus escaping inhibition by aspirin. In fact, aspirin does not inhibit isoprostane formation. Moreover, intraplatelet or extraplatelet thromboxane generation may be only partly inhibited by aspirin under certain pathological conditions, at least at the usual low doses given for cardiovascular protection. TXA(2) receptors (TP) are expressed on several cell types and exert antiatherosclerotic, antivasoconstrictive and antithrombotic effects, depending on the cellular target. Thus, targeting TP receptor, a common downstream pathway for both platelet and extraplatelet TXA(2) as well as for isoprostanes, may be an useful antithrombotic intervention in clinical settings, such as diabetes mellitus characterized by persistently enhanced thromboxane-dependent platelet activation. PMID:20734162

  18. A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

    PubMed Central

    Santhakumar, Vijayalakshmi; Otis, Thomas S.

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  19. A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

    PubMed

    Santhakumar, Vijayalakshmi; Meera, Pratap; Karakossian, Movses H; Otis, Thomas S

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  20. Blocking Fibroblast Growth Factor Receptor Signaling Inhibits Tumor Growth, Lymphangiogenesis, and Metastasis

    PubMed Central

    Larrieu-Lahargue, Frédéric; Welm, Alana L.; Bouchecareilh, Marion; Alitalo, Kari; Li, Dean Y.; Bikfalvi, Andreas; Auguste, Patrick

    2012-01-01

    Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread. PMID:22761819

  1. Targeting colorectal cancer via its microenvironment by inhibiting IGF-1 Receptor-insulin receptor substrate and STAT3 signaling

    PubMed Central

    Sanchez-Lopez, Elsa; Flashner-Abramson, Efrat; Shalapour, Shabnam; Zhong, Zhenyu; Taniguchi, Koji; Levitzki, Alexander; Karin, Michael

    2015-01-01

    The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and Signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer (CRC) development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment. PMID:26364612

  2. Targeting colorectal cancer via its microenvironment by inhibiting IGF-1 receptor-insulin receptor substrate and STAT3 signaling.

    PubMed

    Sanchez-Lopez, E; Flashner-Abramson, E; Shalapour, S; Zhong, Z; Taniguchi, K; Levitzki, A; Karin, M

    2016-05-19

    The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment. PMID:26364612

  3. β-adrenergic receptor agonist, Compound 49b, inhibits TLR4 signaling pathway in diabetic retina

    PubMed Central

    Berger, Elizabeth A.; Carion, Thomas W.; Jiang, Youde; Liu, Li; Chahine, Adam; Walker, Robert Jason; Steinle, Jena J.

    2016-01-01

    Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. While it is clear that tumor necrosis factor- alpha (TNF-α) is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase toll-like receptor 4 (TLR4) signaling, which could be inhibited by a β-adrenergic receptor agonist (Compound 49b) previously shown to have anti-inflammatory actions. In order to investigate β-adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin-injected diabetic mice, as well as human primary retinal endothelial cells (REC) and rat retinal Müller cells (rMC-1) exposed to high glucose (25mM), were treated with a novel β-adrenergic receptor agonist, Compound 49b (50nM), or PBS (control). TLR4 and its downstream signaling partners (MyD88, IRAK1, TRAF6, total and phosphorylated NF-κB) were examined. In addition, we assessed high mobility box group 1 (HMGB1) protein levels. Our data showed that diabetes or high glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in REC and Müller cells grown in high glucose, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway providing a potential mechanism for its protective actions. PMID:26888251

  4. Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis

    PubMed Central

    Nwosu, Lilian N; Mapp, Paul I; Chapman, Victoria; Walsh, David A

    2016-01-01

    Objectives Tropomyosin receptor kinase A (TrkA) mediates nociceptor sensitisation by nerve growth factor (NGF), but it is unknown whether selective TrkA inhibition will be an effective strategy for treating osteoarthritis (OA) pain. We determined the effects of a TrkA inhibitor (AR786) on pain behaviour, synovitis and joint pathology in two rat OA models. Methods Knee OA was induced in rats by intra-articular monosodium-iodoacetate (MIA) injection or meniscal transection (MNX) and compared with saline-injected or sham-operated controls. Pain behaviour was assessed as weight-bearing asymmetry and paw withdrawal threshold to punctate stimulation. Oral doses (30 mg/kg) of AR786 or vehicle were administered twice daily in either preventive (day −1 to –27) or treatment (day 14–28) protocols. Effect maintenance was evaluated for 2 weeks after treatment discontinuation. Alterations in knee structure (cartilage, subchondral bone and synovium) were examined by macroscopic visualisation of articular surfaces and histopathology. Results Preventive AR786 treatment inhibited pain behaviour development and therapeutic treatment attenuated established pain behaviour. Weight-bearing asymmetry increased 1 week after treatment discontinuation, but remained less than in vehicle-treated arthritic rats, whereas paw withdrawal thresholds returned to levels of untreated rats within 5 days of treatment discontinuation. AR786 treatment reduced MIA-induced synovitis and did not significantly affect osteochondral pathology in either model. Conclusions Blocking NGF activity by inhibiting TrkA reduced pain behaviour in two rat models of OA. Analgesia was observed both using preventive and treatment protocols, and was sustained after treatment discontinuation. Selective inhibitors of TrkA therefore hold potential for OA pain relief. PMID:26286016

  5. Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells.

    PubMed

    Dolma, Sonam; Selvadurai, Hayden J; Lan, Xiaoyang; Lee, Lilian; Kushida, Michelle; Voisin, Veronique; Whetstone, Heather; So, Milly; Aviv, Tzvi; Park, Nicole; Zhu, Xueming; Xu, ChangJiang; Head, Renee; Rowland, Katherine J; Bernstein, Mark; Clarke, Ian D; Bader, Gary; Harrington, Lea; Brumell, John H; Tyers, Mike; Dirks, Peter B

    2016-06-13

    Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRβ, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM. PMID:27300435

  6. TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

    PubMed

    Lehnert, Corinna; Weiswange, Maxi; Jeremias, Irmela; Bayer, Carina; Grunert, Michaela; Debatin, Klaus-Michael; Strauss, Gudrun

    2014-10-15

    The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. PMID:25217163

  7. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

    SciTech Connect

    Khan, Shaheen; Liu Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ER{alpha} crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1{alpha} or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NF{kappa}B which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  8. Anthraquinone emodin inhibits human cancer cell invasiveness by antagonizing P2X7 receptors.

    PubMed

    Jelassi, Bilel; Anchelin, Monique; Chamouton, Julie; Cayuela, María Luisa; Clarysse, Lucie; Li, Junying; Goré, Jacques; Jiang, Lin-Hua; Roger, Sébastien

    2013-07-01

    The adenosine 5'-triphosphate (ATP)-gated Ca(2+)-permeable channel P2X7 receptor (P2X7R) is strongly upregulated in many tumors and cancer cells, and has an important role in cancer cell invasion associated with metastases. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone derivative originally isolated from Rheum officinale Baill known for decades to possess anticancer properties. In this study, we examined the effects of emodin on P2X7R-dependent Ca(2+) signaling, extracellular matrix degradation, and in vitro and in vivo cancer cell invasiveness using highly aggressive human cancer cells. Inclusion of emodin at doses ≤10 µM in cell culture had no or very mild effect on the cell viability. ATP elicited increases in intracellular Ca(2+) concentration were reduced by 35 and 60% by 1 and 10 µM emodin, respectively. Emodin specifically inhibited P2X7R-mediated currents with an IC50 of 3 µM and did not inhibit the currents mediated by the other human P2X receptors heterologously expressed in human embryonic kidney (HEK293T) cells. ATP-induced increase in gelatinolytic activity, in cancer cell invasiveness in vitro and in cell morphology changes were prevented by 1 µM emodin. Furthermore, such ATP-evoked effects and inhibition by emodin were almost completely ablated in cancer cells transfected with P2X7R-specific small interfering RNA (siRNA) but not with scrambled siRNA. Finally, the in vivo invasiveness of the P2X7R-positive MDA-MB-435s breast cancer cells, assessed using a zebrafish model of micrometastases, was suppressed by 40 and 50% by 1 and 10 µM emodin. Taken together, these results provide consistent evidence to indicate that emodin inhibits human cancer cell invasiveness by specifically antagonizing the P2X7R. PMID:23524196

  9. The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

    PubMed Central

    Howard, Rebecca J.; Trudell, James R.; Harris, R. Adron

    2012-01-01

    Ionotropic GABAA receptors (GABAARs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABAARs. We used Xenopus laevis oocytes to investigate the 6′ position in the second transmembrane region of GABAARs as a site influencing alcohol inhibition. We asked whether modification of the 6′ position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6′ position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6′ position. We verified a role for the 6′ position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABAARs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABAAR homolog, revealing that the 6′ position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6′ positions in both α2 and β2 GABAAR subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels. PMID:22072732

  10. MiR-503 inhibits hepatocellular carcinoma cell growth via inhibition of insulin-like growth factor 1 receptor

    PubMed Central

    Xiao, Yao; Tian, Qinggang; He, Jiantai; Huang, Ming; Yang, Chao; Gong, Liansheng

    2016-01-01

    MicroRNAs (miRs) have been demonstrated to play key roles in the development and progression of hepatocellular carcinoma (HCC). However, the regulatory mechanism of miR-503 in HCC has not been fully uncovered. In this study, we found that miR-503 was significantly downregulated in HCC tissues compared to nontumorous liver tissues. Moreover, lower miR-503 levels were associated with the malignant progression of HCC, and the expression of miR-503 was also decreased in several common HCC cell lines compared to normal human liver cell line THLE-3. Overexpression of miR-503 inhibited proliferation but induced apoptosis of LM3 and HepG2 cells. Bioinformatical analysis and luciferase reporter assay further identified insulin-like growth factor 1 receptor (IGF-1R) as a novel target of miR-503 in 293T cells. Moreover, overexpression of miR-503 led to a significant decrease in the protein levels of IGF-1R, while knockdown of miR-503 enhanced its protein levels in LM3 and HepG2 cells. Besides, overexpression of IGF-1R reversed the effects of miR-503-mediated HCC cell proliferation and apoptosis, indicating that IGF-1R acts as a downstream effector of miR-503 in HCC cells. Furthermore, IGF-1R was found to be significantly upregulated in HCC tissues compared to nontumorous liver tissues. In addition, the mRNA levels of IGF-1R were inversely correlated to the miR-503 levels in the HCC tissues. Thus, we demonstrate that miR-503 inhibits the proliferation and induces the apoptosis of HCC cells, partly at least, by directly targeting IGF-1R, and suggest that IGF-1R may serve as a promising target for the treatment of HCC. PMID:27366090

  11. Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

    SciTech Connect

    Hisatsune, Akinori; Nakayama, Hideki; Kawasaki, Mitsuru; Horie, Ichiro; Miyata, Takeshi; Isohama, Yoichiro; Kim, Kwang Chul; Katsuki, Hiroshi

    2011-02-18

    Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.

  12. Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway

    PubMed Central

    Liu, Hong-Shuai; Shi, Hai-Lian; Huang, Fei; Peterson, Karin E.; Wu, Hui; Lan, Yun-Yi; Zhang, Bei-Bei; He, Yi-Xin; Woods, Tyson; Du, Min; Wu, Xiao-Jun; Wang, Zheng-Tao

    2016-01-01

    Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases. PMID:26750705

  13. Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

    PubMed

    Tsukahara, Tamotsu; Tsukahara, Ryoko; Fujiwara, Yuko; Yue, Junming; Cheng, Yunhui; Guo, Huazhang; Bolen, Alyssa; Zhang, Chunxiang; Balazs, Louisa; Re, Fabio; Du, Guangwei; Frohman, Michael A; Baker, Daniel L; Parrill, Abby L; Uchiyama, Ayako; Kobayashi, Tetsuyuki; Murakami-Murofushi, Kimiko; Tigyi, Gabor

    2010-08-13

    Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists. PMID:20705243

  14. Class A scavenger receptor activation inhibits endoplasmic reticulum stress-induced autophagy in macrophage.

    PubMed

    Huang, Hanpeng; Li, Xiaoyu; Zhuang, Yan; Li, Nan; Zhu, Xudong; Hu, Jin; Ben, Jingjing; Yang, Qing; Bai, Hui; Chen, Qi

    2014-05-01

    Macrophage death in advanced atherosclerosis promotes plaque necrosis and destabilization. Involvement of autophagy in bulk degradation of cellular components has been recognized recently as an important mechanism for cell survival under endoplasmic reticulum (ER) stress. We previously found that the engagement of class A scavenger receptor (SR-A) triggered JNK-dependent apoptosis in ER-stressed macrophages. However, pro-apoptotic mechanisms mediated by SR-A are not fully understood. Therefore, we sought to see if SR-A mediated apoptosis was associated with autophagy in macrophages. Here, we showed that fucoidan inhibited microtubule-associated protein light chain 3-phospholipid conjugates (LC3-II) formation as well as the number of autophagosomes under ER stress. The inhibition of LC3-II formation was paralleled by the activation of the mTOR pathway, and the inhibition of mTOR allowed LC3-II induction in macrophages treated with thapsigargin plus fucoidan. Furthermore, apoptosis induced by fucoidan was prevented under ER stress by the mTOR inhibitor. We propose that fucoidan, a SR-A agonist, may contribute to macrophage apoptosis during ER stress by inhibiting autophagy. PMID:25013404

  15. Damnacanthal inhibits IgE receptor-mediated activation of mast cells.

    PubMed

    Garcia-Vilas, Javier A; Medina, Miguel A; Melo, Fabio R; Pejler, Gunnar; Garcia-Faroldi, Gianni

    2015-05-01

    Damnacanthal, an anthraquinone obtained from the noni fruit (Morinda citrifolia L.), has been described to possess anti-cancer and anti-inflammatory properties. Since mast cells are key players in various inflammatory conditions as well as in cancer, we considered the possibility that the biological actions of damnacanthal, at least partly, could be due to effects on mast cells. Many of the biological activities of mast cells are mediated by IgE receptor cross-linking, which results in degranulation with release of preformed granule mediators, as well as de novo synthesis and release of additional compounds. Here we show that damnacanthal has profound inhibitory activity on mast cell activation through this pathway. The release of the granule compounds beta-hexosaminidase and tryptase release was completely abrogated by damnacanthal at doses that were non-toxic to mast cells. In addition, damnacanthal inhibited activation-dependent pro-inflammatory gene induction, as well as cytokine/chemokine release in response to mast cell stimulation. The mechanism underlying damnacanthal inhibition was linked to impaired phosphorylation of Syk and Akt. Furthermore, damnacanthal inhibited mast cell activation in response to calcium ionophore A23187. Altogether, the data presented here demonstrate that damnacanthal inhibits mast cell activation induced by different stimuli and open a new window for the use of this compound as a mast cell stabilizer. PMID:25656801

  16. mTOR inhibition improves fibroblast growth factor receptor targeting in hepatocellular carcinoma

    PubMed Central

    Scheller, T; Hellerbrand, C; Moser, C; Schmidt, K; Kroemer, A; Brunner, S M; Schlitt, H J; Geissler, E K; Lang, S A

    2015-01-01

    Background: Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models. Methods: Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used. Results: In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg−1) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis. Conclusions: Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC. PMID:25688743

  17. Presynaptic dopamine D2-like receptors inhibit excitatory transmission onto rat ventral tegmental dopaminergic neurones

    PubMed Central

    Koga, Eiko; Momiyama, Toshihiko

    2000-01-01

    The effects of dopamine (DA) on non-NMDA glutamatergic transmission onto dopaminergic neurones in the ventral tegmental area (VTA) were examined in rat midbrain slices using the whole-cell patch-clamp technique. EPSCs in dopaminergic neurones evoked by focal stimulation within the VTA were reversibly blocked by 5 μm CNQX in the presence of bicuculline (20 μm), strychnine (0.5 μm) and D-amino-5-phosphonopentanoic acid (D-AP5, 25 μm). Bath application of DA reduced the amplitude of EPSCs up to 65.1 ± 9.52% in a concentration-dependent manner between 0.3–1000 μm (IC50, 16.0 μm) without affecting the holding current at −60 mV measured using a Cs+-filled electrode. The effect of DA on evoked EPSCs was mimicked by the D2-like receptor agonist quinpirole but not by the D1-like receptor agonist SKF 81297, and was antagonized by the D2-like receptor antagonist sulpiride (KB, 0.96 μm), but not by the D1-like receptor antagonist SCH 23390 (KB, 228.6 μm). Dopamine (30 μm) reduced the mean frequency of spontaneous miniature EPSCs (mEPSCs) without affecting their mean amplitude, and the DA-induced effect on the mEPSCs was dependent on the external Ca2+ concentration. These results suggest that afferent glutamatergic fibres which terminate on VTA dopaminergic neurones possess presynaptic D2-like receptors, activation of which inhibits glutamate release by reducing Ca2+ influx. PMID:10673553

  18. Inhibition of spontaneous EPSCs and IPSCs by presynaptic GABAB receptors on rat supraoptic magnocellular neurons.

    PubMed Central

    Kabashima, N; Shibuya, I; Ibrahim, N; Ueta, Y; Yamashita, H

    1997-01-01

    1. The function of presynaptic GABA receptors in the regulation of transmitter release in supraoptic nucleus (SON) magnocellular neurons was investigated by recording spontaneous postsynaptic currents from rat magnocellular SON neurons in a slice preparation (150 microns thick, 1.8 mm in diameter) using the whole-cell patch-clamp technique. 2. Both the spontaneous EPSCs and IPSCs were TTX resistant. The EPSCs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the IPSCs were abolished by picrotoxin, suggesting that the EPSCs and IPSCs are synaptic inputs from glutamatergic and GABAergic neurons, respectively. 3. The selective GABAB agonist, baclofen, reduced the frequency of both the EPSCs and IPSCs without affecting the amplitude. The time constant of the decay phase of both the EPSCs and IPSCs remained unchanged after baclofen application. 4. The reduction of the frequency of the synaptic currents by baclofen was dose dependent (10 nM to 100 microM) and the EC50 values were 5.8 and 8.5 microM for the EPSCs and IPSCs, respectively. 5. The effect of baclofen (10 microM) was antagonized by the selective GABAB antagonist, 2-hydroxy-saclofen (2OH-saclofen), at 300 microM. 6. When given alone, 2OH-saclofen (100 microM) increased the frequency of both the EPSCs and IPSCs without affecting their amplitude, suggesting that endogenously released GABA in the slice acts on presynaptic GABAB receptors. 7. The GABAA agonist, muscimol, reduced the frequency of EPSCs, and picrotoxin increased the frequency of the EPSCs, suggesting that GABAA receptors also participate in the presynaptic inhibition of glutamate release. 8. Taken together, these data suggest that GABAB receptors are present on the presynaptic terminals of both GABA and glutamate neurons in the SON, and that these presynaptic GABAB receptors play an important role in the regulation of the neuronal activity in SON magnocellular neurons. Images Figure 1 PMID:9350623

  19. The niacin/butyrate receptor GPR109A suppresses mammary tumorigenesis by inhibiting cell survival

    PubMed Central

    Elangovan, Selvakumar; Pathania, Rajneesh; Ramachandran, Sabarish; Ananth, Sudha; Padia, Ravi N.; Lan, Ling; Singh, Nagendra; Martin, Pamela M.; Hawthorn, Lesleyann; Prasad, Puttur D.; Ganapathy, Vadivel; Thangaraju, Muthusamy

    2014-01-01

    GPR109A, a G-protein-coupled receptor, is activated by niacin and butyrate. Upon activation in colonocytes, GPR109A potentiates anti-inflammatory pathways, induces apoptosis, and protects against inflammation-induced colon cancer. In contrast, GPR109A activation in keratinocytes induces flushing by activation of Cox-2-dependent inflammatory signaling and, the receptor expression is upregulated in human epidermoid carcinoma. Thus, depending on the cellular context and tissue, GPR109A functions either as a tumor suppressor or a tumor promoter. However, the expression status and the functional implications of this receptor in the mammary epithelium are not known. Here we show that GPR109A is expressed in normal mammary tissue and, irrespective of the hormone receptor status, its expression is silenced in human primary breast tumor tissues, breast cancer cell lines, and in tumor tissues of three different murine mammary tumor models. Functional expression of this receptor in human breast cancer cell lines decreases cAMP production, induces apoptosis, and blocks colony formation and mammary tumor growth. Transcriptome analysis revealed that GPR109A activation inhibits genes, which are involved in cell survival and anti-apoptotic signaling, in human breast cancer cells. In addition, deletion of Gpr109a in mice increased tumor incidence and triggered early onset of mammary tumorigenesis with increased lung metastasis in MMTV-Neu mouse model of spontaneous breast cancer. These findings suggest that GPR109A is a tumor suppressor in mammary gland and that pharmacological induction of this gene in tumor tissues followed by its activation with agonists could be an effective therapeutic strategy to treat breast cancer. PMID:24371223

  20. Characterization of prostanoid receptors mediating inhibition of histamine release from anti-IgE-activated rat peritoneal mast cells

    PubMed Central

    Chan, C L; Jones, R L; Lau, H Y A

    2000-01-01

    Prostanoid receptors mediating inhibition of anti-IgE induced histamine release from rat peritoneal mast cells have been characterized pharmacologically. PGD2 and the specific DP receptor agonists BW 245C and ZK 118182 were the most potent inhibitors with half-maximal concentrations of 0.26, 0.06 and 0.02 μM respectively. The maximum inhibition attainable was 60–65% with 10−5 M BW 245C and ZK 118182. Among several EP receptor agonists investigated, only PGE2 and the EP2/EP3 receptor agonist misoprostol induced significant inhibition (46.8±4.7% at 10−4 M and 18.7±6.8% at 10−5 M respectively). The IP receptor agonists cicaprost and iloprost were both less potent than the DP agonists in inhibiting histamine release (45.2±3.3% and 35.1±2.5% inhibition respectively at 10−5 M), whereas PGF2α and the TP receptor agonist U-46619 were only marginally effective. The EP4/TP receptor antagonist AH 23848 failed to affect the inhibitory actions of PGD2 or PGE2 even at 10−5 M, whereas the DP/EP1/EP2 receptor antagonist AH 6809 slightly enhanced the effect of PGD2 at 10−6 M. At concentrations of 3×10−6 to 10−5 M, the putative DP receptor antagonist ZK 138357 dose-dependently suppressed the inhibitory activities of the DP agonists, PGE2 and cicaprost. The antagonism of ZK 138357 against the DP receptor agonists appeared to be competitive with pA2 values of around six. In conclusion, these data support our earlier proposal that an inhibitory DP receptor is the predominant prostanoid receptor in rat peritoneal mast cell. The properties of this receptor in relation to putative DP receptor subtypes reported in the literature are discussed. PMID:10711359

  1. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    SciTech Connect

    Yashima, N.; Wada, A.; Izumi, F.

    1986-04-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of /sup 45/Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of /sup 45/Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of /sup 45/Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the /sup 45/Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the /sup 45/Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of /sup 45/Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of /sup 45/Ca. Based on these findings, the authors suggest that inhibition of the /sup 45/Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels.

  2. Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll Like Receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor

    PubMed Central

    Good, Misty; Sodhi, Chhinder P.; Egan, Charlotte E.; Afrazi, Amin; Jia, Hongpeng; Yamaguchi, Yukihiro; Lu, Peng; Branca, Maria F.; Ma, Congrong; Prindle, Thomas; Mielo, Samantha; Pompa, Anthony; Hodzic, Zerina; Ozolek, John A.; Hackam, David J.

    2015-01-01

    Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease which is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), while selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Over-expression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of GSK3β. Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis. PMID:25899687

  3. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    PubMed Central

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  4. Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling

    PubMed Central

    Son, Dong Ju; Kim, Soo Yeon; Han, Seong Su; Kim, Chan Woo; Kumar, Sandeep; Park, Byeoung Soo; Lee, Sung Eun; Yun, Yeo Pyo; Jo, Hanjoong; Park, Young Hyun

    2012-01-01

    Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-κB) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase C1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-κB—a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo. PMID:22995306

  5. Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling.

    PubMed

    Merkulova, Yulia; Shen, Yue; Parkinson, Leigh G; Raithatha, Sheetal A; Zhao, Hongyan; Westendorf, Kathryn; Sharma, Mehul; Bleackley, Robert Chris; Granville, David J

    2016-09-01

    Chronic non-healing wounds including diabetic, venous, and decubitus skin ulcers are currently lacking effective therapies. Non-healing diabetic ulcers can lead to amputations as progress into a highly chronic state before detection and existing treatments for these wounds often fail. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during excessive or chronic inflammation, GzmB can accumulate in the extracellular milieu, retain its activity, and cleave a number of important extracellular proteins. Epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in cellular processes such as proliferation and migration. EGFR signaling is integral to the wound healing process. The present study investigated the effects of GzmB on keratinocyte cell migration using HaCaT cell line. Using electric cell-substrate impedance sensing and scratch assays, the present study demonstrates that GzmB inhibits keratinocyte migration by interfering with the EGFR pathway. GzmB limited cell transition into a migratory morphology and was found to reduce ligand-induced EGFR phosphorylation. Inhibition of GzmB reversed the aforementioned effects. In summary, data from the present study suggest key role for GzmB in the pathogenesis of impaired wound healing through the impairment of EGFR signaling and cell migration. PMID:27060743

  6. Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

    PubMed

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  7. Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics.

    PubMed

    Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna-Magdalena; Mahrhold, Stefan; Perry, Kay; Cheng, Luisa W; Rummel, Andreas; Jin, Rongsheng

    2015-12-01

    Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes for botulism. BoNT assembles with several auxiliary proteins to survive in the gastrointestinal tract and is subsequently transported through the intestinal epithelium into the general circulation. Several hemagglutinin proteins form a multi-protein complex (HA complex) that recognizes host glycans on the intestinal epithelial cell surface to facilitate BoNT absorption. Blocking carbohydrate binding to the HA complex could significantly inhibit the oral toxicity of BoNT. Here, we identify lactulose, a galactose-containing non-digestible sugar commonly used to treat constipation, as a prototype inhibitor against oral BoNT/A intoxication. As revealed by a crystal structure, lactulose binds to the HA complex at the same site where the host galactose-containing carbohydrate receptors bind. In vitro assays using intestinal Caco-2 cells demonstrated that lactulose inhibits HA from compromising the integrity of the epithelial cell monolayers and blocks the internalization of HA. Furthermore, co-administration of lactulose significantly protected mice against BoNT/A oral intoxication in vivo. Taken together, these data encourage the development of carbohydrate receptor mimics as a therapeutic intervention to prevent BoNT oral intoxication. PMID:26272706

  8. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody.

    PubMed

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L; Ornitz, David M

    2016-05-01

    Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  9. Antipsychotic Drugs Inhibit Platelet Aggregation via P2Y1 and P2Y12 Receptors

    PubMed Central

    Wu, Chang-Chieh; Tsai, Fu-Ming; Chen, Mao-Liang; Wu, Semon; Lee, Ming-Cheng; Tsai, Tzung-Chieh; Wang, Lu-Kai; Wang, Chun-Hua

    2016-01-01

    Antipsychotic drugs (APDs) used to treat clinical psychotic syndromes cause a variety of blood dyscrasias. APDs suppress the aggregation of platelets; however, the underlying mechanism remains unknown. We first analyzed platelet aggregation and clot formation in platelets treated with APDs, risperidone, clozapine, or haloperidol, using an aggregometer and rotational thromboelastometry (ROTEM). Our data indicated that platelet aggregation was inhibited, that clot formation time was increased, and that clot firmness was decreased in platelets pretreated with APDs. We also examined the role two major adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, play in ADP-mediated platelet activation and APD-mediated suppression of platelet aggregation. Our results show that P2Y1 receptor stimulation with ADP-induced calcium influx was inhibited by APDs in human and rats' platelets, as assessed by in vitro or ex vivo approach, respectively. In contrast, APDs, risperidone and clozapine, alleviated P2Y12-mediated cAMP suppression, and the release of thromboxane A2 and arachidonic acid by activated platelets decreased after APD treatment in human and rats' platelets. Our data demonstrate that each APD tested significantly suppressed platelet aggregation via different mechanisms. PMID:27069920

  10. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  11. Evidence that 5-HT1D receptors mediate inhibition of sympathetic ganglionic transmission in anaesthetized cats.

    PubMed Central

    Jones, J. F.; Martin, G. R.; Ramage, A. G.

    1995-01-01

    In anaesthetized cats, 5-carboxamidotryptamine (5-CT) or 5-hydroxytryptamine (5-HT) (0.3-300 micrograms kg-1,i.v.) inhibited the postganglionic compound action potential evoked by preganglionic electrical stimulation (0.5 Hz) with a similar potency in the stellate and splanchnic ganglia. In the 5-HT experiments transmission thorough the inferior mesenteric ganglia was also recorded. The maximal inhibitory effect of 5-HT was greater on the stellate and splanchnic ganglia (60 +/- 4 and 52 +/- 5%) than on the inferior mesenteric (15 +/- 2%). The effects of 5-HT were unaffected by pretreatment with antagonists (1 mg kg-1;i.v.) for 5-HT2 (BW501C67), 5-HT1A (WAY-100635) and 5-HT3 receptors (ondansetron). However, responses to both 5-HT and 5-CT were attenuated significantly by GR127935 (1 mg kg-1) except the responses to 5-HT at the inferior mesenteric ganglia. These results are consistent with the involvement of 5-HT1D receptors mediating inhibition of sympathetic ganglionic transmission in vivo. PMID:8528548

  12. Epigallocatechin gallate inhibits hepatitis B virus via farnesoid X receptor alpha.

    PubMed

    Xu, Jun; Gu, Weizhen; Li, Chaoyan; Li, Xiao; Xing, Guozhen; Li, Yan; Song, Yanhui; Zheng, Wenming

    2016-07-01

    Plants possess various natural antiviral properties. Epigallocatechin-3-gallate (EGCG), a major component of green tea, inhibits a variety of viruses. However, the clinical application of EGCG is currently hindered by a scarcity of information on its molecular mechanism of action. In the present study, we examined the anti-HBV (hepatitis B virus) effects of catechins from green tea at the transcriptional and antigen-expression levels, as well as the associated molecular mechanisms, because HBV-associated liver diseases have become a key public health issue due to their serious impact on human physical and mental health. By using fluorescence quenching and affinity binding, we demonstrated that EGCG is an important transcriptional regulator of the HBV genome, which it achieves by interacting with farnesoid X receptor alpha (FXRα). Luciferase assay showed that EGCG effectively inhibited the transcription of the HBV promoter dose-dependently when expression plasmids of FXRα and retinoid X receptor α (RXRα) were co-transfected into HEK293 cells. These results indicate that the downregulation of the HBV antigen and the decrease in the transcriptional activation of the HBV EnhII/core promoter by FXRα/RXRα are mainly due to the interaction between EGCG and FXRα. Therefore, EGCG, an antagonist of FXRα in liver cells, has the potential to be employed as an effective anti-HBV agent. PMID:26968537

  13. Activation of Cannabinoid Type 2 Receptors Inhibits HIV-1 Envelope Glycoprotein gp120-Induced Synapse Loss

    PubMed Central

    Kim, Hee Jung; Shin, Angela H.

    2011-01-01

    HIV-1 infection of the central nervous system is associated with dendritic and synaptic damage that correlates with cognitive decline in patients with HIV-1-associated dementia (HAD). HAD is due in part to the release of viral proteins from infected cells. Because cannabinoids modulate neurotoxic and inflammatory processes, we investigated their effects on changes in synaptic connections induced by the HIV-1 envelope glycoprotein gp120. Morphology and synapses between cultured hippocampal neurons were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 fused to green fluorescent protein (PSD95-GFP). Twenty-four-hour treatment with gp120 IIIB decreased the number of PSD95-GFP puncta by 37 ± 4%. The decrease was concentration-dependent (EC50 = 153 ± 50 pM). Synapse loss preceded cell death as defined by retention of DsRed2 fluorescence gp120 activated CXCR4 on microglia to evoke interleukin-1β (IL-1β) release. Pharmacological studies determined that sequential activation of CXCR4, the IL-1β receptor, and the N-methyl-d-aspartate receptor was required. Expression of alternative reading frame polypeptide, which inhibits the ubiquitin ligase murine double minute 2, protected synapses, implicating the ubiquitin-proteasome pathway. Cannabimimetic drugs are of particular relevance to HAD because of their clinical and illicit use in patients with AIDS. The cannabinoid receptor full agonist [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt] (Win55,212-2) inhibited gp120-induced IL-1β production and synapse in a manner reversed by a cannabinoid type 2 receptor antagonist. In contrast, Win55,212-2 did not inhibit synapse loss elicited by exposure to the HIV-1 protein Tat. These results indicate that cannabinoids prevent the impairment of network function produced by gp120 and, thus, might have therapeutic potential in HAD. PMID:21670103

  14. The impact of tonic GABAA receptor-mediated inhibition on neuronal excitability varies across brain region and cell type

    PubMed Central

    Lee, Vallent; Maguire, Jamie

    2014-01-01

    The diversity of GABAA receptor (GABAAR) subunits and the numerous configurations during subunit assembly give rise to a variety of receptors with different functional properties. This heterogeneity results in variations in GABAergic conductances across numerous brain regions and cell types. Phasic inhibition is mediated by synaptically-localized receptors with a low affinity for GABA and results in a transient, rapidly desensitizing GABAergic conductance; whereas, tonic inhibition is mediated by extrasynaptic receptors with a high affinity for GABA and results in a persistent GABAergic conductance. The specific functions of tonic versus phasic GABAergic inhibition in different cell types and the impact on specific neural circuits are only beginning to be unraveled. Here we review the diversity in the magnitude of tonic GABAergic inhibition in various brain regions and cell types, and highlight the impact on neuronal excitability in different neuronal circuits. Further, we discuss the relevance of tonic inhibition in various physiological and pathological contexts as well as the potential of targeting these receptor subtypes for treatment of diseases, such as epilepsy. PMID:24550784

  15. Ligand-Mediated cis-Inhibition of Receptor Signaling in the Self-Incompatibility Response of the Brassicaceae1[OPEN

    PubMed Central

    Tantikanjana, Titima; Nasrallah, June B.

    2015-01-01

    The inhibition of self-pollination in self-incompatible Brassicaceae is based on allele-specific trans-activation of the highly polymorphic S-locus receptor kinase (SRK), which is displayed at the surface of stigma epidermal cells, by its even more polymorphic pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. In an attempt to achieve constitutive activation of SRK and thus facilitate analysis of self-incompatibility (SI) signaling, we coexpressed an Arabidopsis lyrata SCR variant with its cognate SRK receptor in the stigma epidermal cells of Arabidopsis (Arabidopsis thaliana) plants belonging to the C24 accession, in which expression of SRK and SCR had been shown to exhibit a robust SI response. Contrary to expectation, however, coexpression of SRK and SCR was found to inhibit SRK-mediated signaling and to disrupt the SI response. This phenomenon, called cis-inhibition, is well documented in metazoans but has not as yet been reported for plant receptor kinases. We demonstrate that cis-inhibition of SRK, like its trans-activation, is based on allele-specific interaction between receptor and ligand. We also show that stigma-expressed SCR causes entrapment of its SRK receptor in the endoplasmic reticulum, thus disrupting the proper targeting of SRK to the plasma membrane, where the receptor would be available for productive interaction with its pollen coat-derived SCR ligand. Although based on an artificial cis-inhibition system, the results suggest novel strategies of pollination control for the generation of hybrid cultivars and large-scale seed production from hybrid plants in Brassicaceae seed crops and, more generally, for inhibiting cell surface receptor function and manipulating signaling pathways in plants. PMID:26269543

  16. Inverse agonist of nuclear receptor ERRγ mediates antidiabetic effect through inhibition of hepatic gluconeogenesis.

    PubMed

    Kim, Don-Kyu; Gang, Gil-Tae; Ryu, Dongryeol; Koh, Minseob; Kim, Yo-Na; Kim, Su Sung; Park, Jinyoung; Kim, Yong-Hoon; Sim, Taebo; Lee, In-Kyu; Choi, Cheol Soo; Park, Seung Bum; Lee, Chul-Ho; Koo, Seung-Hoi; Choi, Hueng-Sik

    2013-09-01

    Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder with diverse pathological manifestations and is often associated with abnormal regulation of hepatic glucose production. Many nuclear receptors known to control the hepatic gluconeogenic program are potential targets for the treatment of T2DM and its complications. Nevertheless, the therapeutic potential of the estrogen-related receptor γ (ERRγ) in T2DM remains unknown. In this study, we show that the nuclear receptor ERRγ is a major contributor to hyperglycemia under diabetic conditions by controlling hepatic glucose production. Hepatic ERRγ expression induced by fasting and diabetic conditions resulted in elevated levels of gluconeogenic gene expression and blood glucose in wild-type mice. Conversely, ablation of hepatic ERRγ gene expression reduced the expression of gluconeogenic genes and normalized blood glucose levels in mouse models of T2DM: db/db and diet-induced obesity (DIO) mice. In addition, a hyperinsulinemic-euglycemic clamp study and long-term studies of the antidiabetic effects of GSK5182, the ERRγ-specific inverse agonist, in db/db and DIO mice demonstrated that GSK5182 normalizes hyperglycemia mainly through inhibition of hepatic glucose production. Our findings suggest that the ability of GSK5182 to control hepatic glucose production can be used as a novel therapeutic approach for the treatment of T2DM. PMID:23775767

  17. ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors.

    PubMed

    Gordts, Philip L S M; Nock, Ryan; Son, Ni-Huiping; Ramms, Bastian; Lew, Irene; Gonzales, Jon C; Thacker, Bryan E; Basu, Debapriya; Lee, Richard G; Mullick, Adam E; Graham, Mark J; Goldberg, Ira J; Crooke, Rosanne M; Witztum, Joseph L; Esko, Jeffrey D

    2016-08-01

    Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III-targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1). ApoC-III ASO treatment lowered plasma TGs in mice lacking lipoprotein lipase (LPL), hepatic heparan sulfate proteoglycan (HSPG) receptors, LDLR, or LRP1 and in animals with combined deletion of the genes encoding HSPG receptors and LDLRs or LRP1. However, the ApoC-III ASO did not lower TG levels in mice lacking both LDLR and LRP1. LDLR and LRP1 were also required for ApoC-III ASO-induced reduction of plasma TGs in mice fed a high-fat diet, in postprandial clearance studies, and when ApoC-III-rich or ApoC-III-depleted lipoproteins were injected into mice. ASO reduction of ApoC-III had no effect on VLDL secretion, heparin-induced TG reduction, or uptake of lipids into heart and skeletal muscle. Our data indicate that ApoC-III inhibits turnover of TG-rich lipoproteins primarily through a hepatic clearance mechanism mediated by the LDLR/LRP1 axis. PMID:27400128

  18. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  19. Histamine H3 receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion.

    PubMed

    Iida, Tomomitsu; Yoshikawa, Takeo; Matsuzawa, Takuro; Naganuma, Fumito; Nakamura, Tadaho; Miura, Yamato; Mohsen, Attayeb S; Harada, Ryuichi; Iwata, Ren; Yanai, Kazuhiko

    2015-07-01

    Histamine is a physiological amine which initiates a multitude of physiological responses by binding to four known G-protein coupled histamine receptor subtypes as follows: histamine H1 receptor (H1 R), H2 R, H3 R, and H4 R. Brain histamine elicits neuronal excitation and regulates a variety of physiological processes such as learning and memory, sleep-awake cycle and appetite regulation. Microglia, the resident macrophages in the brain, express histamine receptors; however, the effects of histamine on critical microglial functions such as chemotaxis, phagocytosis, and cytokine secretion have not been examined in primary cells. We demonstrated that mouse primary microglia express H2 R, H3 R, histidine decarboxylase, a histamine synthase, and histamine N-methyltransferase, a histamine metabolizing enzyme. Both forskolin-induced cAMP accumulation and ATP-induced intracellular Ca(2+) transients were reduced by the H3 R agonist imetit but not the H2 R agonist amthamine. H3 R activation on two ubiquitous second messenger signalling pathways suggests that H3 R can regulate various microglial functions. In fact, histamine and imetit dose-dependently inhibited microglial chemotaxis, phagocytosis, and lipopolysaccharide (LPS)-induced cytokine production. Furthermore, we confirmed that microglia produced histamine in the presence of LPS, suggesting that H3 R activation regulate microglial function by autocrine and/or paracrine signalling. In conclusion, we demonstrate the involvement of histamine in primary microglial functions, providing the novel insight into physiological roles of brain histamine. PMID:25754956

  20. SKF-83566, a D1-dopamine receptor antagonist, inhibits the dopamine transporter.

    PubMed

    Stouffer, Melissa A; Ali, Solav; Reith, Maarten E A; Patel, Jyoti C; Sarti, Federica; Carr, Kenneth D; Rice, Margaret E

    2011-09-01

    Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ∼ 65% in 5 μM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 μM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 μM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 μM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT. PMID:21689106

  1. Inverse Agonist of Nuclear Receptor ERRγ Mediates Antidiabetic Effect Through Inhibition of Hepatic Gluconeogenesis

    PubMed Central

    Kim, Don-Kyu; Gang, Gil-Tae; Ryu, Dongryeol; Koh, Minseob; Kim, Yo-Na; Kim, Su Sung; Park, Jinyoung; Kim, Yong-Hoon; Sim, Taebo; Lee, In-Kyu; Choi, Cheol Soo; Park, Seung Bum; Lee, Chul-Ho; Koo, Seung-Hoi; Choi, Hueng-Sik

    2013-01-01

    Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder with diverse pathological manifestations and is often associated with abnormal regulation of hepatic glucose production. Many nuclear receptors known to control the hepatic gluconeogenic program are potential targets for the treatment of T2DM and its complications. Nevertheless, the therapeutic potential of the estrogen-related receptor γ (ERRγ) in T2DM remains unknown. In this study, we show that the nuclear receptor ERRγ is a major contributor to hyperglycemia under diabetic conditions by controlling hepatic glucose production. Hepatic ERRγ expression induced by fasting and diabetic conditions resulted in elevated levels of gluconeogenic gene expression and blood glucose in wild-type mice. Conversely, ablation of hepatic ERRγ gene expression reduced the expression of gluconeogenic genes and normalized blood glucose levels in mouse models of T2DM: db/db and diet-induced obesity (DIO) mice. In addition, a hyperinsulinemic-euglycemic clamp study and long-term studies of the antidiabetic effects of GSK5182, the ERRγ-specific inverse agonist, in db/db and DIO mice demonstrated that GSK5182 normalizes hyperglycemia mainly through inhibition of hepatic glucose production. Our findings suggest that the ability of GSK5182 to control hepatic glucose production can be used as a novel therapeutic approach for the treatment of T2DM. PMID:23775767

  2. Inhibition of platelet aggregation by vanilloid-like agents is not mediated by transient receptor potential vanilloid-1 channels or cannabinoid receptors.

    PubMed

    Almaghrabi, Safa; Geraghty, Dominic; Ahuja, Kiran; Adams, Murray

    2016-06-01

    Vanilloid-like agents, including capsaicin, N-arachidonoyl-dopamine and N-oleoyldopamine inhibit platelet aggregation, however little is known about the precise mechanism(s) of action. The authors have previously shown that blocking of the capsaicin receptor, transient receptor potential vanilloid-1 (TRPV1), does not interfere with capsaicin action during adenosine diphosphate (ADP)-induced aggregation. This research is extended to investigate the effect of these vanilloid-like-agents on platelet count, and to test whether the effect of these agents is mediated through TRPV1 and/or cannabinoid (CB1 and CB2) receptors in the presence of other agonists, including collagen and arachidonic acid. Incubation of platelets with each of the individual vanilloids, or with receptor antagonists of TRPV1 (SB452533), CB1 (AM251) and CB2 (AM630), for up to 2 h did not significantly affect the platelet count. Similarly, the effect of individual vanilloids on the inhibition of platelet aggregation was not significantly different in the presence of receptor agonists compared to control, irrespective of the agonist used, suggesting that the inhibitory effect of vanilloids on platelet aggregation is independent of TRPV1, CB1 and CB2 receptors. Further research on the antiplatelet activity of vanilloids should focus on mechanisms other than those associated with vanilloid receptors. PMID:26991025

  3. Diurnal inhibition of NMDA-EPSCs at rat hippocampal mossy fibre synapses through orexin-2 receptors

    PubMed Central

    Perin, Martina; Longordo, Fabio; Massonnet, Christine; Welker, Egbert; Lüthi, Anita

    2014-01-01

    Diurnal release of the orexin neuropeptides orexin-A (Ox-A, hypocretin-1) and orexin-B (Ox-B, hypocretin-2) stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically we found sparse but widespread innervation of hippocampal subfields through Ox-A- and Ox-B-containing fibres in young adult rats. The actions of Ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber time (ZT) 4–8, corresponding to 4–8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4–8, exogenous Ox-A (100 nm in bath) inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) at mossy fibre (MF)–CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral–CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 μm), but not by orexin-1 receptor inhibition (SB334867, 1 μm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous Ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 h through i.p. injections of almorexant (100 mg kg−1), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep–wake cycle. PMID:25085886

  4. Cord Blood Stem Cells Inhibit Epidermal Growth Factor Receptor Translocation to Mitochondria in Glioblastoma

    PubMed Central

    Dasari, Venkata Ramesh; Velpula, Kiran Kumar; Alapati, Kiranmai; Gujrati, Meena; Tsung, Andrew J.

    2012-01-01

    Background Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR translocation to mitochondria has been reported recently in different cancer types. However, mechanistic aspects of EGFR translocation to mitochondria in GBM have not been evaluated to date. Methodology/Principle Findings In the present study, we analyzed the expression of EGFR in GBM-patient derived specimens using immunohistochemistry, reverse-transcription based PCR and Western blotting techniques. In clinical samples, EGFR co-localizes with FAK in mitochondria. We evaluated this previous observation in standard glioma cell lines and in vivo mice xenografts. We further analyzed the effect of human umbilical cord blood stem cells (hUCBSC) on the inhibition of EGFR expression and EGFR signaling in glioma cells and xenografts. Treatment with hUCBSC inhibited the expression of EGFR and its co-localization with FAK in glioma cells. Also, hUCBSC inhibited the co-localization of activated forms of EGFR, FAK and c-Src in mitochondria of glioma cells and xenografts. In addition, hUCBSC also inhibited EGFR signaling proteins in glioma cells both in vitro and in vivo. Conclusions/Significance We have shown that hUCBSC treatments inhibit phosphorylation of EGFR, FAK and c-Src forms. Our findings associate EGFR expression and its localization to mitochondria with specific biological functions in GBM cells and provide relevant preclinical information that can be used for the development of effective hUCBSC-based therapies. PMID:22348136

  5. Dopamine receptor antagonist thioridazine inhibits tumor growth in a murine breast cancer model.

    PubMed

    Yin, Tao; He, Sisi; Shen, Guobo; Ye, Tinghong; Guo, Fuchun; Wang, Yongsheng

    2015-09-01

    Neuropsychological factors have been shown to influence tumor progression and therapeutic response. The present study investigated the effect of the dopamine receptor antagonist thioridazine on murine breast cancer. The anti‑tumor efficacy of thioridazine was assessed using a murine breast cancer model. Cell apoptosis and proliferation were analyzed in vitro using flow cytometry (FCM) and the MTT assay, respectively. Western blot analysis was performed to assess Akt, phosphorylated (p)‑Akt, signal transducer and activator of transcription (STAT) 3, p‑STAT3 and p‑p65 in tumor cells following treatment with thioridazine. The Ki67 index and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)‑positive apoptotic cells were assessed in the tumor sections. Thioridazine was found to reduce tumor growth, inhibit tumor cell proliferation and induce apoptosis in a dose‑ and time‑dependent manner in vitro. Thioridazine was also found to markedly inhibit tumor proliferation and induce tumor cell apoptosis in vivo as shown by the lower Ki67 index and increase in TUNEL‑positive cells. In addition, thioridazine was observed to inhibit the activation of the canonical nuclear factor κ‑light‑chain‑enhancer of activated B cells pathway and exert anti‑tumor effects by remodeling the tumor stroma, as well as inhibit angiogenesis in the tumor microenvironment. In conclusion, thioridazine was found to significantly inhibit breast tumor growth and the potential for thioridazine to be used in cancer therapy may be re‑evaluated and investigated in clinical settings. PMID:26095429

  6. Can Structural Features of Kinase Receptors Provide Clues on Selectivity and Inhibition?: A Molecular Modeling Study

    PubMed Central

    Ravichandran, Sarangan; Luke, Brian T.; Collins, Jack R.

    2015-01-01

    Cancer is a complex disease resulting from the uncontrolled proliferation of cell signaling events. Protein kinases have been identified as central molecules that participate overwhelmingly in oncogenic events, thus becoming key targets for anticancer drugs. A majority of studies converged on the idea that ligand-binding pockets of kinases retain clues to the inhibiting abilities and cross-reacting tendencies of inhibitor drugs. Even though these ideas are critical for drug discovery, validating them using experiments is not only difficult, but in some cases infeasible. To overcome these limitations and to test these ideas at the molecular level, we present here the results of receptor-focused in-silico docking of nine marketed drugs to 19 different wild-type and mutated kinases chosen from a wide range of families. This investigation highlights the need for using relevant models to explain the correct inhibition trends and the results are used to make predictions that might be able to influence future experiments. Our simulation studies are able to correctly predict the primary targets for each drug studied in majority of cases and our results agree with the existing findings. Our study shows that the conformations a given receptor acquires during kinase activation, and their micro-environment, defines the ligand partners. Type II drugs display high compatibility and selectivity for DFG-out kinase conformations. On the other hand Type I drugs are less selective and show binding preferences for both the open and closed forms of selected kinases. Using this receptor-focused approach, it is possible to capture the observed fold change in binding affinities between the wild-type and disease-centric mutations in ABL kinase for Imatinib and the second-generation ABL drugs. The effects of mutation are also investigated for two other systems, EGFR and B-Raf. Finally, by including pathway information in the design it is possible to model kinase inhibitors with potentially

  7. microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2.

    PubMed

    Li, Jing; Zhang, Suhua

    2016-08-01

    Transformation of macrophages into foam cells plays a critical role in the pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 μg/ml oxidized low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to prevent macrophage foam cell formation in atherosclerosis. PMID:27216461

  8. Androgen receptor silences thioredoxin-interacting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic β-Cells.

    PubMed

    Harada, Naoki; Katsuki, Takahiro; Takahashi, Yuji; Masuda, Tatsuya; Yoshinaga, Mariko; Adachi, Tetsuya; Izawa, Takeshi; Kuwamura, Mitsuru; Nakano, Yoshihisa; Yamaji, Ryoichi; Inui, Hiroshi

    2015-06-01

    Androgen receptor (AR) is known to bind to the same cis-element that glucocorticoid receptor (GR) binds to. However, the effects of androgen signaling on glucocorticoid signaling have not yet been elucidated. Here, we investigated the effects of testosterone on dexamethasone (DEX, a synthetic glucocorticoid)-induced apoptosis of pancreatic β-cells, which might be involved in the pathogenesis of type 2 diabetes mellitus in males. We used INS-1 #6 cells, which were isolated from the INS-1 pancreatic β-cell line and which express high levels of AR. Testosterone and dihydrotestosterone inhibited apoptosis induced by DEX in INS-1 #6 cells. AR knockdown and the AR antagonist hydroxyflutamide each diminished the anti-apoptotic effects of testosterone. AR was localized in the nucleus of both INS-1 #6 cells and pancreatic β-cells of male rats. Induction of thioredoxin-interacting protein (TXNIP) is known to cause pro-apoptotic effects in β-cells. Testosterone suppressed the DEX-induced increase of TXNIP at the transcriptional level. A Chromatin immunoprecipitation assays showed that both AR and GR competitively bound to the TXNIP promoter in ligand-dependent manners. Recombinant DNA-binding domain of AR bound to the same cis-element of the TXNIP promoter that GR binds to. Our results show that AR and GR competitively bind to the same cis-element of TXNIP promoter as a silencer and enhancer, respectively. These results indicate that androgen signaling functionally competes with glucocorticoid signaling in pancreatic β-cell apoptosis. PMID:25639671

  9. Metformin Disrupts Crosstalk Between G protein-Coupled Receptor and Insulin Receptor Signaling Systems and Inhibits Pancreatic Cancer Growth

    PubMed Central

    Kisfalvi, Krisztina; Eibl, Guido; Sinnett-Smith, James; Rozengurt, Enrique

    2009-01-01

    Recently we identified a novel crosstalk between insulin and G-protein-coupled receptor (GPCR) signaling pathways in human pancreatic cancer cells. Insulin enhanced GPCR signaling through a rapamycin-sensitive mTOR-dependent pathway. Metformin, the most widely used drug in the treatment of type-2 diabetes, activates AMP kinase (AMPK), which negatively regulates mTOR. Here, we determined whether metformin disrupts crosstalk between insulin receptor and GPCR signaling in pancreatic cancer cells. Treatment of human pancreatic cancer cells (PANC-1, MIAPaCa-2, BxPC-3) with insulin (10ng/ml) for 5 min markedly enhanced the increase in intracellular [Ca2+] induced by GPCR agonists (e.g. neurotensin, bradykinin, angiotensin II). Metformin pretreatment completely abrogated insulin-induced potentiation of Ca2+ signaling but did not interfere with the effect of GPCR agonists alone. Insulin also enhanced GPCR agonist-induced growth, measured by DNA synthesis, and numbers of cells cultured in adherent or non-adherent conditions. Low doses of metformin (0.1-0.5 mM) blocked stimulation of DNA synthesis, anchorage-dependent and independent growth induced by insulin and GPCR agonists. Treatment with metformin induced striking and sustained increase in the phosphorylation of AMPK at Thr172 and a selective AMPK inhibitor (compound C, at 5μM) reversed the effects of metformin on [Ca2+]i, and DNA synthesis, indicating that metformin acts through AMPK activation. In view of these results we tested whether metformin inhibits pancreatic cancer growth. Administration of metformin significantly decreased the growth of MIAPaCa-2 and PANC-1 cells xenografted on the flank of nude mice. The results raise the possibility that metformin could be a potential candidate in novel treatment strategies for human pancreatic cancer. PMID:19679549

  10. Coantagonism of Glutamate Receptors and Nicotinic Acetylcholinergic Receptors Disrupts Fear Conditioning and Latent Inhibition of Fear Conditioning

    ERIC Educational Resources Information Center

    Gould, Thomas J.; Lewis, Michael C.

    2005-01-01

    The present study investigated the hypothesis that both nicotinic acetylcholinergic receptors (nAChRs) and glutamate receptors ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) and N-methyl-D-aspartate glutamate receptors (NMDARs)) are involved in fear conditioning, and may modulate similar processes. The effects of the…

  11. Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

    SciTech Connect

    Wang, Junru; Kulkarni, Ashwini; Chintala, Madhu; Fink, Louis M.; Hauer-Jensen, Martin

    2013-01-01

    Purpose: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. Methods and Materials: Rats underwent fractionated X-irradiation (5 Gy Multiplication-Sign 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. Results: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. Conclusion: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

  12. Generation and Characterization of Small Single Domain Antibodies Inhibiting Human Tumor Necrosis Factor Receptor 1*

    PubMed Central

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E.; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-01-01

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named “TNF Receptor-One Silencer” (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients. PMID:25538244

  13. Delphinidin inhibits cell proliferation and invasion via modulation of Met receptor phosphorylation

    SciTech Connect

    Syed, Deeba N.; Afaq, Farrukh; Sarfaraz, Sami; Khan, Naghma; Kedlaya, Rajendra; Setaluri, Vijayasaradhi; Mukhtar, Hasan

    2008-08-15

    The HGF/Met signaling pathway is deregulated in majority of cancers and is associated with poor prognosis in breast cancer. Delphinidin, present in pigmented fruits and vegetables possesses potent anti-oxidant, anti-inflammatory and anti-angiogenic properties. Here, we assessed the anti-proliferative and anti-invasive effects of delphinidin on HGF-mediated responses in the immortalized MCF-10A breast cell line. Treatment of cells with delphinidin prior to exposure to exogenous HGF resulted in the inhibition of HGF-mediated (i) tyrosyl-phosphorylation and increased expression of Met receptor, (ii) phosphorylation of downstream regulators such as FAK and Src and (iii) induction of adaptor proteins including paxillin, Gab-1 and GRB-2. In addition, delphinidin treatment resulted in significant inhibition of HGF-activated (i) Ras-ERK MAPKs and (ii) PI3K/AKT/mTOR/p70S6K pathways. Delphinidin was found to repress HGF-activated NF{kappa}B transcription with a decrease in (i) phosphorylation of IKK{alpha}/{beta} and I{kappa}B{alpha}, and (ii) activation and nuclear translocation of NF{kappa}B/p65. Inhibition of HGF-mediated membrane translocation of PKC{alpha} as well as decreased phosphorylation of STAT3 was further observed in delphinidin treated cells. Finally, decreased cell viability of Met receptor expressing breast cancer cells treated with delphinidin argues for a potential role of the agent in the prevention of HGF-mediated activation of various signaling pathways implicated in breast cancer.

  14. Extracellular Nucleotides Inhibit Insulin Receptor Signaling, Stimulate Autophagy and Control Lipoprotein Secretion

    PubMed Central

    Chatterjee, Cynthia; Sparks, Daniel L.

    2012-01-01

    Hyperglycemia is associated with abnormal plasma lipoprotein metabolism and with an elevation in circulating nucleotide levels. We evaluated how extracellular nucleotides may act to perturb hepatic lipoprotein secretion. Adenosine diphosphate (ADP) (>10 µM) acts like a proteasomal inhibitor to stimulate apoB100 secretion and inhibit apoA-I secretion from human liver cells at 4 h and 24 h. ADP blocks apoA-I secretion by stimulating autophagy. The nucleotide increases cellular levels of the autophagosome marker, LC3-II, and increases co-localization of LC3 with apoA-I in punctate autophagosomes. ADP affects autophagy and apoA-I secretion through P2Y13. Overexpression of P2Y13 increases cellular LC3-II levels by ∼50% and blocks induction of apoA-I secretion. Conversely, a siRNA-induced reduction in P2Y13 protein expression of 50% causes a similar reduction in cellular LC3-II levels and a 3-fold stimulation in apoA-I secretion. P2Y13 gene silencing blocks the effects of ADP on autophagy and apoA-I secretion. A reduction in P2Y13 expression suppresses ERK1/2 phosphorylation, increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, and blocks the inhibition of Akt phosphorylation by TNFα and ADP. Conversely, increasing P2Y13 expression significantly inhibits insulin-induced phosphorylation of insulin receptor (IR-β) and Akt, similar to that observed after treatment with ADP. Nucleotides therefore act through P2Y13, ERK1/2 and insulin receptor signaling to stimulate autophagy and affect hepatic lipoprotein secretion. PMID:22590634

  15. 5-HT1D receptor inhibits renal sympathetic neurotransmission by nitric oxide pathway in anesthetized rats.

    PubMed

    García-Pedraza, José-Ángel; García, Mónica; Martín, María-Luisa; Morán, Asunción

    2015-09-01

    Although serotonin has been shown to inhibit peripheral sympathetic outflow, serotonin regulation on renal sympathetic outflow has not yet been elucidated. This study investigated which 5-HT receptor subtypes are involved. Wistar rats were anesthetized (sodium pentobarbital; 60mg/kg, i.p.), and prepared for in situ autoperfused rat kidney, which allows continuous measurement of systemic blood pressure (SBP), heart rate (HR) and renal perfusion pressure (PP). Electrical stimulation of renal sympathetic nerves resulted in frequency-dependent increases in PP (18.3±1.0, 43.7±2.7 and 66.7±4.0 for 2, 4 and 6Hz, respectively), without altering SBP or HR. 5-HT, 5-carboxamidotryptamine (5-HT1/7 agonist) (0.00000125-0.1μg/kg each) or l-694,247 (5-HT1D agonist; 0.0125μg/kg) i.a. bolus inhibited vasopressor responses by renal nerve electrical stimulation, unlike i.a. bolus of agonists α-methyl-5-HT (5-HT2), 1-PBG (5-HT3), cisapride (5-HT4), AS-19 (5-HT7), CGS-12066B (5-HT1B) or 8-OH-DPAT (5-HT1A) (0.0125μg/kg each). The effect of l-694,247 did not affect the exogenous norepinephrine-induced vasoconstrictions, whereas was abolished by antagonist LY310762 (5-HT1D; 1mg/kg) or l-NAME (nitric oxide; 10mg/kg), but not by indomethacin (COX1/2; 2mg/kg) or glibenclamide (ATP-dependent K(+) channel; 20mg/kg). These results suggest that 5-HT mechanism-induced inhibition of rat vasopressor renal sympathetic outflow is mainly mediated by prejunctional 5-HT1D receptors via nitric oxide release. PMID:26003124

  16. Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization

    PubMed Central

    Gerevich, Z; Zadori, Z; Müller, C; Wirkner, K; Schröder, W; Rubini, P; Illes, P

    2007-01-01

    Background and purpose: The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X3 receptor-channels. Experimental approach: Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X3 receptors (HEK293-hP2X3 cells) and rat dorsal root ganglion (DRG) neurons. Key results: In both cell types, the P2Y1,12,13 receptor agonist, ADP-β-S, inhibited P2X3 currents evoked by the selective agonist, α,β-methylene ATP (α,β-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-β-S, a procedure known to block all G protein heterotrimers. P2X3 currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X3 receptor agonist α,β-meATP. In the presence of intracellular GDP-β-S, ATP- and α,β-meATP-induced currents were identical. Recovery from P2X3 receptor desensitization induced by repetitive ATP application was slower than the recovery from α,β-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-β-S, the recovery from the ATP- and α,β-meATP-induced desensitization were of comparable speed. Conclusions and Implications: Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X3 receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X3 and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity. PMID:17351651

  17. Prostamide F2α receptor antagonism combined with inhibition of FAAH may block the pro-inflammatory mediators formed following selective FAAH inhibition

    PubMed Central

    Ligresti, Alessia; Martos, Jose; Wang, Jenny; Guida, Francesca; Allarà, Marco; Palmieri, Vittoria; Luongo, Livio; Woodward, David; Di Marzo, Vincenzo

    2014-01-01

    Background and PurposeProstamides are lipid mediators formed by COX-2-catalysed oxidation of the endocannabinoid anandamide and eliciting effects often opposed to those caused by anandamide. Prostamides may be formed when hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) is physiologically, pathologically or pharmacologically decreased. Thus, therapeutic benefits of FAAH inhibitors might be attenuated by concomitant production of prostamide F2α. This loss of benefit might be minimized by compounds designed to selectively antagonize prostamide receptors and also inhibiting FAAH. Experimental ApproachInhibition of FAAH by a series of selective antagonists of prostamide receptors, including AGN 204396, AGN 211335 and AGN 211336, was assessed using rat, mouse and human FAAH in vitro, together with affinity for human recombinant CB1 and CB2 receptors. Effects in vivo were measured in a model of formalin-induced inflammatory pain in mice. Key ResultsThe prostamide F2α receptor antagonists were active against mouse and rat FAAH in the low μM range and behaved as non-competitive and plasma membrane-permeant inhibitors. AGN 211335, the most potent inhibitor of rat FAAH (IC50 = 1.2 μM), raised exogenous anandamide levels in intact cells and also bound to cannabinoid CB1 receptors. Both AGN 211335 and AGN 211336 (0.25–1 mg·kg−1, i.p.) inhibited the formalin-induced nociceptive response in mice. Conclusions and ImplicationsSynthetic compounds with indirect agonist activity at cannabinoid receptors and antagonist activity at prostamide receptors can be developed. Such compounds could be used as alternatives to selective FAAH inhibitors to prevent the possibility of prostamide F2α-induced inflammation and pain. Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6 PMID:24102214

  18. Light and hydrogen peroxide inhibit C. elegans Feeding through gustatory receptor orthologs and pharyngeal neurons.

    PubMed

    Bhatla, Nikhil; Horvitz, H Robert

    2015-02-18

    While gustatory sensing of the five primary flavors (sweet, salty, sour, bitter, and savory) has been extensively studied, pathways that detect non-canonical taste stimuli remain relatively unexplored. In particular, while reactive oxygen species cause generalized damage to biological systems, no gustatory mechanism to prevent ingestion of such material has been identified in any organism. We observed that light inhibits C. elegans feeding and used light as a tool to uncover molecular and neural mechanisms for gustation. Light can generate hydrogen peroxide, and we discovered that hydrogen peroxide similarly inhibits feeding. The gustatory receptor family members LITE-1 and GUR-3 are required for the inhibition of feeding by light and hydrogen peroxide. The I2 pharyngeal neurons increase calcium in response to light and hydrogen peroxide, and these responses require GUR-3 and a conserved antioxidant enzyme peroxiredoxin PRDX-2. Our results demonstrate a gustatory mechanism that mediates the detection and blocks ingestion of a non-canonical taste stimulus, hydrogen peroxide. PMID:25640076

  19. Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition.

    PubMed

    Schmidt, Benjamin; Wei, Lan; DePeralta, Danielle K; Hoshida, Yujin; Tan, Poh Seng; Sun, Xiaochen; Sventek, Janelle P; Lanuti, Michael; Tanabe, Kenneth K; Fuchs, Bryan C

    2016-03-15

    A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one-third of all HCC in clinical series. S2 cells express E-cadherin and c-myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non-S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non-S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1-3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1-3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4-MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway. PMID:26481559

  20. Recombinant human vascular endothelial growth factor receptor 1 effectively inhibits angiogenesis in vivo.

    PubMed

    Wang, Jinliang; Shi, Minglei; Xi, Yongyi; Gao, Lihua; Zhang, Guanyi; Shao, Yong; Chen, Huipeng; Hu, Xianwen

    2015-05-01

    Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑121. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro. PMID:25607471

  1. Allicin inhibits lymphangiogenesis through suppressing activation of vascular endothelial growth factor (VEGF) receptor.

    PubMed

    Wang, Weicang; Du, Zheyuan; Nimiya, Yoshiki; Sukamtoh, Elvira; Kim, Daeyoung; Zhang, Guodong

    2016-03-01

    Allicin, the most abundant organosulfur compound in freshly crushed garlic tissues, has been shown to have various health-promoting effects, including anticancer actions. A better understanding of the effects and mechanisms of allicin on tumorigenesis could facilitate development of allicin or garlic products for cancer prevention. Here we found that allicin inhibited lymphangiogenesis, which is a critical cellular process implicated in tumor metastasis. In primary human lymphatic endothelial cells, allicin at 10 μM inhibited capillary-like tube formation and cell migration, and it suppressed phosphorylation of vascular endothelial growth factor receptor 2 and focal adhesion kinase. Using a Matrigel plug assay in mice, addition of 10 μg allicin in Matrigel plug inhibited 40-50% of vascular endothelial growth factor-C-induced infiltration of lymphatic endothelial cells and leukocytes. S-Allylmercaptoglutathione, a major cellular metabolite of allicin, had no effect on lymphangiogenic responses in lymphatic endothelial cells. Together, these results demonstrate the antilymphangiogenic effect of allicin in vitro and in vivo, suggesting a novel mechanism for the health-promoting effects of garlic compounds. PMID:26895668

  2. Pax6 represses androgen receptor-mediated transactivation by inhibiting recruitment of the coactivator SPBP.

    PubMed

    Elvenes, Julianne; Thomassen, Ernst Ivan Simon; Johnsen, Sylvia Sagen; Kaino, Katrine; Sjøttem, Eva; Johansen, Terje

    2011-01-01

    The androgen receptor (AR) has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer. PMID:21935435

  3. Pax6 Represses Androgen Receptor-Mediated Transactivation by Inhibiting Recruitment of the Coactivator SPBP

    PubMed Central

    Johnsen, Sylvia Sagen; Kaino, Katrine; Sjøttem, Eva; Johansen, Terje

    2011-01-01

    The androgen receptor (AR) has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer. PMID:21935435

  4. Light and hydrogen peroxide inhibit C. elegans feeding through gustatory receptor orthologs and pharyngeal neurons

    PubMed Central

    Bhatla, Nikhil; Horvitz, H. Robert

    2015-01-01

    SUMMARY While gustatory sensing of the five primary flavors (sweet, salty, sour, bitter, and savory) has been extensively studied, pathways that detect non-canonical taste stimuli remain relatively unexplored. In particular, while reactive oxygen species cause generalized damage to biological systems, no gustatory mechanism to prevent ingestion of such material has been identified in any organism. We observed that light inhibits C. elegans feeding and used light as a tool to uncover molecular and neural mechanisms for gustation. Light can generate hydrogen peroxide, and we discovered that hydrogen peroxide similarly inhibits feeding. The gustatory receptor family members LITE-1 and GUR-3 are required for the inhibition of feeding by light and hydrogen peroxide. The I2 pharyngeal neurons increase calcium in response to light and hydrogen peroxide, and these responses require GUR-3 and a conserved antioxidant enzyme peroxiredoxin PRDX-2. Our results demonstrate a gustatory mechanism that mediates the detection and blocks ingestion of a non-canonical taste stimulus, hydrogen peroxide. PMID:25640076

  5. Inhibition of protein synthesis by the T cell receptor-inducible human TDAG51 gene product.

    PubMed

    Hinz, T; Flindt, S; Marx, A; Janssen, O; Kabelitz, D

    2001-05-01

    The T cell death associated gene 51 (TDAG51) was shown to be required for T cell receptor (TCR)-dependent induction of Fas/Apo1/CD95 expression in a murine T cell hybridoma. Despite the absence of a nuclear localization sequence and a nucleic acid binding domain, it was suggested to be localized in the nucleus and to function as a transcription factor regulating Fas-expression. However, we demonstrate that the human (h)TDAG51 protein is localized in the cytoplasm and the nucleoli, suggesting a role in ribosome biogenesis and/or translation regulation. Indeed, it strongly inhibited translation of a luciferase mRNA in a reticulocyte translational extract. Furthermore, cotransfection of hTDAG51 and the luciferase gene into 293T cells resulted in a strong inhibition of luciferase mRNA translation. Our findings were further strengthened by isolating in a yeast two-hybrid screen three proteins which are involved in the regulation of translation. We speculate that hTDAG51 couples TCR signaling to inhibition of protein biosynthesis in activated T lymphocytes. PMID:11369516

  6. Enhanced GABAergic inhibition in the premammillary nucleus of photorefractory turkey hens via GABAA receptor upregulation.

    PubMed

    Kosonsiriluk, Sunantha; Chaiworakul, Voravasa; Mauro, Laura J; El Halawani, Mohamed E

    2016-05-01

    The premammillary nucleus (PMM) of the turkey mediobasal hypothalamus, where dopamine-melatonin (DA-Mel) neurons are localized, is a site for photoreception and photoperiodic time measurement, which is essential for the initiation of avian reproductive seasonality. In addition, this area could also be responsible for the onset and maintenance of photorefractoriness at the end of the breeding season due to the enhanced inhibitory effect of γ-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter in the central nervous system which interferes with the photosexual response in the turkey, a seasonally breeding bird. Here, we further characterized the GABAA receptor subunits in the PMM DA-Mel neurons related to reproductive seasonality and the onset of photorefractoriness. GABAA receptor subunits and GABA synthesis enzymes in the PMM of photosensitive and photorefractory turkey hens were identified using real-time qRT-PCR. The upregulation of GABAA receptor α1-3, β2-3, γ1-3, ρ1-3, δ, and θ mRNA expression were observed in the PMM of photorefractory birds when compared to those of photosensitive ones while there is no change observed in the GABA synthesis enzymes, glutamate decarboxylase 1 and 2. Those upregulated GABAA receptor subunits were further examined using immunohistochemical staining and they appeared to be co-localized within the PMM DA-Mel neurons. The upregulation of GABAA receptor subunits observed in the PMM of photorefractory birds coincides with a lack of responsiveness to a light stimulus provided during the photosensitive phase. This is supported by the absence of c-fos induction and TH upregulation in the PMM and a subsequence inhibition of c-fos and GnRH-I expression in the nucleus commissurae pallii. The augmented GABAA receptor subunits expression may mediate an enhancement of inhibitory GABAergic neurotransmission and the subsequent interference with the photosexual response. This could contribute to the state of

  7. Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

    PubMed

    Leong, Ron L; Xing, Hong; Braekman, Jean-Claude; Kem, William R

    2015-10-01

    Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more

  8. Inhibition by synthetic peptides from human IgG Fc of OKT4 binding and Fc receptor binding

    SciTech Connect

    Gaarde, W.A.; McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-05

    Synthetic peptides from the Fc region of human IgG were tested for the ability to inhibit IgG rosette formation, /sup 125/I-IgG binding to Fc receptors on lymphocytes, and expression of the T4 cell determinant. The Fc/sub ..gamma../ peptides inhibited IgG rosette formation and competitively inhibited both /sup 125/I-IgG binding to Fc receptors and OKT4 binding to the T4 cell determinant. Peptides containing D-amino acids did not inhibit IgG rosettes, OKT4 binding or /sup 125/I-IgG binding. OKT4 binding to T4 on MNC was inhibited by heat aggregated IgG but not by monomeric IgG. OKT3, OKT8 and OKT11 binding to their determinants was not altered by Fc/sub ..gamma../ peptides, aggregated IgG or monomeric IgG. These results suggest that T4 antigen, Fc receptor for IgG and the Fc/sub ..gamma../ peptide binding site are in close proximity on the cell surface and when occupied by their respective ligands may sterically hinder binding to other sites. Alternatively, Fc/sub ..gamma../ peptides may indirectly regulate cell surface expression of T4 and/or Fc receptors for IgG. These Fc/sub ..gamma../ peptides inhibit the MLR, inhibit antigen induced T cell proliferation and reverse animal models of autoimmune disease. The immunoregulatory activities of these peptides may be related to their selective action on T4 helper/inducer lymphocytes expressing Fc receptors.

  9. Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth, invasion, and epithelial-to-mesenchymal transition.

    PubMed

    Lin, Jia-Ji; Zhao, Tian-Zhi; Cai, Wen-Ke; Yang, Yong-Xiang; Sun, Chao; Zhang, Zhuo; Xu, Yu-Qiao; Chang, Ting; Li, Zhu-Yi

    2015-07-10

    Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(-)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas. PMID:25940798

  10. Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer.

    PubMed

    Xia, Hui; Zhao, Ying-Nan; Yu, Chang-Hai; Zhao, Yun-Long; Liu, Yang

    2016-07-15

    Metabotropic glutamate receptor 1 (mGlu1 receptor) is expressed in many cancer cell types as compared to normal counterparts underscoring its potential role in tumor behavior. The aim of present study was to test the role of mGlu1 receptor in experimental non-small cell lung cancer (NSCLC). First, protein expression of mGlu1 receptor was higher in human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes, when compared to normal bronchial epithelial cells. Inhibition of mGlu1 receptor by BAY36-7620 (an mGlu1 receptor-specific inhibitor) inhibited tumor growth and prolonged survival of mice with tumors of A549 or H1299. Treatment with BAY36-7620 suppressed AKT phosphorylation in A549 tumors and pre-treatment with BAY36-7620 blocked the L-quisqualate (a potent mGlu1 receptor agonist)-induced AKT phosphorylation in A549 cells. Treatment with BAY36-7620 reduced cellular proliferation of A549 cells. Treatment with BAY36-7620 enhanced cleaved PARP levels and reduced protein expression of bcl-2, HIF-1α, and VEGF. In contrast, treatment with L-quisqualate reduced cleaved PARP levels and enhanced protein expression of bcl-2, HIF-1α, VEGF, and IL-8, which was reversed by co-incubation with MK2206 (an AKT inhibitor). Pre-treatment with BAY36-7620 blocked the VEGF-induced AKT phosphorylation in HUVECs. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was reversed by co-incubation with MK2206. Furthermore, mGlu1 receptor knockdown suppressed tumor growth and prolonged survival of mice with tumors of A549 or H1299. Collectively, inhibition of mGlu1 receptor suppressed tumor growth and angiogenesis in experimental NSCLC. PMID:27132814

  11. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  12. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition

    PubMed Central

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-01-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs. PMID:26358189

  13. MiRNA-101 inhibits breast cancer growth and metastasis by targeting CX chemokine receptor 7

    PubMed Central

    Zhu, Xiao-Shan; Liu, Qin-Qin; Wang, Xiu-Li; Yu, Feng; Liu, Yan-Li; Yang, An-Gang; Gao, Chun-Fang

    2015-01-01

    Whereas miR-101 is involved in the development and progression of breast cancer, the underlying molecular mechanisms remain to be elucidated. Here, we report that miR-101 expression is inversely correlated with the clinical stage, lymph node metastasis and prognosis in breast cancers. Introduction of miR-101 inhibited breast cancer cell proliferation and invasion in vitro and suppressed tumor growth and lung metastasis of in vivo. CX chemokine receptor 7 (CXCR7) is a direct target of miR-101, positively correlating with the histological grade and the incidence of lymph node metastasis in breast cancer patients. The effects of miR-101 were mimicked and counteracted by CXCR7 depletion and overexpression, respectively. STAT3 signaling downstream of CXCR7 is involved in miR-101 regulation of breast cancer cell behaviors. These findings have implications for the potential application of miR-101 in breast cancer treatment. PMID:26360780

  14. Protease-activated receptor 1 and 4 signal inhibition reduces preterm neonatal hemorrhagic brain injury

    PubMed Central

    Lekic, Tim; Klebe, Damon; McBride, Devin W; Manaenko, Anatol; Rolland, William B.; Flores, Jerry J.; Altay, Orhan; Tang, Jiping; Zhang, John H.

    2015-01-01

    Background and Purpose This study examines the role of thrombin’s protease-activated receptors (PAR)-1,-4 in mediating cyclooxygenase (COX)-2 and mammalian target of rapamycin (mTOR) following germinal matrix hemorrhage (GMH). Methods GMH was induced by intraparenchymal infusion of bacterial collagenase into the right ganglionic eminence of P7 rat pups. Animals were treated with either PAR-1, -4, COX-2, or mTOR inhibitors by 1 hour, and up to five days. Results We found increased thrombin activity 6–24 hrs after GMH, and PAR-1, -4, inhibition normalized COX-2 and mTOR by 72 hrs. Early treatment with NS398 or rapamycin substantially improved long-term outcomes in juvenile animals. Conclusions Suppressing early PAR signal transduction, and postnatal NS398 or rapamycin treatment, may help reduce GMH severity in susceptible preterm infants. PMID:25931468

  15. Hypoxia increases exercise heart rate despite combined inhibition of β-adrenergic and muscarinic receptors.

    PubMed

    Siebenmann, C; Rasmussen, P; Sørensen, H; Bonne, T C; Zaar, M; Aachmann-Andersen, N J; Nordsborg, N B; Secher, N H; Lundby, C

    2015-06-15

    Hypoxia increases the heart rate response to exercise, but the mechanism(s) remains unclear. We tested the hypothesis that the tachycardic effect of hypoxia persists during separate, but not combined, inhibition of β-adrenergic and muscarinic receptors. Nine subjects performed incremental exercise to exhaustion in normoxia and hypoxia (fraction of inspired O2 = 12%) after intravenous administration of 1) no drugs (Cont), 2) propranolol (Prop), 3) glycopyrrolate (Glyc), or 4) Prop + Glyc. HR increased with exercise in all drug conditions (P < 0.001) but was always higher at a given workload in hypoxia than normoxia (P < 0.001). Averaged over all workloads, the difference between hypoxia and normoxia was 19.8 ± 13.8 beats/min during Cont and similar (17.2 ± 7.7 beats/min, P = 0.95) during Prop but smaller (P < 0.001) during Glyc and Prop + Glyc (9.8 ± 9.6 and 8.1 ± 7.6 beats/min, respectively). Cardiac output was enhanced by hypoxia (P < 0.002) to an extent that was similar between Cont, Glyc, and Prop + Glyc (2.3 ± 1.9, 1.7 ± 1.8, and 2.3 ± 1.2 l/min, respectively, P > 0.4) but larger during Prop (3.4 ± 1.6 l/min, P = 0.004). Our results demonstrate that the tachycardic effect of hypoxia during exercise partially relies on vagal withdrawal. Conversely, sympathoexcitation either does not contribute or increases heart rate through mechanisms other than β-adrenergic transmission. A potential candidate is α-adrenergic transmission, which could also explain why a tachycardic effect of hypoxia persists during combined β-adrenergic and muscarinic receptor inhibition. PMID:25888515

  16. (-)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites.

    PubMed

    Arias, Hugo R; Ortells, Marcelo O; Feuerbach, Dominik

    2013-11-01

    The interaction of (-)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (-)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca(2+) influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50=3.86±0.49 and 1.92±0.48 μM, respectively, (b) binds to the [(3)H]TCP site with ~13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [(3)H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6' and 13' in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9' is the minimum structural component for (-)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (-)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants. PMID:23917086

  17. Modulators of estrogen receptor inhibit proliferation and migration of prostate cancer cells.

    PubMed

    Piccolella, Margherita; Crippa, Valeria; Messi, Elio; Tetel, Marc J; Poletti, Angelo

    2014-01-01

    In the initial stages, human prostate cancer (PC) is an androgen-sensitive disease, which can be pharmacologically controlled by androgen blockade. This therapy often induces selection of androgen-independent PC cells with increased invasiveness. We recently demonstrated, both in cells and mice, that a testosterone metabolite locally synthetized in prostate, the 5α-androstane-3β, 17β-diol (3β-Adiol), inhibits PC cell proliferation, migration and invasion, acting as an anti-proliferative/anti-metastatic agent. 3β-Adiol is unable to bind androgen receptor (AR), but exerts its protection against PC by specifically interacting with estrogen receptor beta (ERβ). Because of its potential retro-conversion to androgenic steroids, 3β-Adiol cannot be used "in vivo", thus, the aims of this study were to investigate the capability of four ligands of ERβ (raloxifen, tamoxifen, genistein and curcumin) to counteract PC progression by mimicking the 3β-Adiol activity. Our results demonstrated that raloxifen, tamoxifen, genistein and curcumin decreased DU145 and PC3 cell proliferation in a dose-dependent manner; in addition, all four compounds significantly decreased the detachment of cells seeded on laminin or fibronectin. Moreover, raloxifen, tamoxifen, genistein and curcumin-treated DU145 and PC3 cells showed a significant decrease in cell migration. Notably, all these effects were reversed by the anti-estrogen, ICI 182,780, suggesting that their actions are mediated by the estrogenic pathway, via the ERβ, the only isoform present in these PCs. In conclusion, these data demonstrate that by selectively activating the ERβ, raloxifen, tamoxifen, genistein and curcumin inhibit human PC cells proliferation and migration favoring cell adesion. These synthetic and natural modulators of ER action may exert a potent protective activity against the progression of PC even in its androgen-independent status. PMID:24184124

  18. Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling

    SciTech Connect

    Son, Dong Ju; Kim, Soo Yeon; Han, Seong Su; Kim, Chan Woo; Kumar, Sandeep; Park, Byeoung Soo; Lee, Sung Eun; Yun, Yeo Pyo; Jo, Hanjoong; Park, Young Hyun

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Anti-atherogenic effect of PL was examined using partial carotid ligation model in ApoE KO mice. Black-Right-Pointing-Pointer PL prevented atherosclerotic plaque development, VSMCs proliferation, and NF-{kappa}B activation. Black-Right-Pointing-Pointer Piperlongumine reduced vascular smooth muscle cell activation through PDGF-R{beta} and NF-{kappa}B-signaling. Black-Right-Pointing-Pointer PL may serve as a new therapeutic molecule for atherosclerosis treatment. -- Abstract: Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-{kappa}B) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase C{gamma}1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-{kappa}B-a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo.

  19. Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

    PubMed Central

    Jonsson Fagerlund, Malin; Krupp, Johannes; Dabrowski, Michael A.

    2016-01-01

    Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses. PMID:26861354

  20. Endogenous inhibition of the trigeminally evoked neurotransmission to cardiac vagal neurons by muscarinic acetylcholine receptors.

    PubMed

    Gorini, C; Philbin, K; Bateman, R; Mendelowitz, D

    2010-10-01

    Stimulation of the nasal mucosa by airborne irritants or water evokes a pronounced bradycardia accompanied by peripheral vasoconstriction and apnea. The dive response, which includes the trigeminocardiac reflex, is among the most powerful autonomic responses. These responses slow the heart rate and reduce myocardial oxygen consumption. Although normally cardioprotective, exaggeration of this reflex can be detrimental and has been implicated in cardiorespiratory diseases, including sudden infant death syndrome (SIDS). An essential component of the diving response and trigeminocardiac reflex is activation of the parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus that control heart rate. This study examined the involvement of cholinergic receptors in trigeminally evoked excitatory postsynaptic currents in CVNs in an in vitro preparation from rats. CVNs were identified using a retrograde tracer injected into the fat pads at the base of the heart. Application of the acetylcholinesterase inhibitor neostigmine significantly decreased the amplitude of glutamatergic neurotransmission to CVNs on stimulation of trigeminal fibers. Whereas nicotine did not have any effect on the glutamatergic responses, the muscarinic acetylcholine receptor (mAChR) agonist bethanechol significantly decreased the excitatory neurotransmission. Atropine, an mAChR antagonist, facilitated these responses indicating this trigeminally evoked brain stem pathway in vitro is endogenously inhibited by mAChRs. Tropicamide, an m4 mAChR antagonist, prevented the inhibitory action of the muscarinic agonist bethanechol. These results indicate that the glutamatergic synaptic neurotransmission in the trigeminally evoked pathway to CVNs is endogenously inhibited in vitro by m4 mAChRs. PMID:20719927

  1. Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis

    PubMed Central

    Subramani, Ramadevi; Lopez-Valdez, Rebecca; Arumugam, Arunkumar; Nandy, Sushmita; Boopalan, Thiyagarajan; Lakshmanaswamy, Rajkumar

    2014-01-01

    Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor β. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer. PMID:24809702

  2. A selective TSH receptor antagonist inhibits stimulation of thyroid function in female mice.

    PubMed

    Neumann, Susanne; Nir, Eshel A; Eliseeva, Elena; Huang, Wenwei; Marugan, Juan; Xiao, Jingbo; Dulcey, Andrés E; Gershengorn, Marvin C

    2014-01-01

    Because the TSH receptor (TSHR) plays an important role in the pathogenesis of thyroid disease, a TSHR antagonist could be a novel treatment. We attempted to develop a small molecule, drug-like antagonist of TSHR signaling that is selective and active in vivo. We synthesized NCGC00242364 (ANTAG3) by chemical modification of a previously reported TSHR antagonist. We tested its potency, efficacy, and selectivity in a model cell system in vitro by measuring its activity to inhibit stimulation of cAMP production stimulated by TSH, LH, or FSH. We tested the in vivo activity of ANTAG3 by measuring its effects to lower serum free T4 and thyroid gene expression in female BALB/c mice continuously treated with ANTAG3 for 3 days and given low doses of TRH continuously or stimulated by a single administration of a monoclonal thyroid-stimulating antibody M22. ANTAG3 was selective for TSHR inhibition; half-maximal inhibitory doses were 2.1 μM for TSHR and greater than 30 μM for LH and FSH receptors. In mice treated with TRH, ANTAG3 lowered serum free T4 by 44% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 75% and 83%, respectively. In mice given M22, ANTAG3 lowered serum free T4 by 38% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 73% and 40%, respectively. In conclusion, we developed a selective TSHR antagonist that is effective in vivo in mice. This is the first report of a small-molecule TSHR antagonist active in vivo and may lead to a drug to treat Graves' disease. PMID:24169564

  3. Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor

    PubMed Central

    Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S.; Yousif, Mariam H. M.; Benter, Ibrahim F.

    2015-01-01

    Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro7-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications. PMID:26536590

  4. Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor.

    PubMed

    Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S; Yousif, Mariam H M; Benter, Ibrahim F

    2015-01-01

    Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1-7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1-7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1-7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1-7). Ang-(1-7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1-7) that could be prevented by D-Pro7-Ang-(1-7) in VSMC. These data suggest that Ang-(1-7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications. PMID:26536590

  5. Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region.

    PubMed

    Ma, Y; Cunningham, M E; Wang, X; Ghosh, I; Regan, L; Longley, B J

    1999-05-01

    KIT receptor kinase activity is repressed, prior to stem cell factor binding, by unknown structural constraints. Using site-directed mutagenesis, we examined the role of KIT intracellular juxtamembrane residues Met-552 through Ile-563 in controlling receptor autophosphorylation. Alanine substitution for Tyr-553, Trp-557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic alpha-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman algorithm, resulted in substantially increased spontaneous receptor phosphorylation, revealing inhibitory roles for these residues. Alanine substitution for other residues, most of which are on the hydrophilic side of the helix, caused no or slightly increased basal receptor phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no elevation, respectively, in basal KIT phosphorylation, indicating that the phenyl ring of Tyr-553 and the hydrophobicity of Trp-557 are critical for the inhibition. Although alanine substitution for Lys-558 had no effect on receptor phosphorylation, its substitution with proline produced high spontaneous receptor phosphorylation, suggesting that the predicted alpha-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of alpha-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative alpha-helical conformation, exert inhibitory control on the kinase activity of ligand-unoccupied receptor. PMID:10224103

  6. A Comprehensive Optogenetic Pharmacology Toolkit for In Vivo Control of GABA(A) Receptors and Synaptic Inhibition.

    PubMed

    Lin, Wan-Chen; Tsai, Ming-Chi; Davenport, Christopher M; Smith, Caleb M; Veit, Julia; Wilson, Neil M; Adesnik, Hillel; Kramer, Richard H

    2015-12-01

    Exogenously expressed opsins are valuable tools for optogenetic control of neurons in circuits. A deeper understanding of neural function can be gained by bringing control to endogenous neurotransmitter receptors that mediate synaptic transmission. Here we introduce a comprehensive optogenetic toolkit for controlling GABA(A) receptor-mediated inhibition in the brain. We developed a series of photoswitch ligands and the complementary genetically modified GABA(A) receptor subunits. By conjugating the two components, we generated light-sensitive versions of the entire GABA(A) receptor family. We validated these light-sensitive receptors for applications across a broad range of spatial scales, from subcellular receptor mapping to in vivo photo-control of visual responses in the cerebral cortex. Finally, we generated a knockin mouse in which the "photoswitch-ready" version of a GABA(A) receptor subunit genomically replaces its wild-type counterpart, ensuring normal receptor expression. This optogenetic pharmacology toolkit allows scalable interrogation of endogenous GABA(A) receptor function with high spatial, temporal, and biochemical precision. PMID:26606997

  7. The 5-HT3B subunit affects high-potency inhibition of 5-HT3 receptors by morphine

    PubMed Central

    Baptista-Hon, Daniel T; Deeb, Tarek Z; Othman, Nidaa A; Sharp, Douglas; Hales, Tim G

    2012-01-01

    BACKGROUND AND PURPOSE Morphine is an antagonist at 5-HT3A receptors. 5-HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH 5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [3H]GR65630. KEY RESULTS When pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT3A and 5-HT3AB receptors. Morphine also fully displaced [3H]GR65630 from 5-HT3A and 5-HT3AB receptors with similar potency. CONCLUSIONS AND IMPLICATIONS These findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT3 receptors. PMID:21740409

  8. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons.

    PubMed

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (I GABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited I GABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of I GABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation. PMID:27616980

  9. Lactisole inhibits the glucose-sensing receptor T1R3 expressed in mouse pancreatic β-cells.

    PubMed

    Hamano, Kunihisa; Nakagawa, Yuko; Ohtsu, Yoshiaki; Li, Longfei; Medina, Johan; Tanaka, Yuji; Masuda, Katsuyoshi; Komatsu, Mitsuhisa; Kojima, Itaru

    2015-07-01

    Glucose activates the glucose-sensing receptor T1R3 and facilitates its own metabolism in pancreatic β-cells. An inhibitor of this receptor would be helpful in elucidating the physiological function of the glucose-sensing receptor. The present study was conducted to examine whether or not lactisole can be used as an inhibitor of the glucose-sensing receptor. In MIN6 cells, in a dose-dependent manner, lactisole inhibited insulin secretion induced by sweeteners, acesulfame-K, sucralose and glycyrrhizin. The IC50 was ∼4 mmol/l. Lactisole attenuated the elevation of cytoplasmic Ca2+ concentration ([Ca2+]c) evoked by sucralose and acesulfame-K but did not affect the elevation of intracellular cAMP concentration ([cAMP]c) induced by these sweeteners. Lactisole also inhibited the action of glucose in MIN6 cells. Thus, lactisole significantly reduced elevations of intracellular [NADH] and intracellular [ATP] induced by glucose, and also inhibited glucose-induced insulin secretion. To further examine the effect of lactisole on T1R3, we prepared HEK293 cells stably expressing mouse T1R3. In these cells, sucralose elevated both [Ca2+]c and [cAMP]c. Lactisole attenuated the sucralose-induced increase in [Ca2+]c but did not affect the elevation of [cAMP]c. Finally, lactisole inhibited insulin secretion induced by a high concentration of glucose in mouse islets. These results indicate that the mouse glucose-sensing receptor was inhibited by lactisole. Lactisole may be useful in assessing the role of the glucose-sensing receptor in mouse pancreatic β-cells. PMID:25994004

  10. Transient Receptor Potential Vanilloid 4 Inhibits γ-Aminobutyric Acid-Activated Current in Hippocampal Pyramidal Neurons

    PubMed Central

    Hong, Zhiwen; Tian, Yujing; Qi, Mengwen; Li, Yingchun; Du, Yimei; Chen, Lei; Liu, Wentao; Chen, Ling

    2016-01-01

    The balance between excitatory and inhibitory neurotransmitter systems is crucial for the modulation of neuronal excitability in the central nervous system (CNS). The activation of transient receptor potential vanilloid 4 (TRPV4) is reported to enhance the response of hippocampal glutamate receptors, but whether the inhibitory neurotransmitter system can be regulated by TRPV4 remains unknown. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS. Here, we show that application of transient receptor potential vanilloid 4 (TRPV4) synthetic (GSK1016790A or 4α-PDD) or endogenous agonist (5,6-EET) inhibited GABA-activated current (IGABA) in hippocampal CA1 pyramidal neurons, which was blocked by specific antagonists of TRPV4 and of GABAA receptors. GSK1016790A increased the phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylated protein kinase B (p-Akt) protein levels, which was attenuated by removing extracellular calcium or by a calcium/calmodulin-dependent protein kinase kinase-β antagonist. GSK1016790A-induced decrease of p-Akt protein level was sensitive to an AMPK antagonist. GSK1016790A-inhibited IGABA was blocked by an AMPK antagonist or a phosphatidyl inositol 3 kinase (PI3K) agonist. GSK1016790A-induced inhibition of IGABA was also significantly attenuated by a protein kinase C (PKC) antagonist but was unaffected by protein kinase A or calcium/calmodulin-dependent protein kinase II antagonist. We conclude that activation of TRPV4 inhibits GABAA receptor, which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling. Inhibition of GABAA receptors may account for the neuronal hyperexcitability caused by TRPV4 activation.

  11. Targeting tumour vasculature by inhibiting activin receptor-like kinase (ALK)1 function.

    PubMed

    de Vinuesa, Amaya García; Bocci, Matteo; Pietras, Kristian; Ten Dijke, Peter

    2016-08-15

    Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented. PMID:27528762

  12. The bradykinin B1 receptor antagonist R-954 inhibits Ehrlich tumor growth in rodents.

    PubMed

    Fernandes, Patricia Dias; Gomes, Niele de Matos; Sirois, Pierre

    2011-09-01

    The present study investigated the effects of a new bradykinin B(1) receptor antagonist, R-954, on the development of Ehrlich ascitic tumor (EAT) induced by the intraperitoneal inoculation of EAT cells in mice and the formation of a solid tumor by the subcutaneous injection of the cells in rat paw. The development of the tumor was associated with an increase in mouse total cell counts in bone marrow (10.8-fold), ascitic fluid (14.6-fold), and blood (12.6-fold). R-954 (2mg/kg, s.c.) significantly reduced the ascitic fluid volume (63.7%) and the mouse weight gain (30.5%) after 10 consecutive days of treatment. The B(1) antagonist as well as the anti-neoplasic drug vincristine also significantly inhibited the increase in total cell count in bone marrow, ascitic fluid, and blood. R-954 reduced significantly the total protein extravasation (57.3%), the production of nitric oxide (56%), PGE(2) production (82%), and TNFα release (85.7%) in mice peritoneal cavity whereas vincristine reduced the release of these inflammatory mediators by 84-94%. The increase in paw edema after intraplantar injection of EAT cells was reduced by approximately 52% by either R-954 or vincristine treatment. In conclusion, this study presents for the first time the antitumoral activity of a new bradykinin B(1) receptor antagonist on ascitic and solid tumors induced by Ehrlich cell inoculation in mice and rats. PMID:21835216

  13. Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP).

    PubMed

    Lam, Ian P Y; Lee, Leo T O; Choi, Hueng-Sik; Alpini, Gianfranco; Chow, Billy K C

    2009-07-01

    Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids. PMID:19372104

  14. Growth hormone receptor inhibition decreases the growth and metastasis of pancreatic ductal adenocarcinoma

    PubMed Central

    Subramani, Ramadevi; Lopez-Valdez, Rebecca; Salcido, Alyssa; Boopalan, Thiyagarajan; Arumugam, Arunkumar; Nandy, Sushmita; Lakshmanaswamy, Rajkumar

    2014-01-01

    Pancreatic cancer is the only major cancer with very low survival rates (1%). It is the fourth leading cause of cancer-related death. Hyperactivated growth hormone receptor (GHR) levels have been shown to increase the risk of cancer in general and this pathway is a master regulator of key cellular functions like proliferation, apoptosis, differentiation, metastasis, etc. However, to date there is no available data on how GHR promotes pancreatic cancer pathogenesis. Here, we used an RNA interference approach targeted to GHR to determine whether targeting GHR is an effective method for controlling pancreatic cancer growth and metastasis. For this, we used an in vitro model system consisting of HPAC and PANC-1 pancreatic cancer cells lines. GHR is upregulated in both of these cell lines and silencing GHR significantly reduced cell proliferation and viability. Inhibition of GHR also reduced the metastatic potential of pancreatic cancer cells, which was aided through decreased colony-forming ability and reduced invasiveness. Flow cytometric and western blot analyses revealed the induction of apoptosis in GHR silenced cells. GHR silencing affected phosphatidylinositol 3 kinase/AKT, mitogen extracellular signal-regulated kinase/extracellular signal-regulated kinase, Janus kinase/signal transducers and activators of transcription and mammalian target of rapamycin signaling, as well as, epithelial to mesenchymal transition. Interestingly, silencing GHR also suppressed the expression of insulin receptor-β and cyclo-oxygenease-2. Altogether, GHR silencing controls the growth and metastasis of pancreatic cancer and reveals its importance in pancreatic cancer pathogenesis. PMID:25301264

  15. Inhibition of mammillary body neurons by direct activation of Group II metabotropic glutamate receptors

    PubMed Central

    Lee, Charles C.

    2016-01-01

    The mammillary body is an important neural component of limbic circuitry implicated in learning and memory. Excitatory and inhibitory inputs, primarily mediated by glutamate and gamma-amino butyric acid (GABA), respectively, converge and integrate in this region, before sending information to the thalamus. One potentially overlooked mechanism for inhibition of mammillary body neurons is through direct activation of Group II metabotropic glutamate receptors (mGluRs). Here, whole-cell patch clamp recordings of in vitro slice preparations containing the mammillary body nuclei of the mouse were employed to record responses to bath application of pharmacological agents to isolate the direct effect of activating Group II mGluRs. Application of the Group II mGluR specific agonist, APDC, resulted in a hyperpolarization of the membrane potential in mammillary body neurons, likely resulting from the opening of a potassium conductance. These data suggest that glutamatergic inputs to the mammillary body may be attenuated via Group II mGluRs and implicates a functional role for these receptors in memory-related circuits and broadly throughout the central nervous system. PMID:27390777

  16. Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells.

    PubMed

    Trabelsi, Mohamed-Sami; Daoudi, Mehdi; Prawitt, Janne; Ducastel, Sarah; Touche, Véronique; Sayin, Sama I; Perino, Alessia; Brighton, Cheryl A; Sebti, Yasmine; Kluza, Jérôme; Briand, Olivier; Dehondt, Hélène; Vallez, Emmanuelle; Dorchies, Emilie; Baud, Grégory; Spinelli, Valeria; Hennuyer, Nathalie; Caron, Sandrine; Bantubungi, Kadiombo; Caiazzo, Robert; Reimann, Frank; Marchetti, Philippe; Lefebvre, Philippe; Bäckhed, Fredrik; Gribble, Fiona M; Schoonjans, Kristina; Pattou, François; Tailleux, Anne; Staels, Bart; Lestavel, Sophie

    2015-01-01

    Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes. PMID:26134028

  17. Farnesoid X Receptor Inhibits Glucagon-Like Peptide-1 Production by Enteroendocrine L-cells

    PubMed Central

    TRABELSI, Mohamed-Sami; DAOUDI, Mehdi; PRAWITT, Janne; DUCASTEL, Sarah; TOUCHE, Véronique; SAYIN, Sama I.; PERINO, Alessia; BRIGHTON, Cheryl A.; SEBTI, Yasmine; KLUZA, Jérôme; BRIAND, Olivier; DEHONDT, Hélène; VALLEZ, Emmanuelle; DORCHIES, Emilie; BAUD, Grégory; SPINELLI, Valeria; HENNUYER, Nathalie; CARON, Sandrine; BANTUBUNGI, Kadiombo; CAIAZZO, Robert; REIMANN, Frank; MARCHETTI, Philippe; LEFEBVRE, Philippe; BÄCKHED, Fredrik; GRIBBLE, Fiona M.; SCHOONJANS, Kristina; PATTOU, François; TAILLEUX, Anne; STAELS, Bart; LESTAVEL, Sophie

    2015-01-01

    Bile acids (BA) are signalling molecules which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex BA in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces Glucagon-Like Peptide-1 (GLP-1) production by L-cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L-cells and controls GLP-1 production is unknown. Here we show that FXR activation in L-cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR-deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes. PMID:26134028

  18. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    SciTech Connect

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M. . E-mail: laurie.chan@mcgill.ca

    2005-05-15

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

  19. Inhibition of kinin breakdown prolongs retention and action of bradykinin in a myocardial B2 receptor compartment

    PubMed Central

    Dendorfer, Andreas; Folkers, Verena; Klinger, Matthias; Wolfrum, Sebastian; Dominiak, Peter

    2003-01-01

    The high efficacy of ACE inhibitors to potentiate the actions of kinins might be explained by a hypothetical compartment in which B2-receptors are colocalized with kinin degrading enzymes. To demonstrate the functional consequence of such a compartment we compared the myocardial uptake and the persistence of action of bradykinin under the influence of kininase inhibitors. Bradykinin-induced vasodilation and uptake of tritiated bradykinin were studied in perfused rat hearts during inhibition of ACE and aminopeptidase P. B2-receptors were localized by immuno-gold labelling and electron-microscopy. The EC50 of bradykinin-induced vasodilation (5.1±0.8 nM) was shifted to 14 fold lower concentrations during inhibition of both kininases. The maximum persistence of vasodilation after termination of bradykinin application (half-life 112±20 s) was increased by kininase inhibitors to 398±130 s. This prolongation was reversed when B2-receptors were blocked simultaneously with the termination of bradykinin infusion. Tritiated bradykinin (perfused for 1 min) was partially (1.7±0.24%) retained by the myocardium and consecutively released with a half-life of 70±9 s. Kinin uptake was increased during kininase inhibition (7.7±2.6%), and was normalized by HOE 140 (2.0±0.34%), or when a tritiated B2-receptor antagonist (NPC 17731) was used as label. B2-receptors were localized in plasmalemmal and cytosolic vesicles of capillary endothelium. Bradykinin is locally incorporated and can associate with B2-receptors repeatedly when kinin breakdown is inhibited. This is the kinetic and functional consequence of a colocalization of kininases and B2-receptors in a compartment constituted by endothelial membrane vesicles. PMID:12540521

  20. Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing.

    PubMed

    Modi, Meera E; Majchrzak, Mark J; Fonseca, Kari R; Doran, Angela; Osgood, Sarah; Vanase-Frawley, Michelle; Feyfant, Eric; McInnes, Heather; Darvari, Ramin; Buhl, Derek L; Kablaoui, Natasha M

    2016-08-01

    Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non-brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response. PMID:27217590

  1. Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.

    PubMed

    Endo, Toshiaki; Yanagawa, Yuchio; Obata, Kunihiko; Isa, Tadashi

    2005-12-01

    The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for alpha3beta2- and alpha6beta2-containing receptors, alpha-conotoxin MII (alphaCtxMII). A subset of these neurons exhibited a faster alpha-bungarotoxin-sensitive inward current component, indicating the expression of alpha7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABA(A) receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by alphaCtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC. PMID:16107532

  2. Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing

    PubMed Central

    Modi, Meera E.; Majchrzak, Mark J.; Fonseca, Kari R.; Doran, Angela; Osgood, Sarah; Vanase-Frawley, Michelle; Feyfant, Eric; McInnes, Heather; Darvari, Ramin; Buhl, Derek L.

    2016-01-01

    Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non–brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response. PMID:27217590

  3. CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling.

    PubMed

    Wang, Tao; Guo, Shuiming; Liu, Zhuo; Wu, Licheng; Li, Mingchao; Yang, Jun; Chen, Ruibao; Liu, Xiaming; Xu, Hua; Cai, Shaoxin; Chen, Hui; Li, Weiyong; Xu, Shaohua; Wang, Liang; Hu, Zhiquan; Zhuang, Qianyuan; Wang, Liping; Wu, Kongming; Liu, Jihong; Ye, Zhangqun; Ji, Jun-Yuan; Wang, Chenguang; Chen, Ke

    2014-11-15

    Castration resistance is a major obstacle to hormonal therapy for prostate cancer patients. Although androgen independence of prostate cancer growth is a known contributing factor to endocrine resistance, the mechanism of androgen receptor deregulation in endocrine resistance is still poorly understood. Herein, the CAMK2N1 was shown to contribute to the human prostate cancer cell growth and survival through AR-dependent signaling. Reduced expression of CAMK2N1 was correlated to recurrence-free survival of prostate cancer patients with high levels of AR expression in their tumor. CAMK2N1 and AR signaling form an auto-regulatory negative feedback loop: CAMK2N1 expression was down-regulated by AR activation; while CAMK2N1 inhibited AR expression and transactivation through CAMKII and AKT pathways. Knockdown of CAMK2N1 in prostate cancer cells alleviated Casodex inhibition of cell growth, while re-expression of CAMK2N1 in castration-resistant cells sensitized the cells to Casodex treatment. Taken together, our findings suggest that CAMK2N1 plays a tumor suppressive role and serves as a crucial determinant of the resistance of prostate cancer to endocrine therapies. PMID:25296973

  4. Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury.

    PubMed

    Zhang, Si; Ju, Peijun; Tjandra, Editha; Yeap, Yeeshan; Owlanj, Hamed; Feng, Zhiwei

    2016-10-01

    Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages. PMID:26883518

  5. In Vitro Inflammation Inhibition Model Based on Semi-Continuous Toll-Like Receptor Biosensing

    PubMed Central

    Jeon, Jin-Woo; Ha, Un-Hwan; Paek, Se-Hwan

    2014-01-01

    A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an in vivo-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., Pseudomonas aeruginosa) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-κB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests. PMID:25136864

  6. Phasic, Nonsynaptic GABA-A Receptor-Mediated Inhibition Entrains Thalamocortical Oscillations

    PubMed Central

    Rovó, Zita; Mátyás, Ferenc; Barthó, Péter; Slézia, Andrea; Lecci, Sandro; Pellegrini, Chiara; Astori, Simone; Dávid, Csaba; Hangya, Balázs

    2014-01-01

    GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations. PMID:24849349

  7. Angiotensin receptor neprilysin inhibition in heart failure: mechanistic action and clinical impact.

    PubMed

    Buggey, Jonathan; Mentz, Robert J; DeVore, Adam D; Velazquez, Eric J

    2015-09-01

    Heart failure (HF) is an increasingly common syndrome associated with high mortality and economic burden, and there has been a paucity over the past decade of new pharmacotherapies that improve outcomes. However, recent data from a large randomized controlled trial compared the novel agent LCZ696, a dual-acting angiotensin receptor blocker and neprilysin inhibitor (ARNi), with the well established angiotensin-converting enzyme (ACE) inhibitor enalapril and found significant reduction in mortality among the chronic reduced ejection fraction HF population. Preclinical and clinical data suggest that neprilysin inhibition provides beneficial outcomes in HF patients by preventing the degradation of natriuretic peptides and thereby promoting natriuresis and vasodilatation and counteracting the negative cardiorenal effects of the up-regulated renin-angiotensin-aldosterone system. Agents such as omapatrilat combined neprilysin and ACE inhibition but had increased rates of angioedema. Goals of an improved safety profile provided the rationale for the development of the ARNi LCZ696. Along with significant reductions in mortality and hospitalizations, clinical trials suggest that LCZ696 may improve surrogate markers of HF severity. In this paper, we review the preclinical and clinical data that led to the development of LCZ696, the understanding of the underlying mechanistic action, and the robust clinical impact that LCZ696 may have in the near future. PMID:26209000

  8. Curcumin analogues with high activity for inhibiting human prostate cancer cell growth and androgen receptor activation.

    PubMed

    Zhou, Dai-Ying; Ding, Ning; Du, Zhi-Yun; Cui, Xiao-Xing; Wang, Hong; Wei, Xing-Chuan; Conney, Allan H; Zhang, Kun; Zheng, Xi

    2014-09-01

    The androgen receptor (AR) has a critical role in prostate cancer development and progression. Several curcumin analogues (A10, B10, C10, E10 and F10) with different linker groups were investigated for their effects in human prostate cancer CWR‑22Rv1 and LNCaP cell lines. The ability of these compounds to inhibit testosterone (TT)‑ or dihydrotestosterone (DHT)‑induced AR activity was determined by an AR‑linked luciferase assay and by TT‑ or DHT‑induced expression of prostate specific antigen. Compounds F10 and E10 had stronger inhibitory effects on the growth of cultured CWR‑22Rv1 and LNCaP cell lines, and they also had enhanced stimulatory effects on apoptosis compared with curcumin and other curcumin analogues (A10, B10, C10) in CWR‑22Rv1 cells. E10 and F10 were more potent inhibitors of AR activity than curcumin, A10 and B10. The higher activities of E10 and F10 may be correlated with a heteroatom linker. The results indicate that one of the potential mechanisms for the anticancer effect of the curcumin analogues was inhibition of AR pathways in human prostate cancer cells. PMID:25060817

  9. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  10. Emodin isolated from Polygoni Multiflori Ramulus inhibits melanogenesis through the liver X receptor-mediated pathway.

    PubMed

    Kim, Mi Ok; Park, Yong Seek; Nho, Youn Hwa; Yun, Seok Kyun; Kim, Youngsoo; Jung, Eunsun; Paik, Jean Kyung; Kim, Minhee; Cho, Il-Hoon; Lee, Jongsung

    2016-04-25

    Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. We investigated the effects of a Polygoni Multiflori Ramulus extract on melanogenesis and isolated emodin from Polygoni Multiflori as an active compound. In addition, the possible mechanisms of action were examined. We found that emodin inhibited both melanin content and tyrosinase activity concentration and time dependently. Tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 mRNA levels decreased following emodin treatment. However, while the mRNA levels of microphthalmia-associated transcription factor (MITF) were not affected by emodin, emodin reduced MITF protein levels. Furthermore, expression of the liver X-receptor (LXR) α gene, but not the LXR β gene was upregulated by emodin. Moreover, emodin regulated melanogenesis by promoting degradation of the MITF protein by upregulating the LXR α gene. The emodin effects on MITF was found to be mediated by phosphorylation of p42/44 MAPK. Taken together, these findings indicate that the inhibition of melanogenesis by emodin occurs through reduced MITF protein expression, which is mediated by upregulation of the LXR α gene and suggest that emodin may be useful as a hyperpigmentation inhibitor. PMID:26972667

  11. Isoflurane inhibits embryonic stem cell self-renewal through retinoic acid receptor.

    PubMed

    Liu, Sheng; Zhang, Lei; Liu, Yi; Shen, Xia; Yang, Longqiu

    2015-08-01

    The commonly used inhalation anesthetic isoflurane could permeate rapidly through the placental barrier and induce toxicity to the central nervous system of the developing fetus. However, the effects of isoflurane in utero during early gestation are unknown. We therefore treated pregnant mice with 1.4% isoflurane for 2h per day for three days at day3.5 (E3.5) to day6.5 (E6.5) to investigated the toxicity of isoflurane. Pregnant mice were executed and the fetal mice were weighed and observed. Mouse ESCs (E14) was exposed to 2% isoflurane for 6h. Twenty-four hours later, self-renewal was examined with AP staining. Effects of isoflurane on the expression of RAR-γ were examined using Western blot. As a result, anesthesia with 1.4% isoflurane for 2 hour per day for 3 days reduced fetal growth and development. Isoflurane decreased self-renewal and the expression stemness genes (Nanog, Oct4, Sox2, and Lin28) in mESCs. Vitamin A attenuated the effects of isoflurane inducing self-renewal inhibition. In summary, Anesthesia with 1.4% isoflurane for 2h per day for 3 days reduced fetal growth and development. Moreover, isoflurane inhibits mESCs self-renewal through retinoic acid receptor. PMID:26349971

  12. Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

    NASA Astrophysics Data System (ADS)

    Zou, Li-xue; Cui, Shao-yan; Zhong, Jian; Yi, Zong-chun; Sun, Yan; Fan, Yu-bo; Zhuang, Feng-yuan

    2011-07-01

    Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% ( p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO-EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.

  13. Activation of the Aryl Hydrocarbon Receptor by TCDD Inhibits Senescence: A Tumor Promoting Event?

    PubMed Central

    Ray, S.; Swanson, H.I.

    2009-01-01

    Activation of the aryl hydrocarbon receptor (AHR) 1 by the agonist, 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) has been shown to promote tumor formation in both liver and skin. In the liver, but not the skin, the AHR-mediated events that contribute to TCDD’s tumor promoting activities have been studied in some detail and are thought to involve perturbation of cell fate processes. However, studies performed using cultured cells have often resulted in apparent contradictory results indicating that the impact of TCDD on cell fate processes may be cell context dependent. We and others have shown that in primary cultured keratinocytes TCDD increases post-confluent proliferation and increases late differentiation. Further, our studies performed in these cells indicate that TCDD can also inhibit culture-induced senescence. While senescence, a permanent cell cycle arrest, is emerging as an important process regulated by oncogenes and considered to be of therapeutic importance, its role with respect to TCDD/AHR mediated tumor promotion has not been fully considered. The intent of this article is to focus primarily on senescence as a cell process relevant to skin tumorigenesis and explore the idea that the inhibition of senescence by TCDD could be an important mechanism by which it may exert its tumor promoting effects in the skin. PMID:19100242

  14. Chemical inhibition of prometastatic lysyl-tRNA synthetase–laminin receptor interaction

    PubMed Central

    Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L.; Guo, Min; Cho, Hye Young; Mushtaq, AmeeqUl; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon

    2014-01-01

    Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds to KRS, impinged on interaction of KRS with 67LR and suppressed metastasis in 3 different mouse models. The compound inhibited KRS–67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS–67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. PMID:24212136

  15. Inhibition of lymphoma vascularization and dissemination by estrogen receptor β agonists.

    PubMed

    Yakimchuk, Konstantin; Hasni, Mohammad Sharif; Guan, Jiyu; Chao, Mark P; Sander, Birgitta; Okret, Sam

    2014-03-27

    Most lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell-derived lymphoma is repressed following activation of estrogen receptor β (ERβ, ESR2). By using ERβ-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERβ agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERβ reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERβ inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERβ activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERβ is expressed in primary MCL tissue. These results suggest that targeting ERβ with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination. PMID:24470591

  16. [Downregulation of proteinase activated receptor 4 inhibits migration of SW620 human colorectal cancer cells].

    PubMed

    Chen, Lihong; Li, Chunchun; Xie, Yuqiong; Ye, Jingjia; Cao, Jiang

    2016-05-01

    Objective To establish the human colorectal cancer cell model SW620/PAR4D with inducible suppression of proteinase activated receptor 4 (PAR4) expression, and investigate the role PAR4 plays in the proliferation and migration of cancer cells. Methods A human colorectal cancer cell line with tetracycline-inducible expression regulatory system, namely SW620/Tet-on, was established; inducible expression lentiviral vector with artificial microRNA targeting PAR4, pLVX-Tight-Puro-PAR4-miR, was constructed and transfected into SW620/Tet-on to make an inducible PAR4-suppressed cell model SW620/PAR4D. Western blotting was used to confirm the suppression of PAR4 expression after the doxycycline (DOX) treatment. CCK-8 assay was used to evaluate the impact of suppressed PAR4 expression on cell proliferation, and wound-healing assay was used to analyze the migration of the cells. Results The SW620/PAR4D cell model was established successfully. Suppression of PAR4 expression by DOX treatment had no significant impact on the growth/proliferation of SW620/PAR4D cells, but markedly inhibited the cell migration. Conclusion Suppression of PAR4 expression has no significant effect on the proliferation of SW620 cells, but can inhibit the migration of the cells. PMID:27126938

  17. Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition*

    PubMed Central

    Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S.; Conti, Amedeo; Lembo, David

    2015-01-01

    Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. PMID:25814665

  18. An orally administered DNA vaccine targeting vascular endothelial growth factor receptor-3 inhibits lung carcinoma growth.

    PubMed

    Chen, Yan; Liu, Xin; Jin, Cong Guo; Zhou, Yong Chun; Navab, Roya; Jakobsen, Kristine Raaby; Chen, Xiao Qun; Li, Jia; Li, Ting Ting; Luo, Lu; Wang, Xi Cai

    2016-02-01

    Lung cancer is the leading cause of mortality and 5-year survival rate is very low worldwide. Recent studies show that vascular endothelial growth factor receptor-3 (VEGFR-3) signaling pathway contributes to lung cancer progression. So we hypothesize that an oral DNA vaccine that targets VEGFR-3 carried by attenuated Salmonella enterica serovar typhimurium strain SL3261 has impacts on lung cancer progression. In this study, the oral VEGFR-3-based vaccine-immunized mice showed appreciable inhibition of tumor growth and tumor lymphatic microvessels in lung cancer mice model. Moreover, the oral VEGFR-3-based vaccine-immunized mice showed remarkable increases in both VEGFR-3-specific antibody levels and cytotoxic activity. Furthermore, the oral VEGFR-3-based vaccine-immunized mice showed a significant increase in the levels of T helper type 1 (Th1) cell intracellular cytokine expression (IL-2, IFN-γ, and TNF-α). After inoculation with murine Lewis lung carcinoma (LLC) cells, CD4(+) or CD8(+) T cell numbers obviously declined in control groups whereas high levels were maintained in the oral VEGFR-3-based vaccine group. These results demonstrated that the oral VEGFR-3-based vaccine could induce specific humoral and cellular immune responses and then significantly inhibit lung carcinoma growth via suppressing lymphangiogenesis. PMID:26376999

  19. Ligand independent aryl hydrocarbon receptor inhibits lung cancer cell invasion by degradation of Smad4.

    PubMed

    Lee, Chen-Chen; Yang, Wen-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Tsai, Chi-Hao; Kang, Jaw-Jou

    2016-07-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-β signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells. PMID:27060206

  20. Contribution of opioid and metabotropic glutamate receptor mechanisms to inhibition of bladder overactivity by tibial nerve stimulation.

    PubMed

    Matsuta, Yosuke; Mally, Abhijith D; Zhang, Fan; Shen, Bing; Wang, Jicheng; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2013-07-15

    The contribution of metabotropic glutamate receptors (mGluR) and opioid receptors to inhibition of bladder overactivity by tibial nerve stimulation (TNS) was investigated in cats under α-chloralose anesthesia using LY341495 (a group II mGluR antagonist) and naloxone (an opioid receptor antagonist). Slow infusion cystometry was used to measure the volume threshold (i.e., bladder capacity) for inducing a large bladder contraction. After measuring the bladder capacity during saline infusion, 0.25% acetic acid (AA) was infused to irritate the bladder, activate the nociceptive C-fiber bladder afferents, and induce bladder overactivity. AA significantly (P < 0.0001) reduced bladder capacity to 26.6 ± 4.7% of saline control capacity. TNS (5 Hz, 0.2 ms) at 2 and 4 times the threshold (T) intensity for inducing an observable toe movement significantly increased bladder capacity to 62.2 ± 8.3% at 2T (P < 0.01) and 80.8 ± 9.2% at 4T (P = 0.0001) of saline control capacity. LY341495 (0.1-5 mg/kg iv) did not change bladder overactivity, but completely suppressed the inhibition induced by TNS at a low stimulus intensity (2T) and partially suppressed the inhibition at high intensity (4T). Following administration of LY341495, naloxone (0.01 mg/kg iv) completely eliminated the high-intensity TNS-induced inhibition. However, without LY341495 treatment a 10 times higher dose (0.1 mg/kg) of naloxone was required to completely block TNS inhibition. These results indicate that interactions between group II mGluR and opioid receptor mechanisms contribute to TNS inhibition of AA-induced bladder overactivity. Understanding neurotransmitter mechanisms underlying TNS inhibition of bladder overactivity is important for the development of new treatments for bladder disorders. PMID:23576608

  1. Bosentan, a mixed endothelin receptor antagonist, inhibits superoxide anion-induced pain and inflammation in mice.

    PubMed

    Serafim, Karla G G; Navarro, Suelen A; Zarpelon, Ana C; Pinho-Ribeiro, Felipe A; Fattori, Victor; Cunha, Thiago M; Alves-Filho, Jose C; Cunha, Fernando Q; Casagrande, Rubia; Verri, Waldiceu A

    2015-11-01

    Bosentan is a mixed endothelin receptor antagonist widely used to treat patients with pulmonary arterial hypertension, and the emerging literature suggests bosentan as a potent anti-inflammatory drug. Superoxide anion is produced in large amounts during inflammation, stimulates cytokine production, and thus contributes to inflammation and pain. However, it remains to be determined whether endothelin contributes to the inflammatory response triggered by the superoxide anion. The present study investigated the effects of bosentan in a mouse model of inflammation and pain induced by potassium superoxide, a superoxide anion donor. Male Swiss mice were treated with bosentan (10-100 mg/kg) by oral gavage, 1 h before potassium superoxide injection, and the inflammatory response was evaluated locally and at spinal cord (L4-L6) levels. Bosentan (100 mg/kg) inhibited superoxide anion-induced mechanical and thermal hyperalgesia, overt pain-like behavior (abdominal writhings, paw flinching, and licking), paw edema, myeloperoxidase activity (neutrophil marker) in the paw skin, and leukocyte recruitment in the peritoneal cavity. Bosentan also inhibited superoxide anion-induced interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) production, while it enhanced IL-10 production in the paw skin and spinal cord. Bosentan inhibited the reduction of antioxidant capacity (reduced glutathione, ferric reducing antioxidant power, and ABTS radical scavenging ability) induced by the superoxide anion. Finally, we demonstrated that intraplantar injection of potassium superoxide induces the mRNA expression of prepro-endothelin-1 in the paw skin and spinal cord. In conclusion, our results demonstrated that superoxide anion-induced inflammation, pain, cytokine production, and oxidative stress depend on endothelin; therefore, these responses are amenable to bosentan treatment. PMID:26246053

  2. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A) receptor-mediated signaling.

    PubMed

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling. PMID:25419570

  3. Inhibition of melanocortin 1 receptor slows melanoma growth, reduces tumor heterogeneity and increases survival.

    PubMed

    Kansal, Rita G; McCravy, Matthew S; Basham, Jacob H; Earl, Joshua A; McMurray, Stacy L; Starner, Chelsey J; Whitt, Michael A; Albritton, Lorraine M

    2016-05-01

    Melanoma risk is increased in patients with mutations of melanocortin 1 receptor (MC1R) yet the basis for the increased risk remains unknown. Here we report in vivo evidence supporting a critical role for MC1R in regulating melanoma tumor growth and determining overall survival time. Inhibition of MC1R by its physiologically relevant competitive inhibitor, agouti signaling protein (ASIP), reduced melanin synthesis and morphological heterogeneity in murine B16-F10 melanoma cells. In the lungs of syngeneic C57BL/6 mice, mCherry-marked, ASIP-secreting lung tumors inhibited MC1R on neighboring tumors lacking ASIP in a dose dependent manner as evidenced by a proportional loss of pigment in tumors from mice injected with 1:1, 3:1 and 4:1 mixtures of parental B16-F10 to ASIP-expressing tumor cells. ASIP-expressing B16-F10 cells formed poorly pigmented tumors in vivo that correlated with a 20% longer median survival than those bearing parental B16-F10 tumors (p=0.0005). Mice injected with 1:1 mixtures also showed survival benefit (p=0.0054), whereas injection of a 4:1 mixture showed no significant difference in survival. The longer survival time of mice bearing ASIP-expressing tumors correlated with a significantly slower growth rate than parental B16-F10 tumors as judged by quantification of numbers of tumors and total tumor load (p=0.0325), as well as a more homogeneous size and morphology of ASIP-expressing lung tumors. We conclude that MC1R plays an important role in regulating melanoma growth and morphology. Persistent inhibition of MC1R provided a significant survival advantage resulting in part from slower tumor growth, establishing MC1R as a compelling new molecular target for metastatic melanoma. PMID:27028866

  4. The novel Aryl hydrocarbon receptor inhibitor biseugenol inhibits gastric tumor growth and peritoneal dissemination

    PubMed Central

    Lai, De-Wei; Karlsson, Anna Isabella; Wang, Keh-Bin; Chen, Yi-Ching; Shen, Chin-Chang; Wu, Sheng-Mao; Liu, Chia-Yu; Tien, Hsing-Ru; Peng, Yen-Chun; Jan, Yee-Jee; Chao, Te-Hsin; Lan, Keng-Hsin; Arbiser, Jack L.; Sheu, Meei-Ling

    2014-01-01

    Biseugenol (Eug) is known to antiproliferative of cancer cells; however, to date, the antiperitoneal dissemination effects have not been studied in any mouse cancer model. In this study, Aryl hydrocarbon receptor (AhR) expression was associated with lymph node and distant metastasis in patients with gastric cancer and was correlated with clinicolpathological pattern. We evaluated the antiperitoneal dissemination potential of knockdown AhR and Biseugenol in cancer mouse model and assessed mesenchymal characteristics. Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis implanted MKN45 cells were significantly decreased in shAhR and Biseugenol-treated mice and that endoplasmic reticulum (ER) stress was caused. Biseugenol-exposure tumors showed acquired epithelial features such as phosphorylation of E-cadherin, cytokeratin-18 and loss mesenchymal signature Snail, but not vimentin regulation. Snail expression, through AhR activation, is an epithelial-to-mesenchymal transition (EMT) determinant. Moreover, Biseugenol enhanced Calpain-10 (Calp-10) and AhR interaction resulted in Snail downregulation. The effect of shCalpain-10 in cancer cells was associated with inactivation of AhR/Snail promoter binding activity. Inhibition of Calpain-10 in gastric cancer cells by short hairpin RNA or pharmacological inhibitor was found to effectively reduced growth ability and vessel density in vivo. Importantly, knockdown of AhR completed abrogated peritoneal dissemination. Herein, Biseugenol targeting ER stress provokes Calpain-10 activity, sequentially induces reversal of EMT and apoptosis via AhR may involve the paralleling processes. Taken together, these data suggest that Calpain-10 activation and AhR inhibition by Biseugenol impedes both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT. PMID:25226618

  5. Suppression of calcium-sensing receptor ameliorates cardiac hypertrophy through inhibition of autophagy

    PubMed Central

    LIU, LEI; WANG, CHAO; LIN, YAN; XI, YUHUI; LI, HONG; SHI, SA; LI, HONGZHU; ZHANG, WEIHUA; ZHAO, YAJUN; TIAN, YE; XU, CHANGQING; WANG, LINA

    2016-01-01

    The calcium-sensing receptor (CaSR) releases intracellular calcium ([Ca2+]i) by accumulating inositol phosphate. Changes in [Ca2+]i initiate myocardial hypertrophy. Furthermore, autophagy associated with [Ca2+]i. Autophagy has previously been demonstrated to participate in the hypertrophic process. The current study investigated whether suppression of CaSR affects the hypertrophic response via modulating autophagy. Isoproterenol (ISO) was used to induce cardiac hypertrophy in Wistar rats. Hypertrophic status was determined by echocardiographic assessment, hematoxylin and eosin, and Masson's staining. The protein expression levels of CaSR and autophagy level were observed. Changes of hypertrophy and autophagy indicators were observed following intravenous injection of a CaSR inhibitor. An ISO-induced cardiomyocyte hypertrophy model was established and used determine the involvement of GdCl3. [Ca2+]i was determined using Fluo-4/AM dye followed by confocal microscopy. The expression levels of various active proteins were analyzed by western blotting. The size of the heart, expression levels of CaSR and autophagy level were markedly increased in hypertrophic myocardium. In addition, the present study demonstrated that the indicators of hypertrophy and autophagy were effectively suppressed by CaSR inhibitor. Furthermore, similar effects were demonstrated in neonatal rat hypertrophic cardiomyocytes treated with ISO. It was also observed that CaSR regulates the Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway induced by ISO in cardiomyocytes. Furthermore, the AMPK inhibition significantly reduced the autophagy level following CaSR stimulation (P<0.05). The results of the present demonstrated that inhibition of CaSR may ameliorate cardiac hypertrophy induced by ISO and the effect may be associated with the inhibition of autophagy and suppression of the Ca

  6. Valerian Inhibits Rat Hepatocarcinogenesis by Activating GABA(A) Receptor-Mediated Signaling

    PubMed Central

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P+) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2′-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P+ foci by activating GABA(A)R-mediated signaling. PMID:25419570

  7. Characterization of suppressive oligodeoxynucleotides that inhibit Toll-like receptor-9-mediated activation of innate immunity

    PubMed Central

    Peter, Mirjam; Bode, Konrad; Lipford, Grayson B; Eberle, Florian; Heeg, Klaus; Dalpke, Alexander H

    2008-01-01

    Synthetic oligodeoxynucleotides containing unmethylated CpG sequences (CpG-ODNs) stimulate Toll-like receptor-9 (TLR-9), thereby activating innate immunity. Stimulatory CpG-ODNs have been shown to be valuable in modifying immune responses in allergy, infection and cancer. Recently, it has been reported that the stimulation of TLR-9 by endogenous DNA might contribute to the pathogenesis of autoimmune diseases. We here report the identification of a suppressive, guanosine-rich ODN (G-ODN) that inhibited the activation of TLR-9 by stimulatory CpG-ODNs. The G-ODN was suppressive in murine macrophages and dendritic cells as well as in human plasmacytoid dendritic cells in vitro. G-ODN blocked the secretion of tumour necrosis factor-α (TNF-α) and interleukin-12p40 and interfered with the up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecules. G-ODN was inhibitory even at a molar ratio of 1 : 10 (G-ODN:CpG-ODN) and when administered up to 7 hr after stimulation with CpG. G-ODN specifically inhibited TLR-9 but not other TLRs. Inhibition was dependent on a string of five guanosines. G-ODN was also inhibitory in an in vivo model of CpG/galactosamin (GalN) lethal shock. G-ODN interfered with upstream TLR-9 signalling. However, by extensive analysis we can exclude that G-ODN acts at the stage of cellular uptake. G-ODN therefore represents a class of suppressive ODNs that could be of therapeutic use in situations with pathologic TLR-9 activation, as has been proposed for certain autoimmune diseases. PMID:17961163

  8. Suppression of calcium‑sensing receptor ameliorates cardiac hypertrophy through inhibition of autophagy.

    PubMed

    Liu, Lei; Wang, Chao; Lin, Yan; Xi, Yuhui; Li, Hong; Shi, Sa; Li, Hongzhu; Zhang, Weihua; Zhao, Yajun; Tian, Ye; Xu, Changqing; Wang, Lina

    2016-07-01

    The calcium-sensing receptor (CaSR) releases intracellular calcium ([Ca2+]i) by accumulating inositol phosphate. Changes in [Ca2+]i initiate myocardial hypertrophy. Furthermore, autophagy associated with [Ca2+]i. Autophagy has previously been demonstrated to participate in the hypertrophic process. The current study investigated whether suppression of CaSR affects the hypertrophic response via modulating autophagy. Isoproterenol (ISO) was used to induce cardiac hypertrophy in Wistar rats. Hypertrophic status was determined by echocardiographic assessment, hematoxylin and eosin, and Masson's staining. The protein expression levels of CaSR and autophagy level were observed. Changes of hypertrophy and autophagy indicators were observed following intravenous injection of a CaSR inhibitor. An ISO‑induced cardiomyocyte hypertrophy model was established and used determine the involvement of GdCl3. [Ca2+]i was determined using Fluo‑4/AM dye followed by confocal microscopy. The expression levels of various active proteins were analyzed by western blotting. The size of the heart, expression levels of CaSR and autophagy level were markedly increased in hypertrophic myocardium. In addition, the present study demonstrated that the indicators of hypertrophy and autophagy were effectively suppressed by CaSR inhibitor. Furthermore, similar effects were demonstrated in neonatal rat hypertrophic cardiomyocytes treated with ISO. It was also observed that CaSR regulates the Ca2+/calmodulin‑dependent protein kinase kinase β (CaMKKβ)‑AMP‑activated protein kinase (AMPK)‑mammalian target of rapamycin (mTOR) signaling pathway induced by ISO in cardiomyocytes. Furthermore, the AMPK inhibition significantly reduced the autophagy level following CaSR stimulation (P<0.05). The results of the present demonstrated that inhibition of CaSR may ameliorate cardiac hypertrophy induced by ISO and the effect may be associated with the inhibition of autophagy and suppression of the Ca

  9. Dopamine D3 receptor inhibits the ubiquitin-specific peptidase 48 to promote NHE3 degradation

    PubMed Central

    Armando, Ines; Villar, Van Anthony M.; Jones, John E.; Lee, Hewang; Wang, Xiaoyan; Asico, Laureano D.; Yu, Peiying; Yang, Jian; Escano, Crisanto S.; Pascua-Crusan, Annabelle M.; Felder, Robin A.; Jose, Pedro A.

    2014-01-01

    The dopamine D3 receptor (D3R) is crucial in the regulation of blood pressure and sodium balance, in that Drd3 gene ablation in mice results in hypertension and failure to excrete a dietary salt load. The mechanism responsible for the renal sodium retention in these mice is largely unknown. We now offer and describe a novel mechanism by which D3R decreases sodium transport in the long term by inhibiting the deubiquitinylating activity of ubiquitin-specific peptidase 48 (USP48), thereby promoting Na+-H+ exchanger (NHE)-3 degradation. We found that stimulation with the D3R-specific agonist PD128907 (1 μM, 30 min) promoted the interaction and colocalization among D3R, NHE3, and USP48; inhibited USP48 activity (−35±6%, vs. vehicle), resulting in increased ubiquitinylated NHE3 (+140±10%); and decreased NHE3 expression (−50±9%) in human renal proximal tubule cells (hRPTCs). USP48 silencing decreased NHE3's half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t1/2=12.9 h), whereas overexpression of USP48 increased NHE3 half-life (t1/2=21.8 h), indicating that USP48 protects NHE3 from degradation via deubiquitinylation. USP48 accounted for ∼30% of the total deubiquitinylating activity in these cells. Extending our studies in vivo, we found that pharmacologic blockade of D3R via the D3R-specific antagonist GR103691 (1 μg/kg/min, 4 d) in C57Bl/6J mice increased renal NHE3 expression (+310±15%, vs. vehicle), whereas an innovative kidney-restricted Usp48 silencing via siRNA (3 μg/d, 7 d) increased ubiquitinylated NHE3 (+250±30%, vs. controls), decreased total NHE3 (−23±2%), and lowered blood pressure (−24±2 mm Hg), compared with that in control mice that received either the vehicle or nonsilencing siRNA. Our data demonstrate a crucial role for the dynamic interaction between D3R and USP48 in the regulation of NHE3 expression and function.—Armando, I., Villar, V. A. M., Jones J. E., Lee, H., Wang, X., Asico L. D., Yu, P., Yang, J., Escano, C. S. Jr., Pascua

  10. G protein-coupled receptor 30 ligand G-1 increases aryl hydrocarbon receptor signalling by inhibition of tubulin assembly and cell cycle arrest in human MCF-7 cells.

    PubMed

    Tarnow, Patrick; Tralau, Tewes; Luch, Andreas

    2016-08-01

    Regulatory crosstalk between the aryl hydrocarbon receptor (AHR) and oestrogen receptor α (ERα) is well established. Apart from the nuclear receptors ERα and ERβ, oestrogen signalling further involves an unrelated G protein-coupled receptor termed GPR30. In order to investigate potential regulatory crosstalk, this study investigated the influence of G-1 as one of the few GPR30-specific ligands on the AHR regulon in MCF-7 cells. As a well-characterised model system, these human mammary carcinoma cells co-express all three receptors (AHR, ERα and GPR30) and are thus ideally suited to study corresponding regulatory pathway interactions on transcript level. Indeed, treatment with micromolar concentrations of the GPR30-specific agonist G-1 resulted in up-regulation of AHR as well as the transcripts for cytochromes P450 1A1 and 1B1, two well-known targets of the AHR regulon. While this was partly attributable to G-1-mediated inhibition of tubulin assembly and subsequent cell cycle arrest in the G2/M phase, the effects nevertheless required functional AHR. However, G-1-induced up-regulation of CYP 1A1 was not mediated by GPR30, as G15 antagonist treatment as well as a knockdown of GPR30 and AHR failed to inhibit this effect. PMID:26475489

  11. F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling

    SciTech Connect

    Lee, Ha Young; Lee, Sun Young; Shin, Eun Ha; Kim, Sang Doo; Kim, Jung Mo; Lee, Mi-Sook; Ryu, Sung Ho; Bae, Yoe-Sik . E-mail: yoesik@donga.ac.kr

    2007-08-10

    F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of {sup 125}I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.

  12. Hydroxycinnamic acid amides from Scopolia tangutica inhibit the activity of M1 muscarinic acetylcholine receptor in vitro.

    PubMed

    Zhang, Yan; Long, Zhen; Guo, Zhimou; Wang, Zhiwei; Zhang, Xiuli; Ye, Richard D; Liang, Xinmiao; Civelli, Olivier

    2016-01-01

    Scopolia tangutica Maxim (S. tangutica) extracts have been traditionally used as antispasmodic, sedative, and analgesic agents in Tibet and in the Qinghai province of China. Their active compositions are however poorly understood. We have recently isolated five new hydroxycinnamic acid (HCA) amides along with two known HCA amides, one cinnamic acid amide from these extracts. In this study, we evaluate their abilities to inhibit carbacol-induced activity of M1 muscarinic acetylcholine receptor along with the crude extracts. Chinese hamster ovary cells stably expressing the recombinant human M1 receptor (CHO-M1 cells) were employed to evaluate the anticholinergic potentials. Intracellular Ca(2+) changes were monitored using the FLIPR system. Five HCA amides as well as the crude S. tangutica extract displayed dose-dependent inhibitory effects against M1 receptor. These findings demonstrate that HCA amides are part of the M1 receptor-inhibiting principles of S. tangutica. Since blockade of parasympathetic nerve impulse transmission through the inhibition of the M1 receptor lessens smooth muscle spasms, our findings provided a molecular explanation for the traditional use of S. tangutica against spasm. PMID:26586621

  13. Major Histocompatibility Complex Class II Inhibits Fas Antigen-Mediated Gastric Mucosal Cell Apoptosis through Actin-Dependent Inhibition of Receptor Aggregation

    PubMed Central

    Stoicov, Calin; Cai, Xun; Li, Hanchen; Klucevsek, Kristine; Carlson, Jane; Saffari, Reza; Houghton, JeanMarie

    2005-01-01

    Escape from normal apoptotic controls is thought to be essential for the development of cancer. During Helicobacter pylori infection, the leading cause of gastric cancer, activation of the Fas antigen (Fas Ag) apoptotic pathway is responsible for early atrophy and tissue loss. As disease progresses, metaplastic and dysplastic glands arise which express Fas Ag but are resistant to apoptosis and are believed to be the precursor cells for adenocarcinoma. In this report, we show that one mechanism of acquired Fas resistance is inhibition of receptor aggregation via a major histocompatibility complex class II (MHCII)-mediated, actin-dependent mechanism. For these studies we used the well-described C57BL/6 mouse model of Helicobacter pylori and Helicobacter felis infection. Under normal conditions, Fas Ag is expressed at low levels, and MHCII expression on gastric mucosal cells is negligible. With infection and inflammation, both receptors are upregulated, and 6.1% of gastric mucosal cells express MHCII in combination with Fas Ag. Using the rat gastric mucosal cell line RGM-1 transfected with murine Fas Ag and MHCIIαβ chains, we demonstrate that MHCII prevents Fas receptor aggregation and inhibits Fas-mediated signaling through its effects on the actin cytoskeleton. Depolymerization of actin with cytochalasin D allows receptors to aggregate and restores Fas sensitivity. These findings offer one mechanism by which gastric mucosal cells acquire Fas resistance. PMID:16177302

  14. Equol inhibits prostate cancer growth through degradation of androgen receptor by S-phase kinase-associated protein 2.

    PubMed

    Itsumi, Momoe; Shiota, Masaki; Takeuchi, Ario; Kashiwagi, Eiji; Inokuchi, Junichi; Tatsugami, Katsunori; Kajioka, Shunichi; Uchiumi, Takeshi; Naito, Seiji; Eto, Masatoshi; Yokomizo, Akira

    2016-07-01

    Chemopreventive and potential therapeutic effects of soy isoflavones have been shown to be effective in numerous preclinical studies as well as clinical studies in prostate cancer. Although the inhibition of androgen receptor signaling has been supposed as one mechanism underlying their effects, the precise mechanism of androgen receptor inhibition remains unclear. Thus, this study aimed to clarify their mechanism. Among soy isoflavones, equol suppressed androgen receptor as well as prostate-specific antigen expression most potently in androgen-dependent LNCaP cells. However, the inhibitory effect on androgen receptor expression and activity was less prominent in castration-resistant CxR and 22Rv1 cells. Consistently, cell proliferation was suppressed and cellular apoptosis was induced by equol in LNCaP cells, but less so in CxR and 22Rv1 cells. We revealed that the proteasome pathway through S-phase kinase-associated protein 2 (Skp2) was responsible for androgen receptor suppression. Taken together, soy isoflavones, especially equol, appear to be promising as chemopreventive and therapeutic agents for prostate cancer based on the fact that equol augments Skp2-mediated androgen receptor degradation. Moreover, because Skp2 expression was indicated to be crucial for the effect of soy isoflavones, soy isoflavones may be applicable for precancerous and cancerous prostates. PMID:27088761

  15. H-Ras Modulates N-Methyl-d-aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity*

    PubMed Central

    Thornton, Claire; Yaka, Rami; Dinh, Son; Ron, Dorit

    2005-01-01

    Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hip-pocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction. PMID:12695509

  16. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  17. G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

    PubMed

    Hao, Yuan; Chow, Alison W; Yip, Wallace C; Li, Chi H; Wan, Tai F; Tong, Benjamin C; Cheung, King H; Chan, Wood Y; Chen, Yangchao; Cheng, Christopher H; Ko, Wing H

    2016-08-01

    P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia. PMID:27271044

  18. DCP-LA stimulates AMPA receptor exocytosis through CaMKII activation due to PP-1 inhibition.

    PubMed

    Kanno, Takeshi; Yaguchi, Takahiro; Nagata, Tetsu; Tanaka, Akito; Nishizaki, Tomoyuki

    2009-10-01

    The linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) by inhibiting protein phosphatase-1 (PP-1). DCP-LA induced a transient huge facilitation of synaptic transmission monitored from the CA1 region of rat hippocampal slices, which was largely inhibited by the CaMKII inhibitor KN-93. DCP-LA potentiated kainate-evoked whole-cell membrane currents for Xenopus oocytes expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors composed of the GluR1, GluR3, GluR1/GluR2, GluR1/GluR3, and GluR1/GluR2/GluR3 subunits, and the potentiation was significantly inhibited by KN-93. A similar potentiation was still found with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. The GluR1 and GluR2 subunits formed AMPA receptors in the rat hippocampus, and DCP-LA increased expression of both the subunits on the plasma membrane. The DCP-LA action was blocked by KN-93 and the exocytosis inhibitor botulinum toxin type A, but not by the endocytosis inhibitor phenylarsine oxide. DCP-LA, thus, appears to activate CaMKII through PP-1 inhibition, that stimulates AMPA receptor exocytosis to increase expression of the receptors on the plasma membrane, responsible for potentiate AMPA receptor responses and facilitation of hippocampal synaptic transmission. PMID:19492412

  19. Inhibition of the production of mediators of inflammation by corticosteroids is a glucocorticoid receptor-mediated process

    PubMed Central

    Dijk, A. P. M. van; Tak, C. J. A. M.; Wilson, J. H. P.; Zijlstra, F. J.

    1996-01-01

    In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF-∝, IL-6, PGE2 and LTB4 by peripheral blood mononuclear cells (MNC) depends on binding to the glucocorticoid receptor (GR), and whether it is determined by the number or the affinity of the GR of these cells. GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF-∝, IL-6 and PGE2 production but had no effect on LTB4 production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the Kd or receptor number. PMID:18475705

  20. Structure-based design of a bispecific receptor mimic that inhibits T cell responses to a superantigen.

    PubMed

    Lehnert, N M; Allen, D L; Allen, B L; Catasti, P; Shiflett, P R; Chen, M; Lehnert, B E; Gupta, G

    2001-04-10

    Key surface proteins of pathogens and their toxins bind to the host cell receptors in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses. Here we describe a novel strategy for "non-antibody-based" pathogen countermeasure by targeting the very same "alternative mode of host receptor binding" that the pathogen proteins exploit to cause infection and disease. We have chosen the Staphylococcus enterotoxin B (SEB) superantigen as a model pathogen protein to illustrate the principle and application of our strategy. SEB bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen-presenting cell and the T cell receptor. This alternative mode of binding causes massive IL-2 release and T cell proliferation. A normally processed antigen requires all the domains of the receptor complex for its binding, whereas SEB requires only the alpha1 subunit (DRalpha) of the MHC class II receptor and the variable beta subunit (TCRVbeta) of the T cell receptor. This prompted us to design a bispecific chimera, DRalpha-linker-TCRVbeta, that acts as a receptor mimic and prevents the interaction of SEB with its host cell receptors. We have adopted (GSTAPPA)(2) as the linker sequence because it supports synergistic binding of DRalpha and TCRVbeta to SEB and thereby makes DRalpha-(GSTAPPA)(2)-TCRVbeta as effective an SEB binder as the native MHC class II-T cell receptor complex. Finally, we show that DRalpha-(GSTAPPA)(2)-TCRVbeta inhibits SEB-induced IL-2 release and T cell proliferation at nanomolar concentrations. PMID:11284677

  1. Inhibition of pregnane X receptor pathway contributes to the cell growth inhibition and apoptosis of anticancer agents in ovarian cancer cells.

    PubMed

    Masuyama, Hisashi; Nakamura, Keiichiro; Nobumoto, Etsuko; Hiramatsu, Yuji

    2016-09-01

    Epithelial ovarian cancer remains the most devastating gynecologic cancer with drug resistance and rapid recurrence. Pregnane X receptor (PXR) is a nuclear receptor that affects drug metabolism/efflux and drug-drug interaction through control of multiple drug resistance 1 (MDR1), which implies a major role in multidrug resistance, and other genes. We examined whether the inhibition of PXR-mediated pathway using siRNA interference and an antagonist for PXR could influence the paclitaxel and cisplatin cytotoxicity in ovarian cancer cells. PXR agonists, phthalate and pregnenolone had significant positive effects on cytochrome P450 (CYP) 3A4 expression and PXR-mediated transcription through the CYP3A4 promoter, whereas MDR1 expression and PXR-mediated transcription though the MDR1 promoter were significantly increased in the presence of paclitaxel or cisplatin. Downregulation of PXR suppressed the augmented MDR1 expression and PXR-mediated transcription by PXR ligands, and significantly enhanced cell growth inhibition and apoptosis in the presence of paclitaxel or cisplatin. Additionally, ketoconazole, a PXR antagonist, suppressed the augmented MDR1 expression and PXR-mediated transactivation by paclitaxel and cisplatin, and enhanced cell growth inhibition and apoptosis in their presence. In conclusion, inhibition of PXR-mediated pathways could be a novel means of augmenting sensitivity, or overcoming resistance to anticancer agents for ovarian cancer. PMID:27572875

  2. Inhibition of Estrogen Receptor-DNA Binding by the "Pure" Antiestrogen ICI 164,384 Appears to be Mediated by Impaired Receptor Dimerization

    NASA Astrophysics Data System (ADS)

    Fawell, Stephen E.; White, Roger; Hoare, Susan; Sydenham, Mark; Page, Martin; Parker, Malcolm G.

    1990-09-01

    Many estrogen-antagonist and -agonist ligands have been synthesized, some of which have proved clinically important in the treatment of hormone-dependent breast tumors and endocrine disorders. Here we show that the "pure" antiestrogen ICI 164,384 inhibits mouse estrogen receptor-DNA binding in vitro. The effects of this steroid on DNA binding can be overcome by addition of an anti-receptor antibody whose epitope lies N-terminal to the receptor DNA-binding domain. Since this antibody is also capable of restoring DNA-binding activity to receptor mutants that either lack the dimerization domain or bear deleterious mutations within it, we propose that ICI 164,384 reduces DNA binding by interfering with receptor dimerization. In contrast, when complexed with the antagonist/partial agonist tamoxifen, the estrogen receptor is capable of binding to DNA in vitro, but tamoxifen does not promote the agonist-induced conformational change obtained with estradiol. The implications of these data are discussed in relation to the in vivo properties of these drugs.

  3. Inhibition of Coxsackie B Virus Infection by Soluble Forms of Its Receptors: Binding Affinities, Altered Particle Formation, and Competition with Cellular Receptors

    PubMed Central

    Goodfellow, Ian G.; Evans, David J.; Blom, Anna M.; Kerrigan, Dave; Miners, J. Scott; Morgan, B. Paul; Spiller, O. Brad

    2005-01-01

    We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37°C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed. PMID:16140777

  4. COI1, a jasmonate receptor, is involved in ethylene-induced inhibition of Arabidopsis root growth in the light

    PubMed Central

    Adams, Eri; Turner, John

    2010-01-01

    Plant response to stress is orchestrated by hormone signalling pathways including those activated by jasmonates (JAs) and by ethylene, both of which stunt root growth. COI1 is a JA receptor and is required for the known responses to this hormone. It was observed that the coi1 mutant, which is largely unresponsive to growth inhibition by JAs, was also partially unresponsive to growth inhibition by ethylene and by its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in the light but not in the dark. Although COI1 was required for this response to ACC, other components of the JA signal perception pathway were not. Mutants selected for insensitivity to ethylene, including etr1, ein2, and ein3, showed greater ACC-induced root growth inhibition in the light than in the dark. However, the double mutants etr1;coi1, ein2;coi1, and ein3;coi1, and coi1 seedlings treated with silver ions to block the ethylene receptors showed almost complete unresponsiveness to ACC-induced root growth inhibition in the light. The light requirement for the COI1-mediated growth inhibition by ACC was for long photoperiods, and the ACC response was not abolished by mutations in the known photoreceptors. The complementation assay indicated that SCF complex assembly was not required for COI1 function in the ACC response, in contrast to the JA response. It is concluded that COI1 is required for the light-dependent, JA-independent, root growth inhibition by ethylene. PMID:20699268

  5. Sphingosine-1-phosphate receptor inhibition prevents denervation-induced dendritic atrophy.

    PubMed

    Willems, Laurent M; Zahn, Nadine; Ferreirós, Nerea; Scholich, Klaus; Maggio, Nicola; Deller, Thomas; Vlachos, Andreas

    2016-01-01

    A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in which neurons not directly affected by the disease are denervated. These transneuronal effects on the network contribute considerably to the clinical symptoms. Since denervated neurons are viable, they are attractive targets for intervention. Therefore, we studied the role of Sphingosine-1-phosphate (S1P)-receptor signaling, the target of Fingolimod (FTY720), in denervation-induced dendritic atrophy. The entorhinal denervation in vitro model was used to assess dendritic changes of denervated mouse dentate granule cells. Live-cell microscopy of GFP-expressing granule cells in organotypic entorhino-hippocampal slice cultures was employed to follow individual dendritic segments for up to 6 weeks after deafferentation. A set of slice cultures was treated with FTY720 or the S1P-receptor (S1PR) antagonist VPC23019. Lesion-induced changes in S1P (mass spectrometry) and S1PR-mRNA levels (laser microdissection and qPCR) were determined. Denervation caused profound changes in dendritic stability. Dendritic elongation and retraction events were markedly increased, resulting in a net reduction of total dendritic length (TDL) during the first 2 weeks after denervation, followed by a gradual recovery in TDL. These changes were accompanied by an increase in S1P and S1PR1- and S1PR3-mRNA levels, and were not observed in slice cultures treated with FTY720 or VPC23019. We conclude that inhibition of S1PR signaling prevents dendritic destabilization and denervation-induced dendrite loss. These results suggest a novel neuroprotective effect for pharmaceuticals targeting neural S1PR pathways. PMID:27036416

  6. MiR-503 inhibits adipogenesis by targeting bone morphogenetic protein receptor 1a

    PubMed Central

    Man, Xiao-Fei; Tan, Shu-Wen; Tang, Hao-Neng; Guo, Yue; Tang, Chen-Yi; Tang, Jun; Zhou, Ci-La; Zhou, Hou-De

    2016-01-01

    Adipogenesis plays a key role in the regulation of whole-body energy homeostasis and is critically related to obesity. To overcome obesity and its associated disorders, it is necessary to elucidate the molecular mechanisms involved in adipogenesis. An adipogenesis-related miRNA array analysis demonstrated that miR-503 was differentially expressed before and after adipocyte differentiation; however, the exact role of miR-503 in adipocyte differentiation is unclear. Thus, the objective of this study was to further examine miR-503 in adipocyte differentiation. We found significantly decreased expression of miR-503 during adipocyte differentiation process. Using bioinformatic analysis, miR-503 was identified as a potential regulator of Bone Morphogenetic Protein Receptor 1a (BMPR1a). We then validated BMPR1a as the target of miR-503 using a dual luciferase assay, and found decreased miR-503 and increased BMPR1a expression during adipogenesis. Overexpression of miR-503 in preadipocytes repressed expression of BMPR1a and adipogenic-related factors such as CCAAT/enhancer binding protein a (C/EBPα), proliferator-activated receptor-gamma (PPARγ), and adipocyte protein 2 (AP2). In addition, miR-503 overexpression impaired the phosphoinositol-3 kinase (PI3K)/Akt pathway. Inhibition of miR-503 had the opposite effect. Additionally, BMPR1a interference by siRNA attenuated adipocyte differentiation and the accumulation of lipid droplets via downregulating the PI3K/Akt signaling pathway. Our study provides the first evidence of the role miR-503 plays in adipocyte differentiation by regulating BMPR1a via the PI3K/Akt pathway, which may become a novel target for obesity therapy. PMID:27398155

  7. Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex.

    PubMed

    Mateo, Z; Porter, J T

    2007-05-25

    Thalamocortical synapses provide a strong glutamatergic excitation to cortical neurons that is critical for processing sensory information. Unit recordings in vivo indicate that metabotropic glutamate receptors (mGluRs) reduce the effect of thalamocortical input on cortical circuits. However, it is not known whether this reduction is due to a reduction in glutamate release from thalamocortical terminals or from a decrease in cortical neuron excitability. To directly determine whether mGluRs act as autoreceptors on thalamocortical terminals, we examined the effect of mGluR agonists on thalamocortical synapses in slices. Thalamocortical excitatory postsynaptic currents (EPSCs) were recorded in layer IV cortical neurons in developing mouse brain slices. The activation of group II mGluRs with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) reduced thalamocortical EPSCs in both excitatory and inhibitory neurons, while the stimulation of group I or group III mGluRs had no effect on thalamocortical EPSCs. Consistent with a reduction in glutamate release, DCG IV increased the paired pulse ratio and the coefficient of variation of the EPSCs. The reduction induced by DCG IV was reversed by the group II mGluR antagonist, LY341495, and mimicked by another selective group II agonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC). The mGluR2 subtype appears to mediate the reduction of thalamocortical EPSCs, since the selective mGluR3 agonist, N-acetylaspartylglutamate (NAAG), had no effect on the EPSCs. Consistent with this, we showed that mGluR2 is expressed in the barrels. Furthermore, blocking group II mGluRs with LY341495 reduced the synaptic depression induced by a short stimulus train, indicating that synaptically released glutamate activates these receptors. These results indicate that group II mGluRs modulate thalamocortical processing by inhibiting glutamate release from thalamocortical synapses. This inhibition provides a feedback mechanism for

  8. Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival

    PubMed Central

    Vajkoczy, Peter; Knyazev, Pjotr; Kunkel, Andrea; Capelle, Hans-Holger; Behrndt, Sandra; von Tengg-Kobligk, Hendrik; Kiessling, Fabian; Eichelsbacher, Uta; Essig, Marco; Read, Tracy-Ann; Erber, Ralf; Ullrich, Axel

    2006-01-01

    Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, Axl is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition >85%, P < 0.05) and resulted in long-term survival of mice after intracerebral glioma cell implantation when compared with Axl wild-type (AXL-WT) transfected tumor cells (survival times: AXL-WT, 10 days; AXL-DN, >72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P < 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P < 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors. PMID:16585512

  9. Cannabinoid 1 (CB1) receptors coupled to cholinergic motorneurones inhibit neurogenic circular muscle contractility in the human colon

    PubMed Central

    Hinds, Nicholas M; Ullrich, Katja; Smid, Scott D

    2006-01-01

    The effects of cannabinoid subtype 1 (CB1) receptor activation were determined on smooth muscle, inhibitory and excitatory motorneuronal function in strips of human colonic longitudinal muscle (LM) and circular muscle (CM) in vitro. Electrical field stimulation (EFS; 0.5–20 Hz, 50 V) evoked a relaxation in LM and CM precontracted with a neurokinin-2 (NK-2) selective receptor agonist (β-ala8-neurokinin A; 10−6 M) in the presence of atropine (10−6 M); this was unaltered following pretreatment with the CB1-receptor selective agonist arachidonyl-2-chloroethylamide (ACEA; 10−6 M). In the presence of nitric oxide synthase blockade with N-nitro-L-arginine (10−4 M), EFS evoked a frequency-dependent ‘on-contraction' during stimulation and an ‘off-contraction' following stimulus cessation. On-contractions were significantly inhibited in CM strips by pretreatment with ACEA (10−6 M). These inhibitory effects were reversed in the presence of the CB1 receptor-selective antagonist N-(piperidine-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (10−7 M). ACEA did not alter LM or CM contractile responses to acetylcholine or NK-2 receptor-evoked contraction. Immunohistochemical studies revealed a colocalisation of CB1 receptors to cholinergic neurones in the human colon based on colabelling with choline acetyltransferase, in addition to CB1 receptor labelling in unidentified structures in the CM. In conclusion, activation of CB1 receptors coupled to cholinergic motorneurones selectively and reversibly inhibits excitatory nerve transmission in colonic human colonic CM. These results provide evidence of a direct role for cannabinoids in the modulation of motor activity in the human colon by coupling to cholinergic motorneurones. PMID:16520743

  10. Telmisartan inhibits advanced glycation end products (AGEs)-elicited endothelial cell injury by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gammaactivation.

    PubMed

    Yamagishi, Sho-ichi; Matsui, Takanori; Nakamura, Kazuo; Takeuchi, Masayoshi; Inoue, Hiroyoshi

    2008-01-01

    Advanced glycation end products (AGEs)-their receptor (RAGE) axis plays a central role in the pathogenesis of diabetic microangiopathy. Since the pathophysiological crosstalk between the AGEs-RAGE system and angiotensin II has also been associated with diabetic microangiopathy, we examined here whether and how telmisartan, a unique angiotensin II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity, could inhibit the AGEs-elicited endothelial cell injury by suppressing RAGE expression in vitro. Telmisartan suppressed RAGE expression at both mRNA and protein levels in human cultured microvascular endothelial cells (ECs), which were prevented by GW9662, an inhibitor of PPAR-gamma. Further, telmisartan was found to inhibit up-regulation of mRNA levels for monocyte chemoattractant protein-1, intercellular adhesion molecule-1 and vascular endothelial growth factor in AGEs-exposed ECs. These results suggest that telmisartan inhibits the AGEs-elicited EC injury by down-regulating RAGE expression via PPAR-gamma activation. Our present study provides a unique beneficial aspect of telmisartan. Specifically, it could work as an anti-inflammatory agent against AGEs via PPAR-gamma activation and may play a protective role against diabetic microangiopathy. PMID:18855759

  11. Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages

    PubMed Central

    Wex, Katharina; Schmid, Ursula; Just, Sissy; Wang, Xu; Wurm, Rebecca; Naumann, Michael; Schlüter, Dirk; Nishanth, Gopala

    2016-01-01

    Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages. PMID:26834734

  12. Trichostatin A Protects Liver against Septic Injury through Inhibiting Toll-Like Receptor Signaling.

    PubMed

    Kim, So-Jin; Park, Jin-Sook; Lee, Do-Won; Lee, Sun-Mee

    2016-07-01

    Sepsis, a serious clinical problem, is characterized by a systemic inflammatory response to infection and leads to organ failure. Toll-like receptor (TLR) signaling is intimately implicated in hyper-inflammatory responses and tissue injury during sepsis. Histone deacetylase (HDAC) inhibitors have been reported to exhibit anti-inflammatory properties. The aim of this study was to investigate the hepatoprotective mechanisms of trichostatin A (TSA), a HDAC inhibitor, associated with TLR signaling pathway during sepsis. The anti-inflammatory properties of TSA were assayed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP), a clinically relevant model of sepsis. The mice were intraperitoneally received TSA (1, 2 or 5 mg/kg) 30 min before CLP. The serum and liver samples were collected 6 and 24-h after CLP. TSA inhibited the increased production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in LPS-stimulated RAW264.7 cells. TSA improved sepsis-induced mortality, attenuated liver injury and decreased serum TNF-α and IL-6 levels. CLP increased the levels of TLR4, TLR2 and myeloid differentiation primary response protein 88 (MyD88) protein expression and association of MyD88 with TLR4 and TLR2, which were attenuated by TSA. CLP increased nuclear translocation of nuclear factor kappa B and decreased cytosolic inhibitor of kappa B (IκB) protein expression, which were attenuated by TSA. Moreover, CLP decreased acetylation of IκB kinase (IKK) and increased association of IKK with IκB and TSA attenuated these alterations. Our findings suggest that TSA attenuates liver injury by inhibiting TLR-mediated inflammatory response during sepsis. PMID:27068262

  13. Effects of bradykinin B2 receptor antagonism on the hypotensive effects of ACE inhibition.

    PubMed Central

    Bouaziz, H; Joulin, Y; Safar, M; Benetos, A

    1994-01-01

    1. The aim of this study was to determine the participation of endogenous bradykinin (BK) in the antihypertensive effects of the angiotensin converting enzyme inhibitor (ACEI), perindoprilat, in the spontaneously hypertensive rat (SHR) on different salt diets. 2. Conscious SHRs receiving either a low or a high NaCl diet were used in order to evaluate the respective roles of angiotensin II suppression and bradykinin stimulation in the acute hypotensive effects of perindoprilat. Two different B2 receptor antagonists (B 4146 and Hoe 140) were used after bolus administration of 7 mg kg-1 of the ACEI, perindoprilat. In separate animals, Hoe 140 was administered before the injection of perindoprilat. In other experiments, the effects of Hoe 140 on the hypotensive effects of the calcium antagonist, nicardipine, were tested. 3. The different NaCl diets had no effect on baseline blood pressure. Hoe 140 injection before ACE inhibition did not modify blood pressure. Perindoprilat caused more marked hypotension in the low salt-fed rats than in the high salt animals (P < 0.01). Administration of Hoe 140 or B4146 after perindoprilat significantly reduced the antihypertensive effects of perindoprilat in the different groups, but this effect was more pronounced in high salt-fed rats. However, in SHRs receiving Hoe 140 before perindoprilat, the antihypertensive effect of perindoprilat was completely abolished in both high or low salt diet rats. In separate experiments we confirmed that Hoe 140 did not affect the hypotensive efficacy of the calcium antagonist, nicardipine. 4. Our study shows that inhibition of endogenous bradykinin degradation participates in the acute antihypertensive effects of perindoprilat in SHRs. The role of bradykinin is more pronounced following exposure to a high salt diet i.e., when the renin-angiotensin system is suppressed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7858859

  14. Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

    PubMed Central

    Bomben, V. C.; Sontheimer, H. W.

    2009-01-01

    Objectives Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca2+ influx pathway impacting cellular growth. Materials and Methods Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl3, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells’ resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G2 + M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme, the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours. PMID:18211288

  15. Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice.

    PubMed

    Lawson, J A; Fisher, M A; Simmons, C A; Farhood, A; Jaeschke, H

    1999-05-01

    The mechanism of liver cell injury induced by an overdose of the analgesic acetaminophen (AAP) remains controversial. Recently, it was hypothesized that a significant number of hepatocytes die by apoptosis. Since caspases have been implicated as critical signal and effector proteases in apoptosis, we investigated their potential role in the pathophysiology of AAP-induced liver injury. Male C3Heb/FeJ mice were fasted overnight and then treated with 500 mg/kg AAP. Liver injury became apparent at 4 h and was more severe at 6 h (plasma ALT activities: 4110 +/- 320 U/liter; centrilobular necrosis). DNA fragmentation increased parallel to the increase of plasma ALT values. At 6 h there was a 420% increase of DNA fragmentation and a 74-fold increase of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells located predominantly around central veins. However, the activity of the proapoptotic caspase-3 was not increased at any time after AAP. In contrast, injection of the anti-Fas antibody Jo-2 (positive control) caused a 28-fold increase of caspase-3 activity and severe DNA fragmentation before significant ALT release. Treatment with the caspase inhibitor ZVAD-CHF2 had no effect on AAP toxicity but completely prevented Jo-mediated apoptosis. In contrast, Jo-induced caspase activation and apoptosis could be inhibited by AAP treatment in a time- and dose-dependent manner. We conclude that AAP-induced DNA fragmentation does not involve caspases, suggesting a direct activation of endonucleases through elevated Ca2+ levels. In addition, electrophilic metabolites of AAP may inactivate caspases or their activation pathway. This indicates that AAP metabolism has the potential to inhibit signal transduction mechanisms of receptor-mediated apoptosis. PMID:10222310

  16. Trichostatin A Protects Liver against Septic Injury through Inhibiting Toll-Like Receptor Signaling

    PubMed Central

    Kim, So-Jin; Park, Jin-Sook; Lee, Do-Won; Lee, Sun-Mee

    2016-01-01

    Sepsis, a serious clinical problem, is characterized by a systemic inflammatory response to infection and leads to organ failure. Toll-like receptor (TLR) signaling is intimately implicated in hyper-inflammatory responses and tissue injury during sepsis. Histone deacetylase (HDAC) inhibitors have been reported to exhibit anti-inflammatory properties. The aim of this study was to investigate the hepatoprotective mechanisms of trichostatin A (TSA), a HDAC inhibitor, associated with TLR signaling pathway during sepsis. The anti-inflammatory properties of TSA were assayed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP), a clinically relevant model of sepsis. The mice were intraperitoneally received TSA (1, 2 or 5 mg/kg) 30 min before CLP. The serum and liver samples were collected 6 and 24-h after CLP. TSA inhibited the increased production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in LPS-stimulated RAW264.7 cells. TSA improved sepsis-induced mortality, attenuated liver injury and decreased serum TNF-α and IL-6 levels. CLP increased the levels of TLR4, TLR2 and myeloid differentiation primary response protein 88 (MyD88) protein expression and association of MyD88 with TLR4 and TLR2, which were attenuated by TSA. CLP increased nuclear translocation of nuclear factor kappa B and decreased cytosolic inhibitor of kappa B (IκB) protein expression, which were attenuated by TSA. Moreover, CLP decreased acetylation of IκB kinase (IKK) and increased association of IKK with IκB and TSA attenuated these alterations. Our findings suggest that TSA attenuates liver injury by inhibiting TLR-mediated inflammatory response during sepsis. PMID:27068262

  17. Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

    PubMed

    Cordova, Daniel; Benner, Eric A; Schroeder, Mark E; Holyoke, Caleb W; Zhang, Wenming; Pahutski, Thomas F; Leighty, Robert M; Vincent, Daniel R; Hamm, Jason C

    2016-07-01

    Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid. PMID:27130855

  18. Peroxisome Proliferator-Activated ReceptorInhibition Protects Against Doxorubicin-Induced Cardiotoxicity in Mice.

    PubMed

    Rahmatollahi, Mahdieh; Baram, Somayeh Mahmoodi; Rahimian, Reza; Saeedi Saravi, Seyed Soheil; Dehpour, Ahmad Reza

    2016-07-01

    Doxorubicin is an effective chemotherapeutic drug against a considerable number of malignancies. However, its toxic effects on myocardium are confirmed as major limit of utilization. PPAR-α is highly expressed in the heart, and its activation leads to an increased cardiac fatty acid oxidation and cardiomyocyte necrosis. This study was performed to adjust the hypothesis that PPAR-α receptor inhibition protects against doxorubicin-induced cardiac dysfunction in mice. Male Balb/c mice were used in this study. Left atria were isolated, and their contractility was measured in response to electrical field stimulation in a standard organ bath. PPAR-α activity was measured using specific PPAR-α antibody in an ELISA-based system coated with double-strand DNA containing PPAR-α response element sequence. Moreover, cardiac MDA and TNF-α levels were measured by ELISA method. Following incubation with doxorubicin (35 µM), a significant reduction in atrial contractility was observed (P < 0.001). Pretreatment of animals with a selective PPAR-α antagonist, GW6471, significantly improved doxorubicin-induced atrial dysfunction (P < 0.001). Furthermore, pretreatment of the mice with a non-selective cannabinoid agonist, WIN55212-2, significantly decreased PPAR-α activity in cardiac tissue, subsequently leading to significant improvement in doxorubicin-induced atrial dysfunction (P < 0.001). Also, GW6471 and WIN significantly reduced cardiac MDA and TNF-α levels compared with animals receiving doxorubicin (P < 0.001). The study showed that inhibition of PPAR-α is associated with protection against doxorubicin-induced cardiotoxicity in mice, and cannabinoids can potentiate the protection by PPAR-α blockade. Moreover, PPAR-α may be considered as a target to prevent cardiotoxicity induced by doxorubicin in patients undergoing chemotherapy. PMID:26082188

  19. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    SciTech Connect

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  20. Plant lectin can target receptors containing sialic acid, exemplified by podoplanin, to inhibit transformed cell growth and migration.

    PubMed

    Ochoa-Alvarez, Jhon Alberto; Krishnan, Harini; Shen, Yongquan; Acharya, Nimish K; Han, Min; McNulty, Dean E; Hasegawa, Hitoki; Hyodo, Toshinori; Senga, Takeshi; Geng, Jian-Guo; Kosciuk, Mary; Shin, Seung S; Goydos, James S; Temiakov, Dmitry; Nagele, Robert G; Goldberg, Gary S

    2012-01-01

    Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth. PMID:22844530

  1. Prostacyclin Analogue Beraprost Inhibits Cardiac Fibroblast Proliferation Depending on Prostacyclin Receptor Activation through a TGF β-Smad Signal Pathway

    PubMed Central

    Yao, Wenjuan; Zhu, Hongyan; Xu, Xiaole; Meng, Guoliang; Zhang, Wei

    2014-01-01

    Previous studies showed that prostacyclin inhibited fibrosis. However, both receptors of prostacyclin, prostacyclin receptor (IP) and peroxisome proliferator-activated receptor (PPAR), are abundant in cardiac fibroblasts. Here we investigated which receptor was vital in the anti-fibrosis effect of prostacyclin. In addition, the possible mechanism involved in protective effects of prostacyclin against cardiac fibrosis was also studied. We found that beraprost, a prostacyclin analogue, inhibited angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast proliferation in a concentration-dependent and time-dependent manner. Beraprost also suppressed Ang II-induced collagen I mRNA expression and protein synthesis in cardiac fibroblasts. After IP expression was knocked down by siRNA, Ang II-induced proliferation and collagen I synthesis could no longer be rescued by beraprost. However, treating cells with different specific inhibitors of PPAR subtypes prior to beraprost and Ang II stimulation, all of the above attenuating effects of beraprost were still available. Moreover, beraprost significantly blocked transforming growth factor β (TGF β) expression as well as Smad2 phosphorylation and reduced Smad-DNA binding activity. Beraprost also increased phosphorylation of cAMP response element binding protein (CREB) at Ser133 in the nucleus. Co-immunoprecipitation analysis revealed that beraprost increased CREB but decreased Smad2 binding to CREB-binding protein (CBP) in nucleus. In conclusion, beraprost inhibits cardiac fibroblast proliferation by activating IP and suppressing TGF β-Smad signal pathway. PMID:24852754

  2. Quercetin inhibits the 5-hydroxytryptamine type 3 receptor-mediated ion current by interacting with pre-transmembrane domain I.

    PubMed

    Lee, Byung-Hwan; Jeong, Sang-Min; Jung, Sang-Min; Lee, Jun-Ho; Kim, Jong-Hoon; Yoon, In-Soo; Lee, Joon-Hee; Choi, Sun-Hye; Lee, Sang-Mok; Chang, Choon-Gon; Kim, Hyung-Chun; Han, YeSun; Paik, Hyun-Dong; Kim, Yangmee; Nah, Seung-Yeol

    2005-08-31

    The flavonoid, quercetin, is a low molecular weight substance found in apple, tomato and other fruit. Besides its antioxidative effect, quercetin, like other flavonoids, has a wide range of neuropharmacological actions including analgesia, and motility, sleep, anticonvulsant, sedative and anxiolytic effects. In the present study, we investigated its effect on mouse 5-hydroxytryptamine type 3 (5-HT3A) receptor channel activity, which is involved in pain transmission, analgesia, vomiting, and mood disorders. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured with the two-electrode voltage clamp technique. In oocytes injected with 5-HT3A receptor cRNA, quercetin inhibited the 5-HT-induced inward peak current (I(5-HT)) with an IC50 of 64.7 +/- 2.2 microM. Inhibition was competitive and voltage-independent. Point mutations of pre-transmembrane domain 1 (pre-TM1) such as R222T and R222A, but not R222D, R222E and R222K, abolished inhibition, indicating that quercetin interacts with the pre-TM1 of the 5-HT3A receptor. PMID:16258243

  3. Plant Lectin Can Target Receptors Containing Sialic Acid, Exemplified by Podoplanin, to Inhibit Transformed Cell Growth and Migration

    PubMed Central

    Shen, Yongquan; Acharya, Nimish K.; Han, Min; McNulty, Dean E.; Hasegawa, Hitoki; Hyodo, Toshinori; Senga, Takeshi; Geng, Jian-Guo; Kosciuk, Mary; Shin, Seung S.; Goydos, James S.; Temiakov, Dmitry; Nagele, Robert G.; Goldberg, Gary S.

    2012-01-01

    Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth. PMID:22844530

  4. Adenosine deaminase inhibition enhances the inotropic response mediated by A1 adenosine receptor in hyperthyroid guinea pig atrium.

    PubMed

    Kemeny-Beke, Adam; Jakab, Anita; Zsuga, Judit; Vecsernyes, Miklos; Karsai, Denes; Pasztor, Fanni; Grenczer, Maria; Szentmiklosi, Andras Jozsef; Berta, Andras; Gesztelyi, Rudolf

    2007-08-01

    The aim of the present study was to test the hypothesis that inhibition of adenosine deaminase (ADA) enhances the efficiency of signal-transduction of myocardial A1 adenosine receptors in hyperthyroidism. The inotropic response to N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist resistant to ADA, was investigated in the absence or presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an ADA and cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2) inhibitor, or of pentostatin (2'-deoxycoformycin; DCF), an exclusive ADA inhibitor, in left atria isolated from eu- or hyperthyroid guinea pigs. Both ADA inhibitors enhanced the effect of CPA only in hyperthyroid atria. EHNA significantly increased the Emax (mean+/-S.E.M.) from 83.8+/-1.2% to 93.4+/-1.2%, while DCF significantly decreased the logEC50 from -7.5+/-0.07 to -7.83+/-0.07 in hyperthyroid samples. Conversely, EHNA also diminished the logEC50 (from -7.5+/-0.07 to -7.65+/-0.07) and DCF also raised the Emax (from 83.8+/-1.2% to 85.7+/-2%) in hyperthyroidism, but these changes were not significant. In conclusion, ADA inhibition moderately but significantly enhanced the efficiency of A(1) adenosine receptor signaling pathway in the hyperthyroid guinea pig atrium. This suggests that elevated intracellular adenosine level caused by ADA inhibition may improve the suppressed responsiveness to A1 adenosine receptor agonists associated with the hyperthyroid state. Alternatively or in addition, the role of decreased concentration of adenosine degradation products cannot be excluded. Furthermore, in the case of EHNA, inhibition of PDE2 also appears to contribute to the enhanced A1 adenosine receptor signaling in the hyperthyroid guinea pig atrium. PMID:17574432

  5. Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate

    PubMed Central

    Goldman, Corey K.; Kendall, Richard L.; Cabrera, Gustavo; Soroceanu, Liliana; Heike, Yuji; Gillespie, G. Yancey; Siegal, Gene P.; Mao, Xianzhi; Bett, Andrew J.; Huckle, William R.; Thomas, Kenneth A.; Curiel, David T.

    1998-01-01

    Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth. PMID:9671758

  6. Expression of an engineered soluble coxsackievirus and adenovirus receptor by a dimeric AAV9 vector inhibits adenovirus infection in mice.

    PubMed

    Röger, C; Pozzuto, T; Klopfleisch, R; Kurreck, J; Pinkert, S; Fechner, H

    2015-06-01

    Immunosuppressed (IS) patients, such as recipients of hematopoietic stem cell transplantation, occasionally develop severe and fatal adenovirus (Ad) infections. Here, we analyzed the potential of a virus receptor trap based on a soluble coxsackievirus and Ad receptor (sCAR) for inhibition of Ad infection. In vitro, a dimeric fusion protein, sCAR-Fc, consisting of the extracellular domain of CAR and the Fc portion of human IgG1 and a monomeric sCAR lacking the Fc domain, were expressed in cell culture. More sCAR was secreted into the cell culture supernatant than sCAR-Fc, but it had lower Ad neutralization activity than sCAR-Fc. Further investigations showed that sCAR-Fc reduced the Ad infection by a 100-fold and Ad-induced cytotoxicity by ~20-fold. Not only was Ad infection inhibited by sCAR-Fc applied prior to infection, it also inhibited infection when used to treat ongoing Ad infection. In vivo, sCAR-Fc was delivered to IS mice by an AAV9 vector, resulting in persistent and high (>40 μg ml(-1)) sCAR-Fc serum levels. The sCAR-Fc serum concentration was sufficient to significantly inhibit hepatic and cardiac wild-type Ad5 infection. Treatment with sCAR-Fc did not induce side effects. Thus, sCAR-Fc virus receptor trap may be a promising novel therapeutic for treatment of Ad infections. PMID:25786873

  7. GGNBP2 acts as a tumor suppressor by inhibiting estrogen receptor α activity in breast cancer cells.

    PubMed

    Lan, Zi-Jian; Hu, YunHui; Zhang, Sheng; Li, Xian; Zhou, Huaxin; Ding, Jixiang; Klinge, Carolyn M; Radde, Brandie N; Cooney, Austin J; Zhang, Jin; Lei, Zhenmin

    2016-07-01

    Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis. PMID:27357812

  8. Pharmacological evidence that dopamine inhibits the cardioaccelerator sympathetic outflow via D2-like receptors in pithed rats.

    PubMed

    Alcántara-Vázquez, Oscar; Villamil-Hernández, Ma Trinidad; Sánchez-López, Araceli; Centurión, David

    2013-01-01

    It has been suggested that N,N-di-n-propyl-dopamine (dopamine analogue) decreased heart rate in rats through stimulation of dopamine receptors. Nevertheless, the role of prejunctional dopamine D1/2-like receptors or even α2-adrenoceptors to mediate cardiac sympatho-inhibition induced by dopamine remains unclear. Hence, this study identified the pharmacological profile of the cardiac sympatho-inhibition to dopamine in pithed rats. Male Wistar rats were pithed and prepared to stimulate the cardiac sympathetic outflow or to receive i.v. bolus of exogenous noradrenaline. I.v. continuous infusions of dopamine (endogenous ligand) or quinpirole (D2-like agonist) dose-dependently inhibited the tachycardic responses to sympathetic stimulation, but not those to exogenous noradrenaline. In contrast, SKF-38393 (100 μg/kg∙min, D1-like agonist) failed to modify both of these responses. The sympatho-inhibition to dopamine (1.8 μg/kg∙min) or quinpirole (100 μg/kg∙min): i) remained unaltered after saline or the antagonists SCH-23390 (D1-like, 300 μg/kg) and rauwolscine (α2-adrenoceptors, 300 μg/kg); and ii) was significantly antagonized by raclopride (D2-like, 300 μg/kg). These antagonists, at the above doses, failed to modify the sympathetically-induced tachycardic responses. The above results suggest that the inhibition of the cardiac sympathetic outflow to dopamine and quinpirole is primarily mediated by prejunctional D2-like receptors but not D1-like receptors or α2-adrenoceptors. PMID:24225403

  9. Tonic Inhibition of Accumbal Spiny Neurons by Extrasynaptic α4βδ GABAA Receptors Modulates the Actions of Psychostimulants

    PubMed Central

    Maguire, Edward P.; Macpherson, Tom; Swinny, Jerome D.; Dixon, Claire I.; Herd, Murray B.; Belelli, Delia; Stephens, David N.

    2014-01-01

    Within the nucleus accumbens (NAc), synaptic GABAA receptors (GABAARs) mediate phasic inhibition of medium spiny neurons (MSNs) and influence behavioral responses to cocaine. We demonstrate that both dopamine D1- and D2-receptor-expressing MSNs (D-MSNs) additionally harbor extrasynaptic GABAARs incorporating α4, β, and δ subunits that mediate tonic inhibition, thereby influencing neuronal excitability. Both the selective δ-GABAAR agonist THIP and DS2, a selective positive allosteric modulator, greatly increased the tonic current of all MSNs from wild-type (WT), but not from δ−/− or α4−/− mice. Coupling dopamine and tonic inhibition, the acute activation of D1 receptors (by a selective agonist or indirectly by amphetamine) greatly enhanced tonic inhibition in D1-MSNs but not D2-MSNs. In contrast, prolonged D2 receptor activation modestly reduced the tonic conductance of D2-MSNs. Behaviorally, WT and constitutive α4−/− mice did not differ in their expression of cocaine-conditioned place preference (CPP). Importantly, however, mice with the α4 deletion specific to D1-expressing neurons (α4D1−/−) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4−/− or α4D1−/− mice, blocked cocaine enhancement of CPP. In comparison, α4D2−/− mice exhibited normal CPP, but no cocaine enhancement. In conclusion, dopamine modulation of GABAergic tonic inhibition of D1- and D2-MSNs provides an intrinsic mechanism to differentially affect their excitability in response to psychostimulants and thereby influence their ability to potentiate conditioned reward. Therefore, α4βδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors. PMID:24431441

  10. Estrous cycle regulation of extrasynaptic δ-containing GABA(A) receptor-mediated tonic inhibition and limbic epileptogenesis.

    PubMed

    Wu, Xin; Gangisetty, Omkaram; Carver, Chase Matthew; Reddy, Doodipala Samba

    2013-07-01

    The ovarian cycle affects susceptibility to behavioral and neurologic conditions. The molecular mechanisms underlying these changes are poorly understood. Deficits in cyclical fluctuations in steroid hormones and receptor plasticity play a central role in physiologic and pathophysiologic menstrual conditions. It has been suggested that synaptic GABA(A) receptors mediate phasic inhibition in the hippocampus and extrasynaptic receptors mediate tonic inhibition in the dentate gyrus. Here we report a novel role of extrasynaptic δ-containing GABA(A) receptors as crucial mediators of the estrous cycle-related changes in neuronal excitability in mice, with hippocampus subfield specificity. In molecular and immunofluorescence studies, a significant increase occurred in δ-subunit, but not α4- and γ2-subunits, in the dentate gyrus during diestrus. However, δ-subunit upregulation was not evident in the CA1 region. The δ-subunit expression was undiminished by age and ovariectomy and in mice lacking progesterone receptors, but it was significantly reduced by finasteride, a neurosteroid synthesis inhibitor. Electrophysiologic studies confirmed greater potentiation of GABA currents by progesterone-derived neurosteroid allopregnanolone in dissociated dentate gyrus granule cells in diestrus than in CA1 pyramidal cells. The baseline conductance and allopregnanolone potentiation of tonic currents in dentate granule cells from hippocampal slices were higher than in CA1 pyramidal cells. In behavioral studies, susceptibility to hippocampus kindling epileptogenesis was lower in mice during diestrus. These results demonstrate the estrous cycle-related plasticity of neurosteroid-sensitive, δ-containing GABA(A) receptors that mediate tonic inhibition and seizure susceptibility. These findings may provide novel insight on molecular cascades of menstrual disorders like catamenial epilepsy, premenstrual syndrome, and migraine. PMID:23667248

  11. μ-Opioid receptor activation and noradrenaline transport inhibition by tapentadol in rat single locus coeruleus neurons

    PubMed Central

    Sadeghi, Mahsa; Tzschentke, Thomas M; Christie, MacDonald J

    2015-01-01

    BACKGROUND AND PURPOSE Tapentadol is a novel analgesic that combines moderate μ-opioid receptor agonism and noradrenaline reuptake inhibition in a single molecule. Both mechanisms of action are involved in producing analgesia; however, the potency and efficacy of tapentadol in individual neurons has not been characterized. EXPERIMENTAL APPROACH Whole-cell patch-clamp recordings of G-protein-coupled inwardly rectifying K+ (KIR3.x) currents were made from rat locus coeruleus neurons in brain slices to investigate the potency and relative efficacy of tapentadol and compare its intrinsic activity with other clinically used opioids. KEY RESULTS Tapentadol showed agonist activity at μ receptors and was approximately six times less potent than morphine with respect to KIR3.x current modulation. The intrinsic activity of tapentadol was lower than [Met]enkephalin, morphine and oxycodone, but higher than buprenorphine and pentazocine. Tapentadol inhibited the noradrenaline transporter (NAT) with potency similar to that at μ receptors. The interaction between these two mechanisms of action was additive in individual LC neurons. CONCLUSIONS AND IMPLICATIONS Tapentadol displays similar potency for both µ receptor activation and NAT inhibition in functioning neurons. The intrinsic activity of tapentadol at the μ receptor lies between that of buprenorphine and oxycodone, potentially explaining the favourable profile of side effects, related to μ receptors. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24372103

  12. The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

    PubMed Central

    Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

    2016-01-01

    Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

  13. Monoclonal Antibodies Specific for Human IgM Fc Receptor Inhibit Ligand-binding Activity

    PubMed Central

    Kubagawa, Yoshiki; Honjo, Kazuhito; Kang, Dong-Won

    2014-01-01

    A panel of six different murine hybridoma clones secreting IgG monoclonal antibodies (MAbs) specific for the human IgM Fc receptor (FcμR) was generated. All MAbs specifically precipitated a major protein of ∼60 kDa from membrane lysates of FcμR-bearing, but not FcμR-negative, cells as did IgM-ligands. Pre-incubation of membrane lysate of FcμR-bearing cells with these MAbs completely removed the ∼60 kDa IgM-reactive protein. By using recombinant human/mouse chimeric FcμR proteins, the epitope recognized by HM7 and HM10 MAbs was mapped to the Ig-like domain of human FcμR, whereas the other MAbs recognized the stalk region. Pre-incubation of FcμR+ cells with the Ig-like domain-specific MAbs, but not with others, markedly inhibited subsequent IgM-ligand binding. A similar, but much weaker, inhibition was also observed when the incubation order was reversed. When FcμR+ cells were simultaneously incubated with both IgM-ligands and MAbs, HM7 MAb efficiently competed with IgM for FcμR binding. Unlike control Jurkat cells, FcμR-bearing cells were resistant to apoptosis induced by agonistic IgM anti-Fas MAb (CH11); however, addition of the HM7 MAb inhibited the interaction of the Fc portion of CH11 MAb with FcμR, thereby promoting apoptosis of FcμR-bearing Jurkat cells. The variable regions of the HM7 MAb were composed of Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igk3-4 and Igkj2 for the κ light chain. These findings suggest that HM7 MAb efficiently blocks the ligand-binding activity of FcμR. PMID:25545208

  14. Central delta-opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat.

    PubMed

    Dray, A; Nunan, L; Wire, W

    1985-07-01

    The in vivo effects of a number of opioid agonists and antagonists were studied on the spontaneous reflex contractions of the urinary bladder recorded isometrically in the rat anesthetized with urethane. All substances were administered into the central nervous system by the intracereboventricular (i.c.v.) or spinal intrathecal (i.t.) route. The conformationally restricted enkephalin analogues [2-D-penicillamine, 5-L-cysteine] enkephalin (DPLCE), [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE) produced dose-related inhibition of reflex bladder contractions when administered by the i.c.v. or i.t. route. Both the novel delta-opioid receptor antagonist ICI 154,129 (200-600 micrograms) [N,N-bisallyl-Tyr-Gly-Gly-Psi-(CH2S)-Phe-Leu-OH) and ICI 174,864 (1-3 micrograms) [N,N-dially-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] attenuated or abolished the effects of DPLCE, DPLPE and DPDPE when administered by the i.c.v. or i.t. route. The antagonism observed was selective since the equipotent inhibition produced by the mu-opioid receptor agonist [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) was unaffected. Overall, ICI 154,129 was considerably weaker than ICI 174,864 and both antagonists inhibited bladder activity at doses higher than those required to demonstrate delta-receptor antagonism. Further studies of the agonistic effect of ICI 174,864 showed that it was insensitive to low doses of naloxone (2 micrograms, i.c.v. or i.t.) but could be abolished by higher (10-15 micrograms) doses of naloxone. These observations suggested that the agonistic effect of ICI 174,864 was not mediated by mu-opioid receptor. beta-Endorphin (0.2-1.0 micrograms, i.c.v.) inhibited bladder contractions but following recovery from this effect, appeared to prevent the expression of delta-receptor antagonism by ICI 174,864. In addition a previously subthreshold dose of ICI 174,864 now exhibited marked agonistic

  15. Probenecid Inhibits the Human Bitter Taste Receptor TAS2R16 and Suppresses Bitter Perception of Salicin

    PubMed Central

    Greene, Tiffani A.; Alarcon, Suzanne; Thomas, Anu; Berdougo, Eli; Doranz, Benjamin J.; Breslin, Paul A. S.; Rucker, Joseph B.

    2011-01-01

    Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to β-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of

  16. The antinociceptive triterpene β-amyrin inhibits 2-arachidonoylglycerol (2-AG) hydrolysis without directly targeting cannabinoid receptors

    PubMed Central

    Chicca, A; Marazzi, J; Gertsch, J

    2012-01-01

    BACKGROUND AND PURPOSE Pharmacological activation of cannabinoid CB1 and CB2 receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB1 receptors with a subnanomolar Ki value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB1 and CB2 receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system. EXPERIMENTAL APPROACH We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB1 and hCB2 transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV2 cells and pig brain, as well as purified enzymes, were also studied. KEY RESULTS There was no binding of either α- or β-amyrin to hCB receptors in our assays (Ki > 10 µM). The triterpene β-amyrin potently inhibited 2-arachidonoyl glycerol (2-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited 2-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV2 cell and pig brain homogenates. CONCLUSIONS AND IMPLICATIONS We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of 2-AG. LINKED ARTICLES This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8 PMID:22646533

  17. Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction

    PubMed Central

    Jiang, Changtao; Xie, Cen; Lv, Ying; Li, Jing; Krausz, Kristopher W.; Shi, Jingmin; Brocker, Chad N.; Desai, Dhimant; Amin, Shantu G.; Bisson, William H.; Liu, Yulan; Gavrilova, Oksana; Patterson, Andrew D.; Gonzalez, Frank J.

    2015-01-01

    The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-β-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders. PMID:26670557

  18. Dual HER/VEGF receptor targeting inhibits in vivo ovarian cancer tumor growth.

    PubMed

    Becker, Marc A; Farzan, Thahir; Harrington, Sean C; Krempski, James W; Weroha, S John; Hou, Xiaonan; Kalli, Kimberly R; Wong, Tai W; Haluska, Paul

    2013-12-01

    Ovarian cancer mortality ranks highest among all gynecologic cancers with growth factor pathways playing an integral role in tumorigenesis, metastatic dissemination, and therapeutic resistance. The HER and VEGF receptor (VEGFR) are both overexpressed and/or aberrantly activated in subsets of ovarian tumors. While agents targeting either the HER or VEGF pathways alone have been investigated, the impact of these agents have not led to overall survival benefit in ovarian cancer. We tested the hypothesis that cotargeting HER and VEGFR would maximize antitumor efficacy at tolerable doses. To this end, ovarian cancer xenografts grown intraperitoneally in athymic nude mice were tested in response to AC480 (pan-HER inhibitor, "HERi"), cediranib (pan-VEGFR inhibitor "VEGFRi"), or BMS-690514 (combined HER/VEGFR inhibitor "EVRi"). EVRi was superior to both HERi and VEGFRi in terms of tumor growth, final tumor weight, and progression-free survival. Correlative tumor studies employing phosphoproteomic antibody arrays revealed distinct agent-specific alterations, with EVRi inducing the greatest overall effect on growth factor signaling. These data suggest that simultaneous inhibition of HER and VEGFR may benefit select subsets of ovarian cancer tumors. To this end, we derived a novel HER/VEGF signature that correlated with poor overall survival in high-grade, late stage, serous ovarian cancer patient tumors. PMID:24130056

  19. Protective effects of genetic inhibition of Discoidin Domain Receptor 1 in experimental renal disease

    PubMed Central

    Kerroch, Monique; Alfieri, Carlo; Dorison, Aude; Boffa, Jean-Jacques; Chatziantoniou, Christos; Dussaule, Jean-Claude

    2016-01-01

    Chronic kidney disease is a progressive incurable pathology affecting millions of people. Intensive investigations aim to identify targets for therapy. We have previously demonstrated that abnormal expression of the Discoidin Domain Receptor 1 (DDR1) is a key factor of renal disease by promoting inflammation and fibrosis. The present study investigates whether blocking the expression of DDR1 after the initiation of renal disease can delay or arrest the progression of this pathology. Severe renal disease was induced by either injecting nephrotoxic serum (NTS) or performing unilateral ureteral obstruction in mice, and the expression of DDR1 was inhibited by administering antisense oligodeoxynucleotides either at 4 or 8 days after NTS (corresponding to early or more established phases of disease, respectively), or at day 2 after ligation. DDR1 antisense administration at day 4 stopped the increase of proteinuria and protected animals against the progression of glomeruloneprhitis, as evidenced by functional, structural and cellular indexes. Antisense administration at day 8 delayed progression –but to a smaller degree- of renal disease. Similar beneficial effects on renal structure and inflammation were observed with the antisense administration of DDR1 after ureteral ligation. Thus, targeting DDR1 can be a promising strategy in the treatment of chronic kidney disease. PMID:26880216

  20. Impaired estrogen sensitivity in bone by inhibiting both estrogen receptor alpha and beta pathways.

    PubMed

    Ogawa, S; Fujita, M; Ishii, Y; Tsurukami, H; Hirabayashi, M; Ikeda, K; Orimo, A; Hosoi, T; Ueda, M; Nakamura, T; Ouchi, Y; Muramatsu, M; Inoue, S

    2000-07-14

    Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways. PMID:10806217

  1. Protective effects of genetic inhibition of Discoidin Domain Receptor 1 in experimental renal disease.

    PubMed

    Kerroch, Monique; Alfieri, Carlo; Dorison, Aude; Boffa, Jean-Jacques; Chatziantoniou, Christos; Dussaule, Jean-Claude

    2016-01-01

    Chronic kidney disease is a progressive incurable pathology affecting millions of people. Intensive investigations aim to identify targets for therapy. We have previously demonstrated that abnormal expression of the Discoidin Domain Receptor 1 (DDR1) is a key factor of renal disease by promoting inflammation and fibrosis. The present study investigates whether blocking the expression of DDR1 after the initiation of renal disease can delay or arrest the progression of this pathology. Severe renal disease was induced by either injecting nephrotoxic serum (NTS) or performing unilateral ureteral obstruction in mice, and the expression of DDR1 was inhibited by administering antisense oligodeoxynucleotides either at 4 or 8 days after NTS (corresponding to early or more established phases of disease, respectively), or at day 2 after ligation. DDR1 antisense administration at day 4 stopped the increase of proteinuria and protected animals against the progression of glomeruloneprhitis, as evidenced by functional, structural and cellular indexes. Antisense administration at day 8 delayed progression -but to a smaller degree- of renal disease. Similar beneficial effects on renal structure and inflammation were observed with the antisense administration of DDR1 after ureteral ligation. Thus, targeting DDR1 can be a promising strategy in the treatment of chronic kidney disease. PMID:26880216

  2. Pirt reduces bladder overactivity by inhibiting purinergic receptor P2X3.

    PubMed

    Gao, Xiao-Fei; Feng, Ji-Feng; Wang, Wei; Xiang, Zheng-Hua; Liu, Xiu-Jie; Zhu, Chan; Tang, Zong-Xiang; Dong, Xin-Zhong; He, Cheng

    2015-01-01

    Pirt is a transmembrane protein predominantly expressed in peripheral neurons. However, the physiological and pathological roles of Pirt in hollow viscus are largely unknown. Here we show that Pirt deficiency in mice causes bladder overactivity. The density of α,β-meATP-induced currents is significantly reinforced in Pirt-deficient dorsal root ganglion (DRG) neurons. Pirt and P2X3 receptor co-localize in bladder nerve fibres and heterologous Pirt expression significantly reduces P2X3-mediated currents. Pirt interacts with P2X3 through the N-terminal 14 amino-acid residues. TAT-conjugated Pirt(N14) peptide (Pirt(N14)) is sufficient to inhibit P2X3 activation in bladder DRG neurons and to alleviate bladder overactivity in Pirt(-/-) mice. Pirt expression is decreased in the bladder of cyclophosphamide (CYP)-treated mice, a commonly used model of bladder overactivity. Importantly, Pirt(N14) administration reduces the frequency of bladder voiding and restores the voided volume of CYP-treated mice. Therefore, our results demonstrate that Pirt is an endogenous regulator of P2X3 in bladder function. PMID:26151598

  3. The Thyroid Hormone Receptors Inhibit Hepatic Interleukin-6 Signaling During Endotoxemia

    PubMed Central

    Contreras-Jurado, Constanza; Alonso-Merino, Elvira; Saiz-Ladera, Cristina; Valiño, Arturo José; Regadera, Javier; Alemany, Susana; Aranda, Ana

    2016-01-01

    Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-κB and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections. PMID:27484112

  4. The Thyroid Hormone Receptors Inhibit Hepatic Interleukin-6 Signaling During Endotoxemia.

    PubMed

    Contreras-Jurado, Constanza; Alonso-Merino, Elvira; Saiz-Ladera, Cristina; Valiño, Arturo José; Regadera, Javier; Alemany, Susana; Aranda, Ana

    2016-01-01

    Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-κB and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections. PMID:27484112

  5. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid

    PubMed Central

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA. PMID:27057269

  6. Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction.

    PubMed

    Jiang, Changtao; Xie, Cen; Lv, Ying; Li, Jing; Krausz, Kristopher W; Shi, Jingmin; Brocker, Chad N; Desai, Dhimant; Amin, Shantu G; Bisson, William H; Liu, Yulan; Gavrilova, Oksana; Patterson, Andrew D; Gonzalez, Frank J

    2015-01-01

    The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-β-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders. PMID:26670557

  7. Inhibition of metastasis of rhabdomyosarcoma by a novel neutralizing antibody to CXC chemokine receptor-4

    PubMed Central

    Kashima, Kenji; Watanabe, Miho; Sato, Yasuko; Hata, Junichi; Ishii, Nobuya; Aoki, Yuko

    2014-01-01

    Rhabdomyosarcoma is the most common soft tissue sarcoma affecting children, and the overall cure rate of children with metastatic disease remains below 30%. The CXC chemokine receptor-4 (CXCR4)/stromal cell-derived factor-1 (SDF1) axis has been implicated in the promotion of metastatic potential in several tumors. In this study, we developed a novel anti-CXCR4 mAb, CF172, and investigated its antimetastatic activity against rhabdomyosarcoma cells in vitro and in vivo, to evaluate its potential as a therapeutic antibody to treat rhabdomyosarcoma. The CF172 molecule showed a specific binding reactivity against human CXCR4, as well as a specific neutralizing activity against CXCR4/SDF1 signal transduction. Using CF172, we determined that SJCRH30 rhabdomyosarcoma cells expressed high levels of CXCR4. In addition, CF172 was found to inhibit the SDF1-induced migration activity of SJCRH30 cells in vitro. Using xenograft models of SJCRH30 cells, we carried out in vivo efficacy studies for peritoneal and lymph node metastasis, which were clinically observed in rhabdomyosarcoma. These studies indicated that CF172 significantly decreased both types of metastasis of SJCRH30. In conclusion, we found that a novel anti-CXCR4 mAb, CF172, with specific reactivity against human CXCR4, prevented peritoneal metastasis and lymph node metastasis of rhabdomyosarcoma in animal models. These results suggest that CF172 is a potential antimetastasis therapeutic antibody for rhabdomyosarcoma treatment. PMID:25154453

  8. Sensitivity of human granulosa cell tumor cells to epidermal growth factor receptor inhibition.

    PubMed

    Andersson, Noora; Anttonen, Mikko; Färkkilä, Anniina; Pihlajoki, Marjut; Bützow, Ralf; Unkila-Kallio, Leila; Heikinheimo, Markku

    2014-04-01

    Epidermal growth factor receptor (EGFR) is implicated in the progression of many human cancers, but its significance in ovarian granulosa cell tumor (GCT) pathobiology remains poorly understood. We assessed the EGFR gene copy number, surveyed the mRNA and protein expression patterns of EGFR in 90 adult GCTs, and assessed the in vitro sensitivity of GCT cells to EGFR inhibition. Low-level amplification of EGFR gene was observed in five GCTs and high-level amplification in one sample. EGFR mRNA was robustly expressed in GCTs. Most tumors expressed both unphosphorylated and phosphorylated EGFR protein, but the protein expression did not correlate with clinical parameters, including the risk of recurrence. Small-molecule EGFR inhibitors reduced the EGF-induced activation of EGFR and its downstream signaling molecules at nanomolar doses, but cell viability was reduced, and caspase-3/7 was activated in GCT cells only at micromolar doses. Based on the present results, EGFR is active and abundantly expressed in the majority of GCTs, but probably has only minor contribution to GCT cell growth. Given the high doses of EGFR inhibitors required to reduce GCT cell viability in vitro, they are not likely to be effective for GCT treatment as single agents; they should rather be tested as part of combination therapies for these malignancies. PMID:24463098

  9. Toll-like receptor-mediated immune response inhibits prion propagation.

    PubMed

    Kang, Sang-Gyun; Kim, Chiye; Cortez, Leonardo M; Carmen Garza, María; Yang, Jing; Wille, Holger; Sim, Valerie L; Westaway, David; McKenzie, Debbie; Aiken, Judd

    2016-06-01

    Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-β). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity. PMID:26880394

  10. Antagonism of chemokine receptor CXCR3 inhibits osteosarcoma metastasis to lungs.

    PubMed

    Pradelli, Emmanuelle; Karimdjee-Soilihi, Babou; Michiels, Jean-François; Ricci, Jean-Ehrland; Millet, Marie-Ange; Vandenbos, Fanny; Sullivan, Timothy J; Collins, Tassie L; Johnson, Michael G; Medina, Julio C; Kleinerman, Eugenie S; Schmid-Alliana, Annie; Schmid-Antomarchi, Heidy

    2009-12-01

    Metastasis continues to be the leading cause of mortality for patients with cancer. Several years ago, it became clear that chemokines and their receptors could control the tumor progress. CXCR3 has now been identified in many cancers including osteosarcoma and CXCR3 ligands were expressed by lungs that are the primary sites to which this tumor metastasize. This study tested the hypothesis that disruption of the CXCR3/CXCR3 ligands complexes could lead to a decrease in lungs metastasis. The experimental design involved the use of the CXCR3 antagonist, AMG487 and 2 murine models of osteosarcoma lung metastases. After tail vein injection of osteosarcoma cells, mice that were systematically treated with AMG487 according to preventive or curative protocols had a significant reduction in metastatic disease. Treatment of osteosarcoma cells in vitro with AMG487 led to decreased migration, decreased matrix metalloproteinase activity, decreased proliferation/survival and increased caspase-independent death. Taken together, our results support the hypothesis that CXCR3 and their ligands intervene in the initial dissemination of the osteosarcoma cells to the lungs and stimulate the growth and expansion of the metastatic foci in later stages. Moreover, these studies indicate that targeting CXCR3 may specifically inhibit tumor metastasis without adversely affecting antitumoral host response. PMID:19544560

  11. Antagonism of chemokine receptor CXCR3 inhibits osteosarcoma metastasis to lungs

    PubMed Central

    Pradelli, Emmanuelle; Karimdjee-Soilihi, Babou; Michiels, Jean-François; Ricci, Jean-Ehrland; Millet, Marie-Ange; Vandenbos, Fanny; Sullivan, Timothy J.; Collins, Tassie L.; Johnson, Michael G.; Medina, Julio C.; Kleinerman, Eugenie S.; Schmid-Alliana, Annie; Schmid-Antomarchi, Heidy

    2009-01-01

    Metastasis continues to be the leading cause of mortality for patients with cancer. Several years ago, it became clear that chemokines and their receptors could control the tumor progress. CXCR3 has now been identified in many cancers including osteosarcoma and CXCR3 ligands were expressed by lungs that are the primary sites to which this tumor metastasize. This study tested the hypothesis that disruption of the CXCR3/CXCR3 ligands complexes could lead to a decrease in lungs metastasis. The experimental design involved the use of the CXCR3 antagonist, AMG487 and 2 murine models of osteosarcoma lung metastases. After tail vein injection of osteosarcoma cells, mice that were systematically treated with AMG487 according to preventive or curative protocols had a significant reduction in metastatic disease. Treatment of osteosarcoma cells in vitro with AMG487 led to decreased migration, decreased matrix metalloproteinase activity, decreased proliferation/survival and increased caspase-independent death. Taken together, our results support the hypothesis that CXCR3 and their ligands intervene in the initial dissemination of the osteosarcoma cells to the lungs and stimulate the growth and expansion of the metastatic foci in later stages. Moreover, these studies indicate that targeting CXCR3 may specifically inhibit tumor metastasis without adversely affecting antitumoral host response. PMID:19544560

  12. Ethanol acts directly on extrasynaptic subtypes of GABAA receptors to increase tonic inhibition

    PubMed Central

    Santhakumar, Vijayalakshmi; Wallner, Martin; Otis, Thomas S.

    2007-01-01

    Based on the similarity of ethanol intoxication to the behavioral effects of drugs known to target GABAA receptors (GABARs) it has been suspected for decades that ethanol facilitates the activity of GABA. Even so, it has been surprisingly difficult to identify molecular targets of ethanol. Research conducted over the past several years suggests that a subclass of GABARs (those containing δ subunits) responds in a relevant concentration range to ethanol. Although δ subunit-containing GABARs are not ubiquitously expressed at inhibitory synapses like their γ subunit-containing, synaptic counterparts, they are found in many neurons in extrasynaptic locations. Here they give rise to a tonic form of inhibition that can potently suppress neuronal excitability. Studies have shown that both recombinant and native δ subunit-containing GABARs: 1) are modulated by behaviorally-relevant (i.e. low millimolar) concentrations of ethanol, 2) directly bind ethanol over the same concentration range, 3) show altered function upon single amino substitutions linked to changes in behavioral responsiveness to ethanol, and 4) are a site of action of Ro15-4513, a competitive antagonist of ethanol binding and a drug which prevents many of the behavioral aspects of ethanol intoxication. Despite such comprehensive evidence, however, the field is not free from controversy. This review evaluates published data for and against a central role of δ subunit-containing GABARs in ethanol actions and suggests future directions that might help settle points of controversy. PMID:17591544

  13. AB260. Role of µ, κ, and δ opioid receptors in tibial inhibition of bladder overactivity in cats

    PubMed Central

    Zhang, Zhaocun; Jiang, Xuewen; Shang, Zhenhua; Chen, Shouzhen; Tai, Changfeng; Shi, Benkang

    2016-01-01

    Background To study the impact of µ, κ, and δ opioid receptors on tibial inhibition of bladder overactivity in cats Methods In α-chloralose anesthetized cats we examined the role of opioid receptor (OR) subtypes (µ, κ, and δ) intibialnerve stimulation (TNS) induced inhibition of bladder overactivity elicited by intravesical infusion of 0.25% acetic acid (AA). The sensitivity of TNS inhibition to cumulative intravenous doses of selective OR antagonists (cyprodime for µ, nor-binaltorphimine for κ, or naltrindole for δ ORs) was tested. Results Naloxone (1 mg/kg, i.v., an antagonist for µ, κ, and δ ORs) was administered at the end of each experiment. AA caused bladder overactivity and significantly (P<0.01) reduced bladder capacity to 21.1±2.6% of the saline control. TNS at 2 or 4 times threshold (T) intensity for inducing toe movement significantly (P<0.01) restored bladder capacity to 52.9±3.6% or 57.4±4.6% of control, respectively. Cyprodime (0.3–1.0 mg/kg) completely removed TNS inhibition without changing AA control capacity. Nor-binaltorphimine (3–10 mg/kg) also completely reversed TNS inhibition and significantly (P<0.05) increased AA control capacity. Naltrindole(1–10 mg/kg) reduced (P<0.05) TNS inhibition but significantly (P<0.05) increased AA control capacity. Naloxone (1 mg/kg) had no effect in cyprodime pre-treated cats, but reversed the nor-binaltorphimine induced increase in bladder capacity and eliminated the TNS inhibition remaining in naltrindole pre-treated cats. Conclusions These results indicate a major role of µ and κ ORs in TNS inhibition while δORs play a minor role. Meanwhile, κ and δORs also have an excitatory role in irritation-induced bladder overactivity.

  14. 5-HT1B receptors inhibit glutamate release from primary afferent terminals in rat medullary dorsal horn neurons

    PubMed Central

    Choi, I-S; Cho, J-H; An, C-H; Jung, J-K; Hur, Y-K; Choi, J-K; Jang, I-S

    2012-01-01

    BACKGROUND AND PURPOSE Although 5-HT1B receptors are expressed in trigeminal sensory neurons, it is still not known whether these receptors can modulate nociceptive transmission from primary afferents onto medullary dorsal horn neurons. EXPERIMENTAL APPROACH Primary afferent-evoked EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices using a conventional whole-cell patch clamp technique under a voltage-clamp condition. KEY RESULTS CP93129, a selective 5-HT1B receptor agonist, reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, CP93129 reduced the frequency of spontaneous miniature EPSCs without affecting the current amplitude. The CP93129-induced inhibition of EPSCs was significantly occluded by GR55562, a 5-HT1B/1D receptor antagonist, but not LY310762, a 5-HT1D receptor antagonist. Sumatriptan, an anti-migraine drug, also decreased EPSC amplitude, and this effect was partially blocked by either GR55562 or LY310762. On the other hand, primary afferent-evoked EPSCs were mediated by the Ca2+ influx passing through both presynaptic N-type and P/Q-type Ca2+ channels. The CP93129-induced inhibition of EPSCs was significantly occluded by ω-conotoxin GVIA, an N-type Ca2+ channel blocker. CONCLUSIONS AND IMPLICATIONS The present results suggest that the activation of presynaptic 5-HT1B receptors reduces glutamate release from primary afferent terminals onto medullary dorsal horn neurons, and that 5-HT1B receptors could be, at the very least, a potential target for the treatment of pain from orofacial tissues. LINKED ARTICLE This article is commented on by Connor, pp. 353–355 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01963.x PMID:22462474

  15. A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition.

    PubMed

    Philbrook, Alison; Barbagallo, Belinda; Francis, Michael M

    2013-07-01

    Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation. PMID:24778941

  16. Inhibition of antigen receptor-dependent Ca(2+) signals and NF-AT activation by P2X7 receptors in human B lymphocytes.

    PubMed

    Pippel, Anja; Beßler, Björn; Klapperstück, Manuela; Markwardt, Fritz

    2015-04-01

    One of the first intracellular signals after antigen binding by the antigen receptor of B lymphocytes is the increased intracellular Ca(2+) concentration ([Ca(2+)]i), which is followed by several intracellular signaling events like the nuclear translocation of the transcription factor NF-AT controlling the fate of B lymphocytes after their activation. Extracellular ATP, which is released from cells under several pathological conditions, is considered a danger-associated signal serving as an immunomodulator. We investigated the interaction of antigen receptor (BCR) and P2X7 receptor (P2X7R) activation on [Ca(2+)]i signaling and on nuclear translocation of the transcription factor NF-AT in human B lymphocytes. Although the P2X7R is an ATP-gated Ca(2+)-permeable ion channel, P2X7R activation inhibits the BCR-mediated [Ca(2+)]i responses. This effect is mimicked by cell membrane depolarization induced by an increase in the extracellular K(+) concentration or by application of the Na(+) ionophore gramicidin, but is abolished by stabilization of the membrane potential using the K(+) ionophore valinomycin, by extracellular Mg(2+), which is known to inhibit P2X7R-dependent effects, or by replacing Na(+) by the less P2X7R-permeable Tris(+) ion. Furthermore, P2X7R activation by ATP inhibits the BCR-dependent translocation of the transcription factor NF-ATc1 to the nucleus. We therefore conclude that extracellular ATP via the P2X7R mediates inhibitory effects on B cell activation. This may be of relevance for understanding of the activation of the BCR under pathological conditions and for the development of therapeutic strategies targeting human B lymphocytes or P2X7 receptors. PMID:25678443

  17. Tankyrase Inhibitor Sensitizes Lung Cancer Cells to Endothelial Growth Factor Receptor (EGFR) Inhibition via Stabilizing Angiomotins and Inhibiting YAP Signaling.

    PubMed

    Wang, Hui; Lu, Bo; Castillo, Johnny; Zhang, Yue; Yang, Zinger; McAllister, Gregory; Lindeman, Alicia; Reece-Hoyes, John; Tallarico, John; Russ, Carsten; Hoffman, Greg; Xu, Wenqing; Schirle, Markus; Cong, Feng

    2016-07-15

    YAP signaling pathway plays critical roles in tissue homeostasis, and aberrant activation of YAP signaling has been implicated in cancers. To identify tractable targets of YAP pathway, we have performed a pathway-based pooled CRISPR screen and identified tankyrase and its associated E3 ligase RNF146 as positive regulators of YAP signaling. Genetic ablation or pharmacological inhibition of tankyrase prominently suppresses YAP activity and YAP target gene expression. Using a proteomic approach, we have identified angiomotin family proteins, which are known negative regulators of YAP signaling, as novel tankyrase substrates. Inhibition of tankyrase or depletion of RNF146 stabilizes angiomotins. Angiomotins physically interact with tankyrase through a highly conserved motif at their N terminus, and mutation of this motif leads to their stabilization. Tankyrase inhibitor-induced stabilization of angiomotins reduces YAP nuclear translocation and decreases downstream YAP signaling. We have further shown that knock-out of YAP sensitizes non-small cell lung cancer to EGFR inhibitor Erlotinib. Tankyrase inhibitor, but not porcupine inhibitor, which blocks Wnt secretion, enhances growth inhibitory activity of Erlotinib. This activity is mediated by YAP inhibition and not Wnt/β-catenin inhibition. Our data suggest that tankyrase inhibition could serve as a novel strategy to suppress YAP signaling for combinatorial targeted therapy. PMID:27231341

  18. GBR-12909 and fluspirilene potently inhibited binding of ( sup 3 H) (+) 3-PPP to sigma receptors in rat brain

    SciTech Connect

    Contreras, P.C.; Bremer, M.E.; Rao, T.S. )

    1990-01-01

    Fluspirilene and GBR-12909, two compounds structurally similar to BMY-14802 and haloperidol, were assessed for their ability to interact with sigma receptors. Fluspirilene, an antipsychotic agent that interacts potently with dopamine receptors, inhibited the binding of ({sup 3}H)-(+)3-PPP (IC{sub 50} = 380 nM) more potently than rimcazole, a putative sigma antagonist that was tested clinically for antipsychotic activity. GBR-12909, a potent dopamine uptake blocker, also inhibited the binding of ({sup 3}H)-(+)3-PPP with an IC{sub 50} of 48 nM. However, other compounds that block the re-uptake of catecholamines, such as nomifensine, desipramine, imipramine, xylamine, benztropine and cocaine, were much weaker than GBR-12909as sigma ligands. Thus, GBR-12909 and fluspirilene, compounds structurally similar to BMY-14802, are potent sigma ligands.

  19. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    SciTech Connect

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  20. Molecular mechanism for inhibition of G protein-coupled receptor kinase 2 by a selective RNA aptamer

    PubMed Central

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J. G.

    2012-01-01

    SUMMARY Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic αF-αG loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase. PMID:22727813

  1. Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors.

    PubMed

    Castro, Alberto; Aguilar, Justo; González-Ramírez, Ricardo; Loeza-Alcocer, Emanuel; Canto-Bustos, Martha; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2011-08-19

    GABA(A) receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α(5) has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α(5) subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α(5) subunit-containing GABA(A) receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α(4/6) subunit-containing GABA(A) receptors, but is sensitive to L-655708, an antagonist of the GABA(A) receptors containing α(5) subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α(5) subunit in the turtle spinal cord. Together, these results suggest that GABA(A) receptors containing the α(5) subunit mediate the tonic inhibitory currents observed in VHIs. PMID:21798246

  2. Traumatic brain injury causes platelet adenosine diphosphate and arachidonic acid receptor inhibition independent of hemorrhagic shock in humans and rats

    PubMed Central

    Castellino, Francis J.; Chapman, Michael P.; Donahue, Deborah L.; Thomas, Scott; Moore, Ernest E.; Wohlauer, Max V.; Fritz, Braxton; Yount, Robert; Ploplis, Victoria; Davis, Patrick; Evans, Edward; Walsh, Mark

    2014-01-01

    BACKGROUND Coagulopathy in traumatic brain injury (CTBI) is a well-established phenomenon, but its mechanism is poorly understood. Various studies implicate protein C activation related to the global insult of hemorrhagic shock or brain tissue factor release with resultant platelet dysfunction and depletion of coagulation factors. We hypothesized that the platelet dysfunction of CTBI is a distinct phenomenon from the coagulopathy following hemorrhagic shock. METHODS We used thrombelastography with platelet mapping as a measure of platelet function, assessing the degree of inhibition of the adenosine diphosphate (ADP) and arachidonic acid (AA) receptor pathways. First, we studied the early effect of TBI on platelet inhibition by performing thrombelastography with platelet mapping on rats. We then conducted an analysis of admission blood samples from trauma patients with isolated head injury (n = 70). Patients in shock or on clopidogrel or aspirin were excluded. RESULTS In rats, ADP receptor inhibition at 15 minutes after injury was 77.6% ± 6.7% versus 39.0% ± 5.3% for controls (p < 0.0001). Humans with severe TBI (Glasgow Coma Scale [GCS] score ≤ 8) showed an increase in ADP receptor inhibition at 93.1% (interquartile range [IQR], 44.8–98.3%; n = 29) compared with 56.5% (IQR, 35–79.1%; n = 41) in milder TBI and 15.5% (IQR, 13.2–29.1%) in controls (p = 0.0014 and p < 0.0001, respectively). No patient had significant hypotension or acidosis. Parallel trends were noted in AA receptor inhibition. CONCLUSION Platelet ADP and AA receptor inhibition is a prominent early feature of CTBI in humans and rats and is linked to the severity of brain injury in patients with isolated head trauma. This phenomenon is observed in the absence of hemorrhagic shock or multisystem injury. Thus, TBI alone is shown to be sufficient to induce a profound platelet dysfunction. (J Trauma Acute Care Surg. 2014;76: 1169–1176. PMID:24747445

  3. Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling.

    PubMed

    Carneiro de Morais, Carla P; Polidoro, Juliano Z; Ralph, Donna L; Pessoa, Thaissa D; Oliveira-Souza, Maria; Barauna, Valério G; Rebouças, Nancy A; Malnic, Gerhard; McDonough, Alicia A; Girardi, Adriana C C

    2015-10-15

    Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na(+)/H(+) exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na(+)-dependent intracellular pH recovery. We found that 10(-7) M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization. PMID:26246427

  4. Identification of a Small Peptide That Inhibits PCSK9 Protein Binding to the Low Density Lipoprotein Receptor

    PubMed Central

    Zhang, Yingnan; Eigenbrot, Charles; Zhou, Lijuan; Shia, Steven; Li, Wei; Quan, Clifford; Tom, Jeffrey; Moran, Paul; Di Lello, Paola; Skelton, Nicholas J.; Kong-Beltran, Monica; Peterson, Andrew; Kirchhofer, Daniel

    2014-01-01

    PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 μm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å2 largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 μm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the β-strand and a discontinuous short α-helix, and it engaged in the same β-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9. PMID:24225950

  5. Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

    PubMed Central

    Dilger, James P.; Vidal, Ana Maria; Liu, Man; Mettewie, Claire; Suzuki, Takahiro; Pham, Anh; Demazumder, Deeptankar

    2008-01-01

    Background Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α–δ and α–ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α–ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. Methods HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcho-line in the absence and presence of antagonist. Concentration–response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. Results Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. Conclusion Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α–ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α–δ interface. This suggests the possibility of synergistic

  6. Inhibition of Chemokine (C-C Motif) Receptor 7 Sialylation Suppresses CCL19-Stimulated Proliferation, Invasion and Anti-Anoikis

    PubMed Central

    Su, Mei-Lin; Chang, Tsung-Ming; Chiang, Chi-Hsiang; Chang, Han-Chen; Hou, Ming-Feng; Li, Wen-Shan; Hung, Wen-Chun

    2014-01-01

    Chemokine (C-C motif) receptor 7 (CCR7) is involved in lymph-node homing of naive and regulatory T cells and lymphatic metastasis of cancer cells. Sialic acids comprise a group of monosaccharide units that are added to the terminal position of the oligosaccharide chain of glycoproteins by sialyation. Recent studies suggest that aberrant sialylation of receptor proteins contributes to proliferation, motility, and drug resistance of cancer cells. In this study, we addressed whether CCR7 is a sialylated receptor protein and tried to elucidate the effect of sialylation in the regulation of signal transduction and biological function of CCR7. Our results demonstrated that α-2, 3-sialyltransferase which catalyze sialylation reaction in vivo was overexpressed in breast tumor tissues and cell lines. Lectin blot analysis clearly demonstrated that CCR7 receptor was sialyated in breast cancer cells. Chemokine (C-C motif) ligand 19 (CCL19), the cognate ligand for CCR7, induced the activation of extracellular signal-regulated kinase (ERK) and AKT signaling and increased the expression of cell cycle regulatory proteins and proliferation of breast cancer cells. When cells were pre-treated with a sialyltransferase inhibitor AL10 or sialidase, CCL19-induced cell growth was significantly suppressed. CCL19 also increased invasion and prevented anoikis by up-regulating pro-survival proteins Bcl-2 and Bcl-xL. Inhibition of sialylation by AL10 totally abolished these effects. Finally, we showed that AL10 inhibited tumorigenicity of breast cancer in experimental animals. Taken together, we demonstrate for the first time that CCR7 receptor is a sialylated protein and sialylation is important for the paracrine stimulation by its endogenous ligand CCL19. In addition, inhibition of aberrant sialylation of CCR7 suppresses proliferation and invasion and triggers anoikis in breast cancer cells. Targeting of sialylation enzymes may be a novel strategy for breast cancer treatment. PMID:24915301

  7. Harnessing the nuclear receptor PPARγ to inhibit the growth of lung adenocarcinoma by rewiring metabolic circuitries

    PubMed Central

    Yenerall, Paul; Kittler, Ralf

    2015-01-01

    Altered metabolism and nuclear receptor activity have been reported in various cancer types. Here, we discuss our recent finding that the metabolic state of lung adenocarcinoma cells expressing the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) can be modulated by thiazolidinediones, culminating in accumulation of reactive oxygen species and decreased proliferation. PMID:27308443

  8. Harnessing the nuclear receptor PPARγ to inhibit the growth of lung adenocarcinoma by rewiring metabolic circuitries.

    PubMed

    Yenerall, Paul; Kittler, Ralf

    2015-01-01

    Altered metabolism and nuclear receptor activity have been reported in various cancer types. Here, we discuss our recent finding that the metabolic state of lung adenocarcinoma cells expressing the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) can be modulated by thiazolidinediones, culminating in accumulation of reactive oxygen species and decreased proliferation. PMID:27308443

  9. R-Ras protein inhibits autophosphorylation of vascular endothelial growth factor receptor 2 in endothelial cells and suppresses receptor activation in tumor vasculature.

    PubMed

    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-03-27

    Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis. PMID:25645912

  10. The Transient Receptor Potential Channel TRPM8 Is Inhibited via the α2A Adrenoreceptor Signaling Pathway*

    PubMed Central

    Bavencoffe, Alexis; Gkika, Dimitra; Kondratskyi, Artem; Beck, Benjamin; Borowiec, Anne-Sophie; Bidaux, Gabriel; Busserolles, Jérôme; Eschalier, Alain; Shuba, Yaroslav; Skryma, Roman; Prevarskaya, Natalia

    2010-01-01

    The transient receptor potential channel melastatin member 8 (TRPM8) is expressed in sensory neurons, where it constitutes the main receptor of environmental innocuous cold (10–25 °C). Among several types of G protein-coupled receptors expressed in sensory neurons, Gi-coupled α2A-adrenoreceptor (α2A-AR), is known to be involved in thermoregulation; however, the underlying molecular mechanisms remain poorly understood. Here we demonstrated that stimulation of α2A-AR inhibited TRPM8 in sensory neurons from rat dorsal root ganglia (DRG). In addition, using specific pharmacological and molecular tools combined with patch-clamp current recordings, we found that in heterologously expressed HEK-293 (human embryonic kidney) cells, TRPM8 channel is inhibited by the Gi protein/adenylate cyclase (AC)/cAMP/protein kinase A (PKA) signaling cascade. We further identified the TRPM8 S9 and T17 as two key PKA phosphorylation sites regulating TRPM8 channel activity. We therefore propose that inhibition of TRPM8 through the α2A-AR signaling cascade could constitute a new mechanism of modulation of thermosensation in both physiological and pathological conditions. PMID:20110357

  11. Arctigenin reduces neuronal responses in the somatosensory cortex via the inhibition of non-NMDA glutamate receptors.

    PubMed

    Borbély, Sándor; Jócsák, Gergely; Moldován, Kinga; Sedlák, Éva; Preininger, Éva; Boldizsár, Imre; Tóth, Attila; Atlason, Palmi T; Molnár, Elek; Világi, Ildikó

    2016-07-01

    Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. Furthermore, arctigenin can cross the blood-brain barrier and in the brain it interacts with kainate sensitive ionotropic glutamate receptors. These results indicate that arctigenin is a potentially useful new pharmacological tool for the inhibition of glutamate-evoked responses in the central nervous system in vivo. PMID:26972612

  12. Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

    PubMed

    Báez-Pagán, Carlos A; Del Hoyo-Rivera, Natalie; Quesada, Orestes; Otero-Cruz, José David; Lasalde-Dominicci, José A

    2016-08-01

    The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane. PMID:27116687

  13. Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

    PubMed Central

    KE, TIE; LI, RENBIN; CHEN, WENCHANG

    2016-01-01

    Inhibition of the N-methyl-D-aspartate (NMDA) receptor by MK-801 reduces ischemia/reperfusion (I/R) injury in the central nervous system. However, few previous studies have evaluated the neuroprotective effects of MK-801 against peripheral I/R injury. The present study aimed to investigate the protective effects of MK-801 pretreatment against I/R injury in the rat sciatic nerve (SN). Sprague-Dawley rats were subjected to a sham surgery (n=8) or to a 5-h ischemic insult by femoral artery clamping (I/R and I/R+MK-801 groups; n=48 per group). I/R+MK-801 rats were intraperitoneally injected with MK-801 (0.5 ml or 1 mg/kg) at 15 min prior to reperfusion. The rats were sacrificed at 0, 6, 12, 24, 72 h, or 7 days following reperfusion. Plasma malondialdehyde (MDA) and nitric oxide (NO) concentrations, and SN inducible NO synthase (iNOS) protein expression levels, were measured using colorimetry. In addition, the protein expression levels of tumor necrosis factor-α (TNF-α) were measured using immunohistochemistry, and histological analyses of the rat SN were conducted using light and electron microscopy. Alterations in the mRNA expression levels of TNF-α and TNF-α converting enzyme (TACE) in the rat SN were detected using reverse transcription-quantitative polymerase chain reaction. In the I/R group, plasma concentrations of NO (175.3±4.2 µmol/l) and MDA (16.2±1.9 mmol/l), and the levels of iNOS (2.5±0.3) in the SN, peaked at 24 h post-reperfusion. At 24 h, pretreatment with MK-801 significantly reduced plasma NO (107.3±3.6 µmol/l) and MDA (11.8±1.6 mmol/l), and SN iNOS (1.65±0.2) levels (all P<0.01). The mRNA expression levels of TNF-α and TACE in the SN were significantly reduced in the I/R+MK-801 group, as compared with the I/R group (P<0.05). Furthermore, MK-801 pretreatment was shown to have alleviated histological signs of I/R injury, including immune cell infiltration and axon demyelination. The results of the present study suggested that pretreatment

  14. Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling.

    PubMed

    Lamy, Sylvie; Beaulieu, Edith; Labbé, David; Bédard, Valérie; Moghrabi, Albert; Barrette, Stéphane; Gingras, Denis; Béliveau, Richard

    2008-05-01

    Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (ECs) but also smooth muscle cells (SMCs), which contribute to a mature and functional vasculature. We have reported previously that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor-induced phosphorylation of vascular endothelial growth factor receptor-2 and blocks angiogenesis in vitro and in vivo. In the present study, we show that delphinidin also inhibits activation of the platelet-derived growth factor (PDGF)-BB receptor-beta [platelet-derived growth factor receptor-beta (PDGFR-beta)] in SMC and that this inhibition may contribute to its antitumor effect. The inhibitory effect of delphinidin on PDGFR-beta was very rapid and led to the inhibition of PDGF-BB-induced activation of extracellular signal-regulated kinase (ERK)-1/2 signaling and of the chemotactic motility of SMC, as well as the differentiation and stabilization of EC and SMC into capillary-like tubular structures in a three-dimensional coculture system. Using an anthocyan-rich extract of berries, we show that berry extracts were able to suppress the synergistic induction of vessel formation by basic fibroblast growth factor-2 and PDGF-BB in the mouse Matrigel plug assay. Oral administration of the berry extract also significantly retarded tumor growth in a lung carcinoma xenograft model. Taken together, these results provide new insight into the molecular mechanisms underlying the antiangiogenic activity of delphinidin that will be helpful for the development of dietary-based chemopreventive strategies. PMID:18339683

  15. HER kinase axis receptor dimer partner switching occurs in response to EGFR tyrosine kinase inhibition despite failure to block cellular proliferation.

    PubMed

    Jain, Anjali; Penuel, Elicia; Mink, Sheldon; Schmidt, Joanna; Hodge, Amanda; Favero, Kristin; Tindell, Charles; Agus, David B

    2010-03-01

    The human epidermal receptor (HER) axis consists of a dynamic, interconnected family of receptors that make critical contributions to a number of malignancies. Therapeutics targeting epidermal growth factor receptor (EGFR) are unable to effectively inhibit tumor growth in a majority of cases. These tumors are assumed to possess primary resistance to anti-EGFR therapies, but the consequence of inhibiting EGFR in these tumors is unclear. We established isogenic cell lines by prolonged gefitinib treatment at concentrations that are in excess of that which is required for complete EGFR kinase inhibition but only minimally affected growth. Subsequently, we monitored the ligand-dependent HER profiles based on receptor expression, phosphorylation, and dimerization in conjunction with measurements of cellular susceptibility to gefitinib. Chronic EGFR kinase inhibition rapidly switched the HER network from dependence on EGFR to HER2. However, both receptors activated the critical signaling proteins AKT and mitogen-activated protein kinase, and in both cases, HER3 was the common association partner. Remarkably, the switch in receptor dimers caused diminished susceptibility to EGFR-targeted inhibitors gefitinib and cetuximab but acquired susceptibility to the HER2-targeted inhibitor pertuzumab. Overall, our study indicates that the EGFR pathway is responsive to EGFR inhibiting therapies that are not dependent on EGFR for their growth and survival, thus challenging the current definition of primary therapeutic resistance. Furthermore, EGFR kinase inhibition induces HER kinase receptors to engage in alternative dimerization that can ultimately influence therapeutic selection and responsiveness. PMID:20160029

  16. Involvement of multiple µ-opioid receptor subtypes on the presynaptic or postsynaptic inhibition of spinal pain transmission.

    PubMed

    Mizoguchi, Hirokazu; Takagi, Hirokazu; Watanabe, Chizuko; Yonezawa, Akihiko; Sato, Takumi; Sakurada, Tsukasa; Sakurada, Shinobu

    2014-01-01

    The involvement of the μ-opioid receptor subtypes on the presynaptic or postsynaptic inhibition of spinal pain transmission was characterized in ddY mice using endomorphins. Intrathecal treatment with capsaicin, N-methyl-d-aspartate (NMDA) or substance P elicited characteristic nociceptive behaviors that consisted primarily of vigorous biting and/or licking with some scratching. Intrathecal co-administration of endogenous μ-opioid peptide endomorphin-1 or endomorphin-2 resulted in a potent antinociceptive effect against the nociceptive behaviors induced by capsaicin, NMDA or substance P, which was eliminated by i.t. co-administration of the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP). The antinociceptive effect of endomorphin-1 was significantly suppressed by i.t.-co-administration of the μ2-opioid receptor antagonist Tyr-D-Pro-Trp-Phe-NH2 (D-Pro2-endomorphin-1) but not the μ1-opioid receptor antagonist Tyr-D-Pro-Phe-Phe-NH2 (D-Pro2-endomorphin-2) on capsaicin- or NMDA-elicited nociceptive behaviors. In contrast, the antinociceptive effect of endomorphin-2 was significantly suppressed by i.t.-co-administration of D-Pro2-endomorphin-2 but not D-Pro2-endomorphin-1 on capsaicin-, NMDA- or substance P-elicited nociceptive behaviors. Interestingly, regarding substance P-elicited nociceptive behaviors, the antinociceptive effect of endomorphin-1 was significantly suppressed by i.t.-co-administration of another μ2-opioid receptor antagonist, Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), but not D-Pro2-endomorphin-1 or D-Pro2-endomorphin-2. The present results suggest that the multiple μ-opioid receptor subtypes are involved in the presynaptic or postsynaptic inhibition of spinal pain transmission. PMID:24512946

  17. Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

    PubMed Central

    2011-01-01

    Background Late cerebral ischemia carries high morbidity and mortality after subarachnoid hemorrhage (SAH) due to reduced cerebral blood flow (CBF) and the subsequent cerebral ischemia which is associated with upregulation of contractile receptors in the vascular smooth muscle cells (SMC) via activation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide a therapeutic window for treatment of cerebral ischemia after SAH. Results Here we demonstrate that SAH increases the phosphorylation level of ERK1/2 in cerebral vessels and reduces the neurology score in rats in additional with the CBF measured by an autoradiographic method. The intracisternal administration of SB-386023-b, a specific inhibitor of raf, given 6 h after SAH, aborts the receptor changes and protects the brain from the development of late cerebral ischemia at 48 h. This is accompanied by reduced phosphorylation of ERK1/2 in cerebrovascular SMC. SAH per se enhances contractile responses to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and angiotensin II (Ang II), upregulates ETB, 5-HT1B and AT1 receptor mRNA and protein levels. Treatment with SB-386023-b given as late as at 6 h but not at 12 h after the SAH significantly decreased the receptor upregulation, the reduction in CBF and the neurology score. Conclusion These results provide evidence for a role of the ERK1/2 pathway in regulation of expression of cerebrovascular SMC receptors. It is suggested that raf inhibition may reduce late cerebral ischemia after SAH and provides a realistic time window for therapy. PMID:22032648

  18. Estrogen receptors in the medial amygdala inhibit the expression of male prosocial behavior

    PubMed Central

    Cushing, Bruce S.; Perry, Adam; Musatov, Sergei; Ogawa, Sonoko; Papademetriou, Eros

    2008-01-01

    Studies using estrogen receptor alpha (ERα) knockout mice indicate that ERα masculinizes male behavior. Recent studies of ERα and male prosocial behavior have shown an inverse relationship between ERα expression in regions of the brain that regulate social behavior, including the medial amygdala (MeA), and the expression of male prosocial behavior. These studies have lead to the hypothesis that low levels of ERα are necessary to “permit” the expression of high levels of male prosocial behavior. To test this, viral vectors were used to enhance ERα in male prairie voles (Microtus ochrogaster), which display high levels of prosocial behavior and low levels of MeA ERα. Adult male prairie voles were transfected with ERα in the MeA or the caudate putamen (ERα control) or luciferase (MeA - site-specific control), and three weeks later tested for spontaneous alloparental behavior and partner preference. Enhancing ERα in the MeA altered/reduced male prosocial behavior. Only a third of ERα-MeA males, compared to all control males, were alloparental. ERα-MeA males also displayed a significant a preference for a novel female. This is a critical finding as the manipulations of neuropeptides, oxytocin and vasopressin, can inhibit the formation of a partner preference, but do not lead to the formation of a preference for a novel female. The results support the hypothesis that low levels of ERα are necessary for high levels of male prosocial behavior, and provide the first direct evidence that site-specific ERα expression plays a critical role in the expression of male prosocial behavior. PMID:18842899

  19. Inhibition of transferrin receptor 1 transcription by a cell density response element

    PubMed Central

    2005-01-01

    TfR1 (transferrin receptor 1) mediates the uptake of transferrin-bound iron and thereby plays a critical role in cellular iron metabolism. Its expression is coupled to cell proliferation/differentiation and controlled in response to iron levels and other signals by transcriptional and post-transcriptional mechanisms. It is well established that TfR1 levels decline when cultured cells reach a high density and in the present study we have investigated the underlying mechanisms. Consistent with previous findings, we demonstrate that TfR1 expression is attenuated in a cell-density-dependent manner in human lung cancer H1299 cells and in murine B6 fibroblasts as the result of a marked decrease in mRNA content. This response is not associated with alterations in the RNA-binding activity of iron regulatory proteins that are indicative of a transcriptional mechanism. Reporter assays reveal that the human TfR1 promoters contains sequences mediating cell-density-dependent transcriptional inhibition. Mapping of the human and mouse TfR1 promoters identified a conserved hexa-nucleotide 5′-GAGGGC-3′ motif with notable sequence similarity to a previously described element within the IGF-2 (insulin-like growth factor-2) promoter. We show that this motif is necessary for the formation of specific complexes with nuclear extracts and for cell-density-dependent regulation in reporter gene assays. Thus the TfR1 promoter contains a functional ‘cell density response element’ (CDRE). PMID:16092918

  20. Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice.

    PubMed

    Lawlor, Michael W; Read, Benjamin P; Edelstein, Rachel; Yang, Nicole; Pierson, Christopher R; Stein, Matthew J; Wermer-Colan, Ariana; Buj-Bello, Anna; Lachey, Jennifer L; Seehra, Jasbir S; Beggs, Alan H

    2011-02-01

    X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency. PMID:21281811

  1. n-3 Polyunsaturated fatty acids inhibit Fc ε receptor I-mediated mast cell activation.

    PubMed

    Wang, Xiaofeng; Ma, David W L; Kang, Jing X; Kulka, Marianna

    2015-12-01

    In vivo models show that n-3 polyunsaturated fatty acids (PUFA) inhibit some of the processes associated with allergic inflammation but the direct effect of n-3 PUFA on mast cells, the major effector cells in allergy, is poorly understood. We sought to determine the effect and mechanism of n-3 PUFA on Fc ε receptor I (FcεRI)-mediated signal transduction and mast cell activation. Bone marrow-derived mast cells (BMMC) were differentiated from bone marrow obtained from C57BL/6 wild-type (WT) and fat-1 transgenic mice. The fat-1 mice express fatty acid n-3 desaturase and produce endogenous n-3 PUFA. For comparison, exogenous n-3 PUFA were supplemented to WT BMMC and human mast cell (LAD2) cultures. Fat-1 BMMC released less β-hexosaminidase (β-hex) and cysteinyl leukotrienes and produced less tumor necrosis factor and chemokine (C-C motif) ligand 2. n-3 PUFA supplementation reduced LAD2 and BMMC degranulation (β-hex release) following FcεRI activation. Fat-1 BMMC expressed less constitutive Lyn and linker of activated T cells (LAT), and FcεRI-mediated phosphorylation of Lyn, spleen tyrosine kinase and LAT were reduced in fat-1 BMMC. Although the expression of surface and whole cell FcεRI was similar in WT and fat-1 BMMC, unstimulated fat-1 BMMC showed reduced FcεRI localization to lipid rafts, and stimulation with antigen resulted in aberrant FcεRI shuttling to the rafts. Our results show that n-3 PUFA suppress FcεRI-mediated activation of mast cells, which results in reduced mediator release. This effect is associated with a decrease in LAT and Lyn expression as well as abnormal shuttling of FcεRI to lipid rafts. PMID:26363927

  2. H4 histamine receptors inhibit steroidogenesis and proliferation in Leydig cells.

    PubMed

    Abiuso, Adriana María Belén; Berensztein, Esperanza; Pagotto, Romina María; Pereyra, Elba Nora; Medina, Vanina; Martinel Lamas, Diego José; Besio Moreno, Marcos; Pignataro, Omar Pedro; Mondillo, Carolina

    2014-12-01

    The histamine H4 receptor (HRH4), discovered only 13 years ago, is considered a promising drug target for allergy, inflammation, autoimmune disorders and cancer, as reflected by a steadily growing number of scientific publications and patent applications. Although the presence of HRH4 has been evidenced in the testis, its specific localization or its role has not been established. Herein, we sought to identify the possible involvement of HRH4 in the regulation of Leydig cell function. We first evaluated its expression in MA-10 Leydig tumor cells and then assessed the effects of two HRH4 agonists on steroidogenesis and proliferation. We found that HRH4 is functionally expressed in MA-10 cells, and that its activation leads to the inhibition of LH/human chorionic gonadotropin-induced cAMP production and StAR protein expression. Furthermore, we observed decreased cell proliferation after a 24-h HRH4 agonist treatment. We then detected for the sites of HRH4 expression in the normal rat testis, and detected HRH4 immunostaining in the Leydig cells of rats aged 7-240 days, while 21-day-old rats also presented HRH4 expression in male gametes. Finally, we evaluated the effect of HRH4 activation on the proliferation of normal progenitor and immature rat Leydig cell culture, and both proved to be susceptible to the anti-proliferative effect of HRH4 agonists. Given the importance of histamine (2-(1H-imidazol-4-yl)ethanamine) in human (patho)physiology, continued efforts are directed at elucidating the emerging properties of HRH4 and its ligands. This study reveals new sites of HRH4 expression, and should be considered in the design of selective HRH4 agonists for therapeutic purposes. PMID:25253872

  3. Covalent Targeting of Fibroblast Growth Factor Receptor Inhibits Metastatic Breast Cancer.

    PubMed

    Brown, Wells S; Tan, Li; Smith, Andrew; Gray, Nathanael S; Wendt, Michael K

    2016-09-01

    Therapeutic targeting of late-stage breast cancer is limited by an inadequate understanding of how tumor cell signaling evolves during metastatic progression and by the currently available small molecule inhibitors capable of targeting these processes. Herein, we demonstrate that both β3 integrin and fibroblast growth factor receptor-1 (FGFR1) are part of an epithelial-mesenchymal transition (EMT) program that is required to facilitate metastatic outgrowth in response to fibroblast growth factor-2 (FGF2). Mechanistically, β3 integrin physically disrupts an interaction between FGFR1 and E-cadherin, leading to a dramatic redistribution of FGFR1 subcellular localization, enhanced FGF2 signaling and increased three-dimensional (3D) outgrowth of metastatic breast cancer cells. This ability of β3 integrin to drive FGFR signaling requires the enzymatic activity of focal adhesion kinase (FAK). Consistent with these mechanistic data, we demonstrate that FGFR, β3 integrin, and FAK constitute a molecular signature capable of predicting decreased survival of patients with the basal-like subtype of breast cancer. Importantly, covalent targeting of a conserved cysteine in the P-loop of FGFR1-4 with our newly developed small molecule, FIIN-4, more effectively blocks 3D metastatic outgrowth as compared with currently available FGFR inhibitors. In vivo application of FIIN-4 potently inhibited the growth of metastatic, patient-derived breast cancer xenografts and murine-derived metastases growing within the pulmonary microenvironment. Overall, the current studies demonstrate that FGFR1 works in concert with other EMT effector molecules to drive aberrant downstream signaling, and that these events can be effectively targeted using our novel therapeutics for the treatment of the most aggressive forms of breast cancer. Mol Cancer Ther; 15(9); 2096-106. ©2016 AACR. PMID:27371729

  4. Pharmacological inhibition of TGFβ receptor improves Nkx2.5 cardiomyoblast-mediated regeneration

    PubMed Central

    Chen, Wen-Pin; Liu, Yuan-Hung; Ho, Yi-Jin; Wu, Sean M.

    2015-01-01

    Aims Our previous study found that A83-01, a small molecule type 1 TGFβ receptor inhibitor, could induce proliferation of postnatal Nkx2.5+ cardiomyoblasts in vitro and enhance their cardiomyogenic differentiation. The present study addresses whether A83-01 treatment in vivo could increase cardiomyogenesis and improve cardiac function after myocardial infarction through an Nkx2.5+ cardiomyoblast-dependent process. Methods and results To determine the effect of A83-01 on the number of Nkx2.5+ cardiomyoblasts in the heart after myocardial injury, we treated transgenic Nkx2.5 enhancer-GFP reporter mice for 7 days with either A83-01 or DMSO and measured the number of GFP+ cardiomyoblasts in the heart at 1 week after injury by flow cytometry. To determine the degree of new cardiomyocyte formation after myocardial injury and the effect of A83-01 in this process, we employed inducible Nkx2.5 enhancer-Cre transgenic mice to lineage label postnatal Nkx2.5+ cardiomyoblasts and their differentiated progenies after myocardial injury. We also examined the cardiac function of each animal by intracardiac haemodynamic measurements. We found that A83-01 treatment significantly increased the number of Nkx2.5+ cardiomyoblasts at baseline and after myocardial injury, resulting in an increase in newly formed cardiomyocytes. Finally, we showed that A83-01 treatment significantly improved ventricular elastance and stroke work, leading to improved contractility after injury. Conclusion Pharmacological inhibition of TGFβ signalling improved cardiac function in injured mice and promoted the expansion and cardiomyogenic differentiation of Nkx2.5+ cardiomyoblasts. Direct modulation of resident cardiomyoblasts in vivo may be a promising strategy to enhance therapeutic cardiac regeneration. PMID:25362681

  5. Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors

    PubMed Central

    Popova, Maya; Perkins, Daya; Trudell, James R.; Alkana, Ronald L.; Davies, Daryl L.

    2010-01-01

    ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) α-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentration-dependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders. PMID:20543096

  6. Inhibition of Transient Receptor Potential Channel 5 Reverses 5-Fluorouracil Resistance in Human Colorectal Cancer Cells*

    PubMed Central

    Wang, Teng; Chen, Zhen; Zhu, Yifei; Pan, Qiongxi; Liu, Yanjun; Qi, Xiaowei; Jin, Linfang; Jin, Jian; Ma, Xin; Hua, Dong

    2015-01-01

    5-Fluorouracil (5-Fu) is commonly used in the chemotherapy of colorectal cancer (CRC), but resistance to 5-Fu occurs in most cases, allowing cancer progression. Suppressing ABCB1 (ATP-binding cassette, subfamily B, member 1), which is a pump overproduced in cancer cells to export cytotoxic drugs, is an attractive strategy to overcome drug resistance. In the present study, transient receptor potential channel TrpC5 was found to be overproduced at the mRNA and protein levels together with ABCB1 in 5-Fu-resistant human CRC HCT-8 (HCT-8/5-Fu) and LoVo (LoVo/5-Fu) cells. More nuclear-stabilized β-catenin accumulation was found in HCT-8/5-Fu and LoVo/5-Fu cells than in HCT-8 and LoVo cells. Suppressing TrpC5 expression with TrpC5-specific siRNA inhibited the canonical Wnt/β-catenin signal pathway, reduced the induction of ABCB1, weakened the ABCB1 efflux pump, and caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells. On the contrary, enforcing TrpC5 expression resulted in an activated Wnt/β-catenin signal pathway and up-regulation of ABCB1. Taken together, we demonstrated an essential role of TrpC5 in ABCB1 induction and drug resistance in human CRC cells via promoting nuclear β-catenin accumulation. PMID:25404731

  7. Enhanced latent inhibition in dopamine receptor-deficient mice is sex-specific for the D1 but not D2 receptor subtype: implications for antipsychotic drug action.

    PubMed

    Bay-Richter, Cecilie; O'Tuathaigh, Colm M P; O'Sullivan, Gerard; Heery, David M; Waddington, John L; Moran, Paula M

    2009-04-01

    Latent inhibition (LI) is reduced learning to a stimulus that has previously been experienced without consequence. It is an important model of abnormal allocation of salience to irrelevant information in patients with schizophrenia. In rodents LI is abolished by psychotomimetic drugs and in experimental conditions where LI is low in controls, its expression is enhanced by antipsychotic drugs with activity at dopamine (DA) receptors. It is however unclear what the independent contributions of DA receptor subtypes are to these effects. This study therefore examined LI in congenic DA D1 and D2 receptor knockout (D1 KO and D2 KO) mice. Conditioned suppression of drinking was used as the measure of learning in the LI procedure. Both male and female DA D2 KO mice showed clear enhancement of LI reproducing antipsychotic drug effects in the model. Unexpectedly, enhancement was also seen in D1 KO female mice but not in D1 KO male mice. This sex-specific pattern was not replicated in locomotor or motor coordination tasks nor in the effect of DA KOs on baseline learning in control groups indicating some specificity of the effect to LI. These data suggest that the dopaminergic mechanism underlying LI potentiation and possibly antipsychotic action may differ between the sexes, being mediated by D2 receptors in males but by both D1 and D2 receptors in females. These data suggest that the DA D1 receptor may prove an important target for understanding sex differences in the mechanisms of action of antipsychotic drugs and in the aetiology of aberrant salience allocation in schizophrenia. PMID:19012810

  8. Receptor-like glycocompounds in human milk that inhibit classical and El Tor Vibrio cholerae cell adherence (hemagglutination).

    PubMed Central

    Holmgren, J; Svennerholm, A M; Lindblad, M

    1983-01-01

    The two biotypes of Vibrio cholerae were found to have cell-associated hemagglutinins which differ with regard to binding to different species of erythrocytes and inhibition by monosaccharides. A total of 12 classical V. cholerae strains (Inaba or Ogawa) strongly agglutinated human erythrocytes in a reaction specifically inhibited by L-fucose, whereas 12 El Tor strains preferably agglutinated chicken erythrocytes, a reaction reversed by D-mannose or by higher concentrations of D-fructose, D-glucose, alpha-methyl-D-mannoside, or sucrose. Milk from Swedish women inhibited both of these adherence reactions, and the predominating inhibitory activity for each reaction resisted boiling, was destroyed by periodate treatment, and bound a concanavalin A-Sepharose column, suggesting a carbohydrate structure. Further characterization indicated that the inhibitory activity for classical V. cholerae hemagglutination was distributed about equally on glycoprotein and free oligosaccharide, but was not present on glycolipid. The El Tor inhibiting activity, on the other hand, was almost exclusively of a high-molecular-weight glycoprotein nature. These results support our previous suggestion (Holmgren et al., Infect. Immun. 33:136-141, 1981) that human milk may contain receptor-like glycocompounds which can prevent bacterial adherence by competition with receptors on target cells. PMID:6295953

  9. Attenuation of cannabinoid-induced inhibition of medullary dorsal horn neurons by a kappa-opioid receptor antagonist

    PubMed Central

    Okada-Ogawa, Akiko; Kurose, Masayuki; Meng, Ian D.

    2010-01-01

    The kappa-opioid receptor (KOR) antagonist norbinaltorphimine (nor-BNI) attenuates behavioral antinociception produced by spinal administration of the cannabinoid receptor agonist delta-9-tetrahydorcannabinol (THC). The present study examined the ability of nor-BNI to prevent cannabinoid-induced inhibition of medullary dorsal horn (MDH) nociceptive neurons and antinociception produced by the cannabinoid agonist WIN 55,212-2 (WIN-2). Extracellular, single unit recordings of lamina I and lamina V MDH neurons was performed in urethane anesthetized rats. Heat-evoked activity was measured before and after local brainstem application of nor-BNI or vehicle followed by WIN-2. In both lamina I and lamina V neurons, prior application of nor-BNI prevented the inhibition of heat-evoked activity by WIN-2. In separate experiments, the contribution of KOR to cannabinoid-induced increases in heat-evoked head withdrawal latencies was assessed in lightly urethane-anesthetized rats. Antinociception produced by intrathecal administration of WIN-2 and THC was attenuated by prior administration of nor-BNI. In contrast, antinociception produced by the cannabinoid CP55940 remained unaffected by prior administration of nor-BNI. These results indicate that cannabinoid inhibition of nociceptive reflexes produced by WIN-2 and THC may result from inhibition of dorsal horn neurons through a KOR-dependent mechanism. PMID:20807519

  10. Calpain Inhibition Attenuates Angiotensin II-induced Abdominal Aortic Aneurysms and Atherosclerosis in LDL Receptor Deficient Mice

    PubMed Central

    Subramanian, Venkateswaran; Uchida, Haruhito Adam; Ijaz, Talha; Moorleghen, Jessica J.; Howatt, Deborah A.; Balakrishnan, Anju

    2011-01-01

    Chronic infusion of angiotensin II (AngII) augments atherosclerosis and abdominal aortic aneurysm (AAAs) formation in hypercholesterolemic mice. AngII-induced AAAs are associated with medial macrophage accumulation and matrix metalloproteinase (MMP) activation. Inhibition of calpain, a calcium-activated neutral cysteine protease, by overexpression of its endogenous inhibitor, calpastatin, attenuates AngII-induced leukocyte infiltration, perivascular inflammation, and MMP activation in mice. The purpose of this study was to define whether pharmacological inhibition of calpain influences AngII-induced AAAs in hypercholesterolemic mice. Male LDL receptor −/− mice were fed a fat-enriched diet and administered with either vehicle or a calpain-specific inhibitor, BDA-410 (30 mg/kg/day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1,000 ng/kg/min) for 4 weeks. AngII-infusion profoundly increased aortic calpain protein and activity. BDA-410 administration had no effect on plasma cholesterol concentrations or AngII-increased systolic blood pressure. Calpain inhibition significantly attenuated AngII-induced AAA formation and atherosclerosis development. BDA-410 administration attenuated activation of MMP12, pro-inflammatory cytokines (IL-6, MCP-1) and macrophage infiltration into the aorta. BDA-410 administration significantly attenuated thioglycollate-elicited macrophage accumulation in the peritoneal cavity. We conclude that calpain inhibition using BDA-410 attenuated AngII-induced AAA formation and atherosclerosis development in LDL receptor −/− mice. PMID:21964156

  11. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

    PubMed

    Brailoiu, G Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2016-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor. PMID:26467159

  12. [Effect of prednisolone on the inhibition of basal gastric secretion in laboratory rats induced by fats or blockade of H2-receptors].

    PubMed

    Trefilov, A B

    2004-02-01

    Experiments on rats with gastric fistulae for the first time have shown that glucocorticosteroid prednisolone causes weakening of the fat and H2-receptor inhibition of basal gastric secretion. PMID:15143511

  13. Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenylyl cyclase.

    PubMed

    Neve, K A; Cox, B A; Henningsen, R A; Spanoyannis, A; Neve, R L

    1991-06-01

    An aspartate residue corresponding to aspartate-80 of dopamine D2 receptors is strictly conserved among receptors that couple to guanine nucleotide-binding proteins. Mutation of this residue alters the function of several classes of neurotransmitter receptors. Dopamine D2 receptors couple to the guanine nucleotide-binding protein Gi to inhibit adenylyl cyclase (ATP-pyrophosphate-lyase, cyclizing; EC 4.6.1.1). Like other Gi-coupled receptors, the binding of agonists and some antagonists to D2 receptors is sensitive to pH and sodium. In the present report, we demonstrate that substitution of an alanine or glutamate residue for aspartate-80 severely impairs inhibition of adenylyl cyclase by D2 receptors and also abolishes or decreases the regulation of the affinity of D2 receptors for agonists and substituted benzamide antagonists by sodium and pH. Our data support the hypothesis that the conformation of D2 receptors is maintained by interactions of monovalent cations with aspartate-80. The regulation of D2 receptors by this interaction has important consequences for the affinity of D2 receptors for ligands and for signal transduction by D2 receptors. PMID:1828858

  14. TGF-β receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation.

    PubMed

    Lee, Yong-Ung; de Dios Ruiz-Rosado, Juan; Mahler, Nathan; Best, Cameron A; Tara, Shuhei; Yi, Tai; Shoji, Toshihiro; Sugiura, Tadahisa; Lee, Avione Y; Robledo-Avila, Frank; Hibino, Narutoshi; Pober, Jordan S; Shinoka, Toshiharu; Partida-Sanchez, Santiago; Breuer, Christopher K

    2016-07-01

    Stenosis is a critical problem in the long-term efficacy of tissue-engineered vascular grafts (TEVGs). We previously showed that host monocyte infiltration and activation within the graft drives stenosis and that TGF-β receptor 1 (TGF-βR1) inhibition can prevent it, but the latter effect was attributed primarily to inhibition of mesenchymal cell expansion. In this study, we assessed the effects of TGF-βR1 inhibition on the host monocytes. Biodegradable TEVGs were implanted as inferior vena cava interposition conduits in 2 groups of C57BL/6 mice (n = 25/group): unseeded grafts and unseeded grafts with TGF-βR1 inhibitor systemic treatment for the first 2 wk. The TGF-βR1 inhibitor treatment effectively improved TEVG patency at 6 mo compared to the untreated control group (91.7 vs. 48%, P < 0.001), which is associated with a reduction in classic activation of mononuclear phagocytes. Consistent with these findings, the addition of rTGF-β to LPS/IFN-γ-stimulated monocytes enhanced secretion of inflammatory cytokines TNF-α, IL-12, and IL-6; this effect was blocked by TGF-βR1 inhibition (P < 0.0001). These findings suggest that the TGF-β signaling pathway contributes to TEVG stenosis by inducing classic activation of host monocytes. Furthermore, blocking monocyte activation by TGF-βR1 inhibition provides a viable strategy for preventing TEVG stenosis while maintaining neotissue formation.-Lee, Y.-U., de Dios Ruiz-Rosado, J., Mahler, N., Best, C. A., Tara, S., Yi, T., Shoji, T., Sugiura, T., Lee, A. Y., Robledo-Avila, F., Hibino, N., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. TGF-β receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation. PMID:27059717

  15. Vitamin D Receptor Activation Protects Against Myocardial Reperfusion Injury Through Inhibition of Apoptosis and Modulation of Autophagy

    PubMed Central

    Yao, Tianbao; Ying, Xiaoying; Zhao, Yichao; Yuan, Ancai; He, Qing; Tong, Huan; Ding, Song; Liu, Junling; Peng, Xu; Gao, Erhe

    2015-01-01

    Abstract Aims: To determine the roles of vitamin D receptor (VDR) in ischemia/reperfusion-induced myocardial injury and to investigate the underlying mechanisms involved. Results: The endogenous VDR expression was detected in the mouse heart, and myocardial ischemia/reperfusion (MI/R) upregulated VDR expression. Activation of VDR by natural and synthetic agonists reduced myocardial infarct size and improved cardiac function. Mechanistically, VDR activation inhibited endoplasmic reticulum (ER) stress (determined by the reduction of CCAAT/enhancer-binding protein homologous protein expression and caspase-12 activation), attenuated mitochondrial impairment (determined by the decrease of mitochondrial cytochrome c release and caspase-9 activation), and reduced cardiomyocyte apoptosis. Furthermore, VDR activation significantly inhibited MI/R-induced autophagy dysfunction (determined by the inhibition of Beclin 1 over-activation, the reduction of autophagosomes, the LC3-II/LC3-I ratio, p62 protein abundance, and the restoration of autophagy flux). Moreover, VDR activation inhibited MI/R-induced oxidative stress through a metallothionein-dependent mechanism. The cardioprotective effects of VDR agonists mentioned earlier were impaired in the setting of cardiac-specific VDR silencing. In contrast, adenovirus-mediated cardiac VDR overexpression decreased myocardial infarct size and improved cardiac function through attenuating oxidative stress, and inhibiting apoptosis and autophagy dysfunction. Innovation and Conclusion: Our data demonstrate that VDR is a novel endogenous self-defensive and cardioprotective receptor against MI/R injury, via mechanisms (at least in part) reducing oxidative stress, and inhibiting apoptosis and autophagy dysfunction-mediated cell death. Antioxid. Redox Signal. 22, 633–650. PMID:25365634

  16. Anti-epidermal growth factor receptor monoclonal antibody cetuximab inhibits EGFR/HER-2 heterodimerization and activation.

    PubMed

    Patel, Dipa; Bassi, Rajiv; Hooper, Andrea; Prewett, Marie; Hicklin, Daniel J; Kang, Xiaoqiang

    2009-01-01

    Human carcinomas frequently express one or more members of the epidermal growth factor receptor family. Two family members, epidermal growth factor receptor (EGFR) and c-erbB2/neu (HER2), homodimerize or heterodimerize upon activation with ligand and trigger potent mechanisms of cellular proliferation, differentiation and migration. In this study, we examined the effect of the anti-EGFR monoclonal antibody Erbitux (cetuximab) on human tumor cells expressing both EGFR and HER2. Investigation of the effect of cetuximab on the activation of EGFR-EGFR, EGFR-HER2 and HER2-HER2 homodimers and heterodimers was conducted using the NCI-N87 human gastric carcinoma cell line. Treatment of NCI-N87 cells with cetuximab completely inhibited formation of EGFR-EGFR homodimers and EGFR-HER2 heterodimers. Activation of HER2-HER2 homodimers was not appreciably stimulated by exogenous ligand and was not inhibited by cetuximab treatment. Furthermore, cetuximab inhibited EGF-induced EGFR and HER2 phosphorylation in CAL27, NCI-H226 and NCI-N87 cells. The activation of downstream signaling molecules such as AKT, MAPK and STAT-3 were also inhibited by cetuximab in these cells. To examine the effect of cetuximab on the growth of tumors in vivo, athymic mice bearing established NCI-N87 or CAL27 xenografts were treated with cetuximab (1 mg, i.p., q3d). The growth of NCI-N87 and CAL27 tumors was significantly inhibited with cetuximab therapy compared to the control groups (p<0.0001 in both cases). In the CAL27 xenograft model, tumor growth inhibition by cetuximab treatment was similar to that by cetuximab and trastuzumab combination treatment. Immunohistological analysis of cetuximab-treated tumors showed a decrease in EGFR-HER2 signaling and reduced tumor cell proliferation. These results suggest that cetuximab may be useful in the treatment of carcinomas co-expressing EGFR and HER2. PMID:19082474

  17. Co-inhibition of epidermal growth factor receptor and insulin-like growth factor receptor 1 enhances radiosensitivity in human breast cancer cells

    PubMed Central

    2013-01-01

    Background Over-expression of epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor (IGF-1R) have been shown to closely correlate with radioresistance of breast cancer cells. This study aimed to investigate the impact of co-inhibition of EGFR and IGF-1R on the radiosensitivity of two breast cancer cells with different profiles of EGFR and IGF-1R expression. Methods The MCF-7 (EGFR +/−, IGF-1R +++) and MDA-MB-468 (EGFR +++, IGF-1R +++) breast cancer cell lines were used. Radiosensitizing effects were determined by colony formation assay. Apoptosis and cell cycle distribution were measured by flow cytometry. Phospho-Akt and phospho-Erk1/2 were quantified by western blot. In vivo studies were conducted using MDA-MB-468 cells xenografted in nu/nu mice. Results In MDA-MB-468 cells, the inhibition of IGF-1R upregulated the p-EGFR expression. Either EGFR (AG1478) or IGF-1R inhibitor (AG1024) radiosensitized MDA-MB-468 cells. In MCF-7 cells, radiosensitivity was enhanced by AG1024, but not by AG1478. Synergistical radiosensitizing effect was observed by co-inhibition of EGFR and IGF-1R only in MDA-MB-468 cells with a DMF10% of 1.90. The co-inhibition plus irradiation significantly induced more apoptosis and arrested the cells at G0/G1 phase in MDA-MB-468 cells. Only co-inhibition of EGFR and IGF-1R synergistically diminished the expression of p-Akt and p-Erk1/2 in MDA-MB-468 cells. In vivo studies further verified the radiosensitizing effects by co-inhibition of both pathways in a MDA-MB-468 xenograft model. Conclusion Our data suggested that co-inhibition of EGFR and IGF-1R synergistically radiosensitized breast cancer cells with both EGFR and IGF-1R high expression. The approach may have an important therapeutic implication in the treatment of breast cancer patients with high expression of EGFR and IGF-1R. PMID:23777562

  18. Neuropeptide Y1 receptor inhibits cell growth through inactivating mitogen-activated protein kinase signal pathway in human hepatocellular carcinoma.

    PubMed

    Lv, Xiufang; Zhao, Fengbo; Huo, Xisong; Tang, Weidong; Hu, Baoying; Gong, Xiu; Yang, Juan; Shen, Qiujin; Qin, Wenxin

    2016-07-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers, and its incidence is increasing worldwide. Neuropeptide Y (NPY) broadly expressed in the central and peripheral nervous system. It participates in multiple physiological and pathological processes through specific receptors. Evidences are accumulating that NPY is involved in development and progression in neuro- or endocrine-related cancers. However, little is known about the potential roles and underlying mechanisms of NPY receptors in HCC. In this study, we analyzed the expression of NPY receptors by real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Correlation between NPY1R levels and clinicopathological characteristics, and survival of HCC patients were explored, respectively. Cell proliferation was researched by CCK-8 in vitro, and tumor growth was studied by nude mice xenografts in vivo. We found that mRNA and protein level of NPY receptor Y1 subtype (NPY1R) significantly decreased in HCC tissues. Low expression of NPY1R closely correlated with poor prognosis in HCC patients. Proliferation of HCC cells was significantly inhibited by recombinant NPY protein in vitro. This inhibitory effect could be blocked by selected NPY1R antagonist BIBP3226. Furthermore, overexpression of NPY1R could significantly inhibit HCC cell proliferation. Knockdown of NPY1R promoted cell multiplication in vitro and increased tumorigenicity and tumor growth in vivo. NPY1R was found to participate in the inhibition of cell proliferation via inactivating mitogen-activated protein kinase signal pathway in HCC cells. Collectively, NPY1R plays an inhibitory role in tumor growth and may be a promising therapeutic target for HCC. PMID:27262566

  19. Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating kappa-opioid and cannabinoid receptors.

    PubMed

    Fichna, J; Schicho, R; Andrews, C N; Bashashati, M; Klompus, M; McKay, D M; Sharkey, K A; Zjawiony, J K; Janecka, A; Storr, M A

    2009-12-01

    The major active ingredient of the plant Salvia divinorum, salvinorin A (SA) has been used to treat gastrointestinal (GI) symptoms. As the action of SA on the regulation of colonic function is unknown, our aim was to examine the effects of SA on mouse colonic motility and secretion in vitro and in vivo. The effects of SA on GI motility were studied using isolated preparations of colon, which were compared with preparations from stomach and ileum. Colonic epithelial ion transport was evaluated using Ussing chambers. Additionally, we studied GI motility in vivo by measuring colonic propulsion, gastric emptying, and upper GI transit. Salvinorin A inhibited contractions of the mouse colon, stomach, and ileum in vitro, prolonged colonic propulsion and slowed upper GI transit in vivo. Salvinorin A had no effect on gastric emptying in vivo. Salvinorin A reduced veratridine-, but not forskolin-induced epithelial ion transport. The effects of SA on colonic motility in vitro were mediated by kappa-opioid receptors (KORs) and cannabinoid (CB) receptors, as they were inhibited by the antagonists nor-binaltorphimine (KOR), AM 251 (CB(1) receptor) and AM 630 (CB(2) receptor). However, in the colon in vivo, the effects were largely mediated by KORs. The effects of SA on veratridine-mediated epithelial ion transport were inhibited by nor-binaltorphimine and AM 630. Salvinorin A slows colonic motility in vitro and in vivo and influences neurogenic ion transport. Due to its specific regional action, SA or its derivatives may be useful drugs in the treatment of lower GI disorders associated with increased GI transit and diarrhoea. PMID:19650775

  20. Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

    PubMed

    Lee, Gregory; Ge, Bixia

    2010-07-01

    As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations. PMID:20182875

  1. Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

    SciTech Connect

    Reif, J.S.; Schwartz, A.L.; Fallon, R.J. )

    1991-02-01

    Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.

  2. Insulin-like growth factor-1 inhibits adult supraoptic neurons via complementary modulation of mechanoreceptors and glycine receptors.

    PubMed

    Ster, Jeanne; Colomer, Claude; Monzo, Cécile; Duvoid-Guillou, Anne; Moos, Françoise; Alonso, Gérard; Hussy, Nicolas

    2005-03-01

    In the CNS, insulin-like growth factor-1 (IGF-1) is mainly known for its trophic effect both during development and in adulthood. Here, we show than in adult rat supraoptic nucleus (SON), IGF-1 receptor immunoreactivity is present in neurons, whereas IGF-1 immunoreactivity is found principally in astrocytes and more moderately in neurons. In vivo application of IGF-1 within the SON acutely inhibits the activity of both vasopressin and oxytocin neurons, the two populations of SON neuroendocrine cells. Recordings of acutely isolated SON neurons showed that this inhibition occurs through two rapid and reversible mechanisms, both involving the neuronal IGF-1 receptor but different intracellular messengers. IGF-1 inhibits Gd3+-sensitive and osmosensitive mechanoreceptor cation current via phosphatidylinositol-3 (PI3) kinase activation. IGF-1 also potentiates taurine-activated glycine receptor (GlyR) Cl- currents by increasing the agonist sensitivity through a extremely rapid (within a second) PI3 kinase-independent mechanism. Both mechanoreceptor channels and GlyR, which form the excitatory and inhibitory components of SON neuron osmosensitivity, are active at rest, and their respective inhibition and potentiation will both be inhibitory, leading to strong decrease in neuronal activity. It will be of interest to determine whether IGF-1 is released by neurons, thus participating in an inhibitory autocontrol, or astrocytes, then joining the growing family of glia-to-neuron transmitters that modulate neuronal and synaptic activity. Through the opposite and complementary acute regulation of mechanoreceptors and GlyR, IGF-1 appears as a new important neuromodulator in the adult CNS, participating in the complex integration of neural messages that regulates the level of neuronal excitability. PMID:15745952

  3. Noncompetitive Inhibition of 5-HT3 Receptors by Citral, Linalool, and Eucalyptol Revealed by Nonlinear Mixed-Effects Modeling.

    PubMed

    Jarvis, Gavin E; Barbosa, Roseli; Thompson, Andrew J

    2016-03-01

    Citral, eucalyptol, and linalool are widely used as flavorings, fragrances, and cosmetics. Here, we examined their effects on electrophysiological and binding properties of human 5-HT3 receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. Data were analyzed using nonlinear mixed-effects modeling to account for random variance in the peak current response between oocytes. The oils caused an insurmountable inhibition of 5-HT-evoked currents (citral IC50 = 120 µM; eucalyptol = 258 µM; linalool = 141 µM) and did not compete with fluorescently labeled granisetron, suggesting a noncompetitive mechanism of action. Inhibition was not use-dependent but required a 30-second preapplication. Compound washout caused a slow (∼180 seconds) but complete recovery. Coapplication of the oils with bilobalide or diltiazem indicated they did not bind at the same locations as these channel blockers. Homology modeling and ligand docking predicted binding to a transmembrane cavity at the interface of adjacent subunits. Liquid chromatography coupled to mass spectrometry showed that an essential oil extracted from Lippia alba contained 75.9% citral. This inhibited expressed 5-HT3 receptors (IC50 = 45 µg ml(-1)) and smooth muscle contractions in rat trachea (IC50 = 200 µg ml(-1)) and guinea pig ileum (IC50 = 20 µg ml(-1)), providing a possible mechanistic explanation for why this oil has been used to treat gastrointestinal and respiratory ailments. These results demonstrate that citral, eucalyptol, and linalool inhibit 5-HT3 receptors, and their binding to a conserved cavity suggests a valuable target for novel allosteric modulators. PMID:26669427

  4. ML-18 is a non-peptide bombesin receptor subtype-3 antagonist which inhibits lung cancer growth.

    PubMed

    Moody, Terry W; Mantey, Samuel A; Moreno, Paola; Nakamura, Taichi; Lacivita, Enza; Leopoldo, Marcello; Jensen, Robert T

    2015-02-01

    Bombesin receptor subtype (BRS)-3 is a G protein coupled receptor (GPCR) for the bombesin (BB)-family of peptides. BRS-3 is an orphan GPCR and little is known of its physiological role due to the lack of specific agonists and antagonists. PD168368 is a nonpeptide antagonist for the neuromedin B (NMB) receptor (R) whereas PD176252 is a nonpeptide antagonist for the gastrin releasing peptide (GRP) R and NMBR but not BRS-3. Here nonpeptide analogs of PD176252 e.g. the S-enantiomer ML-18, and the R-enantiomer, EMY-98, were investigated as BRS-3 antagonists using lung cancer cells. ML-18 and EMY-98 inhibited specific (125)I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB(6-14) binding to NCI-H1299 lung cancer cells stably transfected with BRS-3 with IC50 values of 4.8 and >100μM, respectively. In contrast, ML-18 bound with lower affinity to the GRPR and NMBR with IC50 values of 16 and >100μM, respectively. ML-18 (16μM), but not its enantiomer EMY-98, inhibited the ability of 10nM BA1 to elevate cytosolic Ca(2+) in a reversible manner using lung cancer cells loaded with FURA2-AM. ML-18 (16μM), but not EMY-98, inhibited the ability of 100nM BA1 to cause tyrosine phosphorylation of the EGFR and ERK in lung cancer cells. ML-18 but not EMY-98 inhibited the proliferation of lung cancer cells. The results indicate that ML-18 is a nonpeptide BRS-3 antagonist that should serve as a template to improve potency and selectivity. PMID:25554218

  5. Identification of approved drugs that inhibit the binding of amyloid β oligomers to ephrin type-B receptor 2.

    PubMed

    Suzuki, Koichiro; Aimi, Takahiro; Ishihara, Tomoaki; Mizushima, Tohru

    2016-05-01

    Ephrin type-B receptor 2 (EphB2) is a member of the receptor tyrosine kinase family and plays an important role in learning and memory functions. In patients with Alzheimer's disease (AD) and in mouse models of AD, a reduction in the hippocampal EphB2 level is observed. It was recently reported that normalization of the EphB2 level in t