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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 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

  14. 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

  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 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

  4. 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

  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. 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

  16. 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

  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. 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

  10. 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

  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. 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

  5. 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

  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

    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

  10. 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

  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. 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

  8. 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

  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. 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

  17. 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

  18. 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

  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. 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

  19. 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

  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. 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

  7. 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

  8. 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

  9. 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.

  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. 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

  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. 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.

  3. 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

  4. 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

  5. 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

  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. 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

  9. 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.

  10. 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

  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. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  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. 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

  2. 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.

  3. 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

  4. 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

  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. 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

  8. 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

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  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. 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.

  7. 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

  8. 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

  9. 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

  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. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  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. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  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. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  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. 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

  5. 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

  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. 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.

  12. 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.

  13. 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

  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. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  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