Protection against methamphetamine-induced neurotoxicity to neostriatal dopaminergic neurons by adenosine receptor activation.

Science.gov (United States)

Delle Donne, K T; Sonsalla, P K

1994-12-01

Methamphetamine (METH)-induced neurotoxicity to nigrostriatal dopaminergic neurons in experimental animals appears to have a glutamatergic component because blockade of N-methyl-D-aspartate receptors prevents the neuropathologic consequences. Because adenosine affords neuroprotection against various forms of glutamate-mediated neuronal damage, the present studies were performed to investigate whether adenosine plays a protective role in METH-induced toxicity. METH-induced decrements in neostriatal dopamine content and tyrosine hydroxylase activity in mice were potentiated by concurrent treatment with caffeine, a nonselective adenosine antagonist that blocks both A1 and A2 adenosine receptors. In contrast, chronic treatment of mice with caffeine through their drinking water for 4 weeks, which increased the number of adenosine A1 receptors in the neostriatum and frontal cortex, followed by drug washout, prevented the neurochemical changes produced by the treatment of mice with METH treatment. In contrast, this treatment did not prevent 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine-induced dopaminergic neurotoxicity. Furthermore, concurrent administration of cyclopentyladenosine, an adenosine A1 receptor agonist, attenuated the METH-induced neurochemical changes. This protection by cyclopentyladenosine was blocked by cyclopentyltheophylline, an A1 receptor antagonist. These results indicate that activation of A1 receptors can protect against METH-induced neurotoxicity in mice.

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

DEFF Research Database (Denmark)

Barnathan, E S; Kuo, A; Karikó, K

1990-01-01

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence...

Modulation of β-catenin signaling by glucagon receptor activation.

Directory of Open Access Journals (Sweden)

Jiyuan Ke

Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

Energy Technology Data Exchange (ETDEWEB)

Silva, Jose Alberto Alves da

2009-07-01

Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

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

Science.gov (United States)

Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul; Melikyan, Gregory B

2015-09-01

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

Blocking beta 2-adrenergic receptor inhibits dendrite ramification in a mouse model of Alzheimer's disease.

Science.gov (United States)

Wu, Qin; Sun, Jin-Xia; Song, Xiang-He; Wang, Jing; Xiong, Cun-Quan; Teng, Fei-Xiang; Gao, Cui-Xiang

2017-09-01

Dendrite ramification affects synaptic strength and plays a crucial role in memory. Previous studies revealed a correlation between beta 2-adrenergic receptor dysfunction and Alzheimer's disease (AD), although the mechanism involved is still poorly understood. The current study investigated the potential effect of the selective β 2 -adrenergic receptor antagonist, ICI 118551 (ICI), on Aβ deposits and AD-related cognitive impairment. Morris water maze test results demonstrated that the performance of AD-transgenic (TG) mice treated with ICI (AD-TG/ICI) was significantly poorer compared with NaCl-treated AD-TG mice (AD-TG/NaCl), suggesting that β 2 -adrenergic receptor blockage by ICI might reduce the learning and memory abilities of mice. Golgi staining and immunohistochemical staining revealed that blockage of the β 2 -adrenergic receptor by ICI treatment decreased the number of dendritic branches, and ICI treatment in AD-TG mice decreased the expression of hippocampal synaptophysin and synapsin 1. Western blot assay results showed that the blockage of β 2 -adrenergic receptor increased amyloid-β accumulation by downregulating hippocampal α-secretase activity and increasing the phosphorylation of amyloid precursor protein. These findings suggest that blocking the β 2 -adrenergic receptor inhibits dendrite ramification of hippocampal neurons in a mouse model of AD.

In vitro H1-receptor antagonist activity of methanolic extract of tuber of Stephania glabra

Directory of Open Access Journals (Sweden)

Nisar Ahmad Khan

2010-06-01

Full Text Available In the present study, methanolic extract of tuber of Stephania glabra was evaluated for H1-bloker activity by employing in vitro screening models of guinea pig ileum and goat tracheal chain preparation. Goat isolated trachea and guinea pig ileum contracted to histamine in a dose-dependent manner while chlorpheniramine blocked this effect. The methanolic extract produced significant dose-dependent H1-receptor antagonist activity by blocking histamine-induced contraction.

Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

Science.gov (United States)

Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

2015-10-01

Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

Naltrexone pretreatment blocks microwave-induced changes in central cholinergic receptors

Energy Technology Data Exchange (ETDEWEB)

Lai, H.; Carino, M.A.; Wen, Y.F.; Horita, A.; Guy, A.W. (Univ. of Washington School of Medicine, Seattle (USA))

1991-01-01

Repeated exposure of rats to pulsed, circularly polarized microwaves (2,450-MHz, 2-microseconds pulses at 500 pps, power density 1 mW/cm2, at an averaged, whole-body SAR of 0.6 W/kg) induced biphasic changes in the concentration of muscarinic cholinergic receptors in the central nervous system. An increase in receptor concentration occurred in the hippocampus of rats subjected to ten 45-min sessions of microwave exposure, whereas a decrease in concentration was observed in the frontal cortex and hippocampus of rats exposed to ten 20-min sessions. These findings, which confirm earlier work in the authors' laboratory, were extended to include pretreatment of rats with the narcotic antagonist naltrexone (1 mg/kg, IP) before each session of exposure. The drug treatment blocked the microwave-induced changes in cholinergic receptors in the brain. These data further support the authors' hypothesis that endogenous opioids play a role in the effects of microwaves on central cholinergic systems.

Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

Energy Technology Data Exchange (ETDEWEB)

Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

1987-08-01

The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

Reactivation of desensitized formyl peptide receptors by platelet activating factor: a novel receptor cross talk mechanism regulating neutrophil superoxide anion production.

Directory of Open Access Journals (Sweden)

Huamei Forsman

Full Text Available Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPRdes can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPRdes back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPRdes as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPRdes also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPRdes initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.

Blocking mineralocorticoid receptors impairs, blocking glucocorticoid receptors enhances memory retrieval in humans.

Science.gov (United States)

Rimmele, Ulrike; Besedovsky, Luciana; Lange, Tanja; Born, Jan

2013-04-01

Memory retrieval is impaired at very low as well as very high cortisol levels, but not at intermediate levels. This inverted-U-shaped relationship between cortisol levels and memory retrieval may originate from different roles of the mineralocorticoid (MR) and glucocorticoid receptor (GR) that bind cortisol with distinctly different affinity. Here, we examined the role of MRs and GRs in human memory retrieval using specific receptor antagonists. In two double-blind within-subject, cross-over designed studies, young healthy men were asked to retrieve emotional and neutral texts and pictures (learnt 3 days earlier) between 0745 and 0915 hours in the morning, either after administration of 400 mg of the MR blocker spironolactone vs placebo (200 mg at 2300 hours and 200 mg at 0400 hours, Study I) or after administration of the GR blocker mifepristone vs placebo (200 mg at 2300 hours, Study II). Blockade of MRs impaired free recall of both texts and pictures particularly for emotional material. In contrast, blockade of GRs resulted in better memory retrieval for pictures, with the effect being more pronounced for neutral than emotional materials. These findings indicate indeed opposing roles of MRs and GRs in memory retrieval, with optimal retrieval at intermediate cortisol levels likely mediated by high MR but concurrently low GR activation.

Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

DEFF Research Database (Denmark)

Xiang, B; Yu, G H; Guo, J

2001-01-01

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

Galangin suppresses HepG2 cell proliferation by activating the TGF-β receptor/Smad pathway

International Nuclear Information System (INIS)

Wang, Yajun; Wu, Jun; Lin, Biyun; Li, Xv; Zhang, Haitao; Ding, Hang; Chen, Xiaoyi; Lan, Liubo; Luo, Hui

2014-01-01

Galangin can suppress hepatocellular carcinoma (HCC) cell proliferation. In this study, we demonstrated that galangin induced autophagy by activating the transforming growth factor (TGF)-β receptor/Smad pathway and increased TGF-β receptor I (RI), TGF-βRII, Smad1, Smad2, Smad3 and Smad4 levels but decreased Smad6 and Smad7 levels. Autophagy induced by galangin appears to depend on the TGF-β receptor/Smad signalling pathway because the down-regulation of Smad4 by siRNA or inhibition of TGF-β receptor activation by LY2109761 blocked galangin-induced autophagy. The down-regulation of Beclin1, autophagy-related gene (ATG) 16L, ATG12 and ATG3 restored HepG2 cell proliferation and prevented galangin-induced apoptosis. Our findings indicate a novel mechanism for galangin-induced autophagy via activation of the TGF-β receptor/Smad pathway. The induction of autophagy thus reflects the anti-proliferation effect of galangin on HCC cells

AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity.

Science.gov (United States)

Reim, Tina; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang; Thamm, Markus; Scheiner, Ricarda

2017-01-01

The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP] i ). Type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP] i . The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient. The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

International Nuclear Information System (INIS)

Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

2009-01-01

The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPAR-γ). Although nifedipine did not affect expression levels of PPAR-γ, it increased the PPAR-γ transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-γ activation.

Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression

International Nuclear Information System (INIS)

Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X.; Walterscheid, Jeffrey P.; Ullrich, Stephen E.

2004-01-01

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependant manner. The release of biological response modifiers, particularly prostaglandin E 2 (PGE 2 ), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE 2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE 2 secretion. Jet fuel-induced PGE 2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin

Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

Science.gov (United States)

Battistello, Elena; Katanayeva, Natalya; Dheilly, Elie; Tavernari, Daniele; Donaldson, Maria C; Bonsignore, Luca; Thome, Margot; Christie, Amanda L; Murakami, Mark A; Michielin, Olivier; Ciriello, Giovanni; Zoete, Vincent; Oricchio, Elisa

2018-05-24

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients. © 2018 by The American Society of Hematology.

The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src.

Directory of Open Access Journals (Sweden)

Shiva Akbarzadeh

2008-03-01

Full Text Available The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS and dominant acting Brachydactyly type B (BDB. Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells.

Science.gov (United States)

Karasawa, Jun-ichi; Yamamoto, Hideko; Yamamoto, Toshifumi; Sagi, Naoki; Horikomi, Kazutoshi; Sora, Ichiro

2002-02-22

MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca2+ influx with fura-2/ AM loaded cells. MS-377 showed no effects on the basal Ca2+ concentration and NMDA-induced Ca2+ influx by itself PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca2+ concentration. Pre-incubation of 1 microM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca2+ influx. Second, the effect of MS-377 on [3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [3H]MK-801 binding, although MS-377 showed no effect by itself Pre-treatment of MS-377 markedly reversed the inhibition of [3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.

Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

Science.gov (United States)

Bhattarai, Janardhan Prasad; Park, Soo Joung; Han, Seong Kyu

2013-01-01

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM) and almost completely blocked by strychnine (2 μM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABAA receptor (GABAAR)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons. PMID:24379976

Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

Directory of Open Access Journals (Sweden)

Thi Thanh Hoang Nguyen

2013-01-01

Full Text Available The substantia gelatinosa (SG of the trigeminal subnucleus caudalis (Vc has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM and almost completely blocked by strychnine (2 μM, suggesting that taurine-mediated responses are via glycine receptor (GlyR activation. In addition, taurine (1 mM activated extrasynaptic GABAA receptor (GABAAR-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons.

Activation of the ζ receptor 1 suppresses NMDA responses in rat retinal ganglion cells.

Science.gov (United States)

Zhang, X-J; Liu, L-L; Jiang, S-X; Zhong, Y-M; Yang, X-L

2011-03-17

The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights

The changes in drug binding activity of GABA receptor and animal neural-behavior after gamma irradiation

International Nuclear Information System (INIS)

Zheng Hui; Zhen Rong; Zhao Naikun; Xue Hong; Wang Zihui

2004-01-01

Objective: The purpose of this study was to investigate the effect of irradiation on gamma-aminobutyric-acid receptor (GABA-R) as well as behavioral changes after brain 60 Co γ-irradiation. Methods: The mice were irradiated with gamma rays (20 Gy; 10 Gy and 5 Gy) . The drug binding activity of GABA receptor in brain receptor was measured by fluorescence anisotropy (FA) and equilibrium dissociation constants. The behavioral changes were observed by the locomotor activity test, elevated plus-maze test and hole-board test at 1, 10, 24 and 48 hr after irradiation. Results: 1. The drug binding activity of the GABA receptor was decreased and the equilibrium dissociation constant (K d ) was significantly increased compared with the negative control group 2 hr after irradiation, and a spike value appeared at 24 hr. It showed that the irradiation might damage or decrease the binding activity and the bio-activity of GABA receptor. 2. The animal experiment confirmed that the irradiated animal model showed neural-behavioral changes of anxiety or depression. 3. The decreased binding activity of GABA receptor and changes in behavior of irradiated animal were dependent on radiation intensity. 4. The changes of behavior was similar to the blocked GABA receptor group. It suggests the relationship of radiation and GABA receptor. Conclusion: These results suggest that GABA receptor may be involved in radiation injury. The functional changes of GABA receptor may be an induction factor of behavioral disorder. The article also discussed the effect of anxiety and results obtained from the point of view of GABA receptor system involvement in the changes observed after irradiation. (authors)

The undesirable effects of neuromuscular blocking drugs

DEFF Research Database (Denmark)

Claudius, C; Garvey, L H; Viby-Mogensen, J

2009-01-01

Neuromuscular blocking drugs are designed to bind to the nicotinic receptor at the neuromuscular junction. However, they also interact with other acetylcholine receptors in the body. Binding to these receptors causes adverse effects that vary with the specificity for the cholinergic receptor...... in question. Moreover, all neuromuscular blocking drugs may cause hypersensitivity reactions. Often the symptoms are mild and self-limiting but massive histamine release can cause systematic reactions with circulatory and respiratory symptoms and signs. At the end of anaesthesia, no residual effect...... of a neuromuscular blocking drug should be present. However, the huge variability in response to neuromuscular blocking drugs makes it impossible to predict which patient will suffer postoperative residual curarization. This article discusses the undesirable effects of the currently available neuromuscular blocking...

Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

Directory of Open Access Journals (Sweden)

Maraziotis Theodore

2007-10-01

Full Text Available Abstract Background Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Methods Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK and c-Jun (pc-Jun were used. The labeling index (LI, defined as the percentage of positive (labeled cells out of the total number of tumor cells counted, was determined. Results Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1% in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK and c-Jun (pc-Jun were

Estradiol-induced increase in the magnitude of long-term potentiation is prevented by blocking NR2B-containing receptors.

Science.gov (United States)

Smith, Caroline C; McMahon, Lori L

2006-08-16

Estradiol, through activation of genomic estrogen receptors, induces changes in synaptic morphology and function in hippocampus, a brain region important for memory acquisition. Specifically, this hormone increases CA1 pyramidal cell dendritic spine density, NMDA receptor (NMDAR)-mediated transmission, and the magnitude of long-term potentiation (LTP) at CA3-CA1 synapses. We recently reported that the estradiol-induced increase in LTP magnitude occurs only when there is a simultaneous increase in the fractional contribution of NMDAR-mediated transmission relative to AMPA receptor transmission, suggesting a direct role for the increase in NMDAR transmission to the heightened LTP magnitude. Estradiol has been shown to increase expression of the NMDAR subunit NR2B, but whether this translates into an increase in function of NR2B-containing receptors remains to be determined. Here we show that not only is the estradiol-induced increase in NMDAR transmission mediated by NR2B-containing receptors, but blocking these receptors using RO25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol] (0.5 microM), an NR2B selective antagonist, prevents the estradiol-induced increase in LTP magnitude. Thus, our data show a causal link between the estradiol-induced increase in transmission mediated by NR2B-containing NMDARs and the increase in LTP magnitude.

Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake.

Directory of Open Access Journals (Sweden)

Jae Hoon Jeong

2018-04-01

Full Text Available Proopiomelanocortin (POMC neurons in the arcuate nucleus of the hypothalamus (ARC respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR, immunohistochemistry, electrophysiology, TRPV1 knock-out (KO, and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5 carrying a Cre-dependent channelrhodopsin-2 (ChR2-enhanced yellow fluorescent protein (eYFP expression cassette under the control of the two neuronal POMC enhancers (nPEs. Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.

Acute serotonin 2A receptor blocking alters the processing of fearful faces in the orbitofrontal cortex and amygdala

DEFF Research Database (Denmark)

Hornboll, Bettina; Macoveanu, Julian; Rowe, James

2013-01-01

judging the gender of neutral, fearful and angry faces. Methods: 5-HT2A receptors were blocked with ketanserin to a variable degree across subjects by adjusting the time between ketanserin-infusion and onset of the fMRI protocol. Neocortical 5-HT2A receptor binding in terms of the binding potential (BPp...... blockade reduced the neural response to fearful faces in the medial orbitofrontal cortex (OFC), independently of 5-HT2A receptor occupancy or neocortical 5-HT2A receptor BPp . The medial OFC also showed increased functional coupling with the left amygdala during processing of fearful faces depending...

Activation of the GABAB receptor prevents nicotine-induced locomotor stimulation in mice

Directory of Open Access Journals (Sweden)

Carla eLobina

2011-12-01

Full Text Available Recent studies demonstrated that activation of the GABAB receptor, either by means of orthosteric agonists or positive allosteric modulators (PAMs, inhibited different nicotine-related behaviors, including intravenous self-administration and conditioned place preference, in rodents. The present study investigated whether the anti-nicotine effects of the GABAB receptor agonist, baclofen, and GABAB PAMs, CGP7930 and GS39783, extend to nicotine stimulant effects. To this end, CD1 mice were initially treated with baclofen (0, 1.25, and 2.5 mg/kg, i.p., CGP7930 (0, 25, and 50 mg/kg, i.g., or GS39783 (0, 25, and 50 mg/kg, i.g., then treated with nicotine (0 and 0.05 mg/kg, s.c., and finally exposed to an automated apparatus for recording of locomotor activity. Pretreatment with doses of baclofen, CGP7930, or GS39783 that did not alter locomotor activity when given with nicotine vehicle fully prevented hyperlocomotion induced by 0.05 mg/kg nicotine. These data extend to nicotine stimulant effects the capacity of baclofen and GABAB PAMs to block the reinforcing, motivational, and rewarding properties of nicotine. These data strengthen the hypothesis that activation of the GABAB receptor may represent a potentially useful, anti-smoking therapeutic strategy.

Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

DEFF Research Database (Denmark)

Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe

2013-01-01

after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor

Science.gov (United States)

1990-01-01

Considerable evidence implicates gp90MEL as a lymphocyte homing receptor mediating lymphocyte attachment to high endothelial venules of lymph nodes in mouse. The protein appears to function as a calcium- dependent, lectin-like receptor as inferred primarily by the ability of specific carbohydrates to block its function and by the presence of a calcium-type lectin domain in its primary sequence. An ELISA assay is described which provides the first demonstration that the isolated protein has lectin activity and allows a further definition of its carbohydrate specificity. In addition to the monosaccharides mannose-6- phosphate and fructose-1-phosphate, ligand activity is shown for the sulfated glycolipid, sulfatide, and for two sulfated fucose-containing polysaccharides (fucoidin and egg jelly coat) from nonmammalian sources. PMID:2202735

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

Science.gov (United States)

González, Alfredo; Crittenden, Elizabeth L; García, Dana M

2004-07-13

In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

Directory of Open Access Journals (Sweden)

Crittenden Elizabeth L

2004-07-01

Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Insulin/insulin like growth factors in cancer: new roles for the aryl hydrocarbon receptor, tumor resistance mechanisms and new blocking strategies

Directory of Open Access Journals (Sweden)

Travis B Salisbury

2015-02-01

Full Text Available The insulin-like growth factor 1 receptor (IGF1R and the insulin receptor (IR are receptor tyrosine kinases (RTKs that are expressed in cancer cells. The results of different studies indicate that tumor proliferation and survival is dependent on the IGF1R and IR, and that their inhibition leads to reductions in proliferation and increases in cell death. Molecular targeting therapies that have been used in solid tumors include: anti-IGF1R antibodies, anti-IGF1/IGF2 antibodies and small molecule inhibitors that suppress IGF1R and IR kinase activity. New advances in the molecular basis of anti-IGF1R blocking antibodies reveal they are biased agonists and promote the binding of IGF1 to integrin β3 receptors in some cancer cells. Our recent reports indicate that pharmacological aryl hydrocarbon receptor (AHR ligands inhibit breast cancer cell responses to IGFs, suggesting that targeting AHR may have benefit in cancers whose proliferation and survival are dependent on insulin/IGF signaling. Novel aspects of IGF1R/IR in cancer, such as biased agonism, integrin β3 signaling, AHR and new therapeutic targeting strategies will be discussed.

Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

International Nuclear Information System (INIS)

Assimakopoulou, Martha; Kondyli, Maria; Gatzounis, George; Maraziotis, Theodore; Varakis, John

2007-01-01

Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75 NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75 NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75 NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75 NTR , and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75 NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75 NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor

The Glycolytic Metabolite, Fructose-1,6-bisphosphate, Blocks Epileptiform Bursts by Attenuating Voltage-Activated Calcium Currents in Hippocampal Slices

Directory of Open Access Journals (Sweden)

Li-Rong Shao

2018-06-01

Full Text Available Manipulation of metabolic pathways (e.g., ketogenic diet (KD, glycolytic inhibition alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a “branching” metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP. Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5–5 mM blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min, suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531, nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA. In contrast, F1,6BP caused moderate reduction (~50% in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 μM, suggesting that these bursts are calcium dependent. Altogether, these data

Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

Directory of Open Access Journals (Sweden)

Paul David Whissell

2013-09-01

Full Text Available Extrasynaptic γ-aminobutyric acid type A (GABAA receptors that contain the δ subunit (δGABAA receptors are expressed in several brain regions including the dentate gyrus (DG and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p. on memory performance in wild-type (WT and δGABAA receptor null mutant (Gabrd–/– mice. Additionally, the effects of THIP on long-term potentiation (LTP, a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd–/– mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd–/– mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity.

Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

Science.gov (United States)

Whissell, Paul D.; Eng, Dave; Lecker, Irene; Martin, Loren J.; Wang, Dian-Shi; Orser, Beverley A.

2013-01-01

Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABAA receptor null mutant (Gabrd−/−) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd−/− mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd−/− mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity. PMID:24062648

Synaptic activity regulates AMPA receptor trafficking through different recycling pathways

Science.gov (United States)

Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

2015-01-01

Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. DOI: http://dx.doi.org/10.7554/eLife.06878.001 PMID:25970033

Group III mGlu receptor agonists potentiate the anticonvulsant effect of AMPA and NMDA receptor block.

Science.gov (United States)

De Sarro, Giovambattista; Chimirri, Alba; Meldrum, Brian S

2002-09-06

We report the anticonvulsant action in DBA/2 mice of two mGlu Group III receptor agonists: (R,S)-4-phosphonophenylglycine, (R,S)-PPG, a compound with moderate mGlu8 selectivity, and of (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid, ACPT-1, a selective agonist for mGlu4alpha receptors. Both compounds, given intracerebroventricularly at doses which did not show marked anticonvulsant activity, produced a consistent shift to the left of the dose-response curves (i.e. enhanced the anticonvulsant properties) of 1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one hydrochloride, CFM-2, a noncompetitive AMPA receptor antagonist, and 3-((+/-)-2-carboxypiperazin-4-yl)-1-phosphonic acid, CPPene, a competitive NMDA receptor antagonist, in DBA/2 mice. In addition, (R,S)-PPG and ACPT-1 administered intracerebroventricularly prolonged the time course of the anticonvulsant properties of CFM-2 (33 micromol/kg, i.p.) and CPPene (3.3 micromol/kg, i.p.) administered intraperitoneally. We conclude that modest reduction of synaptic glutamate release by activation of Group III metabotropic receptors potentiates the anticonvulsant effect of AMPA and NMDA receptor blockade. Copyright 2002 Elsevier Science B.V.

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods

DEFF Research Database (Denmark)

Collin, Caitlin Alexis; Hauser, Frank; Gonzalez de Valdivia, Ernesto I

2013-01-01

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5......). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M......) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked...

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1 by Diacylglycerol (DAG

Directory of Open Access Journals (Sweden)

Oh Seog

2008-10-01

Full Text Available Abstract The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1, is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC. However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG, a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C.

Blocking of beta-2 adrenergic receptors hastens recovery from hypoglycemia-associated social withdrawal.

Science.gov (United States)

Park, Min Jung; Guest, Christopher B; Barnes, Meredith B; Martin, Jonathan; Ahmad, Uzma; York, Jason M; Freund, Gregory G

2008-11-01

Hypoglycemia is associated with a variety of adverse behaviors including fatigue, confusion and social withdrawal. While these clinical symptoms are well characterized, the mechanism of their cause is not understood. Here we investigated how insulin-induced hypoglycemia causes social withdrawal. Male 8-12-week-old C57BL/6J mice were injected intraperitoneally (IP) with or without and/or insulin, norepinephrine (NE) and epinephrine (Epi), terbutaline and butoxamine with subsequent measurement of blood glucose, social withdrawal and plasma catecholamines. Insulin generated (0.75h post-injection) significant hypoglycemia with blood glucose nadirs of 64+/-4 and 48+/-5mg/dl for 0.8 and 1.2units/kg of insulin, respectively. Insulin (0.8 or 1.2units/kg) caused near total social withdrawal at 0.75h with full recovery not occurring until 4h (0.8units/kg) or 8h (1.2units/kg) post-insulin injection. Insulin also caused a marked elevation in plasma catecholamines. Basal 12h fasting NE and Epi were 287+/-38 and 350+/-47pg/ml, respectively. Insulin at 0.8units/kg increased plasma NE and Epi to 994+/-73 and 1842+/-473pg/ml, respectively. Administration of exogenous NE or Epi caused social withdrawal similar in magnitude to insulin. Importantly, administration of the beta-2 adrenergic receptor agonist terbutaline also caused social withdrawal while administration of the beta-2 adrenergic receptor antagonist butoxamine blocked NE-induced social withdrawal. Finally, butoxamine blocked insulin-induced social withdrawal. These data demonstrate that hypoglycemia-associated social withdrawal is dependent on catecholamines via a beta-2 receptor-mediated pathway.

Analysis of Onset Mechanisms of a Sphingosine 1-Phosphate Receptor Modulator Fingolimod-Induced Atrioventricular Conduction Block and QT-Interval Prolongation

Energy Technology Data Exchange (ETDEWEB)

Yagi, Yukihiro [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama, Kanagawa 222–8567 (Japan); Nakamura, Yuji [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Kitahara, Ken [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Division of Cardiovascular Medicine, Department of Internal Medicine, Faculty of Medicine, Toho University, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143–8541 (Japan); Harada, Takuma [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Kato, Kazuhiko; Ninomiya, Tomohisa [Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama, Kanagawa 222–8567 (Japan); Cao, Xin [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Ohara, Hiroshi [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Division of Cardiovascular Medicine, Department of Internal Medicine, Faculty of Medicine, Toho University, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143–8541 (Japan); Izumi-Nakaseko, Hiroko [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); Suzuki, Kokichi [Pharmaceutical Research Center, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama, Kanagawa 222–8567 (Japan); Ando, Kentaro [Department of Pharmacology, Faculty of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143–8540 (Japan); and others

2014-11-15

Fingolimod, a sphingosine 1-phosphate (S1P) receptor subtype 1, 3, 4 and 5 modulator, has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular conduction block and/or QT-interval prolongation have been reported in some patients after the first dose. In this study, we directly compared the electropharmacological profiles of fingolimod with those of siponimod, a modulator of sphingosine 1-phosphate receptor subtype 1 and 5, using in vivo guinea-pig model and in vitro human ether-a-go-go-related gene (hERG) assay to better understand the onset mechanisms of the clinically observed adverse events. Fingolimod (0.01 and 0.1 mg/kg) or siponimod (0.001 and 0.01 mg/kg) was intravenously infused over 10 min to the halothane-anaesthetized guinea pigs (n = 4), whereas the effects of fingolimod (1 μmol/L) and siponimod (1 μmol/L) on hERG current were examined (n = 3). The high doses of fingolimod and siponimod induced atrioventricular conduction block, whereas the low dose of siponimod prolonged PR interval, which was not observed by that of fingolimod. The high dose of fingolimod prolonged QT interval, which was not observed by either dose of siponimod. Meanwhile, fingolimod significantly inhibited hERG current, which was not observed by siponimod. These results suggest that S1P receptor subtype 1 in the heart could be one of the candidates for fingolimod- and siponimod-induced atrioventricular conduction block since S1P receptor subtype 5 is localized at the brain, and that direct I{sub Kr} inhibition may play a key role in fingolimod-induced QT-interval prolongation. - Highlights: • Fingolimod and siponimod are S1P{sub 1,3,4,5} and S1P{sub 1,5} receptor modulators, respectively. • Fingolimod and siponimod induced AV block in the halothane-anesthetized guinea pigs. • S1P{sub 1} in the hearts may be the target of fingolimod- and siponimod-induced AV block. • Fingolimod directly inhibited hERG current, which was not

Analysis of Onset Mechanisms of a Sphingosine 1-Phosphate Receptor Modulator Fingolimod-Induced Atrioventricular Conduction Block and QT-Interval Prolongation

International Nuclear Information System (INIS)

Yagi, Yukihiro; Nakamura, Yuji; Kitahara, Ken; Harada, Takuma; Kato, Kazuhiko; Ninomiya, Tomohisa; Cao, Xin; Ohara, Hiroshi; Izumi-Nakaseko, Hiroko; Suzuki, Kokichi; Ando, Kentaro

2014-01-01

Fingolimod, a sphingosine 1-phosphate (S1P) receptor subtype 1, 3, 4 and 5 modulator, has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular conduction block and/or QT-interval prolongation have been reported in some patients after the first dose. In this study, we directly compared the electropharmacological profiles of fingolimod with those of siponimod, a modulator of sphingosine 1-phosphate receptor subtype 1 and 5, using in vivo guinea-pig model and in vitro human ether-a-go-go-related gene (hERG) assay to better understand the onset mechanisms of the clinically observed adverse events. Fingolimod (0.01 and 0.1 mg/kg) or siponimod (0.001 and 0.01 mg/kg) was intravenously infused over 10 min to the halothane-anaesthetized guinea pigs (n = 4), whereas the effects of fingolimod (1 μmol/L) and siponimod (1 μmol/L) on hERG current were examined (n = 3). The high doses of fingolimod and siponimod induced atrioventricular conduction block, whereas the low dose of siponimod prolonged PR interval, which was not observed by that of fingolimod. The high dose of fingolimod prolonged QT interval, which was not observed by either dose of siponimod. Meanwhile, fingolimod significantly inhibited hERG current, which was not observed by siponimod. These results suggest that S1P receptor subtype 1 in the heart could be one of the candidates for fingolimod- and siponimod-induced atrioventricular conduction block since S1P receptor subtype 5 is localized at the brain, and that direct I Kr inhibition may play a key role in fingolimod-induced QT-interval prolongation. - Highlights: • Fingolimod and siponimod are S1P 1,3,4,5 and S1P 1,5 receptor modulators, respectively. • Fingolimod and siponimod induced AV block in the halothane-anesthetized guinea pigs. • S1P 1 in the hearts may be the target of fingolimod- and siponimod-induced AV block. • Fingolimod directly inhibited hERG current, which was not observed by

Acutely increasing δGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus.

Science.gov (United States)

Whissell, Paul D; Eng, Dave; Lecker, Irene; Martin, Loren J; Wang, Dian-Shi; Orser, Beverley A

2013-01-01

Extrasynaptic γ-aminobutyric acid type A (GABA(A)) receptors that contain the δ subunit (δGABA(A) receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABA(A) receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABA(A) receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABA(A) receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABA(A) receptor null mutant (Gabrd(-/-)) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd(-/-) mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd(-/-) mice, an effect that was blocked by GABA(A) receptor antagonist bicuculline. Thus, acutely increasing δGABA(A) receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABA(A) receptor activity.

Platelet-activating factor and group I metabotropic glutamate receptors interact for full development and maintenance of long-term potentiation in the rat medial vestibular nuclei.

Science.gov (United States)

Grassi, S; Francescangeli, E; Goracci, G; Pettorossi, V E

1999-01-01

In rat brainstem slices, we investigated the interaction between platelet-activating factor and group I metabotropic glutamate receptors in mediating long-term potentiation within the medial vestibular nuclei. We analysed the N1 field potential wave evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation. The group I metabotropic glutamate receptor antagonist, (R,S)-1-aminoindan-1,5-dicarboxylic acid, prevented long-term potentiation induced by a platelet-activating factor analogue [1-O-hexadecyl-2-O-(methylcarbamyl)-sn-glycero-3-phosphocholine], as well as the full development of potentiation, induced by high-frequency stimulation under the blocking agent for synaptosomal platelet-activating factor receptors (ginkolide B), at drug washout. However, potentiation directly induced by the group I glutamate metabotropic receptor agonist, (R,S)-3,5-dihydroxyphenylglycine, was reduced by ginkolide B. These findings suggest that platelet-activating factor, whether exogenous or released following potentiation induction, exerts its effect through presynaptic group I metabotropic glutamate receptors, mediating the increase of glutamate release. In addition, we found that this mechanism, which led to full potentiation through presynaptic group I metabotropic glutamate receptor activation, was inactivated soon after application of potentiation-inducing stimulus. In fact, the long-lasting block of the platelet-activating factor and metabotropic glutamate receptors prevented the full potentiation development and the induced potentiation progressively declined to null. Moreover, ginkolide B, given when high-frequency-dependent potentiation was established, only reduced it within 5 min after potentiation induction. We conclude that to fully develop vestibular long-term potentiation requires presynaptic events. Platelet-activating factor, released after the activation of postsynaptic mechanisms which induce potentiation, is necessary

Evidence that central dopamine receptors modulate sympathetic neuronal activity to the adrenal medulla to alter glucoregulatory mechanisms.

Science.gov (United States)

Arnerić, S P; Chow, S A; Bhatnagar, R K; Webb, R L; Fischer, L J; Long, J P

1984-02-01

Previous reports suggest that analogs of dopamine (DA) can produce hyperglycemia in rats by interacting with DA receptors. Experiments reported here indicate the site of action and describe the metabolic sequalae associated with the hyperglycemic effect of apomorphine (APO), produced in conscious unrestrained rats. Apomorphine was more potent when administered by intracerebroventricular (i.c.v.) injection than when given subcutaneously (s.c.). Very small doses of the DA receptor antagonist pimozide, given intraventricularly, blocked the hyperglycemic effect of apomorphine administered subcutaneously. Sectioning of the spinal cord at thoracic vertebra T1-2 or sectioning the greater splanchnic nerve blocked apomorphine-induced hyperglycemia; whereas section of the superior colliculus or section at T5-6 had no effect. A dose of apomorphine or epinephrine (EPI) producing a similar degree of hyperglycemia elevated the concentration of EPI in serum to a similar degree, and the increase in EPI in serum preceded the increase in glucose in serum. Fasting animals for 2 or 18 hr had no significant effect on EPI- or apomorphine-induced hyperglycemia despite a reduction (91-93%) of the glycogen content of liver and skeletal muscle during the 18 hr fast. 5-Methoxyindole-2-carboxylic acid (MICA), an inhibitor of gluconeogenesis, blocked EPI- and apomorphine-induced hyperglycemia in rats fasted for 18 hr. However, 5-methoxyindole-2-carboxylic acid was ineffective in blocking hyperglycemia in animals fasted for 2 hr. Changes in insulin or glucagon in serum alone cannot account for the hyperglycemic action of apomorphine. These data demonstrate that apomorphine interacts with central DA receptors located in the hindbrain to activate sympathetic neuronal activity to the adrenal gland which subsequently releases epinephrine to alter homeostasis of glucose. Epinephrine may then, depending on the nutritional status, facilitate glycogenolytic or gluconeogenic processes to produce

Transcriptional activation of peroxisome proliferator-activated receptor-γ requires activation of both protein kinase A and Akt during adipocyte differentiation

International Nuclear Information System (INIS)

Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

2010-01-01

Research highlights: → Elevated cAMP activates both PKA and Epac. → PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. → Akt modulates PPAR-γ transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

Palmitoylation regulates 17β-estradiol-induced estrogen receptor-α degradation and transcriptional activity.

Science.gov (United States)

La Rosa, Piergiorgio; Pesiri, Valeria; Leclercq, Guy; Marino, Maria; Acconcia, Filippo

2012-05-01

The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17β-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK/ERK and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions.

Antinociceptive Action of Isolated Mitragynine from Mitragyna Speciosa through Activation of Opioid Receptor System

Directory of Open Access Journals (Sweden)

Mohamad Aris Mohd Moklas

2012-09-01

Full Text Available Cannabinoids and opioids systems share numerous pharmacological properties and antinociception is one of them. Previous findings have shown that mitragynine (MG, a major indole alkaloid found in Mitragyna speciosa (MS can exert its antinociceptive effects through the opioids system. In the present study, the action of MG was investigated as the antinociceptive agent acting on Cannabinoid receptor type 1 (CB1 and effects on the opioids receptor. The latency time was recorded until the mice showed pain responses such as shaking, licking or jumping and the duration of latency was measured for 2 h at every 15 min interval by hot plate analysis. To investigate the beneficial effects of MG as antinociceptive agent, it was administered intraperitoneally 15 min prior to pain induction with a single dosage (3, 10, 15, 30, and 35 mg/kg b.wt. In this investigation, 35 mg/kg of MG showed significant increase in the latency time and this dosage was used in the antagonist receptor study. The treated groups were administered with AM251 (cannabinoid receptor-1 antagonist, naloxone (non-selective opioid antagonist, naltrindole (δ-opioid antagonist naloxonazine (µ1-receptor antagonist and norbinaltorpimine (κ-opioid antagonist respectively, prior to administration of MG (35 mg/kg. The results showed that the antinociceptive effect of MG was not antagonized by AM251; naloxone and naltrindole were effectively blocked; and norbinaltorpimine partially blocked the antinociceptive effect of MG. Naloxonazine did inhibit the effect of MG, but it was not statistically significant. These results demonstrate that CB1 does not directly have a role in the antinociceptive action of MG where the effect was observed with the activation of opioid receptor.

NMDA receptor blockade in the prelimbic cortex activates the mesolimbic system and dopamine-dependent opiate reward signaling.

Science.gov (United States)

Tan, Huibing; Rosen, Laura G; Ng, Garye A; Rushlow, Walter J; Laviolette, Steven R

2014-12-01

N-Methyl-D-aspartate (NMDA) receptors in the medial prefrontal cortex (mPFC) are involved in opiate reward processing and modulate sub-cortical dopamine (DA) activity. NMDA receptor blockade in the prelimbic (PLC) division of the mPFC strongly potentiates the rewarding behavioural properties of normally sub-reward threshold doses of opiates. However, the possible functional interactions between cortical NMDA and sub-cortical DAergic motivational neural pathways underlying these effects are not understood. This study examines how NMDA receptor modulation in the PLC influences opiate reward processing via interactions with sub-cortical DAergic transmission. We further examined whether direct intra-PLC NMDA receptor modulation may activate DA-dependent opiate reward signaling via interactions with the ventral tegmental area (VTA). Using an unbiased place conditioning procedure (CPP) in rats, we performed bilateral intra-PLC microinfusions of the competitive NMDA receptor antagonist, (2R)-amino-5-phosphonovaleric acid (AP-5), prior to behavioural morphine place conditioning and challenged the rewarding effects of morphine with DA receptor blockade. We next examined the effects of intra-PLC NMDA receptor blockade on the spontaneous activity patterns of presumptive VTA DA or GABAergic neurons, using single-unit, extracellular in vivo neuronal recordings. We show that intra-PLC NMDA receptor blockade strongly activates sub-cortical DA neurons within the VTA while inhibiting presumptive non-DA GABAergic neurons. Behaviourally, NMDA receptor blockade activates a DA-dependent opiate reward system, as pharmacological blockade of DA transmission blocked morphine reward only in the presence of intra-PLC NMDA receptor antagonism. These findings demonstrate a cortical NMDA-mediated mechanism controlling mesolimbic DAergic modulation of opiate reward processing.

Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

International Nuclear Information System (INIS)

Semino, Carlos E.; Kamm, Roger D.; Lauffenburger, Douglas A.

2006-01-01

We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures

Cyprodinil as an activator of aryl hydrocarbon receptor

International Nuclear Information System (INIS)

Fang, Chien-Chung; Chen, Fei-Yun; Chen, Chang-Rong; Liu, Chien-Chiang; Wong, Liang-Chi; Liu, Yi-Wen; Su, Jyan-Gwo Joseph

2013-01-01

Highlights: ► Cyprodinil activated the aryl hydrocarbon receptor (AHR). ► Cyprodinil induced nuclear translocation of the AHR, and the expression of CYP1A1. ► Cyprodinil enhanced dexamethasone-induced gene expression. ► Cyprodinil phosphorylated ERK, indicating its deregulation of ERK activity. -- Abstract: Cyprodinil is a pyrimidinamine fungicide, used worldwide by agriculture. It is used to protect fruit plants and vegetables from a wide range of pathogens. Benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are toxic environmental pollutants and are prototypes of aryl hydrocarbon receptor (AHR) ligands. Although the structure of cyprodinil distinctly differs from those of BaP and TCDD, our results show that cyprodinil induced nuclear translocation of the AHR, and induced the transcriptional activity of aryl hydrocarbon response element (AHRE). Cyprodinil induced the expression of cytochrome P450 (CYP) 1A1, a well-known AHR-targeted gene, in ovarian granulosa cells, HO23, and hepatoma cells, Hepa-1c1c7. Its induction did not appear in AHR signal-deficient cells, and was blocked by the AHR antagonist, CH-223191. Cyprodinil decreased AHR expression in HO23 cells, resulting in CYP1A1 expression decreasing after it peaked at 9 h of treatment in HO23 cells. Dexamethasone is a synthetic agonist of glucocorticoids. Cyprodinil enhanced dexamethasone-induced gene expression, and conversely, its induction of CYP1A1 expression was decreased by dexamethasone in HO23 cells, indicating its induction of crosstalk between the AHR and glucocorticoid receptor and its role as a potential endocrine disrupter. In addition to BaP, TCDD, and an AHR agonist, β-NF, cyprodinil also phosphorylated extracellular signal-regulated kinase (ERK) in HO23 and Hepa-1c1c7 cells, indicating its deregulation of ERK activity. In summary, our results demonstrate that cyprodinil, similar to BaP, acts as an AHR activator, a potential endocrine disrupter, and an ERK disrupter

Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

DEFF Research Database (Denmark)

Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

2002-01-01

that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

Science.gov (United States)

Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

1992-01-01

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

Substance P receptor desensitization requires receptor activation but not phospholipase C

International Nuclear Information System (INIS)

Sugiya, Hiroshi; Putney, J.W. Jr.

1988-01-01

Previous studies have shown that exposure of parotid acinar cells to substance P at 37 degree C results in activation of phospholipase C, formation of [ 3 H]inositol 1,4,5-trisphosphate (IP 3 ), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to ∼20% of control values, IP 3 formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to ∼5% of control values, and both IP 3 formation and desensitization were blocked. A series of substance P-related peptides increased the formation of [ 3 H]IP 3 and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, [D-Pro 2 , D-Try 7,9 ]-substance P, inhibited substance P-induced IP 3 formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP 3 formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization

LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation

Directory of Open Access Journals (Sweden)

Rainer Kraehenmann

2017-11-01

Full Text Available Rationale: Stimulation of serotonin 2A (5-HT2A receptors by lysergic acid diethylamide (LSD and related compounds such as psilocybin has previously been shown to increase primary process thinking – an ontologically and evolutionary early, implicit, associative, and automatic mode of thinking which is typically occurring during altered states of consciousness such as dreaming. However, it is still largely unknown whether LSD induces primary process thinking under placebo-controlled, standardized experimental conditions and whether these effects are related to subjective experience and 5-HT2A receptor activation. Therefore, this study aimed to test the hypotheses that LSD increases primary process thinking and that primary process thinking depends on 5-HT2A receptor activation and is related to subjective drug effects.Methods: Twenty-five healthy subjects performed an audio-recorded mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally. The main outcome variable in this study was primary index (PI, a formal measure of primary process thinking in the imagery reports. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC rating scale.Results: LSD, compared with placebo, significantly increased primary index (p < 0.001, Bonferroni-corrected. The LSD-induced increase in primary index was positively correlated with LSD-induced disembodiment (p < 0.05, Bonferroni-corrected, and blissful state (p < 0.05, Bonferroni-corrected on the 5D-ASC. Both LSD-induced increases in primary index and changes in state of consciousness were fully blocked by ketanserin.Conclusion: LSD induces primary process thinking via activation of 5-HT2A receptors and in relation to disembodiment and blissful state. Primary process thinking appears to crucially organize inner experiences during both dreams and

LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation

Science.gov (United States)

Kraehenmann, Rainer; Pokorny, Dan; Aicher, Helena; Preller, Katrin H.; Pokorny, Thomas; Bosch, Oliver G.; Seifritz, Erich; Vollenweider, Franz X.

2017-01-01

Rationale: Stimulation of serotonin 2A (5-HT2A) receptors by lysergic acid diethylamide (LSD) and related compounds such as psilocybin has previously been shown to increase primary process thinking – an ontologically and evolutionary early, implicit, associative, and automatic mode of thinking which is typically occurring during altered states of consciousness such as dreaming. However, it is still largely unknown whether LSD induces primary process thinking under placebo-controlled, standardized experimental conditions and whether these effects are related to subjective experience and 5-HT2A receptor activation. Therefore, this study aimed to test the hypotheses that LSD increases primary process thinking and that primary process thinking depends on 5-HT2A receptor activation and is related to subjective drug effects. Methods: Twenty-five healthy subjects performed an audio-recorded mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). The main outcome variable in this study was primary index (PI), a formal measure of primary process thinking in the imagery reports. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) rating scale. Results: LSD, compared with placebo, significantly increased primary index (p LSD-induced increase in primary index was positively correlated with LSD-induced disembodiment (p LSD-induced increases in primary index and changes in state of consciousness were fully blocked by ketanserin. Conclusion: LSD induces primary process thinking via activation of 5-HT2A receptors and in relation to disembodiment and blissful state. Primary process thinking appears to crucially organize inner experiences during both dreams and psychedelic states of consciousness. PMID:29167644

Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β.

Science.gov (United States)

Jana, Malabendu; Pahan, Kalipada

2012-08-01

Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug, in primary human microglia. Bacterial lipopolysachharides (LPS) induced the expression of various proinflammatory molecules and upregulated the expression of microglial surface marker CD11b in human microglia. However, gemfibrozil markedly suppressed proinflammatory molecules and CD11b in LPS-stimulated microglia. Human microglia expressed PPAR-β and -γ, but not PPAR-α. Interestingly, either antisense knockdown of PPAR-β or antagonism of PPAR-β by a specific chemical antagonist abrogated gemfibrozil-mediated inhibition of microglial activation. On the other hand, blocking of PPAR-α and -γ had no effect on gemfibrozil-mediated anti-inflammatory effect in microglia. These results highlight the fact that gemfibrozil regulates microglial activation by inhibiting inflammatory gene expression in a PPAR-β dependent pathway and further reinforce its therapeutic application in several neuroinflammatory and neurodegenerative diseases.

{delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

Energy Technology Data Exchange (ETDEWEB)

Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

2009-07-15

{delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

International Nuclear Information System (INIS)

Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

2009-01-01

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

Extracellular polysaccharides produced by Ganoderma formosanum stimulate macrophage activation via multiple pattern-recognition receptors

Directory of Open Access Journals (Sweden)

Wang Cheng-Li

2012-08-01

Full Text Available Abstract Background The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2. Results PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3, laminarin, or piceatannol (a spleen tyrosine kinase inhibitor, suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4. PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression. Conclusions Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α.

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

Science.gov (United States)

Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (pAPC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Science.gov (United States)

Nuñez, S B; Medin, J A; Braissant, O; Kemp, L; Wahli, W; Ozato, K; Segars, J H

1997-03-14

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

Science.gov (United States)

Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

2013-05-07

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

International Nuclear Information System (INIS)

Wei, Kuo-Liang; Chen, Fei-Yun; Lin, Chih-Yi; Gao, Guan-Lun; Kao, Wen-Ya; Yeh, Chi-Hui; Chen, Chang-Rong; Huang, Hao-Chun; Tsai, Wei-Ren; Jong, Koa-Jen; Li, Wan-Jung; Su, Jyan-Gwo Joseph

2016-01-01

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG 0 /G 1 population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy. - Highlights: • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element. • Carbendazim induced nuclear translocation of the aryl hydrocarbon

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

Energy Technology Data Exchange (ETDEWEB)

Wei, Kuo-Liang [Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan, ROC (China); College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan, ROC (China); Chen, Fei-Yun; Lin, Chih-Yi [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Gao, Guan-Lun [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Department of Biological Resources, National Chiayi University, Chiayi, 60004, Taiwan, ROC (China); Kao, Wen-Ya [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Yeh, Chi-Hui [Department of Environmental Engineering, College of Engineering, Da-Yeh University, Dacun, Changhua 51591, Taiwan, ROC (China); Chen, Chang-Rong [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Huang, Hao-Chun [Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan, ROC (China); Tsai, Wei-Ren [Division of Applied Toxicology, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture, Executive Yuan, Taichung 41358, Taiwan, ROC (China); Jong, Koa-Jen [Department of Biological Resources, National Chiayi University, Chiayi, 60004, Taiwan, ROC (China); Li, Wan-Jung [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Su, Jyan-Gwo Joseph, E-mail: jgjsu@mail.ncyu.edu.tw [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China)

2016-09-01

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG{sub 0}/G{sub 1} population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy. - Highlights: • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element. • Carbendazim induced nuclear translocation of the aryl

GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

Directory of Open Access Journals (Sweden)

Pengmin eQin

2012-12-01

Full Text Available Recent imaging studies have demonstrated that levels of resting GABA in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC and eyes open (EO state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: An EO and EC block design, allowing the modelling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicts the change of functional connectivity between visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

In-vivo characteristics of high and low specific activity radioiodinated (+)-2-[4-(4-iodophenyl) piperidino] cyclohexanol [(+)-pIV] for imaging sigma-1 receptor in brain

International Nuclear Information System (INIS)

Akhter, Nasima; Kinuya, Seigo; Nakajima, Kenichi; Shiba, Kazuhiro; Ogawa, Kazuma; Mori, Hirofumi

2007-01-01

Full text: In this study, (+)-enantiomer of radioiodinated 2-[4-(4- iodophenyl)piperidino]cyclohexanol ((+)-[ 125 I]-p- iodovesamicol) [(+)-[ 125 I]pIV], which is reported to bind with high affinity to the sigma-1 receptor both in vitro and in vivo, was tested to compare the in vivo characteristics between high and low specific activity (+)-[ 125 I]pIV to image sigma-1 receptor in the central nervous system. In the biodistribution study, no significant difference was observed between two methods. Accumulation of (+)- [ 125 I]pIV in rat brain was significant (approximately 3% of the injected dose) and its retention was prolonged. In the blocking study, the accumulation of (+)-[ 125 I] pIV in the rat brain was significantly reduced by the co-administration of sigma ligands such as pentazocine, haloperidol or SA4503 in both methods. But the blocking effect was relatively stronger in the study using high specific activity radioiodinated (+)pIV. Though, the distribution of high and low specific activity (+)-[ 125 I] pIV was more or less similar to bind to sigma-1 receptor in the central nervous system in vivo, high specific activity radioiodinated (+) pIV might have a better specificity to bind sigma-1 receptor in brain. (author)

Long-term activation upon brief exposure to xanomleline is unique to M1 and M4 subtypes of muscarinic acetylcholine receptors.

Directory of Open Access Journals (Sweden)

Eva Šantrůčková

Full Text Available Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

Directory of Open Access Journals (Sweden)

Yuko Nakagawa

Full Text Available BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+ ([Ca(2+](c and cAMP ([cAMP](c were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+](c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+](c response. The effect of sucralose on [Ca(2+](c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q inhibitor. Sucralose also induced sustained elevation of [cAMP](c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+ and cAMP-dependent mechanisms.

The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

Energy Technology Data Exchange (ETDEWEB)

Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

2015-11-30

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

International Nuclear Information System (INIS)

Bellamy-McIntyre, Anna K.; Baer, Severine; Ludlow, Louise; Drummer, Heidi E.; Poumbourios, Pantelis

2010-01-01

The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1 QH1549.13 blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.

Transient Receptor Potential Vanilloid 4 Activation-Induced Increase in Glycine-Activated Current in Mouse Hippocampal Pyramidal Neurons

Directory of Open Access Journals (Sweden)

Mengwen Qi

2018-02-01

Full Text Available Background/Aims: Glycine plays an important role in regulating hippocampal inhibitory/ excitatory neurotransmission through activating glycine receptors (GlyRs and acting as a co-agonist of N-methyl-d-aspartate-type glutamate receptors. Activation of transient receptor potential vanilloid 4 (TRPV4 is reported to inhibit hippocampal A-type γ-aminobutyric acid receptor, a ligand-gated chloride ion channel. GlyRs are also ligand-gated chloride ion channels and this paper aimed to explore whether activation of TRPV4 could modulate GlyRs. Methods: Whole-cell patch clamp recording was employed to record glycine-activated current (IGly and Western blot was conducted to assess GlyRs subunits protein expression. Results: Application of TRPV4 agonist (GSK1016790A or 5,6-EET increased IGly in mouse hippocampal CA1 pyramidal neurons. This action was blocked by specific antagonists of TRPV4 (RN-1734 or HC-067047 and GlyR (strychnine, indicating that activation of TRPV4 increases strychnine-sensitive GlyR function in mouse hippocampal pyramidal neurons. GSK1016790A-induced increase in IGly was significantly attenuated by protein kinase C (PKC (BIM II or D-sphingosine or calcium/calmodulin-dependent protein kinase II (CaMKII (KN-62 or KN-93 antagonists but was unaffected by protein kinase A or protein tyrosine kinase antagonists. Finally, hippocampal protein levels of GlyR α1 α2, α3 and β subunits were not changed by treatment with GSK1016790A for 30 min or 1 h, but GlyR α2, α3 and β subunits protein levels increased in mice that were intracerebroventricularly (icv. injected with GSK1016790A for 5 d. Conclusion: Activation of TRPV4 increases GlyR function and expression, and PKC and CaMKII signaling pathways are involved in TRPV4 activation-induced increase in IGly. This study indicates that GlyRs may be effective targets for TRPV4-induced modulation of hippocampal inhibitory neurotransmission.

Blocking p75 (NTR) receptors alters polyinnervationz of neuromuscular synapses during development.

Science.gov (United States)

Garcia, Neus; Tomàs, Marta; Santafe, Manel M; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2011-09-01

High-resolution immunohistochemistry shows that the receptor protein p75(NTR) is present in the nerve terminal, muscle cell, and glial Schwann cell at the neuromuscular junction (NMJ) of postnatal rats (P4-P6) during the synapse elimination period. Blocking the receptor with the antibody anti-p75-192-IgG (1-5 μg/ml, 1 hr) results in reduced endplate potentials (EPPs) in mono- and polyinnervated synapses ex vivo, but the mean number of functional inputs per NMJ does not change for as long as 3 hr. Incubation with exogenous brain-derived neurotrophic factor (BDNF) for 1 hr (50 nM) resulted in a significant increase in the size of the EPPs in all nerve terminals, and preincubation with anti-p75-192-IgG prevented this potentiation. Long exposure (24 hr) in vivo of the NMJs to the antibody anti-p75-192-IgG (1-2 μg/ml) results in a delay of postnatal synapse elimination and even some regrowth of previously withdrawn axons, but also in some acceleration of the morphologic maturation of the postsynaptic nicotinic acetylcholine receptor (nAChR) clusters. The results indicate that p75(NTR) is involved in both ACh release and axonal retraction during postnatal axonal competition and synapse elimination. Copyright © 2011 Wiley-Liss, Inc.

Cis-urocanic acid, a sunlight-induced immunosuppressive factor, activates immune suppression via the 5-HT2A receptor

Science.gov (United States)

Walterscheid, Jeffrey P.; Nghiem, Dat X.; Kazimi, Nasser; Nutt, Leta K.; McConkey, David J.; Norval, Mary; Ullrich, Stephen E.

2006-01-01

Exposure to UV radiation induces skin cancer and suppresses the immune response. To induce immune suppression, the electromagnetic energy of UV radiation must be absorbed by an epidermal photoreceptor and converted into a biologically recognizable signal. Two photoreceptors have been recognized: DNA and trans-urocanic acid (UCA). Trans-UCA is normally found in the outermost layer of skin and isomerizes to the cis isomer upon exposure to UV radiation. Although UCA was identified as a UV photoreceptor years ago, and many have documented its ability to induce immune suppression, its exact mode of action remains elusive. Particularly vexing has been the identity of the molecular pathway by which cis-UCA mediates immune suppression. Here we provide evidence that cis-UCA binds to the serotonin [5-hydroxytryptamine (5-HT)] receptor with relatively high affinity (Kd = 4.6 nM). Anti-cis-UCA antibody precipitates radiolabeled 5-HT, and the binding is inhibited by excess 5-HT and/or excess cis-UCA. Similarly, anti-5-HT antibody precipitates radiolabeled cis-UCA, and the binding is inhibited by excess 5-HT or excess cis-UCA. Calcium mobilization was activated when a mouse fibroblast line, stably transfected with the human 5-HT2A receptor, was treated with cis-UCA. Cis-UCA-induced calcium mobilization was blocked with a selective 5-HT2A receptor antagonist. UV- and cis-UCA-induced immune suppression was blocked by antiserotonin antibodies or by treating the mice with 5-HT2A receptor antagonists. Our findings identify cis-UCA as a serotonin receptor ligand and indicate that the immunosuppressive effects of cis-UCA and UV radiation are mediated by activation of the 5-HT2A receptor. PMID:17085585

Acute Cocaine Induces Fast Activation of D1 Receptor and Progressive Deactivation of D2 Receptor Strial Neurons: In Vivo Optical Microprobe [Ca2+]i Imaging

International Nuclear Information System (INIS)

Du, C.; Luo, Z.; Volkow, N.D.; Heintz, N.; Pan, Y.; Du, C.

2011-01-01

Cocaine induces fast dopamine increases in brain striatal regions, which are recognized to underlie its rewarding effects. Both dopamine D1 and D2 receptors are involved in cocaine's reward but the dynamic downstream consequences of cocaine effects in striatum are not fully understood. Here we used transgenic mice expressing EGFP under the control of either the D1 receptor (D1R) or the D2 receptor (D2R) gene and microprobe optical imaging to assess the dynamic changes in intracellular calcium ([Ca 2+ ] i ) responses (used as marker of neuronal activation) to acute cocaine in vivo separately for D1R- versus D2R-expressing neurons in striatum. Acute cocaine (8 mg/kg, i.p.) rapidly increased [Ca 2+ ] i in D1R-expressing neurons (10.6 ± 3.2%) in striatum within 8.3 ± 2.3 min after cocaine administration after which the increases plateaued; these fast [Ca 2+ ] i increases were blocked by pretreatment with a D1R antagonist (SCH23390). In contrast, cocaine induced progressive decreases in [Ca 2+ ] i in D2R-expressing neurons (10.4 ± 5.8%) continuously throughout the 30 min that followed cocaine administration; these slower [Ca 2+ ] i decreases were blocked by pretreatment with a D2R antagonist (raclopride). Since activation of striatal D1R-expressing neurons (direct-pathway) enhances cocaine reward, whereas activation of D2R expressing neurons suppresses it (indirect-pathway) (Lobo et al., 2010), this suggests that cocaine's rewarding effects entail both its fast stimulation ofD1R (resulting in abrupt activation of direct-pathway neurons) and a slower stimulation of D2R (resulting in longer-lasting deactivation of indirect-pathway neurons). We also provide direct in vivo evidence of D2R and D1R interactions in the striatal responses to acute cocaine administration.

Dopamine D4 receptor activation increases hippocampal gamma oscillations by enhancing synchronization of fast-spiking interneurons.

Directory of Open Access Journals (Sweden)

Richard Andersson

Full Text Available BACKGROUND: Gamma oscillations are electric activity patterns of the mammalian brain hypothesized to serve attention, sensory perception, working memory and memory encoding. They are disrupted or altered in schizophrenic patients with associated cognitive deficits, which persist in spite of treatment with antipsychotics. Because cognitive symptoms are a core feature of schizophrenia it is relevant to explore signaling pathways that potentially regulate gamma oscillations. Dopamine has been reported to decrease gamma oscillation power via D1-like receptors. Based on the expression pattern of D4 receptors (D4R in hippocampus, and pharmacological effects of D4R ligands in animals, we hypothesize that they are in a position to regulate gamma oscillations as well. METHODOLOGY/PRINCIPAL FINDINGS: To address this hypothesis we use rat hippocampal slices and kainate-induced gamma oscillations. Local field potential recordings as well as intracellular recordings of pyramidal cells, fast-spiking and non-fast-spiking interneurons were carried out. We show that D4R activation with the selective ligand PD168077 increases gamma oscillation power, which can be blocked by the D4R-specific antagonist L745,870 as well as by the antipsychotic drug Clozapine. Pyramidal cells did not exhibit changes in excitatory or inhibitory synaptic current amplitudes, but inhibitory currents became more coherent with the oscillations after application of PD168077. Fast-spiking, but not non-fast spiking, interneurons, increase their action potential phase-coupling and coherence with regard to ongoing gamma oscillations in response to D4R activation. Among several possible mechanisms we found that the NMDA receptor antagonist AP5 also blocks the D4R mediated increase in gamma oscillation power. CONCLUSIONS/SIGNIFICANCE: We conclude that D4R activation affects fast-spiking interneuron synchronization and thereby increases gamma power by an NMDA receptor-dependent mechanism. This

Subregion-specific modulation of excitatory input and dopaminergic output in the striatum by tonically activated glycine and GABAA receptors

Directory of Open Access Journals (Sweden)

Louise eAdermark

2011-10-01

Full Text Available The flow of cortical information through the basal ganglia is a complex spatiotemporal pattern of increased and decreased firing. The striatum is the biggest input nucleus to the basal ganglia and the aim of this study was to assess the role of inhibitory GABAA and glycine receptors in regulating synaptic activity in the dorsolateral (DLS and ventral striatum (nucleus accumbens, nAc. Local field potential recordings from coronal brain slices of juvenile and adult Wistar rats showed that GABAA receptors and strychnine-sensitive glycine receptors are tonically activated and inhibit excitatory input to the DLS and to the nAc. Strychnine-induced disinhibition of glutamatergic transmission was insensitive to the muscarinic receptor inhibitor scopolamine (10 µM, inhibited by the nicotinic acetylcholine receptor antagonist mecamylamine (10 µM and blocked by GABAA receptor inhibitors, suggesting that tonically activated glycine receptors depress excitatory input to the striatum through modulation of cholinergic and GABAergic neurotransmission. As an end-product example of striatal GABAergic output in vivo we measured dopamine release in the DLS and nAc by microdialysis in the awake and freely moving rat. Reversed dialysis of bicuculline (50 μM in perfusate only increased extrasynaptic dopamine levels in the nAc, while strychnine administered locally (200 μM in perfusate decreased dopamine output by 60% in both the DLS and nAc. Our data suggest that GABAA and glycine receptors are tonically activated and modulate striatal transmission in a partially sub-region specific manner.

GABA(A) receptors in visual and auditory cortex and neural activity changes during basic visual stimulation.

Science.gov (United States)

Qin, Pengmin; Duncan, Niall W; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J; Northoff, Georg

2012-01-01

Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABA(A) receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [(18)F]Flumazenil PET to measure GABA(A) receptor binding potentials. It was demonstrated that the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABA(A) receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

OpenAIRE

Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

1995-01-01

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

β-Adrenergic receptor-mediated suppression of interleukin 2 receptors in human lymphocytes

International Nuclear Information System (INIS)

Feldman, R.D.; Hunninghake, G.W.; McArdle, W.L.

1987-01-01

Adrenergic receptor agonists are know to attenuate the proliferative response of human lymphocytes after activation; however, their mechanism of action is unknown. Since expression of interleukin 2 (IL-2) receptors is a prerequisite for proliferation, the effect of β-adrenergic receptor agonists on lymphocyte IL-2 receptors was studied on both mitogen-stimulated lymphocytes and IL-2-dependent T lymphocyte cell lines. In both cell types the β-adrenergic receptor agonist isoproterenol blocked the expression of IL-2 receptors, as determined with the IL-2 receptor anti-TAC antibody. To determine the effect of β-adrenergic agonists on expression of the high affinity IL-2 receptors, [ 125 I]IL-2 binding studies were performed at concentrations selective for high affinity sites. No significant effect of β-adrenergic agonists on high affinity IL-2 receptor sites could be detected. The data demonstrate that β-adrenergic receptor agonists down-regulate IL-2 receptors primarily affecting low affinity sites

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

Science.gov (United States)

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

2013-01-01

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

Chapter 8. Activation mechanisms of chemokine receptors

DEFF Research Database (Denmark)

Jensen, Pia C; Rosenkilde, Mette M

2009-01-01

binding. Attempts to unravel the activation mechanism of 7TM receptors have led to the conclusion that activation involves movements of the transmembrane segments VI and VII in particular, as recently gathered in the Global Toggle Switch Model. However, to understand the activation mechanism completely......, more research has to be done in this field. Chemokine receptors are interesting tools in this matter. First, the chemokine system has a high degree of promiscuity that allows several chemokines to target one receptor in different ways, as well as a single chemokine ligand to target several receptors...

Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

Science.gov (United States)

Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang

2017-12-01

Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.

Excessive D1 Dopamine Receptor Activation in the Dorsal Striatum Promotes Autistic-Like Behaviors.

Science.gov (United States)

Lee, Yunjin; Kim, Hannah; Kim, Ji-Eun; Park, Jin-Young; Choi, Juli; Lee, Jung-Eun; Lee, Eun-Hwa; Han, Pyung-Lim

2018-07-01

The dopamine system has been characterized in motor function, goal-directed behaviors, and rewards. Recent studies recognize various dopamine system genes as being associated with autism spectrum disorder (ASD). However, how dopamine system dysfunction induces ASD pathophysiology remains unknown. In the present study, we demonstrated that mice with increased dopamine functions in the dorsal striatum via the suppression of dopamine transporter expression in substantia nigra neurons or the optogenetic stimulation of the nigro-striatal circuitry exhibited sociability deficits and repetitive behaviors relevant to ASD pathology in animal models, while these behavioral changes were blocked by a D1 receptor antagonist. Pharmacological activation of D1 dopamine receptors in normal mice or the genetic knockout (KO) of D2 dopamine receptors also produced typical autistic-like behaviors. Moreover, the siRNA-mediated inhibition of D2 dopamine receptors in the dorsal striatum was sufficient to replicate autistic-like phenotypes in D2 KO mice. Intervention of D1 dopamine receptor functions or the signaling pathways-related D1 receptors in D2 KO mice produced anti-autistic effects. Together, our results indicate that increased dopamine function in the dorsal striatum promotes autistic-like behaviors and that the dorsal striatum is the neural correlate of ASD core symptoms.

The role of neonatal NMDA receptor activation in defeminization and masculinization of sex behavior in the rat

Science.gov (United States)

Schwarz, Jaclyn M.; McCarthy, Margaret M.

2008-01-01

Normal development of the male rat brain involves two distinct processes, masculinization and defeminization, that occur during a critical period of brain sexual differentiation. Masculinization allows for the capacity to express male sex behavior in adulthood, and defeminization eliminates or suppresses the capacity to express female sex behavior in adulthood. Despite being separate processes, both masculinization and defeminization are induced by neonatal estradiol exposure. Though the mechanisms underlying estradiol-mediated masculinization of behavior during development have been identified, the mechanisms underlying defeminization are still unknown. We sought to determine whether neonatal activation of glutamate NMDA receptors is a necessary component of estradiol-induced defeminization of behavior. We report here that antagonizing glutamate receptors during the critical period of sexual differentiation blocks estradiol-induced defeminization but not masculinization of behavior in adulthood. However, enhancing NMDA receptor activation during the same critical period mimics estradiol to permanently induce both defeminization and masculinization of sexual behavior. PMID:18687334

Histamine H3 Receptors Decrease Dopamine Release in the Ventral Striatum by Reducing the Activity of Striatal Cholinergic Interneurons.

Science.gov (United States)

Varaschin, Rafael Koerich; Osterstock, Guillaume; Ducrot, Charles; Leino, Sakari; Bourque, Marie-Josée; Prado, Marco A M; Prado, Vania Ferreira; Salminen, Outi; Rannanpää Née Nuutinen, Saara; Trudeau, Louis-Eric

2018-04-15

Histamine H 3 receptors are widely distributed G i -coupled receptors whose activation reduces neuronal activity and inhibits release of numerous neurotransmitters. Although these receptors are abundantly expressed in the striatum, their modulatory role on activity-dependent dopamine release is not well understood. Here, we observed that histamine H 3 receptor activation indirectly diminishes dopamine overflow in the ventral striatum by reducing cholinergic interneuron activity. Acute brain slices from C57BL/6 or channelrhodopsin-2-transfected DAT-cre mice were obtained, and dopamine transients evoked either electrically or optogenetically were measured by fast-scan cyclic voltammetry. The H 3 agonist α-methylhistamine significantly reduced electrically- evoked dopamine overflow, an effect blocked by the nicotinic acetylcholine receptor antagonist dihydro-β-erythroidine, suggesting involvement of cholinergic interneurons. None of the drug treatments targeting H 3 receptors affected optogenetically evoked dopamine overflow, indicating that direct H 3 -modulation of dopaminergic axons is unlikely. Next, we used qPCR and confirmed the expression of histamine H 3 receptor mRNA in cholinergic interneurons, both in ventral and dorsal striatum. Activation of H 3 receptors by α-methylhistamine reduced spontaneous firing of cholinergic interneurons in the ventral, but not in the dorsal striatum. Resting membrane potential and number of spontaneous action potentials in ventral-striatal cholinergic interneurons were significantly reduced by α-methylhistamine. Acetylcholine release from isolated striatal synaptosomes, however, was not altered by α-methylhistamine. Together, these results indicate that histamine H 3 receptors are important modulators of dopamine release, specifically in the ventral striatum, and that they do so by decreasing the firing rate of cholinergic neurons and, consequently, reducing cholinergic tone on dopaminergic axons. Copyright © 2018 IBRO

Activation of neurotensin receptors and purinoceptors in human colonic adenocarcinoma cells detected with the microphysiometer.

Science.gov (United States)

Richards, M; van Giersbergen, P; Zimmermann, A; Lesur, B; Hoflack, J

1997-10-01

Activation of endogenous neurotensin (NT) receptors and P2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), NT[8-13] (Arg-Arg-Pro-Tyr-Ile-Leu), NT[9-13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (N alpha methyl-Arg-Lys-Pro-Trp-Tle-Leu [Tle = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-1-oxo-2-[[(phenylmethoxy)carbonyl]-amino]hex yl]amino]phenyl]-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-isoleucyl]-L-le ucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may

5-HT4-receptors modulate induction of long-term depression but not potentiation at hippocampal output synapses in acute rat brain slices.

Directory of Open Access Journals (Sweden)

Matthias Wawra

Full Text Available The subiculum is the principal target of CA1 pyramidal cells and mediates hippocampal output to various cortical and subcortical regions of the brain. The majority of subicular pyramidal cells are burst-spiking neurons. Previous studies indicated that high frequency stimulation in subicular burst-spiking cells causes presynaptic NMDA-receptor dependent long-term potentiation (LTP whereas low frequency stimulation induces postsynaptic NMDA-receptor-dependent long-term depression (LTD. In the present study, we investigate the effect of 5-hydroxytryptamine type 4 (5-HT4 receptor activation and blockade on both forms of synaptic plasticity in burst-spiking cells. We demonstrate that neither activation nor block of 5-HT4 receptors modulate the induction or expression of LTP. In contrast, activation of 5-HT4 receptors facilitates expression of LTD, and block of the 5-HT4 receptor prevents induction of short-term depression and LTD. As 5-HT4 receptors are positively coupled to adenylate cyclase 1 (AC1, 5-HT4 receptors might modulate PKA activity through AC1. Since LTD is blocked in the presence of 5-HT4 receptor antagonists, our data are consistent with 5-HT4 receptor activation by ambient serotonin or intrinsically active 5-HT4 receptors. Our findings provide new insight into aminergic modulation of hippocampal output.

Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells

Science.gov (United States)

Hasebe, Masaharu

2016-01-01

The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na+-activated K+ (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells. PMID:26984419

In vivo interactions between α7 nicotinic acetylcholine receptor and nuclear peroxisome proliferator-activated receptor-α: Implication for nicotine dependence.

Science.gov (United States)

Jackson, Asti; Bagdas, Deniz; Muldoon, Pretal P; Lichtman, Aron H; Carroll, F Ivy; Greenwald, Mark; Miles, Michael F; Damaj, M Imad

2017-05-15

Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric α7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPARα as a possible mediator of the effect of α7 nAChR activation in nicotine dependence. Our results demonstrate the PPARα antagonist GW6471 blocks actions of the α7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that α7 nAChR activation attenuates nicotine CPP in a PPARα-dependent manner. To evaluate PPARα activation in nicotine dependence we used the selective and potent PPARα agonist, WY-14643 and the clinically used PPARα activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPARα in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPARα plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of α7 nAChRs in nicotine dependence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural basis for activation of G-protein-coupled receptors

DEFF Research Database (Denmark)

Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

2002-01-01

into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where...... changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co...

Plasma homovanillic acid, plasma anti-D1 and -D2 dopamine-receptor activity, and negative symptoms in chronically mediated schizophrenia.

Science.gov (United States)

Suzuki, E; Kanba, S; Nibuya, M; Koshikawa, H; Nakaki, T; Yagi, G

1992-02-15

We have investigated the relationship between the concentration of homovanillic acid in human plasma (pHVA) and plasma anti-D1 and anti-D2 dopamine receptor activity in chronic schizophrenic patients whose neuroleptic dosage was changed. The change in pHVA level correlated with that in anti-D1, not anti-D2 activity, thus suggesting that the neuroleptic-induced changes in pHVA concentration may be associated with the blocking of D1- as well as D2- receptors. The change of scores on the Scale for the Assessment of Negative Symptoms did not significantly correlate with changes in anti-D1 or anti-D2 activity, but did so correlated with the change in pHVA level.

Levodopa acts centrally to induce an antinociceptive action against colonic distension through activation of D2 dopamine receptors and the orexinergic system in the brain in conscious rats

Directory of Open Access Journals (Sweden)

Toshikatsu Okumura

2016-02-01

Subcutaneously (80 mg/rat or intracisternally (2.5 μg/rat administered levodopa significantly increased the threshold of colonic distension-induced AWR in conscious rats. The dose difference to induce the antinociceptive action suggests levodopa acts centrally to exert its antinociceptive action against colonic distension. While neither sulpiride, a D2 dopamine receptor antagonist, nor SCH23390, a D1 dopamine receptor antagonist by itself changed the threshold of colonic distension-induced AWR, the intracisternally injected levodopa-induced antinociceptive action was significantly blocked by pretreatment with subcutaneously administered sulpiride but not SCH23390. Treatment with intracisternal SB334867, an orexin 1 receptor antagonist, significantly blocked the subcutaneously administered levodopa-induced antinociceptive action. These results suggest that levodopa acts centrally to induce an antinociceptive action against colonic distension through activation of D2 dopamine receptors and the orexinergic system in the brain.

Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

Directory of Open Access Journals (Sweden)

Cintia Ferreira Marinho

Full Text Available In dengue virus (DENV infection, complement system (CS activation appears to have protective and pathogenic effects. In severe dengue fever (DF, the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b, CR4 (CD11c and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b or CR3 (CD11b/CD18 reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b or CR3 (CD11b/CD18 blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

Nicotine Receptor Subtype-Specific Effects on Auditory Evoked Oscillations and Potentials

Science.gov (United States)

Featherstone, Robert E.; Phillips, Jennifer M.; Thieu, Tony; Ehrlichman, Richard S.; Halene, Tobias B.; Leiser, Steven C.; Christian, Edward; Johnson, Edwin; Lerman, Caryn; Siegel, Steven J.

2012-01-01

Background Individuals with schizophrenia show increased smoking rates which may be due to a beneficial effect of nicotine on cognition and information processing. Decreased amplitude of the P50 and N100 auditory event-related potentials (ERPs) is observed in patients. Both measures show normalization following administration of nicotine. Recent studies identified an association between deficits in auditory evoked gamma oscillations and impaired information processing in schizophrenia, and there is evidence that nicotine normalizes gamma oscillations. Although the role of nicotine receptor subtypes in augmentation of ERPs has received some attention, less is known about how these receptor subtypes regulate the effect of nicotine on evoked gamma activity. Methodology/Principal Findings We examined the effects of nicotine, the α7 nicotine receptor antagonist methyllycaconitine (MLA) the α4β4/α4β2 nicotine receptor antagonist dihydro-beta-erythroidine (DHβE), and the α4β2 agonist AZD3480 on P20 and N40 amplitude as well as baseline and event-related gamma oscillations in mice, using electrodes in hippocampal CA3. Nicotine increased P20 amplitude, while DHβE blocked nicotine-induced enhancements in P20 amplitude. Conversely, MLA did not alter P20 amplitude either when presented alone or with nicotine. Administration of the α4β2 specific agonist AZD3480 did not alter any aspect of P20 response, suggesting that DHβE blocks the effects of nicotine through a non-α4β2 receptor specific mechanism. Nicotine and AZD3480 reduced N40 amplitude, which was blocked by both DHβE and MLA. Finally, nicotine significantly increased event-related gamma, as did AZD3480, while DHβE but not MLA blocked the effect of nicotine on event-related gamma. Conclusions/Significance These results support findings showing that nicotine-induced augmentation of P20 amplitude occurs via a DHβE sensitive mechanism, but suggests that this does not occur through activation of α4β2

TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch.

Science.gov (United States)

Jian, Tunyu; Yang, Niuniu; Yang, Yan; Zhu, Chan; Yuan, Xiaolin; Yu, Guang; Wang, Changming; Wang, Zhongli; Shi, Hao; Tang, Min; He, Qian; Lan, Lei; Wu, Guanyi; Tang, Zongxiang

2016-01-01

Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG) neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip)-a histamine H4 receptor special agonist under cutaneous injection-obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3-50 μM) could also induce a dose-dependent increase in intracellular Ca(2+) ([Ca(2+)]i) of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca(2+) responses. In addition, immepip-induced [Ca(2+)]i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons' responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation.

TRPV1 and PLC Participate in Histamine H4 Receptor-Induced Itch

Directory of Open Access Journals (Sweden)

Tunyu Jian

2016-01-01

Full Text Available Histamine H4 receptor has been confirmed to play a role in evoking peripheral pruritus. However, the ionic and intracellular signaling mechanism of activation of H4 receptor on the dorsal root ganglion (DRG neurons is still unknown. By using cell culture and calcium imaging, we studied the underlying mechanism of activation of H4 receptor on the DRG neuron. Immepip dihydrobromide (immepip—a histamine H4 receptor special agonist under cutaneous injection—obviously induced itch behavior of mice. Immepip-induced scratching behavior could be blocked by TRPV1 antagonist AMG9810 and PLC pathway inhibitor U73122. Application of immepip (8.3–50 μM could also induce a dose-dependent increase in intracellular Ca2+ (Ca2+i of DRG neurons. We found that 77.8% of the immepip-sensitized DRG neurons respond to the TRPV1 selective agonist capsaicin. U73122 could inhibit immepip-induced Ca2+ responses. In addition, immepip-induced Ca2+i increase could be blocked by ruthenium red, capsazepine, and AMG9810; however it could not be blocked by TRPA1 antagonist HC-030031. These results indicate that TRPV1 but not TRPA1 is the important ion channel to induce the DRG neurons’ responses in the downstream signaling pathway of histamine H4 receptor and suggest that TRPV1 may be involved in the mechanism of histamine-induced itch response by H4 receptor activation.

Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

Science.gov (United States)

Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

2014-01-01

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

Endogenous fatty acid ethanolamides suppress nicotine-induced activation of mesolimbic dopamine neurons through nuclear receptors.

Science.gov (United States)

Melis, Miriam; Pillolla, Giuliano; Luchicchi, Antonio; Muntoni, Anna Lisa; Yasar, Sevil; Goldberg, Steven R; Pistis, Marco

2008-12-17

Nicotine stimulates the activity of mesolimbic dopamine neurons, which is believed to mediate the rewarding and addictive properties of tobacco use. Accumulating evidence suggests that the endocannabinoid system might play a major role in neuronal mechanisms underlying the rewarding properties of drugs of abuse, including nicotine. Here, we investigated the modulation of nicotine effects by the endocannabinoid system on dopamine neurons in the ventral tegmental area with electrophysiological techniques in vivo and in vitro. We discovered that pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme that catabolizes fatty acid ethanolamides, among which the endocannabinoid anandamide (AEA) is the best known, suppressed nicotine-induced excitation of dopamine cells. Importantly, this effect was mimicked by the administration of the FAAH substrates oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), but not methanandamide, the hydrolysis resistant analog of AEA. OEA and PEA are naturally occurring lipid signaling molecules structurally related to AEA, but devoid of affinity for cannabinoid receptors. They blocked the effects of nicotine by activation of the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a nuclear receptor transcription factor involved in several aspects of lipid metabolism and energy balance. Activation of PPAR-alpha triggered a nongenomic stimulation of tyrosine kinases, which might lead to phosphorylation and negative regulation of neuronal nicotinic acetylcholine receptors. These data indicate for the first time that the anorexic lipids OEA and PEA possess neuromodulatory properties as endogenous ligands of PPAR-alpha in the brain and provide a potential new target for the treatment of nicotine addiction.

Interaction of GABAA receptors with purinergic P2X2 receptors

International Nuclear Information System (INIS)

Shrivastava, A.

2010-01-01

GABA A Rs in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X 2 Rs are also expressed in spinal cord neurons and are known to cross-talk with GABA A Rs. Here we investigated a possible 'dynamic' interaction between GABA A Rs and P2X 2 Rs using co-immunoprecipitation and FRET studies in HEK cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X 2 Rs forms a transient complex with GABA A Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface. P2X 2 Rs and GABA A Rs are then co-inserted into the cell membrane and are primarily located extra-synaptically. Furthermore, agonist induced activation of P2X 2 Rs results in disassembly of the receptor complex and destabilization of GABA A Rs whereas P2X 2 Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2XRs with other receptors could be used for specific targeting to the neuronal membrane, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA A Rs.(author) (author) [de

Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

Directory of Open Access Journals (Sweden)

Lasse Henriksen

Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

Science.gov (United States)

Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

2013-01-01

The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

Design of block-copolymer-based micelles for active and passive targeting

NARCIS (Netherlands)

Lebouille, Jérôme G J L; Leermakers, Frans A M; Cohen Stuart, Martien A.; Tuinier, Remco

2016-01-01

A self-consistent field study is presented on the design of active and passive targeting block-copolymeric micelles. These micelles form in water by self-assembly of triblock copolymers with a hydrophilic middle block and two hydrophobic outer blocks. A minority amount of diblock copolymers with the

Design of block-copolymer-based micelles for active and passive targeting

NARCIS (Netherlands)

Lebouille, Jérôme G.J.L.; Leermakers, Frans A.M.; Cohen Stuart, Martien A.; Tuinier, Remco

2016-01-01

A self-consistent field study is presented on the design of active and passive targeting block-copolymeric micelles. These micelles form in water by self-assembly of triblock copolymers with a hydrophilic middle block and two hydrophobic outer blocks. A minority amount of diblock copolymers with

A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

Energy Technology Data Exchange (ETDEWEB)

Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

2017-08-07

Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on H_CA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

Curcumin blocks interleukin-1 signaling in chondrosarcoma cells.

Directory of Open Access Journals (Sweden)

Thomas Kalinski

Full Text Available Interleukin (IL-1 signaling plays an important role in inflammatory processes, but also in malignant processes. The essential downstream event in IL-1 signaling is the activation of nuclear factor (NF-κB, which leads to the expression of several genes that are involved in cell proliferation, invasion, angiogenesis and metastasis, among them VEGF-A. As microenvironment-derived IL-1β is required for invasion and angiogenesis in malignant tumors, also in chondrosarcomas, we investigated IL-1β-induced signal transduction and VEGF-A expression in C3842 and SW1353 chondrosarcoma cells. We additionally performed in vitro angiogenesis assays and NF-κB-related gene expression analyses. Curcumin is a substance which inhibits IL-1 signaling very early by preventing the recruitment of IL-1 receptor associated kinase (IRAK to the IL-1 receptor. We demonstrate that IL-1 signaling and VEGF-A expression are blocked by Curcumin in chondrosarcoma cells. We further show that Curcumin blocks IL-1β-induced angiogenesis and NF-κB-related gene expression. We suppose that IL-1 blockade is an additional treatment option in chondrosarcoma, either by Curcumin, its derivatives or other IL-1 blocking agents.

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

Science.gov (United States)

Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

2008-01-01

Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

Leukotriene E4 induces airflow obstruction and mast cell activation through the cysteinyl leukotriene type 1 receptor.

Science.gov (United States)

Lazarinis, Nikolaos; Bood, Johan; Gomez, Cristina; Kolmert, Johan; Lantz, Ann-Sofie; Gyllfors, Pär; Davis, Andy; Wheelock, Craig E; Dahlén, Sven-Erik; Dahlén, Barbro

2018-03-05

Leukotriene (LT) E 4 is the final active metabolite among the cysteinyl leukotrienes (CysLTs). Animal studies have identified a distinct LTE 4 receptor, suggesting that current cysteinyl leukotriene type 1 (CysLT 1 ) receptor antagonists can provide incomplete inhibition of CysLT responses. We tested this hypothesis by assessing the influence of the CysLT 1 antagonist montelukast on responses induced by means of inhalation of LTE 4 in asthmatic patients. Fourteen patients with mild intermittent asthma and 2 patients with aspirin-exacerbated respiratory disease received 20 mg of montelukast twice daily and placebo for 5 to 7 days in a randomized, double-blind, crossover study (NCT01841164). The PD 20 value was determined at the end of each treatment period based on an increasing dose challenge. Measurements included lipid mediators in urine and sputum cells 4 hours after LTE 4 challenge. Montelukast completely blocked LTE 4 -induced bronchoconstriction. Despite tolerating an at least 10 times higher dose of LTE 4 after montelukast, there was no difference in the percentage of eosinophils in sputum. Urinary excretion of all major lipid mediators increased after LTE 4 inhalation. Montelukast blocked release of the mast cell product prostaglandin (PG) D 2 , as well as release of PGF 2α and thromboxane (Tx) A 2 , but not increased excretion of PGE 2 and its metabolites or isoprostanes. LTE 4 induces airflow obstruction and mast cell activation through the CysLT 1 receptor. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Selective pharmacological blockade of the 5-HT7 receptor attenuates light and 8-OH-DPAT induced phase shifts of mouse circadian wheel running activity

Directory of Open Access Journals (Sweden)

Jonathan eShelton

2015-01-01

Full Text Available Recent reports have illustrated a reciprocal relationship between circadian rhythm disruption and mood disorders. The 5-HT7 receptor may provide a crucial link between the two sides of this equation since the receptor plays a critical role in sleep, depression, and circadian rhythm regulation. To further define the role of the 5-HT7 receptor as a potential pharmacotherapy to correct circadian rhythm disruptions, the current study utilized the selective 5-HT7 antagonist JNJ-18038683 (10 mg/kg in three different circadian paradigms. While JNJ-18038683 was ineffective at phase shifting the onset of wheel running activity in mice when administered at different circadian time (CT points across the circadian cycle, pretreatment with JNJ-18038683 blocked non-photic phase advance (CT6 induced by the 5-HT1A/7 receptor agonist 8-OH-DPAT (3 mg/kg. Since light induced phase shifts in mammals are partially mediated via the modulation of the serotonergic system, we determined if JNJ-18038683 altered phase shifts induced by a light pulse at times known to phase delay (CT15 or advance (CT22 wheel running activity in free running mice. Light exposure resulted in a robust shift in the onset of activity in vehicle treated animals at both times tested. Administration of JNJ-18038683 significantly attenuated the light-induced phase delay and completely blocked the phase advance. The current study demonstrates that pharmacological blockade of the 5-HT7 receptor by JNJ-18038683 blunts both non-photic and photic phase shifts of circadian wheel running activity in mice. These findings highlight the importance of the 5-HT7 receptor in modulating circadian rhythms. Due to the opposite modulating effects of light resetting between diurnal and nocturnal species, pharmacotherapy targeting the 5-HT7 receptor in conjunction with bright light therapy may prove therapeutically beneficial by correcting the desynchronization of internal rhythms observed in depressed individuals.

Tamoxifen and ICI 182,780 activate hypothalamic G protein-coupled estrogen receptor 1 to rapidly facilitate lordosis in female rats.

Science.gov (United States)

Long, Nathan; Long, Bertha; Mana, Asma; Le, Dream; Nguyen, Lam; Chokr, Sima; Sinchak, Kevin

2017-03-01

In the female rat, sexual receptivity (lordosis) can be facilitated by sequential activation of estrogen receptor (ER) α and G protein-coupled estrogen receptor 1 (GPER) by estradiol. In the estradiol benzoate (EB) primed ovariectomized (OVX) rat, EB initially binds to ERα in the plasma membrane that complexes with and transactivates metabotropic glutamate receptor 1a to activate β-endorphin neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). This activates MPN μ-opioid receptors (MOP), inhibiting lordosis. Infusion of non-esterified 17β-estradiol into the ARH rapidly reduces MPN MOP activation and facilitates lordosis via GPER. Tamoxifen (TAM) and ICI 182,780 (ICI) are selective estrogen receptor modulators that activate GPER. Therefore, we tested the hypothesis that TAM and ICI rapidly facilitate lordosis via activation of GPER in the ARH. Our first experiment demonstrated that injection of TAM intraperitoneal, or ICI into the lateral ventricle, deactivated MPN MOP and facilitated lordosis in EB-primed rats. We then tested whether TAM and ICI were acting rapidly through a GPER dependent pathway in the ARH. In EB-primed rats, ARH infusion of either TAM or ICI facilitated lordosis and reduced MPN MOP activation within 30min compared to controls. These effects were blocked by pretreatment with the GPER antagonist, G15. Our findings demonstrate that TAM and ICI deactivate MPN MOP and facilitate lordosis in a GPER dependent manner. Thus, TAM and ICI may activate GPER in the CNS to produce estrogenic actions in neural circuits that modulate physiology and behavior. Published by Elsevier Inc.

Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion.

Directory of Open Access Journals (Sweden)

Haifeng Wang

Full Text Available Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF activation and ultimately interferon (IFN production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV, which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1/IkappaB kinase-epsilon (IKKepsilon, the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKepsilon activity by disrupting the interaction between IKKepsilon and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKepsilon activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.

Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

International Nuclear Information System (INIS)

Miao, H.-Q.; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping

2006-01-01

Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies

Production of antibodies which recognize opiate receptors on murine leukocytes

Energy Technology Data Exchange (ETDEWEB)

Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

1988-01-01

An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

Activation of glucocorticoid receptors increases 5-HT2A receptor levels

DEFF Research Database (Denmark)

Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa

2009-01-01

an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased......Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker...... of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish...

Designer interface peptide grafts target estrogen receptor alpha dimerization

International Nuclear Information System (INIS)

Chakraborty, S.; Asare, B.K.; Biswas, P.K.; Rajnarayanan, R.V.

2016-01-01

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide “I-box” derived from ER residues 503–518 specifically blocks ER dimerization. Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479–485), LQQQHQRLAQ (residues 497–506), and LSHIRHMSNK (residues 511–520) and reported the suitability of using LQQQHQRLAQ (ER 497–506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. - Highlights: • Designer peptide grafts retain core molecular recognition motif during MD simulations. • Designer peptide grafts with Poly-ALA helix form stable

Designer interface peptide grafts target estrogen receptor alpha dimerization

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, S. [Laboratory of Computational Biophysics & Bioengineering, Department of Physics, Tougaloo College, Tougaloo, MS 39174 (United States); Asare, B.K. [Department of Pharmacology and Toxicology, University of Buffalo, Buffalo, NY 14214 (United States); Biswas, P.K., E-mail: pbiswas@tougaloo.edu [Laboratory of Computational Biophysics & Bioengineering, Department of Physics, Tougaloo College, Tougaloo, MS 39174 (United States); Rajnarayanan, R.V., E-mail: rajendra@buffalo.edu [Department of Pharmacology and Toxicology, University of Buffalo, Buffalo, NY 14214 (United States)

2016-09-09

The nuclear transcription factor estrogen receptor alpha (ERα), triggered by its cognate ligand estrogen, regulates a variety of cellular signaling events. ERα is expressed in 70% of breast cancers and is a widely validated target for anti-breast cancer drug discovery. Administration of anti-estrogen to block estrogen receptor activation is still a viable anti-breast cancer treatment option but anti-estrogen resistance has been a significant bottle-neck. Dimerization of estrogen receptor is required for ER activation. Blocking ERα dimerization is therefore a complementary and alternative strategy to combat anti-estrogen resistance. Dimer interface peptide “I-box” derived from ER residues 503–518 specifically blocks ER dimerization. Recently using a comprehensive molecular simulation we studied the interaction dynamics of ERα LBDs in a homo-dimer. Based on this study, we identified three interface recognition peptide motifs LDKITDT (ERα residues 479–485), LQQQHQRLAQ (residues 497–506), and LSHIRHMSNK (residues 511–520) and reported the suitability of using LQQQHQRLAQ (ER 497–506) as a template to design inhibitors of ERα dimerization. Stability and self-aggregation of peptide based therapeutics poses a significant bottle-neck to proceed further. In this study utilizing peptide grafted to preserve their pharmacophoric recognition motif and assessed their stability and potential to block ERα mediated activity in silico and in vitro. The Grafted peptides blocked ERα mediated cell proliferation and viability of breast cancer cells but did not alter their apoptotic fate. We believe the structural clues identified in this study can be used to identify novel peptidometics and small molecules that specifically target ER dimer interface generating a new breed of anti-cancer agents. - Highlights: • Designer peptide grafts retain core molecular recognition motif during MD simulations. • Designer peptide grafts with Poly-ALA helix form stable

Deflation-activated receptors, not classical inflation-activated receptors, mediate the Hering-Breuer deflation reflex.

Science.gov (United States)

Yu, Jerry

2016-11-01

Many airway sensory units respond to both lung inflation and deflation. Whether those responses to opposite stimuli come from one sensor (one-sensor theory) or more than one sensor (multiple-sensor theory) is debatable. One-sensor theory is commonly presumed in the literature. This article proposes a multiple-sensor theory in which a sensory unit contains different sensors for sensing different forces. Two major types of mechanical sensors operate in the lung: inflation- and deflation-activated receptors (DARs). Inflation-activated sensors can be further divided into slowly adapting receptors (SARs) and rapidly adapting receptors (RARs). Many SAR and RAR units also respond to lung deflation because they contain DARs. Pure DARs, which respond to lung deflation only, are rare in large animals but are easily identified in small animals. Lung deflation-induced reflex effects previously attributed to RARs should be assigned to DARs (including pure DARs and DARs associated with SARs and RARs) if the multiple-sensor theory is accepted. Thus, based on the information, it is proposed that activation of DARs can attenuate lung deflation, shorten expiratory time, increase respiratory rate, evoke inspiration, and cause airway secretion and dyspnea.

Dynamic transition on the seizure-like neuronal activity by astrocytic calcium channel block

International Nuclear Information System (INIS)

Li, Jiajia; Wang, Rong; Du, Mengmeng; Tang, Jun; Wu, Ying

2016-01-01

The involvement of astrocytes in neuronal firing dynamics is becoming increasingly evident. In this study, we used a classical hippocampal tripartite synapse model consisting of soma-dendrite coupled neuron models and a Hodgkin–Huxley-like astrocyte model, to investigate the seizure-like firing in the somatic neuron induced by the over-expressed neuronal N-methyl-d-aspartate (NMDA) receptors. Based on this model, we further investigated the effect of the astrocytic channel block on the neuronal firing through a bifurcation analysis. Results show that blocking inositol-1,4,5-triphosphate(IP3)-dependent calcium channel in astrocytes efficiently suppresses the astrocytic calcium oscillation, which in turn suppresses the seizure-like firing in the neuron.

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

Science.gov (United States)

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

2007-05-01

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

Role of Dopamine Receptors in the Anticancer Activity of ONC201.

Science.gov (United States)

Kline, Christina Leah B; Ralff, Marie D; Lulla, Amriti R; Wagner, Jessica M; Abbosh, Phillip H; Dicker, David T; Allen, Joshua E; El-Deiry, Wafik S

2018-01-01

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201's interaction with DRD2 plays a role in ONC201's anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type-specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201's anticancer effects. Although ONC201's anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201's anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201's ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity. Copyright © 2018 The Authors

Role of Dopamine Receptors in the Anticancer Activity of ONC201

Directory of Open Access Journals (Sweden)

Christina Leah B. Kline

2018-01-01

Full Text Available ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201’s interaction with DRD2 plays a role in ONC201’s anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type–specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201’s anticancer effects. Although ONC201’s anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201’s anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201’s ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity.

Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

Directory of Open Access Journals (Sweden)

Yasuko Kitagishi

2013-10-01

Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

Energy Technology Data Exchange (ETDEWEB)

Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

2013-10-21

Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

International Nuclear Information System (INIS)

Matsuda, Satoru; Kitagishi, Yasuko

2013-01-01

Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

Science.gov (United States)

Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

Fear potentiated startle increases phospholipase D (PLD) expression/activity and PLD-linked metabotropic glutamate receptor mediated post-tetanic potentiation in rat amygdala.

Science.gov (United States)

Krishnan, Balaji; Scott, Michael T; Pollandt, Sebastian; Schroeder, Bradley; Kurosky, Alexander; Shinnick-Gallagher, Patricia

2016-02-01

Long-term memory (LTM) of fear stores activity dependent modifications that include changes in amygdala signaling. Previously, we identified an enhanced probability of release of glutamate mediated signaling to be important in rat fear potentiated startle (FPS), a well-established translational behavioral measure of fear. Here, we investigated short- and long-term synaptic plasticity in FPS involving metabotropic glutamate receptors (mGluRs) and associated downstream proteomic changes in the thalamic-lateral amygdala pathway (Th-LA). Aldolase A, an inhibitor of phospholipase D (PLD), expression was reduced, concurrent with significantly elevated PLD protein expression. Blocking the PLD-mGluR signaling significantly reduced PLD activity. While transmitter release probability increased in FPS, PLD-mGluR agonist and antagonist actions were occluded. In the unpaired group (UNP), blocking the PLD-mGluR increased while activating the receptor decreased transmitter release probability, consistent with decreased synaptic potentials during tetanic stimulation. FPS Post-tetanic potentiation (PTP) immediately following long-term potentiation (LTP) induction was significantly increased. Blocking PLD-mGluR signaling prevented PTP and reduced cumulative PTP probability but not LTP maintenance in both groups. These effects are similar to those mediated through mGluR7, which is co-immunoprecipitated with PLD in FPS. Lastly, blocking mGluR-PLD in the rat amygdala was sufficient to prevent behavioral expression of fear memory. Thus, our study in the Th-LA pathway provides the first evidence for PLD as an important target of mGluR signaling in amygdala fear-associated memory. Importantly, the PLD-mGluR provides a novel therapeutic target for treating maladaptive fear memories in posttraumatic stress and anxiety disorders. Published by Elsevier Inc.

Dreamlike effects of LSD on waking imagery in humans depend on serotonin 2A receptor activation.

Science.gov (United States)

Kraehenmann, Rainer; Pokorny, Dan; Vollenweider, Leonie; Preller, Katrin H; Pokorny, Thomas; Seifritz, Erich; Vollenweider, Franz X

2017-07-01

Accumulating evidence indicates that the mixed serotonin and dopamine receptor agonist lysergic acid diethylamide (LSD) induces an altered state of consciousness that resembles dreaming. This study aimed to test the hypotheses that LSD produces dreamlike waking imagery and that this imagery depends on 5-HT2A receptor activation and is related to subjective drug effects. Twenty-five healthy subjects performed an audiorecorded guided mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). Cognitive bizarreness of guided mental imagery reports was quantified as a standardised formal measure of dream mentation. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) questionnaire. LSD, compared with placebo, significantly increased cognitive bizarreness (p < 0.001). The LSD-induced increase in cognitive bizarreness was positively correlated with the LSD-induced loss of self-boundaries and cognitive control (p < 0.05). Both LSD-induced increases in cognitive bizarreness and changes in state of consciousness were fully blocked by ketanserin. LSD produced mental imagery similar to dreaming, primarily via activation of the 5-HT2A receptor and in relation to loss of self-boundaries and cognitive control. Future psychopharmacological studies should assess the differential contribution of the D2/D1 and 5-HT1A receptors to cognitive bizarreness.

Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

DEFF Research Database (Denmark)

Thiele, Stefanie; Rosenkilde, Mette Marie

2014-01-01

and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity......-contributing interactions are attributed to different domains and known to occur in two steps. Here, knowledge on chemokine and receptor domains involved in the first binding-step and the second activation-step is reviewed. A mechanism comprising at least two steps seems consistent; however, several intermediate...... interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly...

Dopamine receptor activation increases HIV entry into primary human macrophages.

Directory of Open Access Journals (Sweden)

Peter J Gaskill

Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

BDNF Up-Regulates α7 Nicotinic Acetylcholine Receptor Levels on Subpopulations of Hippocampal Interneurons

Science.gov (United States)

Massey, Kerri A.; Zago, Wagner M.; Berg, Darwin K.

2006-01-01

In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of α7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABAA receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased α7-nAChR clusters were most prominent on interneuron subtypes known to innervate directly excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling α7-nAChR levels. PMID:17029981

The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

Science.gov (United States)

Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

2014-01-01

Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

Stress-restress evokes sustained iNOS activity and altered GABA levels and NMDA receptors in rat hippocampus

DEFF Research Database (Denmark)

Harvey, Brian H; Oosthuizen, Frasia; Brand, Linda

2004-01-01

. The NOS isoform involved, and the role of stress-mediated corticosterone release in NOS activation, was verified with the administration of selective iNOS and nNOS inhibitors, aminoguanidine (50 mg/kg/day i.p.) and 7-nitroindazole (12.5 mg/kg/day i.p.), and the steroid synthesis inhibitor, ketoconazole...... (24 mg/kg/day i.p.), administered for 21 days prior to and during the stress procedure. RESULTS: Stress evoked a sustained increase in NOS activity, but reduced NMDA receptor density and total GABA levels. Aminoguanidine or ketoconazole, but not 7-nitroindazole or saline, blocked stress-induced NOS...

An immunocapture/scintillation proximity analysis of G alpha q/11 activation by native serotonin (5-HT)2A receptors in rat cortex: blockade by clozapine and mirtazapine.

Science.gov (United States)

Mannoury La Cour, C; Chaput, C; Touzard, M; Millan, M J

2009-02-01

Though transduction mechanisms recruited by heterologously expressed 5-HT(2A) receptors have been extensively studied, their interaction with specific subtypes of G-protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5-HT, the prototypical 5-HT(2A) agonist, DOI, and Ro60,0175 all enhanced [(35)S]GTPgammaS binding to G alpha q/11 in rat cortex with pEC(50) values of 6.22, 7.24 and 6.35, respectively. No activation of G o or G s/olf was seen at equivalent concentrations of DOI. Stimulation of G alpha q/11 by 5-HT (30 microM) and DOI (30 microM) was abolished by the selective 5-HT(2A) vs. 5-HT(2C)/5-HT(2B) antagonists, ketanserin (pK(B) values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5-HT-induced [(35)S]GTPgammaS binding to G alpha q/11 was only weakly inhibited by the preferential 5-HT(2C) receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5-HT(2B) receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5-HT(2A) receptors, blocked the recruitment of G alpha q/11 by 5-HT and DOI with pK(B) values of 8.54 and 8.14, respectively. Its actions were mimicked by the "atypical" antidepressant and 5-HT(2A) receptor antagonist, mirtazapine, which likewise blocked 5-HT and DOI-induced G alpha q/11 protein activation with pK(B) values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5-HT(2A) receptors in rat frontal cortex specifically recruit G alpha q/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5-HT(2A) receptor-mediated G alpha q/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent. 2008 Wiley-Liss, Inc.

Acute Cocaine Induces Fast Activation of D1 Receptor and Progressive Deactivation of D2 Receptor Strial Neurons: In Vivo Optical Microprobe [Ca(superscript)2+]subscript)i Imaging

Energy Technology Data Exchange (ETDEWEB)

Du, C.; Luo, Z.; Volkow, N.D.; Heintz, N.; Pan, Y.; Du, C.

2011-09-14

Cocaine induces fast dopamine increases in brain striatal regions, which are recognized to underlie its rewarding effects. Both dopamine D1 and D2 receptors are involved in cocaine's reward but the dynamic downstream consequences of cocaine effects in striatum are not fully understood. Here we used transgenic mice expressing EGFP under the control of either the D1 receptor (D1R) or the D2 receptor (D2R) gene and microprobe optical imaging to assess the dynamic changes in intracellular calcium ([Ca{sup 2+}]{sub i} ) responses (used as marker of neuronal activation) to acute cocaine in vivo separately for D1R- versus D2R-expressing neurons in striatum. Acute cocaine (8 mg/kg, i.p.) rapidly increased [Ca{sup 2+}]{sub i} in D1R-expressing neurons (10.6 {+-} 3.2%) in striatum within 8.3 {+-} 2.3 min after cocaine administration after which the increases plateaued; these fast [Ca{sup 2+}]{sub i} increases were blocked by pretreatment with a D1R antagonist (SCH23390). In contrast, cocaine induced progressive decreases in [Ca{sup 2+}]{sub i} in D2R-expressing neurons (10.4 {+-} 5.8%) continuously throughout the 30 min that followed cocaine administration; these slower [Ca{sup 2+}]{sub i} decreases were blocked by pretreatment with a D2R antagonist (raclopride). Since activation of striatal D1R-expressing neurons (direct-pathway) enhances cocaine reward, whereas activation of D2R expressing neurons suppresses it (indirect-pathway) (Lobo et al., 2010), this suggests that cocaine's rewarding effects entail both its fast stimulation ofD1R (resulting in abrupt activation of direct-pathway neurons) and a slower stimulation of D2R (resulting in longer-lasting deactivation of indirect-pathway neurons). We also provide direct in vivo evidence of D2R and D1R interactions in the striatal responses to acute cocaine administration.

A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation.

Directory of Open Access Journals (Sweden)

Bridgette Janine Connell

Full Text Available Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1-7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS as well as to an antibody (mAb 17b directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1.

β-Adrenergic Receptor Mediation of Stress-Induced Reinstatement of Extinguished Cocaine-Induced Conditioned Place Preference in Mice: Roles for β1 and β2 Adrenergic Receptors

Science.gov (United States)

Vranjkovic, Oliver; Hang, Shona; Baker, David A.

2012-01-01

Stress can trigger the relapse of drug use in recovering cocaine addicts and reinstatement in rodent models through mechanisms that may involve norepinephrine release and β-adrenergic receptor activation. The present study examined the role of β-adrenergic receptor subtypes in the stressor-induced reinstatement of extinguished cocaine-induced (15 mg/kg i.p.) conditioned place preference in mice. Forced swim (6 min at 22°C) stress or activation of central noradrenergic neurotransmission by administration of the selective α2 adrenergic receptor antagonist 2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL-44,408) (10 mg/kg i.p.) induced reinstatement in wild-type, but not β- adrenergic receptor-deficient Adrb1/Adrb2 double-knockout, mice. In contrast, cocaine administration (15 mg/kg i.p.) resulted in reinstatement in both wild-type and β-adrenergic receptor knockout mice. Stress-induced reinstatement probably involved β2 adrenergic receptors. The β2 adrenergic receptor antagonist -(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI-118,551) (1 or 2 mg/kg i.p.) blocked reinstatement by forced swim or BRL-44,408, whereas administration of the nonselective β-adrenergic receptor agonist isoproterenol (2 or 4 mg/kg i.p.) or the β2 adrenergic receptor-selective agonist clenbuterol (2 or 4 mg/kg i.p.) induced reinstatement. Forced swim-induced, but not BRL-44,408-induced, reinstatement was also blocked by a high (20 mg/kg) but not low (10 mg/kg) dose of the β1 adrenergic receptor antagonist betaxolol, and isoproterenol-induced reinstatement was blocked by pretreatment with either ICI-118,551 or betaxolol, suggesting a potential cooperative role for β1 and β2 adrenergic receptors in stress-induced reinstatement. Overall, these findings suggest that targeting β-adrenergic receptors may represent a promising pharmacotherapeutic strategy for preventing drug relapse, particularly in cocaine addicts whose drug use is stress

Enhancement of bradykinin and resensitization of its B2 receptor.

Science.gov (United States)

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

1999-03-01

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

Comparison of the effectiveness and safety between lymphocytes scavenger and IL-2 receptor blocking agent induction in living kidney transplantation

Directory of Open Access Journals (Sweden)

Ning-bo QIN

2013-03-01

Full Text Available Objective 　To compare the safety of two antibody inductors, namely lymphocytes scavenger and IL-2 receptor blocking agent, in living kidney transplantation. Methods 　The data of 191 patients, who received living kidney transplant in our hospital from Feb. 2007 to Jul. 2012, were retrospectively analyzed, and grouped according to the inductors they received as: a lymphocytes scavenger group (n=56, with rabbit antithymocyte immunoglobulin (rATG, 4 cases and porcine antihuman T-lymphocyte immunoglobulin (pATG, 52 cases served as the inductor; b IL -2 receptor blocking agent group (n=54, with basiliximab (40 cases and daclizumab (14 cases served as the inductor; and c control group (n=81. The incidence of rejection and infection, and the survival rate of patient/allograft within one year were then compared among the three groups. Results 　Within one year after the transplantation, the incidence of acute rejection in lymphocytes scavenger group, IL-2 receptor blocking agent group and control group was 12.5%, 11.1% and 28.4%, respectively. There was a significant difference between the two inductor groups and control group (P=0.003, but no significant difference was found between the two inductor groups (P>0.05. The incidence of delayed graft function (DGF in the three groups was 8.9%, 7.4% and 13.6%, respectively, with no statistical significance (P>0.05. Also there was no significant difference among the three groups in the incidence of infection and the survival rate of patient/allograft within one year after transplantation (P>0.05. Conclusion 　Both inductors may significantly reduce the incidence of acute rejection within one year without increasing the incidence of infection and other adverse events, nor affect the postoperative patient/graft survival, so they are both safe and effective.

Histamine Excites Rat Superior Vestibular Nuclear Neurons via Postsynaptic H1 and H2 Receptors in vitro

Directory of Open Access Journals (Sweden)

Qian-Xing Zhuang

2012-09-01

Full Text Available The superior vestibular nucleus (SVN, which holds a key position in vestibulo-ocular reflexes and nystagmus, receives direct hypothalamic histaminergic innervations. By using rat brainstem slice preparations and extracellular unitary recordings, we investigated the effect of histamine on SVN neurons and the underlying receptor mechanisms. Bath application of histamine evoked an excitatory response of the SVN neurons, which was not blocked by the low-Ca2+/high-Mg2+ medium, indicating a direct postsynaptic effect of the amine. Selective histamine H1 receptor agonist 2-pyridylethylamine and H2 receptor agonist dimaprit, rather than VUF8430, a selective H4 receptor agonist, mimicked the excitation of histamine on SVN neurons. In addition, selective H1 receptor antagonist mepyramine and H2 receptor antagonist ranitidine, but not JNJ7777120, a selective H4 receptor antagonist, partially blocked the excitatory response of SVN neurons to histamine. Moreover, mepyramine together with ranitidine nearly totally blocked the histamine-induced excitation. Immunostainings further showed that histamine H1 and H2 instead of H4 receptors existed in the SVN. These results demonstrate that histamine excites the SVN neurons via postsynaptic histamine H1 and H2 receptors, and suggest that the central histaminergic innervation from the hypothalamus may actively bias the SVN neuronal activity and subsequently modulate the SVN-mediated vestibular functions and gaze control.

Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

International Nuclear Information System (INIS)

Dai, Guodong; Peng, Tao; Zhou, Xuhong; Zhu, Jun; Kong, Zhihua; Ma, Li; Xiong, Zhi; Yuan, Yulin

2013-01-01

Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and

Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

Energy Technology Data Exchange (ETDEWEB)

Dai, Guodong [Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071 (China); Peng, Tao; Zhou, Xuhong [Department of Otolaryngology-Head and Neck Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Zhu, Jun; Kong, Zhihua; Ma, Li; Xiong, Zhi [Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071 (China); Yuan, Yulin, E-mail: yuanyulin19620120@126.com [Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071 (China)

2013-11-01

Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

Science.gov (United States)

Yao, Yongxue; Harrison, Kathleen A; Al-Hassani, Mohammed; Murphy, Robert C; Rezania, Samin; Konger, Raymond L; Travers, Jeffrey B

2012-03-16

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

Science.gov (United States)

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Activation of β-Adrenoceptors by Dobutamine May Induce a Higher Expression of Peroxisome Proliferator-Activated Receptors δ (PPARδ in Neonatal Rat Cardiomyocytes

Directory of Open Access Journals (Sweden)

Ming-Ting Chou

2012-01-01

Full Text Available Recent evidence showed the role of peroxisome proliferator-activated receptors (PPARs in cardiac function. Cardiac contraction induced by various agents is critical in restoring the activity of peroxisome proliferator-activated receptors δ (PPARδ in cardiac myopathy. Because dobutamine is an agent widely used to treat heart failure in emergency setting, this study is aimed to investigate the change of PPARδ in response to dobutamine. Neonatal rat cardiomyocytes were used to examine the effects of dobutamine on PPARδ expression levels and cardiac troponin I (cTnI phosphorylation via Western blotting analysis. We show that treatment with dobutamine increased PPARδ expression and cTnI phosphorylation in a time- and dose-dependent manner in neonatal rat cardiomyocytes. These increases were blocked by the antagonist of β1-adrenoceptors. Also, the action of dobutamine was related to the increase of calcium ions and diminished by chelating intracellular calcium. Additionally, dobutamine-induced action was reduced by the inhibition of downstream messengers involved in this calcium-related pathway. Moreover, deletion of PPARδ using siRNA generated the reduction of cTnI phosphorylation in cardiomyocytes treated with dobutamine. Thus, we concluded that PPARδ is increased by dobutamine in cardiac cells.

Pharmacological Modulation of 5-HT2C Receptor Activity Produces Bidirectional Changes in Locomotor Activity, Responding for a Conditioned Reinforcer, and Mesolimbic DA Release in C57BL/6 Mice.

Science.gov (United States)

Browne, Caleb J; Ji, Xiaodong; Higgins, Guy A; Fletcher, Paul J; Harvey-Lewis, Colin

2017-10-01

Converging lines of behavioral, electrophysiological, and biochemical evidence suggest that 5-HT 2C receptor signaling may bidirectionally influence reward-related behavior through an interaction with the mesolimbic dopamine (DA) system. Here we directly test this hypothesis by examining how modulating 5-HT 2C receptor activity affects DA-dependent behaviors and relate these effects to changes in nucleus accumbens (NAc) DA release. In C57BL/6 mice, locomotor activity and responding for a conditioned reinforcer (CRf), a measure of incentive motivation, were examined following treatment with three 5-HT 2C receptor ligands: the agonist CP809101 (0.25-3 mg/kg), the antagonist SB242084 (0.25-1 mg/kg), or the antagonist/inverse agonist SB206553 (1-5 mg/kg). We further tested whether doses of these compounds that changed locomotor activity and responding for a CRf (1 mg/kg CP809101, 0.5 mg/kg SB242084, or 2.5 mg/kg SB206553) also altered NAc DA release using in vivo microdialysis in anesthetized mice. CP809101 reduced locomotor activity, responding for a CRf, and NAc DA release. In contrast, both SB242084 and SB206553 enhanced locomotor activity, responding for a CRf, and NAc DA release, although higher doses of SB206553 produced opposite behavioral effects. Pretreatment with the non-selective DA receptor antagonist α-flupenthixol prevented SB242084 from enhancing responding for a CRf. Thus blocking tonic 5-HT 2C receptor signaling can release serotonergic inhibition of mesolimbic DA activity and enhance reward-related behavior. The observed bidirectional effects of 5-HT 2C receptor ligands may have important implications when considering the 5-HT 2C receptor as a therapeutic target for psychiatric disorders, particularly those presenting with motivational dysfunctions.

Differential trafficking of AMPA receptors following activation of NMDA receptors and mGluRs

Directory of Open Access Journals (Sweden)

Sanderson Thomas M

2011-07-01

Full Text Available Abstract The removal of AMPA receptors from synapses is a major component of long-term depression (LTD. How this occurs, however, is still only partially understood. To investigate the trafficking of AMPA receptors in real-time we previously tagged the GluA2 subunit of AMPA receptors with ecliptic pHluorin and studied the effects of NMDA receptor activation. In the present study we have compared the effect of NMDA receptor and group I mGluR activation, using GluA2 tagged with super ecliptic pHluorin (SEP-GluA2 expressed in cultured hippocampal neurons. Surprisingly, agonists of the two receptors, which are both able to induce chemical forms of LTD, had clearly distinct effects on AMPA receptor trafficking. In agreement with our previous work we found that transient NMDA receptor activation results in an initial decrease in surface GluA2 from extrasynaptic sites followed by a delayed reduction in GluA2 from puncta (putative synapses. In contrast, transient activation of group I mGluRs, using DHPG, led to a pronounced but more delayed decrease in GluA2 from the dendritic shafts. Surprisingly, there was no average change in the fluorescence of the puncta. Examination of fluorescence at individual puncta, however, indicated that alterations did take place, with some puncta showing an increase and others a decrease in fluorescence. The effects of DHPG were, like DHPG-induced LTD, prevented by treatment with a protein tyrosine phosphatase (PTP inhibitor. The electrophysiological correlate of the effects of DHPG in the SEP-GluA2 infected cultures was a reduction in mEPSC frequency with no change in amplitude. The implications of these findings for the initial mechanisms of expression of both NMDA receptor- and mGluR-induced LTD are discussed.

Activation of Adenosine Receptor A2A Increases HSC Proliferation and Inhibits Death and Senescence by Down-regulation of p53 and Rb

Directory of Open Access Journals (Sweden)

Md. Kaimul eAhsan

2014-04-01

Full Text Available Background & Aims: During fibrosis hepatic stellate cells (HSC undergo activation, proliferation and senescence but the regulation of these important processes is poorly understood. The adenosine A2A receptor (A2A is known to be present on HSC, and its activation results in liver fibrosis. In this study, we tested if A2A has a role in the regulation of HSC proliferation, apoptosis, senescence, and the relevant molecular mechanism.Methods: The ability of adenosine to regulate p53 and Rb protein levels, proliferation, apoptosis and senescence was tested in the human HSC cell line LX-2 and rat primary HSC.Results: Adenosine receptor activation down-regulates p53 and Rb protein levels, increases BrdU incorporation and increases cell survival in LX-2 cells and in primary rat HSC. These effects of NECA were reproduced by an adenosine A2A receptor specific agonist (CGS21680 and blocked by a specific antagonist (ZM241385. By day twenty-one of culture primary rat HSC entered senescence and expressed -gal which was significantly inhibited by NECA. Furthermore, NECA induced down regulation of p53 and Rb and Rac1, and decreased phosphorylation of p44-42 MAP Kinase in LX-2 cells and primary rat HSC. These effects were reproduced by the cAMP analog 8-Bromo-cAMP, and the adenylyl cyclase activator forskolin, and were blocked by PKA inhibitors.Conclusions: These results demonstrate that A2A receptor regulates a number of HSC fate decisions and induces greater HSC proliferation, reduces apoptosis and senescence by decreasing p53 and Rb through cAMP-PKA/Rac1/p38 MAPK pathway. This provides a mechanism for adenosine induced HSC regulation and liver fibrosis.

Silver-enhanced block copolymer membranes with biocidal activity

KAUST Repository

Madhavan, Poornima

2014-11-12

Silver nanoparticles were deposited on the surface and pore walls of block copolymer membranes with highly ordered pore structure. Pyridine blocks constitute the pore surfaces, complexing silver ions and promoting a homogeneous distribution. Nanoparticles were then formed by reduction with sodium borohydride. The morphology varied with the preparation conditions (pH and silver ion concentration), as confirmed by field emission scanning and transmission electron microscopy. Silver has a strong biocide activity, which for membranes can bring the advantage of minimizing the growth of bacteria and formation of biofilm. The membranes with nanoparticles prepared under different pH values and ion concentrations were incubated with Pseudomonas aeruginosa and compared with the control. The strongest biocidal activity was achieved with membranes containing membranes prepared under pH 9. Under these conditions, the best distribution with small particle size was observed by microscopy.

Silver-enhanced block copolymer membranes with biocidal activity

KAUST Repository

Madhavan, Poornima; Hong, Pei-Ying; Sougrat, Rachid; Nunes, Suzana Pereira

2014-01-01

Silver nanoparticles were deposited on the surface and pore walls of block copolymer membranes with highly ordered pore structure. Pyridine blocks constitute the pore surfaces, complexing silver ions and promoting a homogeneous distribution. Nanoparticles were then formed by reduction with sodium borohydride. The morphology varied with the preparation conditions (pH and silver ion concentration), as confirmed by field emission scanning and transmission electron microscopy. Silver has a strong biocide activity, which for membranes can bring the advantage of minimizing the growth of bacteria and formation of biofilm. The membranes with nanoparticles prepared under different pH values and ion concentrations were incubated with Pseudomonas aeruginosa and compared with the control. The strongest biocidal activity was achieved with membranes containing membranes prepared under pH 9. Under these conditions, the best distribution with small particle size was observed by microscopy.

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae

DEFF Research Database (Denmark)

Schöller, Susanne; Belmont, Martin; Cazzamali, Giuseppe

2005-01-01

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two...... previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10...... identification of a mosquito neuropeptide receptor....

Morphine withdrawal enhances constitutive μ-opioid receptor activity in the ventral tegmental area.

Science.gov (United States)

Meye, Frank J; van Zessen, Ruud; Smidt, Marten P; Adan, Roger A H; Ramakers, Geert M J

2012-11-14

μ-Opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive activity is highly relevant in the mouse VTA, as it regulates GABAergic input to dopamine neurons. Specifically, suppression of MOR constitutive activity with the inverse agonist KC-2-009 enhanced GABAergic neurotransmission onto VTA dopamine neurons. This inverse agonistic effect was fully blocked by the specific MOR neutral antagonist CTOP, which had no effect on GABAergic transmission itself. We next show that withdrawal from chronic morphine further increases the magnitude of inverse agonistic effects at the MOR, suggesting enhanced MOR constitutive activity. We demonstrate that this increase can be an adaptive response to the detrimental elevation in cAMP levels known to occur during morphine withdrawal. These findings offer important insights in the physiological occurrence and function of MOR constitutive activity, and have important implications for therapeutic strategies aimed at normalizing MOR signaling during addiction and opioid overdose.

Waking action of ursodeoxycholic acid (UDCA involves histamine and GABAA receptor block.

Directory of Open Access Journals (Sweden)

Yevgenij Yanovsky

Full Text Available Since ancient times ursodeoxycholic acid (UDCA, a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer's disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA(AR antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC(50 = 70 µM and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA(A receptors composed of α1, β1-3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA(A receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA(AR potentiation by several neurosteroids, had no effect on GABA(AR inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA(A receptors.

PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

Directory of Open Access Journals (Sweden)

Hyo-Sup Kim

Full Text Available BACKGROUND: It has been reported that peroxisome proliferator-activated receptor (PPAR-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells. METHODS: Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ. RESULTS: PPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION: These observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

Lamotrigine blocks NMDA receptor-initiated arachidonic acid signalling in rat brain: Implications for its efficacy in bipolar disorder

Science.gov (United States)

Ramadan, Epolia; Basselin, Mireille; Rao, Jagadeesh S.; Chang, Lisa; Chen, Mei; Ma, Kaizong; Rapoport, Stanley I.

2011-01-01

An upregulated brain arachidonic acid (AA) cascade and a hyperglutamatergic state characterize bipolar disorder (BD). Lamotrigine (LTG), a mood stabilizer approved for treating BD, is reported to interfere with glutamatergic neurotransmission involving N-methyl-D-aspartate receptors (NMDARs). NMDARs allow extracellular calcium into the cell, thereby stimulating calcium-dependent cytosolic phospholipase A2 (cPLA2) to release arachidonic acid (AA) from membrane phospholipid. We hypothesized that LTG, like other approved mood stabilizers, would reduce NMDAR-mediated AA signaling in rat brain. An acute subconvulsant dose of NMDA (25 mg/kg) or saline was administered intraperitoneally to unanesthetized rats that had been treated p.o. daily for 42 days with vehicle or a therapeutically relevant dose of LTG (10 mg/kg/.d). Regional brain AA incorporation coefficients k* and rates Jin, AA signals, were measured using quantitative autoradiography after intravenous [1-14C]AA infusion, as were other AA cascade markers. In chronic vehicle-treated rats, acute NMDA compared to saline increased k* and Jin in widespread regions of the brain, as well as prostaglandin (PG)E2 and thromboxane B2 concentrations. Chronic LTG treatment compared to vehicle reduced brain cyclooxygenase (COX) activity, PGE2 concentration, and DNA binding activity of the COX-2 transcription factor, NF-κB. Pretreatment with chronic LTG blocked the acute NMDA effects on AA cascade markers. In summary, chronic LTG like other mood stabilizers blocks NMDA-mediated signaling involving the AA metabolic cascade. Since markers of the AA cascade and of NMDAR signaling are up-regulated in the postmortem BD brain, mood stabilizers generally may be effective in BD by dampening NMDAR signalling and the AA cascade. PMID:21733229

Exercise restores decreased physical activity levels and increases markers of autophagy and oxidative capacity in myostatin/activin-blocked mdx mice.

Science.gov (United States)

Hulmi, Juha J; Oliveira, Bernardo M; Silvennoinen, Mika; Hoogaars, Willem M H; Pasternack, Arja; Kainulainen, Heikki; Ritvos, Olli

2013-07-15

The importance of adequate levels of muscle size and function and physical activity is widely recognized. Myostatin/activin blocking increases skeletal muscle mass but may decrease muscle oxidative capacity and can thus be hypothesized to affect voluntary physical activity. Soluble activin receptor IIB (sActRIIB-Fc) was produced to block myostatin/activins. Modestly dystrophic mdx mice were injected with sActRIIB-Fc or PBS with or without voluntary wheel running exercise for 7 wk. Healthy mice served as controls. Running for 7 wk attenuated the sActRIIB-Fc-induced increase in body mass by decreasing fat mass. Running also enhanced/restored the markers of muscle oxidative capacity and autophagy in mdx mice to or above the levels of healthy mice. Voluntary running activity was decreased by sActRIIB-Fc during the first 3-4 wk correlating with increased body mass. Home cage physical activity of mice, quantified from the force plate signal, was decreased by sActRIIB-Fc the whole 7-wk treatment in sedentary mice. To understand what happens during the first weeks after sActRIIB-Fc administration, when mice are less active, healthy mice were injected with sActRIIB-Fc or PBS for 2 wk. During the sActRIIB-Fc-induced rapid 2-wk muscle growth period, oxidative capacity and autophagy were reduced, which may possibly explain the decreased running activity. These results show that increased muscle size and decreased markers of oxidative capacity and autophagy during the first weeks of myostatin/activin blocking are associated with decreased voluntary activity levels. Voluntary exercise in dystrophic mice enhances the markers of oxidative capacity and autophagy to or above the levels of healthy mice.

Orexin-A increases the firing activity of hippocampal CA1 neurons through orexin-1 receptors.

Science.gov (United States)

Chen, Xin-Yi; Chen, Lei; Du, Yi-Feng

2017-07-01

Orexins including two peptides, orexin-A and orexin-B, are produced in the posterior lateral hypothalamus. Much evidence has indicated that central orexinergic systems play numerous functions including energy metabolism, feeding behavior, sleep/wakefulness, and neuroendocrine and sympathetic activation. Morphological studies have shown that the hippocampal CA1 regions receive orexinergic innervation originating from the hypothalamus. Positive orexin-1 (OX 1 ) receptors are detected in the CA1 regions. Previous behavioral studies have shown that microinjection of OX 1 receptor antagonist into the hippocampus impairs acquisition and consolidation of spatial memory. However, up to now, little has been known about the direct electrophysiological effects of orexin-A on hippocampal CA1 neurons. Employing multibarrel single-unit extracellular recordings, the present study showed that micropressure administration of orexin-A significantly increased the spontaneous firing rate from 2.96 ± 0.85 to 8.45 ± 1.86 Hz (P neurons in male rats. Furthermore, application of the specific OX 1 receptor antagonist SB-334867 alone significantly decreased the firing rate from 4.02 ± 1.08 to 2.11 ± 0.58 Hz in 7 out of the 17 neurons (P neurons. Coapplication of SB-334867 completely blocked orexin-A-induced excitation of hippocampal CA1 neurons. The PLC pathway may be involved in activation of OX 1 receptor-induced excitation of CA1 neurons. Taken together, the present study's results suggest that orexin-A produces excitatory effects on hippocampal neurons via OX 1 receptors. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nanomimics of host cell membranes block invasion and expose invasive malaria parasites.

Science.gov (United States)

Najer, Adrian; Wu, Dalin; Bieri, Andrej; Brand, Françoise; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang

2014-12-23

The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics-polymersomes presenting receptors required for parasite attachment to host cells-were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response.

The hallucinogen d-lysergic diethylamide (LSD) decreases dopamine firing activity through 5-HT1A, D2 and TAAR1 receptors.

Science.gov (United States)

De Gregorio, Danilo; Posa, Luca; Ochoa-Sanchez, Rafael; McLaughlin, Ryan; Maione, Sabatino; Comai, Stefano; Gobbi, Gabriella

2016-11-01

d-lysergic diethylamide (LSD) is a hallucinogenic drug that interacts with the serotonin (5-HT) system binding to 5-HT 1 and 5-HT 2 receptors. Little is known about its potential interactions with the dopamine (DA) neurons of the ventral tegmental area (VTA). Using in-vivo electrophysiology in male adult rats, we evaluated the effects of cumulative doses of LSD on VTA DA neuronal activity, compared these effects to those produced on 5-HT neurons in the dorsal raphe nucleus (DRN), and attempted to identify the mechanism of action mediating the effects of LSD on VTA DA neurons. LSD, at low doses (5-20μg/kg, i.v.) induced a significant decrease of DRN 5-HT firing activity through 5-HT 2A and D 2 receptors. At these low doses, LSD did not alter VTA DA neuronal activity. On the contrary, at higher doses (30-120μg/kg, i.v.), LSD dose-dependently decreased VTA DA firing activity. The depletion of 5-HT with p-chlorophenylalanine did not modulate the effects of LSD on DA firing activity. The inhibitory effects of LSD on VTA DA firing activity were prevented by the D 2 receptor antagonist haloperidol (50μg/kg, i.v.) and by the 5-HT 1A receptor antagonist WAY-100,635 (500μg/kg, i.v.). Notably, pretreatment with the trace amine-associate receptor 1 (TAAR 1 ) antagonist EPPTB (5mg/kg, i.v.) blocked the inhibitory effect of LSD on VTA DA neurons. These results suggest that LSD at high doses strongly affects DA mesolimbic neuronal activity in a 5-HT independent manner and with a pleiotropic mechanism of action involving 5-HT 1A, D 2 and TAAR 1 receptors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Common structural basis for constitutive activity of the ghrelin receptor family

DEFF Research Database (Denmark)

Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

2004-01-01

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand......-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves' Disease.

Science.gov (United States)

Cho, Sun Wook; Bae, Jae Hyun; Noh, Gyeong Woon; Kim, Ye An; Moon, Min Kyong; Park, Kyoung Un; Song, Junghan; Yi, Ka Hee; Park, Do Joon; Chung, June-Key; Cho, Bo Youn; Park, Young Joo

2015-01-01

Osteoporosis-related fractures are one of the complications of Graves' disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR) antibodies, both stimulating and blocking activities in Graves' disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves' disease patients were enrolled (n = 93) and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83) and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10). From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII) to the blocking activity group were further classified as stimulating activity-matched control (n = 11). Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves' disease.

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves' Disease.

Directory of Open Access Journals (Sweden)

Sun Wook Cho

Full Text Available Osteoporosis-related fractures are one of the complications of Graves' disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR antibodies, both stimulating and blocking activities in Graves' disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves' disease patients were enrolled (n = 93 and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83 and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10. From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII to the blocking activity group were further classified as stimulating activity-matched control (n = 11. Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves' disease.

Acute stress-induced sensitization of the pituitary-adrenal response to heterotypic stressors: independence of glucocorticoid release and activation of CRH1 receptors.

Science.gov (United States)

Belda, Xavier; Daviu, Núria; Nadal, Roser; Armario, Antonio

2012-09-01

A single exposure to some severe stressors causes sensitization of the hypothalamic-pituitary-adrenal (HPA) response to novel stressors. However, the putative factors involved in stress-induced sensitization are not known. In the present work we studied in adult male rats the possible role of glucocorticoids and CRH type 1 receptor (CRH-R1), using an inhibitor of glucocorticoid synthesis (metyrapone, MET), the glucocorticoid receptor (GR) antagonist RU38486 (mifepristone) and the non-peptide CRH-R1 antagonist R121919. In a first experiment we demonstrated with different doses of MET (40-150 mg/kg) that the highest dose acted as a pharmacological stressor greatly increasing ACTH release and altering the normal circadian pattern of HPA hormones, but no dose affected ACTH responsiveness to a novel environment as assessed 3 days after drug administration. In a second experiment, we found that MET, at a dose (75 mg/kg) that blocked the corticosterone response to immobilization (IMO), did not alter IMO-induced ACTH sensitization. Finally, neither the GR nor the CRH-R1 antagonists blocked IMO-induced ACTH sensitization on the day after IMO. Thus, a high dose of MET, in contrast to IMO, was unable to sensitize the HPA response to a novel environment despite the huge activation of the HPA axis caused by the drug. Neither a moderate dose of MET that markedly reduced corticosterone response to IMO, nor the blockade of GR or CRH-R1 receptors was able to alter stress-induced HPA sensitization. Therefore, stress-induced sensitization is not the mere consequence of a marked HPA activation and does not involve activation of glucocorticoid or CRH-R1 receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

Hippocampal activation during face-name associative memory encoding: blocked versus permuted design

International Nuclear Information System (INIS)

De Vogelaere, Frederick; Vingerhoets, Guy; Santens, Patrick; Boon, Paul; Achten, Erik

2010-01-01

The contribution of the hippocampal subregions to episodic memory through the formation of new associations between previously unrelated items such as faces and names is established but remains under discussion. Block design studies in this area of research generally tend to show posterior hippocampal activation during encoding of novel associational material while event-related studies emphasize anterior hippocampal involvement. We used functional magnetic resonance imaging to assess the involvement of anterior and posterior hippocampus in the encoding of novel associational material compared to the viewing of previously seen associational material. We used two different experimental designs, a block design and a permuted block design, and applied it to the same associative memory task to perform valid statistical comparisons. Our results indicate that the permuted design was able to capture more anterior hippocampal activation compared to the block design, which emphasized more posterior hippocampal involvement. These differences were further investigated and attributed to a combination of the polymodal stimuli we used and the experimental design. Activation patterns during encoding in both designs occurred along the entire longitudinal axis of the hippocampus, but with different centers of gravity. The maximal activated voxel in the block design was situated in the posterior half of the hippocampus while in the permuted design this was located in the anterior half. (orig.)

Hippocampal activation during face-name associative memory encoding: blocked versus permuted design

Energy Technology Data Exchange (ETDEWEB)

De Vogelaere, Frederick; Vingerhoets, Guy [Ghent University, Laboratory for Neuropsychology, Department of Neurology, Ghent (Belgium); Santens, Patrick; Boon, Paul [Ghent University Hospital, Department of Neurology, Ghent (Belgium); Achten, Erik [Ghent University Hospital, Department of Radiology, Ghent (Belgium)

2010-01-15

The contribution of the hippocampal subregions to episodic memory through the formation of new associations between previously unrelated items such as faces and names is established but remains under discussion. Block design studies in this area of research generally tend to show posterior hippocampal activation during encoding of novel associational material while event-related studies emphasize anterior hippocampal involvement. We used functional magnetic resonance imaging to assess the involvement of anterior and posterior hippocampus in the encoding of novel associational material compared to the viewing of previously seen associational material. We used two different experimental designs, a block design and a permuted block design, and applied it to the same associative memory task to perform valid statistical comparisons. Our results indicate that the permuted design was able to capture more anterior hippocampal activation compared to the block design, which emphasized more posterior hippocampal involvement. These differences were further investigated and attributed to a combination of the polymodal stimuli we used and the experimental design. Activation patterns during encoding in both designs occurred along the entire longitudinal axis of the hippocampus, but with different centers of gravity. The maximal activated voxel in the block design was situated in the posterior half of the hippocampus while in the permuted design this was located in the anterior half. (orig.)

The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

Science.gov (United States)

Reissmann, Eva; Jörnvall, Henrik; Blokzijl, Andries; Andersson, Olov; Chang, Chenbei; Minchiotti, Gabriella; Persico, M. Graziella; Ibáñez, Carlos F.; Brivanlou, Ali H.

2001-01-01

Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling. PMID:11485994

p-Coumaric acid activates the GABA-A receptor in vitro and is orally anxiolytic in vivo.

Science.gov (United States)

Scheepens, Arjan; Bisson, Jean-Francois; Skinner, Margot

2014-02-01

The increasing prevalence and social burden of subclinical anxiety in the western world represents a significant psychosocial and financial cost. Consumers are favouring a more natural and nonpharmacological approach for alleviating the effects of everyday stress and anxiety. The gamma-aminobutyric acid (GABA) receptor is the primary mediator of central inhibitory neurotransmission, and GABA-receptor agonists are well known to convey anxiolytic effects. Using an in vitro screening approach to identify naturally occurring phytochemical GABA agonists, we discovered the plant secondary metabolite p-coumaric acid to have significant GABAergic activity, an effect that could be blocked by co-administration of the specific GABA-receptor antagonist, picrotoxin. Oral administration of p-coumaric acid to rodents induced a significant anxiolytic effect in vivo as measured using the elevated plus paradigm, in line with the effects of oral diazepam. Given that p-coumaric acid is reasonably well absorbed following oral consumption in man and is relatively nontoxic, it may be suitable for the formulation of a safe and effective anxiolytic functional food. Copyright © 2013 John Wiley & Sons, Ltd.

An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1.

Science.gov (United States)

Skinner, Camille M; Ivanov, Nikita S; Barr, Sarah A; Chen, Yan; Skalsky, Rebecca L

2017-11-01

Epstein-Barr virus (EBV) encodes >44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in Epstein-Barr virus (EBV)-positive tumors, and manipulate several biological processes, including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-κB activation following treatment with proinflammatory cytokines, specifically interleukin-1β (IL-1β). Analysis of EBV PAR-CLIP miRNA targetome data sets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways. By further analyzing changes in cellular gene expression patterns, we identified the IL-1 receptor 1 (IL1R1) as a direct target of miR-BHRF1-2-5p. Targeting the IL1R1 3' untranslated region (UTR) by EBV miR-BHRF1-2-5p was confirmed using 3'-UTR luciferase reporter assays and Western blot assays. Manipulation of EBV BHRF1-2 miRNA activity in latently infected B cells altered steady-state cytokine levels and disrupted IL-1β responsiveness. These studies demonstrate functionally relevant BHRF1-2 miRNA interactions during EBV infection, which is an important step in understanding their roles in pathogenesis. IMPORTANCE IL-1 signaling plays an important role in inflammation and early activation of host innate immune responses following virus infection. Here, we demonstrate that a viral miRNA downregulates the IL-1 receptor 1 during EBV infection, which consequently alters the responsiveness of cells to IL-1 stimuli and changes the cytokine expression levels within infected cell populations. We postulate that this viral miRNA activity not only disrupts IL-1 autocrine and paracrine signaling loops that can alert effector cells to sites of infection but also provides a survival advantage by dampening excessive inflammation that may be detrimental to the infected cell. Copyright © 2017 American Society for Microbiology.

Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

Science.gov (United States)

Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

2009-08-17

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.

Science.gov (United States)

Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

2018-01-01

Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.

Dynamic regulation of Drosophila nuclear receptor activity in vivo.

Science.gov (United States)

Palanker, Laura; Necakov, Aleksandar S; Sampson, Heidi M; Ni, Ruoyu; Hu, Chun; Thummel, Carl S; Krause, Henry M

2006-09-01

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a 'ligand sensor' system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control.

Selectivity of beta-adrenergic stimulating and blocking agents.

Science.gov (United States)

Löfdahl, C G; Svedmyr, N

1984-01-01

Studies have been performed to answer two questions: whether there are subgroups of beta 2-receptors separating effects in bronchial and skeletal muscle and whether beta 1-receptors in asthmatic airways mediate bronchoconstriction. Asthmatic patients have been studied in randomised cross-over trials. Effects on FEV1, heart rate and skeletal muscle tremor have been monitored. In some experimental studies, two new compounds, D2343 and QH-25, have shown a selectivity for beta 2-receptors in bronchial muscle compared to skeletal muscle. Studies in asthmatics did not confirm this. Thus, the beta 2-receptors in the two organs appear to be identical. The clinical effect of beta 1-receptors in the the airways was studied by giving selective beta 1-receptor blocking agents. It was shown that pafenolol , a beta-blocker more beta 1-selective than metoprolol, had less effect on FEV1 than metoprolol given in equipotent beta 1-blocking doses. Beta 1-receptor stimulation with a new selective beta 1-stimulating agent, prenalterol, did not give bronchodilation in doses which gave a significant increase of heart rate. Thus, beta 1-receptors do not contribute to bronchodilation in asthmatic patients.

The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells

International Nuclear Information System (INIS)

Cherukuri, Durga Prasad; Chen, Xiao B.O.; Goulet, Anne-Christine; Young, Robert N.; Han, Yongxin; Heimark, Ronald L.; Regan, John W.; Meuillet, Emmanuelle; Nelson, Mark A.

2007-01-01

Accumulating evidence indicates that elevated levels of prostaglandin E 2 (PGE 2 ) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE 2 exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE 2 to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE 2 -induced ERK phosphorylation and cell proliferation of HCA-7 cells. In order to identify downstream target genes of ERK1/2 signaling, we found that PGE 2 induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE 2 treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE 2 driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE 2 in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer

Receptor activity-independent recruitment of βarrestin2 reveals specific signalling modes

Science.gov (United States)

Terrillon, Sonia; Bouvier, Michel

2004-01-01

The roles of βarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, βarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with βarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the βarrestin/receptor complexes. Interestingly, βarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating βarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of βarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by βarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that βarrestin2 can act as a ‘bonafide' signalling molecule even in the absence of activated receptor. PMID:15385966

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

International Nuclear Information System (INIS)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

Blocking opioid receptors alters short-term feed intake and oro-sensorial preferences in weaned calves.

Science.gov (United States)

Montoro, C; Ipharraguerre, I R; Bach, A

2012-05-01

Opioid peptides may participate in the control of feed intake through mechanisms involving pleasure reward linked to consumption of palatable feed. The objective of this study was to determine whether blocking opioid receptors might void oro-sensorial preferences of calves, and affect circulating glucose, insulin, and anorexigenic hormones in fasted and fed calves. Two experiments involved 32 Holstein calves [body weight (BW)=86.5±1.73 kg, age=72±0.6 d]. In experiment 1, all calves received an ad libitum choice of the same feed either unflavored or flavored with a sweetener (Luctarom SFS-R, Lucta, Montornès del Vallès, Spain). Feed consumption was recorded every 2 h from 0800 to 1400 h for 3 consecutive days to verify the establishment of an oro-sensorial preference for sweet feed (SF). The SF was preferred over the control feed (CF) at all recorded times. In experiment 2, calves were subjected to a 2 × 2 factorial design to study the interaction between opioid activity and metabolic state. Half of the calves were fasted for 14 h (FAS), whereas the other half remained well fed (FED). Within each of these groups, at feeding time (0800 h), half of the calves received an i.v. injection of naloxone (NAL, an opioid receptor antagonist; 1 mg/kg of BW) and the other half was injected with saline solution (SAL; 0.9% NaCl). Therefore, treatments were FED-NAL, FED-SAL, FAS-NAL, and FAS-SAL. Blood samples were taken at -10, 20, 180, and 240 min relative to NAL or SAL injections. As expected, cumulative consumption of starter feed was greater in FAS than in FED calves. Total feed consumption 2 h after feeding was lower in NAL than in SAL calves. Calves in the FAS group did not discern between CF and SF during the first 4 h after feed offer. Preference for SF was greater in SAL than in NAL calves. Calves in the FED-SAL treatment preferred SF at 2 and 6 h after feed offer and tended to prefer SF at 4 h after feeding. However, FED-NAL calves did not discern between SF and CF

ERK, Akt, and STAT5 are differentially activated by the two growth hormone receptors subtypes of a teleost fish (Oncorhynchus mykiss

Directory of Open Access Journals (Sweden)

Jeffrey eKittilson

2011-09-01

Full Text Available Previously, we found that the teleost fish, rainbow trout, possesses two growth hormone receptor (GHR subtypes that display distinct ligand binding and agonist-induced regulation features. In this study, we used Chinese hamster ovary-K1 cells stably transfected individually with the two trout GHR subtypes, GHR1 and GHR2, to elucidate receptor-effector pathway linkages. Growth hormone (GH stimulated rapid (5-10 min phosphorylation of ERK, Akt, JAk2, and STAT5 in both GHR1- and GHR2-expressing cells; however; STAT5 was activated to a greater extent through GHR1 than through GHR2, whereas ERK and Akt were activated to a greater through GHR2 than through GHR1. Although blockade of the ERK pathway had no effect on the activation of Akt, inhibition of PI3k-Akt partially prevented activation of ERK, suggesting cross-talk between the ERK and PI3K-Akt pathways. JAK2 inhibition completely blocked activation of ERK, Akt, and STAT5, suggesting that all of these pathways link to GHR1 and GHR2 via JAK2. These findings establish important receptor-effector pathway linkages and suggest that the GHR subtypes of teleost fish may be functionally distinct.

Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

Directory of Open Access Journals (Sweden)

Freeman Robert S

2011-02-01

Full Text Available Abstract Background Thrombolytic therapy with tissue plasminogen activator (tPA benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult. Results We show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant AktK179A and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM, we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates

The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation

DEFF Research Database (Denmark)

Dagil, Robert; Knudsen, Maiken J.; Olsen, Johan Gotthardt

2012-01-01

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane...

Mincle suppresses Toll-like receptor 4 activation.

Science.gov (United States)

Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

2016-07-01

Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation. © Society for Leukocyte Biology.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

Directory of Open Access Journals (Sweden)

Thomas Pleli

2018-03-01

Full Text Available Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2 inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors. In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Science.gov (United States)

Kowalczyk-Quintas, Christine; Willen, Laure; Dang, Anh Thu; Sarrasin, Heidi; Tardivel, Aubry; Hermes, Katharina; Schneider, Holm; Gaide, Olivier; Donzé, Olivier; Kirby, Neil; Headon, Denis J; Schneider, Pascal

2014-02-14

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia*

Science.gov (United States)

Kowalczyk-Quintas, Christine; Willen, Laure; Dang, Anh Thu; Sarrasin, Heidi; Tardivel, Aubry; Hermes, Katharina; Schneider, Holm; Gaide, Olivier; Donzé, Olivier; Kirby, Neil; Headon, Denis J.; Schneider, Pascal

2014-01-01

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated. PMID:24391090

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present and biochemically active in the central nervous system of the pond snail Lymnaea stagnalis.

Science.gov (United States)

Pirger, Zsolt; Laszlo, Zita; Hiripi, Laszlo; Hernadi, Laszlo; Toth, Gabor; Lubics, Andrea; Reglodi, Dora; Kemenes, Gyorgy; Mark, Laszlo

2010-11-01

PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

Science.gov (United States)

Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

2003-01-01

Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539

Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ in vitro

Directory of Open Access Journals (Sweden)

Dionisi Mauro

2012-05-01

Full Text Available Abstract Background Oleamide (ODA is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs as potential targets for ODA action. Results Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. Conclusions We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

The role of GH receptor tyrosine phosphorylation in Stat5 activation

DEFF Research Database (Denmark)

Hansen, J A; Hansen, L H; Wang, X

1997-01-01

Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

The anti-influenza drug oseltamivir evokes hypothermia in mice through dopamine D2 receptor activation via central actions

Directory of Open Access Journals (Sweden)

Akihiro Fukushima

2018-01-01

Full Text Available Oseltamivir has a hypothermic effect in mice when injected intraperitoneally (i.p. and intracerebroventricularly (i.c.v.. Here we show that the hypothermia evoked by i.c.v.-oseltamivir is inhibited by non-selective dopamine receptor antagonists (sulpiride and haloperidol and the D2-selective antagonist L-741,626, but not by D1/D5-selective and D3-selective antagonists (SCH-23390 and SB-277011-A, respectively. The hypothermic effect of i.p.-administered oseltamivir was not inhibited by sulpiride, haloperidol, L-741,626 and SCH-23390. In addition, neither sulpiride, haloperidol nor SCH-23390 blocked hypothermia evoked by i.c.v.-administered oseltamivir carboxylate (a hydrolyzed metabolite of oseltamivir. These results suggest that oseltamivir in the brain induces hypothermia through activation of dopamine D2 receptors.

Sigma-1 receptor agonists directly inhibit Nav1.2/1.4 channels.

Directory of Open Access Journals (Sweden)

Xiao-Fei Gao

Full Text Available (+-SKF 10047 (N-allyl-normetazocine is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+-SKF 10047 inhibits K(+, Na(+ and Ca2+ channels via sigma-1 receptor activation. We found that (+-SKF 10047 inhibited Na(V1.2 and Na(V1.4 channels independently of sigma-1 receptor activation. (+-SKF 10047 equally inhibited Na(V1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+-SKF 10047 inhibition of Na(V1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM and 1,3-di-o-tolyl-guanidine (DTG also inhibited Na(V1.2 currents through a sigma-1 receptor-independent pathway. The (+-SKF 10047 inhibition of Na(V1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe(1764 and Tyr(1771 in the IV-segment 6 domain of the Na(V1.2 channel and Phe(1579 in the Na(V1.4 channel for (+-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit Na(V1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies.

Assembly and activation of neurotrophic factor receptor complexes.

Science.gov (United States)

Simi, Anastasia; Ibáñez, Carlos F

2010-04-01

Neurotrophic factors play important roles in the development and function of both neuronal and glial elements of the central and peripheral nervous systems. Their functional diversity is in part based on their ability to interact with alternative complexes of receptor molecules. This review focuses on our current understanding of the mechanisms that govern the assembly and activation of neurotrophic factor receptor complexes. The realization that many, if not the majority, of these complexes exist in a preassembled form at the plasma membrane has forced the revision of classical ligand-mediated oligomerization models, and led to the discovery of novel mechanisms of receptor activation and generation of signaling diversity which are likely to be shared by many different classes of receptors.

Evidence for a cyclic AMP-dependent pathway in angiotensin AT1-receptor activation of human omental arteries

Directory of Open Access Journals (Sweden)

Hoa Ytterberg

2001-03-01

Full Text Available Enhanced responses to vasoconstriction induced by neuropeptide Y and α2-adrenoceptor agonists have been seen following pharmacological activation of the adenylyl cyclase (AC system. Since preliminary studies revealed only minor responses to angiotensin II (Ang II in human omental arteries, we have investigated whether enhanced activity of AC may unravel further functional Ang II receptors. Human omental arteries were obtained in conjunction with elective gut surgery. After dissection of the vessel, the endothelium was removed by 10 sec of Triton X-100 treatment. Ring segments (1—2 mm long were mounted on a myograph and studied. Ang II produced small contractions, 27±5% relative to the response elicited by 60 mM K+. However, enhanced Ang II (105±10%, p<0.001 responses were seen during AC activation by forskolin (0.1—1 µM. This enhanced contractile response to Ang II was not inhibited by the angiotensin II type 2 (AT2-receptor antagonist PD 123319 (0.1 µM, but was blocked in an insurmountable way by the angiotensin II type 1 (AT1-receptor antagonist candesartan (1 nM and in a surmountable manner by losartan (0.1 µM and irbesartan (0.1 µM. Pertussis toxin (a Gi-protein blocker and the protein kinase C inhibitor, RO31—8220 (0.01, 0.1 and 1 µM, markedly reduced this response, while the protein kinase A inhibitor, H89 (1, 10 µM, had no effect. RT-PCR provided evidence for the presence of mRNA for both AT1- and AT2-receptors. The results suggest that both a cAMP-dependent and a cAMP-independent mechanism are involved in the contractile responses to Ang II in human omental arteries and that both responses are mediated via the AT1-receptor.

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Science.gov (United States)

Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

1995-03-01

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits.

Science.gov (United States)

Wang, Lan; Yin, Jie; Wang, Xuefei; Shao, Miaomiao; Duan, Fangfang; Wu, Weicheng; Peng, Peike; Jin, Jing; Tang, Yue; Ruan, Yuanyuan; Sun, Yihong; Gu, Jianxin

2016-05-01

C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition

Circulating Estradiol Regulates Brain-Derived Estradiol via Actions at GnRH Receptors to Impact Memory in Ovariectomized Rats.

Science.gov (United States)

Nelson, Britta S; Black, Katelyn L; Daniel, Jill M

2016-01-01

Systemic estradiol treatment enhances hippocampus-dependent memory in ovariectomized rats. Although these enhancements are traditionally thought to be due to circulating estradiol, recent data suggest these changes are brought on by hippocampus-derived estradiol, the synthesis of which depends on gonadotropin-releasing hormone (GnRH) activity. The goal of the current work is to test the hypothesis that peripheral estradiol affects hippocampus-dependent memory through brain-derived estradiol regulated via hippocampal GnRH receptor activity. In the first experiment, intracerebroventricular infusion of letrozole, which prevents the synthesis of estradiol, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task. In the second experiment, hippocampal infusion of antide, a long-lasting GnRH receptor antagonist, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory. In the third experiment, hippocampal infusion of GnRH enhanced hippocampus-dependent memory, the effects of which were blocked by letrozole infusion. Results indicate that peripheral estradiol-induced enhancement of cognition is mediated by brain-derived estradiol via hippocampal GnRH receptor activity.

Induction of an Olfactory Memory by the Activation of a Metabotropic Glutamate Receptor

Science.gov (United States)

Kaba, Hideto; Hayashi, Yasunori; Higuchi, Takashi; Nakanishi, Shigetada

1994-07-01

Female mice form an olfactory memory of male pheromones at mating; exposure to the pheromones of a strange male after that mating will block pregnancy. The formation of this memory is mediated by the accessory olfactory system, in which an increase in norepinephrine after mating reduces inhibitory transmission of γ-aminobutyric acid from the granule cells to the mitral cells. This study shows that the activation of mGluR2, a metabotropic glutamate receptor that suppresses the γ-aminobutyric acid inhibition of the mitral cells, permits the formation of a specific olfactory memory without the occurrence of mating by infusion of mGluR2 agonists into the female's accessory olfactory bulb. This memory faithfully reflects the memory formed at mating.

Estradiol suppresses ingestive response evoked by activation of 5-HT1A receptors in the lateral hypothalamus of ovariectomized rats.

Science.gov (United States)

Taschetto, Ana P D; Levone, Brunno R; Kochenborger, Larissa; da Silva, Eduardo S; Flores, Rafael A; Faria, Moacir S; Paschoalini, Marta A

2018-03-08

The present study investigated the effects of estradiol (E2) on ingestive behavior after activation of 5-HT1A receptors in the lateral hypothalamus (LH) of female rats habituated to eat a wet mash diet. Ovariectomized rats treated with corn oil (OVX) or estradiol cypionate (OVX+E) received local injections into the LH of vehicle or an agonist of 5-HT1A receptors, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT; at a dose of 6 nmol). To determine the involvement of these receptors in food intake, some animals were pretreated with N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide maleate (WAY-100635, a 5-HT1A receptor full antagonist, at a dose of 0.37 nmol), followed by the injection of the agonist 8-OH-DPAT or its vehicle. The results showed that the injection of 8-OH-DPAT into the LH of OVX rats significantly increased food intake, and the duration and frequency of this behavior. The pretreatment with E2 suppressed the hyperphagic response induced by 8-OH-DPAT in OVX animals. The inhibition of 5-HT1A receptors after pretreatment with WAY-100635 blocked the hyperphagic effects evoked by 8-OH-DPAT in OVX. These results indicate that the activity of LH 5-HT1A receptors could be affected by blood E2 levels.

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

International Nuclear Information System (INIS)

Raufman, Jean-Pierre; Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng

2011-01-01

Highlights: ► Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. ► Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. ► Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers – this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

Energy Technology Data Exchange (ETDEWEB)

Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States); Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States)

2011-11-18

Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre

Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements.

Science.gov (United States)

Keller, H; Givel, F; Perroud, M; Wahli, W

1995-07-01

Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are nuclear hormone receptors that are activated by fatty acids and 9-cis-retinoic acid, respectively. PPARs and RXRs form heterodimers that activate transcription by binding to PPAR response elements (PPREs) in the promoter of target genes. The PPREs described thus far consist of a direct tandem repeat of the AGGTCA core element with one intervening nucleotide. We show here that the vitellogenin A2 estrogen response element (ERE) can also function as a PPRE and is bound by a PPAR/RXR heterodimer. Although this heterodimer can bind to several other ERE-related palindromic response elements containing AGGTCA half-sites, only the ERE is able to confer transactivation of test reporter plasmids, when the ERE is placed either close to or at a distance from the transcription initiation site. Examination of natural ERE-containing promoters, including the pS2, very-low-density apolipoprotein II and vitellogenin A2 genes, revealed considerable differences in the binding of PPAR/RXR heterodimers to these EREs. In their natural promoter context, these EREs did not allow transcriptional activation by PPARs/RXRs. Analysis of this lack of stimulation of the vitellogenin A2 promoter demonstrated that PPARs/RXRs bind to the ERE but cannot transactivate due to a nonpermissive promoter structure. As a consequence, PPARs/RXRs inhibit transactivation by the estrogen receptor through competition for ERE binding. This is the first example of signaling cross-talk between PPAR/RXR and estrogen receptor.

On the mechanism of TBA block of the TRPV1 channel.

Science.gov (United States)

Oseguera, Andrés Jara; Islas, León D; García-Villegas, Refugio; Rosenbaum, Tamara

2007-06-01

The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselective cation channel activated by capsaicin and responsible for thermosensation. To date, little is known about the gating characteristics of these channels. Here we used tetrabutylammonium (TBA) to determine whether this molecule behaves as an ion conduction blocker in TRPV1 channels and to gain insight into the nature of the activation gate of this protein. TBA belongs to a family of classic potassium channel blockers that have been widely used as tools for determining the localization of the activation gate and the properties of the pore of several ion channels. We found TBA to be a voltage-dependent pore blocker and that the properties of block are consistent with an open-state blocker, with the TBA molecule binding to multiple open states, each with different blocker affinities. Kinetics of channel closure and burst-length analysis in the presence of blocker are consistent with a state-dependent blocking mechanism, with TBA interfering with closing of an activation gate. This activation gate may be located cytoplasmically with respect to the binding site of TBA ions, similar to what has been observed in potassium channels. We propose an allosteric model for TRPV1 activation and block by TBA, which explains our experimental data.

Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor.

Science.gov (United States)

Armour, S L; Foord, S; Kenakin, T; Chen, W J

1999-12-01

Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.

Sympathetic activity induced by naloxone-precipitated morphine withdrawal is blocked in genetically engineered mice lacking functional CRF1 receptor

International Nuclear Information System (INIS)

García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

2015-01-01

There is large body evidence indicating that stress can lead to cardiovascular disease. However, the exact brain areas and the mechanisms involved remain to be revealed. Here, we performed a series of experiments to characterize the role of CRF1 receptor (CRF1R) in the stress response induced by naloxone-precipitated morphine withdrawal. The experiments were performed in the hypothalamic paraventricular nucleus (PVN) ventrolateral medulla (VLM), brain regions involved in the regulation of cardiovascular activity, and in the right ventricle by using genetically engineered mice lacking functional CRF1R levels (KO). Mice were treated with increasing doses of morphine and withdrawal was precipitated by naloxone administration. Noradrenaline (NA) turnover, c-Fos, expression, PKA and TH phosphorylated at serine 40, was evaluated by high-performance liquid chromatography (HPLC), immunohistochemistry and immunoblotting. Morphine withdrawal induced an enhancement of NA turnover in PVN in parallel with an increase in TH neurons expressing c-Fos in VLM in wild-type mice. In addition we have demonstrated an increase in NA turnover, TH phosphorylated at serine 40 and PKA levels in heart. The main finding of the present study was that NA turnover, TH positive neurons that express c-Fos, TH phosphorylated at serine 40 and PKA expression observed during morphine withdrawal were significantly inhibited in CRF1R KO mice. Our results demonstrate that CRF/CRF1R activation may contribute to the adaptive changes induced by naloxone-precipitated withdrawal in the heart and in the brain areas which modulate the cardiac sympathetic function and suggest that CRF/CRF1R pathways could be contributing to cardiovascular disease associated to opioid addiction. - Highlights: • Naloxone-precipitated morphine withdrawal increases sympathetic activity in the PVN and heart. • Co-localization of TH phosphorylated at serine 40/c-Fos in the VLM after morphine withdrawal • Naloxone

Sympathetic activity induced by naloxone-precipitated morphine withdrawal is blocked in genetically engineered mice lacking functional CRF1 receptor

Energy Technology Data Exchange (ETDEWEB)

García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

2015-02-15

There is large body evidence indicating that stress can lead to cardiovascular disease. However, the exact brain areas and the mechanisms involved remain to be revealed. Here, we performed a series of experiments to characterize the role of CRF1 receptor (CRF1R) in the stress response induced by naloxone-precipitated morphine withdrawal. The experiments were performed in the hypothalamic paraventricular nucleus (PVN) ventrolateral medulla (VLM), brain regions involved in the regulation of cardiovascular activity, and in the right ventricle by using genetically engineered mice lacking functional CRF1R levels (KO). Mice were treated with increasing doses of morphine and withdrawal was precipitated by naloxone administration. Noradrenaline (NA) turnover, c-Fos, expression, PKA and TH phosphorylated at serine 40, was evaluated by high-performance liquid chromatography (HPLC), immunohistochemistry and immunoblotting. Morphine withdrawal induced an enhancement of NA turnover in PVN in parallel with an increase in TH neurons expressing c-Fos in VLM in wild-type mice. In addition we have demonstrated an increase in NA turnover, TH phosphorylated at serine 40 and PKA levels in heart. The main finding of the present study was that NA turnover, TH positive neurons that express c-Fos, TH phosphorylated at serine 40 and PKA expression observed during morphine withdrawal were significantly inhibited in CRF1R KO mice. Our results demonstrate that CRF/CRF1R activation may contribute to the adaptive changes induced by naloxone-precipitated withdrawal in the heart and in the brain areas which modulate the cardiac sympathetic function and suggest that CRF/CRF1R pathways could be contributing to cardiovascular disease associated to opioid addiction. - Highlights: • Naloxone-precipitated morphine withdrawal increases sympathetic activity in the PVN and heart. • Co-localization of TH phosphorylated at serine 40/c-Fos in the VLM after morphine withdrawal • Naloxone

Potentiation of glycine-gated NR1/NR3A NMDA receptors relieves Ca2+-dependent outward rectification

Directory of Open Access Journals (Sweden)

Christian Madry

2010-03-01

Full Text Available Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named excitatory glycine receptors. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I-V dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I-V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I-V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors.

D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

Science.gov (United States)

Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

2014-09-15

Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. Copyright © 2014 the American Physiological Society.

Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

Science.gov (United States)

Hempstead, Andrew D; Isberg, Ralph R

2015-12-08

Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

Opportunistic activation of TRP receptors by endogenous lipids: exploiting lipidomics to understand TRP receptor cellular communication.

Science.gov (United States)

Bradshaw, Heather B; Raboune, Siham; Hollis, Jennifer L

2013-03-19

Transient receptor potential channels (TRPs) form a large family of ubiquitous non-selective cation channels that function as cellular sensors and in many cases regulate intracellular calcium. Identification of the endogenous ligands that activate these TRP receptors is still under intense investigation with the majority of these channels still remaining "orphans." That these channels respond to a variety of external stimuli (e.g. plant-derived lipids, changes in temperature, and changes in pH) provides a framework for their abilities as cellular sensors, however, the mechanism of direct activation is still under much debate and research. In the cases where endogenous ligands (predominately lipids) have shown direct activation of a channel, multiple ligands have been shown to activate the same channel suggesting that these receptors are "promiscuous" in nature. Lipidomics of a growing class of endogenous lipids, N-acyl amides, the most famous of which is N-arachidonoyl ethanolamine (the endogenous cannabinoid, Anandamide) is providing a novel set of ligands that have been shown to activate some members of the TRP family and have the potential to deorphanize many more. Here it is argued that activation of TRPV receptors, a subset of the larger family of TRPs, by multiple endogenous lipids that are structurally analogous is a model system to drive our understanding that many TRP receptors are not promiscuous, but are more characteristically "opportunistic" in nature; exploiting the structural similarity and biosynthesis of a narrow range of analogous endogenous lipids. In addition, this manuscript will compare the activation properties of TRPC5 to the activity profile of an "orphan" lipid, N-palmitoyl glycine; further demonstrating that lipidomics aimed at expanding our knowledge of the family of N-acyl amides has the potential to provide novel avenues of research for TRP receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity

Directory of Open Access Journals (Sweden)

Darya V. Bazovkina

2015-01-01

Full Text Available In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors.

Antagonism of brain insulin-like growth factor-1 receptors blocks estradiol effects on memory and levels of hippocampal synaptic proteins in ovariectomized rats

Science.gov (United States)

Nelson, Britta S.; Springer, Rachel C.; Daniel, Jill M.

2013-01-01

Rationale Treatment with estradiol, the primary estrogen produced by the ovaries, enhances hippocampus-dependent spatial memory and increases levels of hippocampal synaptic proteins in ovariectomized rats. Increasing evidence indicates that the ability of estradiol to impact the brain and behavior is dependent upon its interaction with insulin-like growth factor-1 (IGF-1). Objectives The goal of the current experiment was to test the hypothesis that the ability of estradiol to impact hippocampus-dependent memory and levels of hippocampal synaptic proteins is dependent on its interaction with IGF-1. Methods Adult rats were ovariectomized and implanted with estradiol or control capsules and trained on a radial-maze spatial memory task. After training, rats were implanted with intracerebroventricular cannulae attached to osmotic minipumps (flow rate 0.15 μl/hr). Half of each hormone treatment group received continuous delivery of JB1 (300 μg/ml), an IGF-1 receptor antagonist, and half received delivery of aCSF vehicle. Rats were tested on trials in the radial-arm maze during which delays were imposed between the 4th and 5th arm choices. Hippocampal levels of synaptic proteins were measured by western blotting. Results Estradiol treatment resulted in significantly enhanced memory. JB1 blocked that enhancement. Estradiol treatment resulted in significantly increased hippocampal levels of postsynaptic density protein 95 (PSD-95), spinophilin, and synaptophysin. JB1 blocked the estradiol-induced increase of PSD-95 and spinophilin and attenuated the increase of synaptophysin. Conclusions Results support a role for IGF-1 receptor activity in estradiol-induced enhancement of spatial memory that may be dependent on changes in synapse structure in the hippocampus brought upon by estradiol/IGF-1 interactions. PMID:24146138

Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

Directory of Open Access Journals (Sweden)

Francesco Marras

2011-01-01

Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

Activation of serotonin 2A receptors underlies the psilocybin-induced effects on α oscillations, N170 visual-evoked potentials, and visual hallucinations.

Science.gov (United States)

Kometer, Michael; Schmidt, André; Jäncke, Lutz; Vollenweider, Franz X

2013-06-19

Visual illusions and hallucinations are hallmarks of serotonergic hallucinogen-induced altered states of consciousness. Although the serotonergic hallucinogen psilocybin activates multiple serotonin (5-HT) receptors, recent evidence suggests that activation of 5-HT2A receptors may lead to the formation of visual hallucinations by increasing cortical excitability and altering visual-evoked cortical responses. To address this hypothesis, we assessed the effects of psilocybin (215 μg/kg vs placebo) on both α oscillations that regulate cortical excitability and early visual-evoked P1 and N170 potentials in healthy human subjects. To further disentangle the specific contributions of 5-HT2A receptors, subjects were additionally pretreated with the preferential 5-HT2A receptor antagonist ketanserin (50 mg vs placebo). We found that psilocybin strongly decreased prestimulus parieto-occipital α power values, thus precluding a subsequent stimulus-induced α power decrease. Furthermore, psilocybin strongly decreased N170 potentials associated with the appearance of visual perceptual alterations, including visual hallucinations. All of these effects were blocked by pretreatment with the 5-HT2A antagonist ketanserin, indicating that activation of 5-HT2A receptors by psilocybin profoundly modulates the neurophysiological and phenomenological indices of visual processing. Specifically, activation of 5-HT2A receptors may induce a processing mode in which stimulus-driven cortical excitation is overwhelmed by spontaneous neuronal excitation through the modulation of α oscillations. Furthermore, the observed reduction of N170 visual-evoked potentials may be a key mechanism underlying 5-HT2A receptor-mediated visual hallucinations. This change in N170 potentials may be important not only for psilocybin-induced states but also for understanding acute hallucinatory states seen in psychiatric disorders, such as schizophrenia and Parkinson's disease.

Mu-opioid receptor knockout mice show diminished food-anticipatory activity

NARCIS (Netherlands)

Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

Modulation of inhibitory activity markers by intermittent theta-burst stimulation in rat cortex is NMDA-receptor dependent.

Science.gov (United States)

Labedi, Adnan; Benali, Alia; Mix, Annika; Neubacher, Ute; Funke, Klaus

2014-01-01

Intermittent theta-burst stimulation (iTBS) applied via transcranial magnetic stimulation has been shown to increase cortical excitability in humans. In the rat brain it strongly reduced the number of neurons expressing the 67-kD isoform of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) and those expressing the calcium-binding proteins parvalbumin (PV) and calbindin (CB), specific markers of fast-spiking (FS) and non-FS inhibitory interneurons, respectively, an indication of modified cortical inhibition. Since iTBS effects in humans have been shown to be NMDA receptor sensitive, we wondered whether the iTBS-induced changes in the molecular phenotype of interneurons may be also sensitive to glutamatergic synaptic transmission mediated by NMDA receptors. In a sham-controlled fashion, five iTBS-blocks of 600 stimuli were applied to rats either lightly anesthetized by only urethane or by an additional low (subnarcotic) or high dose of the NMDA receptor antagonist ketamine before immunohistochemical analysis. iTBS reduced the number of neurons expressing GAD67, PV and CB. Except for CB, a low dose of ketamine partially prevented these effects while a higher dose almost completely abolished the iTBS effects. Our findings indicate that iTBS modulates the molecular, and likely also the electric, activity of cortical inhibitory interneurons and that the modulation of FS-type but less that of non-FS-type neurons is mediated by NMDA receptors. A combination of iTBS with pharmacological interventions affecting distinct receptor subtypes may thus offer options to enhance its selectivity in modulating the activity of distinct cell types and preventing others from being modulated. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Energy Technology Data Exchange (ETDEWEB)

Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

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.

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

International Nuclear Information System (INIS)

Wang, Feng; Yang, Yong

2014-01-01

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

Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

Directory of Open Access Journals (Sweden)

Mei-Fei Yueh

Full Text Available Triclocarban (3,4,4'-trichlorocarbanilide, TCC is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs. To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR and estrogen receptor alpha (ERα activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR because no induction occurred in hUGT1Car(-/- mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for

β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

Science.gov (United States)

Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

2010-01-01

Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

Science.gov (United States)

Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

2016-05-01

The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses*

Science.gov (United States)

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

2010-01-01

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy. PMID:20018848

Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

Science.gov (United States)

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

2010-02-19

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Androgen insensitivity syndrome: gonadal androgen receptor activity

International Nuclear Information System (INIS)

Coulam, C.B.; Graham, M.L.; Spelsberg, T.C.

1984-01-01

To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant . 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons

Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors.

Directory of Open Access Journals (Sweden)

Mohammad Harun-Or-Rashid

Full Text Available Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR system and extracellular signal-regulated kinase (ERK signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173 EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2, EGFR-inhibitor (AG1478, EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001, consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in

Crosstalk between the peroxisome proliferator-activated receptor γ (PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25-dihydroxyvitamin D3 mediated transactivation

International Nuclear Information System (INIS)

Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R.; Knethen, Andreas von; Choubey, Divaker; Mehta, Rajendra G.

2012-01-01

Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D 3 (1,25D 3 ) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D 3 transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D 3 action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways. -- Highlights: PPARγ's role on 1α,25-dihydroxyvitamin D 3 transcriptional activity is examined. ► PPARγ physically binds to VDR and inhibits 1α,25-dihydroxyvitamin D 3 action. ► PPARγ's hinge and ligand binding domains are important for this inhibitory effect. ► PPARγ competes with VDR for the availability of their binding partner, RXRα.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe

2018-01-22

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe; Wheeler, Janet I; Marondedze, Claudius; Gehring, Christoph A; Irving, Helen R

2018-01-01

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L

NARCIS (Netherlands)

Real, PJ; Benito, A; Cuevas, J; Berciano, MT; de Juan, A; Coffer, P; Gomez-Roman, J; Lafarga, M; Lopez-Vega, JM; Fernandez-Luna, JL

2005-01-01

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and β-adrenergic receptor signaling pathways

International Nuclear Information System (INIS)

Shin, Vivian Yvonne; Jin, H.C.; Ng, Enders K.O.; Yu Jun; Leung, W.K.; Cho, C.H.; Sung, J.J.Y.

2008-01-01

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor (α7 nAChR) and β-adrenergic receptors. Treatment of cells with α-bungarotoxin (α-BTX, α7nAChR antagonist) or propranolol (β-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE 2 and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE 2 induction can only be suppressed by propranolol, but not α-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis

A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism

Science.gov (United States)

Hasbi, Ahmed; Perreault, Melissa L.; Shen, Maurice Y. F.; Zhang, Lucia; To, Ryan; Fan, Theresa; Nguyen, Tuan; Ji, Xiaodong; O'Dowd, Brian F.; George, Susan R.

2014-01-01

Although the dopamine D1-D2 receptor heteromer has emerging physiological relevance and a postulated role in different neuropsychiatric disorders, such as drug addiction, depression, and schizophrenia, there is a need for pharmacological tools that selectively target such receptor complexes in order to analyze their biological and pathophysiological functions. Since no selective antagonists for the D1-D2 heteromer are available, serial deletions and point mutations were used to precisely identify the amino acids involved in an interaction interface between the receptors, residing within the carboxyl tail of the D1 receptor that interacted with the D2 receptor to form the D1-D2 receptor heteromer. It was determined that D1 receptor carboxyl tail residues 404Glu and 405Glu were critical in mediating the interaction with the D2 receptor. Isolated mutation of these residues in the D1 receptor resulted in the loss of agonist activation of the calcium signaling pathway mediated through the D1-D2 receptor heteromer. The physical interaction between the D1 and D2 receptor could be disrupted, as shown by coimmunoprecipitation and BRET analysis, by a small peptide generated from the D1 receptor sequence that contained these amino acids, leading to a switch in G-protein affinities and loss of calcium signaling, resulting in the inhibition of D1-D2 heteromer function. The use of the D1-D2 heteromer-disrupting peptide in vivo revealed a pathophysiological role for the D1-D2 heteromer in the modulation of behavioral despair. This peptide may represent a novel pharmacological tool with potential therapeutic benefits in depression treatment.—Hasbi, A., Perreault, M. L., Shen, M. Y. F., Zhang, L., To, R., Fan, T., Nguyen, T., Ji, X., O'Dowd, B. F., George, S. R. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism. PMID:25063849

NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes.

Science.gov (United States)

Lalo, Ulyana; Pankratov, Yuri; Kirchhoff, Frank; North, R Alan; Verkhratsky, Alexei

2006-03-08

Chemical transmission between neurons and glial cells is an important element of integration in the CNS. Here, we describe currents activated by NMDA in cortical astrocytes, identified in transgenic mice that express enhanced green fluorescent protein under control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp either in slices or after gentle nonenzymatic mechanical dissociation. Acutely isolated astrocytes showed a three-component response to glutamate. The initial rapid component was blocked by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), which is an antagonist of AMPA receptors (IC50, 2 microM), and the NMDA receptor antagonist D-AP-5 blocked the later sustained component (IC50, 0.6 microM). The third component of glutamate application response was sensitive to D,L-threo-beta-benzyloxyaspartate, a glutamate transporter blocker. Fast application of NMDA evoked concentration-dependent inward currents (EC50, 0.3 microM); these showed use-dependent block by (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801). These NMDA-evoked currents were linearly dependent on membrane potential and were not affected by extracellular magnesium at concentrations up to 10 mM. Electrical stimulation of axons in layer IV-VI induced a complex inward current in astrocytes situated in the cortical layer II, part of which was sensitive to MK-801 at holding potential -80 mV and was not affected by the AMPA glutamate receptor antagonist NBQX. The fast miniature spontaneous currents were observed in cortical astrocytes in slices as well. These currents exhibited both AMPA and NMDA receptor-mediated components. We conclude that cortical astrocytes express functional NMDA receptors that are devoid of Mg2+ block, and these receptors are involved in neuronal-glial signal transmission.

Family C 7TM receptor dimerization and activation

DEFF Research Database (Denmark)

Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

2006-01-01

The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug...... to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors.......-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational...

Differences in signal activation by LH and hCG are mediated by the LH/CG receptor`s extracellular hinge region

Directory of Open Access Journals (Sweden)

Paul eGrzesik

2015-09-01

Full Text Available The human lutropin/choriogonadotropin receptor (LHCGR can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG - secreted by the placenta, and lutropin (LH - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich repeat domain (LRRD, as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting mutations. These helix preserving modifications showed no effect on hormone induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10 deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region s. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region

Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies

Science.gov (United States)

Morshed, S. A.; Davies, T. F.

2016-01-01

The immunologic processes involved in Graves' disease (GD) have one unique characteristic – the autoantibodies to the TSH receptor (TSHR) – which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called “neutral” antibodies and which we now characterize as autoantibodies to the “cleavage” region of the TSHR ectodomain. PMID:26361259

Anthropogenic Changes in Mid-latitude Storm and Blocking Activities from Observations and Climate Models

Science.gov (United States)

Li, D.

2017-12-01

Fingerprints of anthropogenic climate change can be most readily detected in the high latitudes of Northern Hemisphere, where temperature has been rising faster than the rest of the globe and sea ice cover has shrunk dramatically over recent decades. Reducing the meridional temperature gradient, this amplified warming over the high latitudes influences weather in the middle latitudes by modulating the jet stream, storms, and atmospheric blocking activities. Whether observational records have revealed significant changes in mid-latitude storms and blocking activities, however, has remained a subject of much debate. Buried deep in strong year-to-year variations, the long-term dynamic responses of the atmosphere are more difficult to identify, compared with its thermodynamic responses. Variabilities of decadal and longer timescales further obscure any trends diagnosed from satellite observations, which are often shorter than 40 years. Here, new metrics reflecting storm and blocking activities are developed using surface air temperature and pressure records, and their variations and long-term trends are examined. This approach gives an inkling of the changes in storm and blocking activities since the Industrial Revolution in regions with abundant long-term observational records, e.g. Europe and North America. The relationship between Atlantic Multi-decadal Oscillation and variations in storm and blocking activities across the Atlantic is also scrutinized. The connection between observed centennial trends and anthropogenic forcings is investigated using a hierarchy of numerical tools, from highly idealized to fully coupled atmosphere-ocean models. Pre-industrial control simulations and a set of large ensemble simulations forced by increased CO2 are analyzed to evaluate the range of natural variabilities, which paves the way to singling out significant anthropogenic changes from observational records, as well as predicting future changes in mid-latitude storm and

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

Science.gov (United States)

Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

2017-01-01

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

DEFF Research Database (Denmark)

Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren

2002-01-01

delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

Structural mechanism of ligand activation in human calcium-sensing receptor

Energy Technology Data Exchange (ETDEWEB)

Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P.; Brennan, Sarah C.; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X.; Cao, Baohua; Chang, Donald D.; Quick, Matthias; Conigrave, Arthur D.; Colecraft, Henry M.; McDonald, Patricia; Fan, Qing R.

2016-07-19

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca²⁺homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca²⁺and PO₄^3-ions. Both ions are crucial for structural integrity of the receptor. While Ca²⁺ions stabilize the active state, PO₄^3-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

Plasticizers May Activate Human Hepatic Peroxisome Proliferator-Activated Receptor α Less Than That of a Mouse but May Activate Constitutive Androstane Receptor in Liver

Science.gov (United States)

Ito, Yuki; Nakamura, Toshiki; Yanagiba, Yukie; Ramdhan, Doni Hikmat; Yamagishi, Nozomi; Naito, Hisao; Kamijima, Michihiro; Gonzalez, Frank J.; Nakajima, Tamie

2012-01-01

Dibutylphthalate (DBP), di(2-ethylhexyl)phthalate (DEHP), and di(2-ethylhexyl)adipate (DEHA) are used as plasticizers. Their metabolites activate peroxisome proliferator-activated receptor (PPAR) α, which may be related to their toxicities. However, species differences in the receptor functions between rodents and human make it difficult to precisely extrapolate their toxicity from animal studies to human. In this paper, we compared the species differences in the activation of mouse and human hepatic PPARα by these plasticizers using wild-type (mPPARα) and humanized PPARα (hPPARα) mice. At 12 weeks old, each genotyped male mouse was classified into three groups, and fed daily for 2 weeks per os with corn oil (vehicle control), 2.5 or 5.0 mmol/kg DBP (696, 1392 mg/kg), DEHP (977, 1953 mg/kg), and DEHA (926, 1853 mg/kg), respectively. Generally, hepatic PPARα of mPPARα mice was more strongly activated than that of hPPARα mice when several target genes involving β-oxidation of fatty acids were evaluated. Interestingly, all plasticizers also activated hepatic constitutive androstane receptor (CAR) more in hPPARα mice than in mPPARα mice. Taken together, these plasticizers activated mouse and human hepatic PPARα as well as CAR. The activation of PPARα was stronger in mPPARα mice than in hPPARα mice, while the opposite was true of CAR. PMID:22792086

Plasticizers May Activate Human Hepatic Peroxisome Proliferator-Activated Receptor α Less Than That of a Mouse but May Activate Constitutive Androstane Receptor in Liver

Directory of Open Access Journals (Sweden)

Yuki Ito

2012-01-01

Full Text Available Dibutylphthalate (DBP, di(2-ethylhexylphthalate (DEHP, and di(2-ethylhexyladipate (DEHA are used as plasticizers. Their metabolites activate peroxisome proliferator-activated receptor (PPAR α, which may be related to their toxicities. However, species differences in the receptor functions between rodents and human make it difficult to precisely extrapolate their toxicity from animal studies to human. In this paper, we compared the species differences in the activation of mouse and human hepatic PPARα by these plasticizers using wild-type (mPPARα and humanized PPARα (hPPARα mice. At 12 weeks old, each genotyped male mouse was classified into three groups, and fed daily for 2 weeks per os with corn oil (vehicle control, 2.5 or 5.0 mmol/kg DBP (696, 1392 mg/kg, DEHP (977, 1953 mg/kg, and DEHA (926, 1853 mg/kg, respectively. Generally, hepatic PPARα of mPPARα mice was more strongly activated than that of hPPARα mice when several target genes involving β-oxidation of fatty acids were evaluated. Interestingly, all plasticizers also activated hepatic constitutive androstane receptor (CAR more in hPPARα mice than in mPPARα mice. Taken together, these plasticizers activated mouse and human hepatic PPARα as well as CAR. The activation of PPARα was stronger in mPPARα mice than in hPPARα mice, while the opposite was true of CAR.

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

Energy Technology Data Exchange (ETDEWEB)

Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

2011-10-14

Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

International Nuclear Information System (INIS)

Teutschbein, Janka; Haydn, Johannes M; Samans, Birgit; Krause, Michael; Eilers, Martin; Schartl, Manfred; Meierjohann, Svenja

2010-01-01

Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

Directory of Open Access Journals (Sweden)

Krause Michael

2010-07-01

Full Text Available Abstract Background Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1, early growth response 1 (Egr1, osteopontin (Opn, insulin-like growth factor binding protein 3 (Igfbp3, dual-specificity phosphatase 4 (Dusp4, and tumor-associated antigen L6 (Taal6. Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute

Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on delta-opioid receptor stimulation.

Science.gov (United States)

Panneerselvam, Mathivadhani; Tsutsumi, Yasuo M; Bonds, Jacqueline A; Horikawa, Yousuke T; Saldana, Michelle; Dalton, Nancy D; Head, Brian P; Patel, Piyush M; Roth, David M; Patel, Hemal H

2010-11-01

Epicatechin, a flavonoid, is a well-known antioxidant linked to a variety of protective effects in both humans and animals. In particular, its role in protection against cardiovascular disease has been demonstrated by epidemiologic studies. Low-dose epicatechin, which does not have significant antioxidant activity, is also protective; however, the mechanism by which low-dose epicatechin induces this effect is unknown. Our laboratory tested the hypothesis that low-dose epicatechin mediates cardiac protection via opioid receptor activation. C57BL/6 mice were randomly assigned to 1 of 10 groups: control, epicatechin, naloxone (nonselective opioid receptor antagonist), epicatechin + naloxone, naltrindole (δ-specific opioid receptor antagonist), epicatechin + naltrindole, norbinaltorphimine (nor-BNI, κ-specific opioid receptor antagonist), epicatechin + nor-BNI, 5-hydroxydecanoic acid [5-HD, ATP-sensitive potassium channel antagonist], and epicatechin + 5-HD. Epicatechin (1 mg/kg) or other inhibitors (5 mg/kg) were administered by oral gavage or intraperitoneal injection, respectively, daily for 10 days. Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion, and infarct size was determined via planimetry. Whole heart homogenates were assayed for downstream opioid receptor signaling targets. Infarct size was significantly reduced in epicatechin- and epicatechin + nor-BNI-treated mice compared with control mice. This protection was blocked by naloxone, naltrindole, and 5-HD. Epicatechin and epicatechin + nor-BNI increased the phosphorylation of Src, Akt, and IκBα, while simultaneously decreasing the expression of c-Jun NH(2)-terminal kinase and caspase-activated DNase. All signaling effects are consistent with opioid receptor stimulation and subsequent cardiac protection. Naloxone, naltrindole, and 5-HD attenuated these effects. In conclusion, epicatechin acts via opioid receptors and more specifically through the δ-opioid receptor to

Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

Science.gov (United States)

Suhara, W; Koide, H; Okuzawa, T; Hayashi, D; Hashimoto, T; Kojo, H

2009-09-01

The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

A synthetic peptide blocking TRPV1 activation inhibits UV-induced skin responses.

Science.gov (United States)

Kang, So Min; Han, Sangbum; Oh, Jang-Hee; Lee, Young Mee; Park, Chi-Hyun; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

2017-10-01

Transient receptor potential type 1 (TRPV1) can be activated by ultraviolet (UV) irradiation, and mediates UV-induced matrix metalloproteinase (MMP)-1 and proinflammatory cytokines in keratinocytes. Various chemicals and compounds targeting TRPV1 activation have been developed, but are not in clinical use mostly due to their safety issues. We aimed to develop a novel TRPV1-targeting peptide to inhibit UV-induced responses in human skin. We designed and generated a novel TRPV1 inhibitory peptide (TIP) which mimics the specific site in TRPV1 (aa 701-709: Gln-Arg-Ala-Ile-Thr-Ile-Leu-Asp-Thr, QRAITILDT), Thr 705 , and tested its efficacy of blocking UV-induced responses in HaCaT, mouse, and human skin. TIP effectively inhibited capsaicin-induced calcium influx and TRPV1 activation. Treatment of HaCaT with TIP prevented UV-induced increases of MMP-1 and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α. In mouse skin in vivo, TIP inhibited UV-induced skin thickening and prevented UV-induced expression of MMP-13 and MMP-9. Moreover, TIP attenuated UV-induced erythema and the expression of MMP-1, MMP-2, IL-6, and IL-8 in human skin in vivo. The novel synthetic peptide targeting TRPV1 can ameliorate UV-induced skin responses in vitro and in vivo, providing a promising therapeutic approach against UV-induced inflammation and photoaging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

FMRFamide receptors of Helix aspersa

International Nuclear Information System (INIS)

Payza, K.

1988-01-01

A receptor binding assay and an isolated heart bioassay were used to identify and characterize the FMRFamide receptors in Helix. In the heart bioassay, FMRFamide increased myocardial contraction force. A potent FMRFamide analog, desaminoTyr-Phe-norLeu-arg-Phe-amide (daYFnLRFamide), was used as a radioiodinated receptor ligand. The high affinity binding of 125 I-daYFnLRFamide at 0 degree C to Helix brain membranes was reversible, saturable, pH-dependent and specific, with a K D of 13-14 nM. A lower affinity (245 nM) site was also observed. Radioligand binding sites were also identified in the heart, male reproductive organs and digestive organs. The structure-activity relations (SAR) of cardiostimulation correlated with the specificity of 125 I-daYFnLRFamide binding to brain and heart receptors. The SAR were similar to those of other molluscan FMRFamide bioassays, except that they showed a marked preference for some analogs with blocked amino-terminals

Effect of propofol on androgen receptor activity in prostate cancer cells.

Science.gov (United States)

Tatsumi, Kenichiro; Hirotsu, Akiko; Daijo, Hiroki; Matsuyama, Tomonori; Terada, Naoki; Tanaka, Tomoharu

2017-08-15

Androgen receptor is a nuclear receptor and transcription factor activated by androgenic hormones. Androgen receptor activity plays a pivotal role in the development and progression of prostate cancer. Although accumulating evidence suggests that general anesthetics, including opioids, affect cancer cell growth and impact patient prognosis, the effect of those drugs on androgen receptor in prostate cancer is not clear. The purpose of this study was to investigate the effect of the general anesthetic propofol on androgen receptor activity in prostate cancer cells. An androgen-dependent human prostate cancer cell line (LNCaP) was stimulated with dihydrotestosterone (DHT) and exposed to propofol. The induction of androgen receptor target genes was investigated using real-time reverse transcription polymerase chain reaction, and androgen receptor protein levels and localization patterns were analyzed using immunoblotting and immunofluorescence assays. The effect of propofol on the proliferation of LNCaP cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Propofol significantly inhibited DHT-induced expression of androgen receptor target genes in a dose- and time-dependent manner, and immunoblotting and immunofluorescence assays indicated that propofol suppressed nuclear levels of androgen receptor proteins. Exposure to propofol for 24h suppressed the proliferation of LNCaP cells, whereas 4h of exposure did not exert significant effects. Together, our results indicate that propofol suppresses nuclear androgen receptor protein levels, and inhibits androgen receptor transcriptional activity and proliferation in LNCaP cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

LENUS (Irish Health Repository)

Gleeson, Eimear M

2013-07-19

Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

Regulation of extinction-related plasticity by opioid receptors in the ventrolateral periaqueductal gray matter

Directory of Open Access Journals (Sweden)

Ryan Parsons

2010-08-01

Full Text Available Recent work has led to a better understanding of the neural mechanisms underlying the extinction of Pavlovian fear conditioning. Long-term synaptic changes in the medial prefrontal cortex (mPFC are critical for extinction learning, but very little is currently known about how the mPFC and other brain areas interact during extinction. The current study examined the effect of drugs that impair the extinction of fear conditioning on the activation of the extracellular-related kinase/mitogen-activated protein kinase (ERK/MAPK in brain regions that likely participate in the consolidation of extinction learning. Inhibitors of opioid and N-methyl-D-aspartic acid (NMDA receptors were applied to the ventrolateral periaqueductal gray matter (vlPAG and amygdala shortly before extinction training. Results from these experiments show that blocking opioid receptors in the vlPAG prevented the formation of extinction memory, whereas NMDA receptor blockade had no effect. Conversely, blocking NMDA receptors in the amygdala disrupted the formation of fear extinction memory, but opioid receptor blockade in the same brain area did not. Subsequent experiments tested the effect of these drug treatments on the activation of the ERK/MAPK signaling pathway in various brain regions following extinction training. Only opioid receptor blockade in the vlPAG disrupted ERK phosphorylation in the mPFC and amygdala. These data support the idea that opiodergic signaling derived from the vlPAG affects plasticity across the brain circuit responsible for the formation of extinction memory.

Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer metastasis.

Science.gov (United States)

Kenny, Hilary A; Leonhardt, Payton; Ladanyi, Andras; Yamada, S Diane; Montag, Anthony; Im, Hae Kyung; Jagadeeswaran, Sujatha; Shaw, David E; Mazar, Andrew P; Lengyel, Ernst

2011-02-01

To understand the functional and preclinical efficacy of targeting the urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in 3 (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cell lines with the u-PAR antibody inhibited cell invasion, migration, and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α(5)-integrin and u-PAR colocalization on primary human omental extracellular matrix. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, β(3)-integrin, and fibroblast growth factor receptor-1 both in vitro and in vivo. This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α(5)-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication. ©2010 AACR.

Quantum chemical study of agonist-receptor vibrational interactions for activation of the glutamate receptor.

Science.gov (United States)

Kubo, M; Odai, K; Sugimoto, T; Ito, E

2001-06-01

To understand the mechanism of activation of a receptor by its agonist, the excitation and relaxation processes of the vibrational states of the receptor should be examined. As a first approach to this problem, we calculated the normal vibrational modes of agonists (glutamate and kainate) and an antagonist (6-cyano-7-nitroquinoxaline-2,3-dione: CNQX) of the glutamate receptor, and then investigated the vibrational interactions between kainate and the binding site of glutamate receptor subunit GluR2 by use of a semiempirical molecular orbital method (MOPAC2000-PM3). We found that two local vibrational modes of kainate, which were also observed in glutamate but not in CNQX, interacted through hydrogen bonds with the vibrational modes of GluR2: (i) the bending vibration of the amine group of kainate, interacting with the stretching vibration of the carboxyl group of Glu705 of GluR2, and (ii) the symmetric stretching vibration of the carboxyl group of kainate, interacting with the bending vibration of the guanidinium group of Arg485. We also found collective modes with low frequency at the binding site of GluR2 in the kainate-bound state. The vibrational energy supplied by an agonist may flow from the high-frequency local modes to the low-frequency collective modes in a receptor, resulting in receptor activation.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

Science.gov (United States)

Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

2015-04-28

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

AMP is an adenosine A1 receptor agonist.

Science.gov (United States)

Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

2012-02-17

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

Science.gov (United States)

Taupin, J L; Anderson, P

1994-12-01

The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

Melanocortin-4 receptor activation stimulates hypothalamic brain-derived neurotrophic factor release to regulate food intake, body temperature and cardiovascular function.

Science.gov (United States)

Nicholson, J R; Peter, J-C; Lecourt, A-C; Barde, Y-A; Hofbauer, K G

2007-12-01

In the present study, we aimed to investigate the neuromodulatory role played by hypothalamic brain-derived neurotrophic factor (BDNF) in the regulation of acute cardiovascular and feeding responses to melanocortin-4 receptor (MC4R) activation. In vitro, a selective MC4R agonist, MK1, stimulated BDNF release from isolated rat hypothalami and this effect was blocked by preincubation with the MC3/4R antagonist SHU-9119. In vivo, peripheral administration of MK1 decreased food intake in rats and this effect was blocked by pretreatment with an anti-BDNF antibody administered into the third ventricle. When anorexia was induced with the cannabinoid-1 receptor (CB1R) antagonist AM251, the anti-BDNF antibody did not prevent the reduction in food intake. Peripheral administration of MK1 also increased mean arterial pressure, heart rate and body temperature. These effects were prevented by pretreatment with the anti-BDNF antibody whereas the intracerebroventricular administration of BDNF caused changes similar to those of MK1. These findings demonstrate for the first time that activation of MC4R leads to an acute release of BDNF in the hypothalamus. This release is a prerequisite for MC4R-induced effects on appetite, body temperature and cardiovascular function. By contrast, CB1R antagonist-mediated anorexia is independent of the MC4R/BDNF pathway. Overall, these results show that BDNF is an important downstream mediator of the MC4R pathway.

Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

KAUST Repository

Muleya, Victor

2014-09-23

Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

Proteinase-activated receptors - mediators of early and delayed normal tissue radiation responses

International Nuclear Information System (INIS)

Hauer-Jensen, M.

2003-01-01

Proteinase-activated receptors (PARs) are G-protein coupled receptors that are activated by proteolytic exposure of a receptor-tethered ligand. The discovery of this receptor family represents one of the most intriguing recent developments in signal transduction. PARs are involved in the regulation of many normal and pathophysiological processes, notably inflammatory and fibroproliferative responses to injury. Preclinical studies performed in our laboratory suggest that proteinase-activated receptor-1 (PAR-1) plays a critical role in the mechanism of chronicity of radiation fibrosis, while proteinase-activated receptor-2 (PAR-2) may mediate important fibroproliferative responses in irradiated intestine. Specifically, activation of PAR-1 by thrombin, and PAR-2 by pancreatic trypsin and mast cell proteinases, appears to be involved in acute radiation-induced inflammation, as well as in subsequent extracellular matrix deposition, leading to the development of intestinal wall fibrosis and clinical complications. Pharmacological modulators of PAR-1 or PAR-2 expression or activation would be potentially useful as preventive or therapeutic agents in patients who receive radiation therapy, especially if blockade could be targeted to specific tissues or cellular compartments

Baclofen blocks the acquisition and expression of mitragynine-induced conditioned place preference in rats.

Science.gov (United States)

Yusoff, Nurul H M; Mansor, Sharif M; Müller, Christian P; Hassan, Zurina

2018-06-01

Mitragynine is the major alkaloid found in the leaves of M. speciosa Korth (Rubiaceae), a plant that is native to Southeast Asia. This compound has been used, either traditionally or recreationally, due to its psychostimulant and opioid-like effects. Recently, mitragynine has been shown to exert conditioned place preference (CPP), indicating the rewarding and motivational properties of M. speciosa. Here, the involvement of GABA B receptors in mediating mitragynine reward is studied using a CPP paradigm in rats. First, we examined the effects of GABA B receptor agonist baclofen (1.25, 2.5 and 5 mg/kg) on the acquisition of mitragynine (10 mg/kg)-induced CPP. Second, the involvement of GABA B receptors in the expression of mitragynine-induced CPP was tested. We found that the acquisition of mitragynine-induced CPP could be blocked by higher doses (2.5 and 5 mg/kg) of baclofen. Baclofen at a high dose inhibited locomotor activity and caused a CPP. Furthermore, we found that baclofen (2.5 and 5 mg/kg) also blocked the expression of mitragynine-induced CPP. These findings suggest that both, the acquisition and expression of mitragynine's reinforcing properties is controlled by the GABA B receptor. Copyright © 2018 Elsevier B.V. All rights reserved.

Endogenous activation of adenosine A(1) receptors accelerates ischemic suppression of spontaneous electrocortical activity

DEFF Research Database (Denmark)

Ilie, Andrei; Ciocan, Dragos; Zagrean, Ana-Maria

2006-01-01

Cerebral ischemia induces a rapid suppression of spontaneous brain rhythms prior to major alterations in ionic homeostasis. It was found in vitro during ischemia that the rapidly formed adenosine, resulting from the intracellular breakdown of ATP, may inhibit synaptic transmission via the A(1......) receptor subtype. The link between endogenous A(1) receptor activation during ischemia and the suppression of spontaneous electrocortical activity has not yet been established in the intact brain. The aim of this study was to investigate in vivo the effects of A(1) receptor antagonism by 8-cyclopentyl-1...

Designing peptide inhibitor of insulin receptor to induce diabetes mellitus type 2 in animal model Mus musculus.

Science.gov (United States)

Permatasari, Galuh W; Utomo, Didik H; Widodo

2016-10-01

A designing peptide as agent for inducing diabetes mellitus type 2 (T2DM) in an animal model is challenging. The computational approach provides a sophisticated tool to design a functional peptide that may block the insulin receptor activity. The peptide that able to inhibit the binding between insulin and insulin receptor is a warrant for inducing T2DM. Therefore, we designed a potential peptide inhibitor of insulin receptor as an agent to generate T2DM animal model by bioinformatics approach. The peptide has been developed based on the structure of insulin receptor binding site of insulin and then modified it to obtain the best properties of half life, hydrophobicity, antigenicity, and stability binding into insulin receptor. The results showed that the modified peptide has characteristics 100h half-life, high-affinity -95.1±20, and high stability 28.17 in complex with the insulin receptor. Moreover, the modified peptide has molecular weight 4420.8g/Mol and has no antigenic regions. Based on the molecular dynamic simulation, the complex of modified peptide-insulin receptor is more stable than the commercial insulin receptor blocker. This study suggested that the modified peptide has the promising performance to block the insulin receptor activity that potentially induce diabetes mellitus type 2 in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

Directory of Open Access Journals (Sweden)

Carmen A Banuelos

Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block

Science.gov (United States)

Brenneis, C; Kistner, K; Puopolo, M; Jo, S; Roberson, DP; Sisignano, M; Segal, D; Cobos, EJ; Wainger, BJ; Labocha, S; Ferreirós, N; Hehn, C; Tran, J; Geisslinger, G; Reeh, PW; Bean, BP; Woolf, C J

2014-01-01

Background and Purpose: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. Experimental Approach: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. Key Results: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. Conclusions and Implications: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful. PMID:24117225

Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

Science.gov (United States)

Jeske, Judith; Windshügel, Björn; Thasler, Wolfgang E; Schwab, Matthias; Burk, Oliver

2017-06-01

Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

Extrapunitive and intropunitive individuals activate different parts of the prefrontal cortex under an ego-blocking frustration.

Science.gov (United States)

Minamoto, Takehiro; Osaka, Mariko; Yaoi, Ken; Osaka, Naoyuki

2014-01-01

Different people make different responses when they face a frustrating situation: some punish others (extrapunitive), while others punish themselves (intropunitive). Few studies have investigated the neural structures that differentiate extrapunitive and intropunitive individuals. The present fMRI study explored these neural structures using two different frustrating situations: an ego-blocking situation which blocks a desire or goal, and a superego-blocking situation which blocks self-esteem. In the ego-blocking condition, the extrapunitive group (n = 9) showed greater activation in the bilateral ventrolateral prefrontal cortex, indicating that these individuals prefer emotional processing. On the other hand, the intropunitive group (n = 9) showed greater activation in the left dorsolateral prefrontal cortex, possibly reflecting an effortful control for anger reduction. Such patterns were not observed in the superego-blocking condition. These results indicate that the prefrontal cortex is the source of individual differences in aggression direction in the ego-blocking situation.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

Energy Technology Data Exchange (ETDEWEB)

Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Mirshahi, Faridoddin [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Grider, John R. [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Murthy, Karnam S., E-mail: skarnam@vcu.edu [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Sanyal, Arun J., E-mail: asanyal@mcvh-vcu.edu [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States)

2012-10-26

Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells

International Nuclear Information System (INIS)

Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

2012-01-01

Highlights: ► G protein coupled receptor TGR5 is expressed in mouse and human islets. ► TGR5 is coupled to activation of Gs and Ca 2+ release via cAMP/Epac/PLC-ε pathway. ► Activation of TGR5 by bile salts and selective ligands causes insulin secretion. ► TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gα s and caused an increase in intracellular cAMP and Ca 2+ . OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gα s inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G s /cAMP/Ca 2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

The epidermal growth factor receptor as a target for gastrointestinal cancer therapy.

Science.gov (United States)

Tedesco, Karen L; Lockhart, A Craig; Berlin, Jordan D

2004-10-01

The epidermal growth factor receptor (EGFR) is a member of the family of transmembrane protein kinase receptors known as the erbB or HER receptor family. When activated, EGFR phosphorylates and activates other intracellular proteins that affect cell signaling pathways, cellular proliferation, control of apoptosis and angiogenesis. EGFR signaling is best thought of as a network of activating and inactivating proteins with EGFR as the entry point into the network. EGFR overexpression occurs in most GI malignancies and while data are not entirely consistent, EGFR overexpression often confers a poor prognosis in those GI malignancies that have been studied. It often correlates with poorly differentiated histology, more advanced stage and other known poor prognostic markers. The EGFR is a tempting target because of its presence and overexpression on so many tumor types. However, downstream of the EGFR are several proteins that may be activated without EGFR thus allowing blockade to be overcome. Therefore, while blocking the activity of the EGFR protein appears to be a promising anticancer strategy, a simplistic strategy of blocking only EGFR is likely to only impact a minority of patients. It is time for the laboratory and clinical researchers to work closely together to develop this treatment strategy, moving back and forth from clinical to laboratory to best understand how to block this network effectively enough to produce a broader antitumor effect. While multiple methods of targeting the EGFR pathway are under development, including the inhibition of downstream proteins, only two modalities have entered clinical trials in GI malignancies: small molecule inhibitors of the intracellular kinase domain of EGFR and antibodies designed to block the extracellular ligand-binding domain of EGFR. EGFR inhibitors are still experimental in every GI malignancy with the notable exception of cetuximab that is approved for second or third-line therapy of metastatic colorectal

Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions.

Science.gov (United States)

Perianes-Cachero, A; Burgos-Ramos, E; Puebla-Jiménez, L; Canelles, S; Frago, L M; Hervás-Aguilar, A; de Frutos, S; Toledo-Lobo, M V; Mela, V; Viveros, M P; Argente, J; Chowen, J A; Arilla-Ferreiro, E; Barrios, V

2013-11-12

Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5 μg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain

Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

Directory of Open Access Journals (Sweden)

Ahnen Dennis

2005-01-01

Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

Directory of Open Access Journals (Sweden)

Farhad Dehkhoda

2018-02-01

Full Text Available The growth hormone receptor (GHR, although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK–signal transducer and activator of transcription (STAT signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling.

The metabotropic glutamate receptors: structure, activation mechanism and pharmacology.

Science.gov (United States)

Pin, Jean-Philippe; Acher, Francine

2002-06-01

The metabotropic glutamate receptors are G-protein coupled receptors (GPCR) involved in the regulation of many synapses, including most glutamatergic fast excitatory synapses. Eight subtypes have been identified that can be classified into three groups. The molecular characterization of these receptors revealed proteins much more complex than any other GPCRs. They are composed of a Venus Flytrap (VFT) module where glutamate binds, connected to a heptahelical domain responsible for G-protein coupling. Recent data including the structure of the VFT module determined with and without glutamate, indicate that these receptors function as dimers. Moreover a number of intracellular proteins can regulate their targeting and transduction mechanism. Such structural features of mGlu receptors offer multiple possibilities for synthetic compounds to modulate their activity. In addition to agonists and competitive antagonists acting at the glutamate binding site, a number of non-competitive antagonists with inverse agonist activity, and positive allosteric modulators have been discovered. These later compounds share specific properties that make them good candidates for therapeutic applications. First, their non-amino acid structure makes them pass more easily the blood brain barrier. Second, they are much more selective than any other compound identified so far, being the first subtype selective molecules. Third, for the negative modulators, their non competitive mechanism of action makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g. stroke, epilepsy, neuropathic pain, etc.). Fourth, like the benzodiazepines acting at the GABA(A) receptors, the positive modulators offer a new way to increase the activity of these receptors in vivo, with a low risk of inducing their desensitization. The present review article focuses on the specific structural features of these receptors and highlights the various possibilities these

Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents.

Science.gov (United States)

Mesnage, Robin; Phedonos, Alexia; Biserni, Martina; Arno, Matthew; Balu, Sucharitha; Corton, J Christopher; Ugarte, Ricardo; Antoniou, Michael N

2017-10-01

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (-4.10 kcal mol -1 ) compared to estradiol (-25.79 kcal mol -1 ), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Kaempferol inhibits the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes by blocking activation of the MAPK pathway.

Science.gov (United States)

Pan, Dongmei; Li, Nan; Liu, Yanyan; Xu, Qiang; Liu, Qingping; You, Yanting; Wei, Zhenquan; Jiang, Yubao; Liu, Minying; Guo, Tianfeng; Cai, Xudong; Liu, Xiaobao; Wang, Qiang; Liu, Mingling; Lei, Xujie; Zhang, Mingying; Zhao, Xiaoshan; Lin, Changsong

2018-02-01

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) play an essential role in cartilage destruction. Aggressive migration and invasion by FLSs significantly affect RA pathology. Kaempferol has been shown to inhibit cancer cell migration and invasion. However, the effects of kaempferol on RA FLSs have not been investigated. Our study aimed to determine the effects of kaempferol on RA both in vitro and in vivo. In vitro, cell migration and invasion were measured using scratch assays and the Boyden chamber method, respectively. The cytoskeletal reorganization of RA FLSs was evaluated by immunofluorescence staining. Matrix metalloproteinase (MMP) levels were measured by real-time PCR, and protein expression levels were measured by western blotting. In vivo, the effects of kaempferol were evaluated in mice with CIA. The results showed that kaempferol reduced migration, invasion and MMP expression in RA FLSs. In addition, we demonstrated that kaempferol inhibited reorganization of the actin cytoskeleton during cell migration. Moreover, kaempferol dramatically suppressed tumor necrosis factor (TNF)-α-induced MAPK activation without affecting the expression of TNF-α receptors. We also demonstrated that kaempferol attenuated the severity of arthritis in mice with CIA. Taken together, these results suggested that kaempferol inhibits the migration and invasion of FLSs in RA by blocking MAPK pathway activation without affecting the expression of TNF-α receptors. Copyright © 2017 Elsevier B.V. All rights reserved.

Extrapunitive and intropunitive individuals activate different parts of the prefrontal cortex under an ego-blocking frustration.

Directory of Open Access Journals (Sweden)

Takehiro Minamoto

Full Text Available Different people make different responses when they face a frustrating situation: some punish others (extrapunitive, while others punish themselves (intropunitive. Few studies have investigated the neural structures that differentiate extrapunitive and intropunitive individuals. The present fMRI study explored these neural structures using two different frustrating situations: an ego-blocking situation which blocks a desire or goal, and a superego-blocking situation which blocks self-esteem. In the ego-blocking condition, the extrapunitive group (n = 9 showed greater activation in the bilateral ventrolateral prefrontal cortex, indicating that these individuals prefer emotional processing. On the other hand, the intropunitive group (n = 9 showed greater activation in the left dorsolateral prefrontal cortex, possibly reflecting an effortful control for anger reduction. Such patterns were not observed in the superego-blocking condition. These results indicate that the prefrontal cortex is the source of individual differences in aggression direction in the ego-blocking situation.

CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

Data.gov (United States)

U.S. Environmental Protection Agency — Data from a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating using predictive computational...

Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

Science.gov (United States)

Pang, Yefei; Dong, Jing; Thomas, Peter

2015-05-15

Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways. Copyright © 2015 the American Physiological Society.

Rapid resensitization of purinergic receptor function in human platelets.

Science.gov (United States)

Mundell, S J; Barton, J F; Mayo-Martin, M B; Hardy, A R; Poole, A W

2008-08-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

Seizures, refractory status epilepticus, and depolarization block as endogenous brain activities

Science.gov (United States)

El Houssaini, Kenza; Ivanov, Anton I.; Bernard, Christophe; Jirsa, Viktor K.

2015-01-01

Epilepsy, refractory status epilepticus, and depolarization block are pathological brain activities whose mechanisms are poorly understood. Using a generic mathematical model of seizure activity, we show that these activities coexist under certain conditions spanning the range of possible brain activities. We perform a detailed bifurcation analysis and predict strategies to escape from some of the pathological states. Experimental results using rodent data provide support of the model, highlighting the concept that these pathological activities belong to the endogenous repertoire of brain activities.

3-[2,4-Dimethoxybenzylidene]anabaseine (DMXB) selectively activates rat alpha7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner.

Science.gov (United States)

Meyer, E M; Tay, E T; Papke, R L; Meyers, C; Huang, G L; de Fiebre, C M

1997-09-12

The alpha7 nicotinic receptor agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB; GTS-21) was investigated for its ability to: (1) activate a variety of nicotinic receptor subtypes in Xenopus oocytes; (2) improve passive avoidance and spatial Morris water task performances in mecamylamine-sensitive manners in bilaterally nucleus basalis lesioned rats; and (3) elevate high-affinity [3H]acetylcholine (ACh) and high-affinity alpha-[125I]bungarotoxin binding in rat neocortex following 2 weeks of daily injections. DMXB (100 microM) activated alpha7 homo-oligomeric receptors, without significant activity at alpha2-, alpha3- and alpha4-containing subtypes. Mecamylamine blocked rat alpha7 receptors weakly if co-administered with agonist, but much more potently when pre-applied. Bilateral ibotenic acid lesions of the nucleus basalis interfered with passive avoidance and spatial memory-related behaviors. DMXB (0.5 mg/kg, i.p.) improved passive avoidance behavior in lesioned animals in a mecamylamine-sensitive manner. DMXB (0.5 mg/kg 15 min before each session) also improved performance in the training and probe components of the Morris water task. DMXB-induced improvement in the probe component but not the training phase was mecamylamine-sensitive. [3H]ACh binding was elevated after 14 days of daily i.p. injections with 0.2 mg/kg nicotine but not after 1 mg/kg DMXB. Neither drug elevated high-affinity alpha-[125I]bungarorotoxin binding over this interval.

The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus

Science.gov (United States)

Rojas, Asheebo; Gueorguieva, Paoula; Lelutiu, Nadia; Quan, Yi; Shaw, Renee; Dingledine, Raymond

2014-01-01

Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response. PMID:24952362

Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

DEFF Research Database (Denmark)

Jacob, K K; Sap, J; Stanley, F M

1998-01-01

A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

Activation instead of blocking mesolimbic dopaminergic reward circuitry is a preferred modality in the long term treatment of reward deficiency syndrome (RDS: a commentary

Directory of Open Access Journals (Sweden)

Waite Roger L

2008-11-01

Full Text Available Abstract Background and hypothesis Based on neurochemical and genetic evidence, we suggest that both prevention and treatment of multiple addictions, such as dependence to alcohol, nicotine and glucose, should involve a biphasic approach. Thus, acute treatment should consist of preferential blocking of postsynaptic Nucleus Accumbens (NAc dopamine receptors (D1-D5, whereas long term activation of the mesolimbic dopaminergic system should involve activation and/or release of Dopamine (DA at the NAc site. Failure to do so will result in abnormal mood, behavior and potential suicide ideation. Individuals possessing a paucity of serotonergic and/or dopaminergic receptors, and an increased rate of synaptic DA catabolism due to high catabolic genotype of the COMT gene, are predisposed to self-medicating any substance or behavior that will activate DA release, including alcohol, opiates, psychostimulants, nicotine, gambling, sex, and even excessive internet gaming. Acute utilization of these substances and/or stimulatory behaviors induces a feeling of well being. Unfortunately, sustained and prolonged abuse leads to a toxic" pseudo feeling" of well being resulting in tolerance and disease or discomfort. Thus, a reduced number of DA receptors, due to carrying the DRD2 A1 allelic genotype, results in excessive craving behavior; whereas a normal or sufficient amount of DA receptors results in low craving behavior. In terms of preventing substance abuse, one goal would be to induce a proliferation of DA D2 receptors in genetically prone individuals. While in vivo experiments using a typical D2 receptor agonist induce down regulation, experiments in vitro have shown that constant stimulation of the DA receptor system via a known D2 agonist results in significant proliferation of D2 receptors in spite of genetic antecedents. In essence, D2 receptor stimulation signals negative feedback mechanisms in the mesolimbic system to induce mRNA expression causing

The non-biphenyl-tetrazole angiotensin AT1 receptor antagonist eprosartan is a unique and robust inverse agonist of the active state of the AT1 receptor.

Science.gov (United States)

Takezako, Takanobu; Unal, Hamiyet; Karnik, Sadashiva S; Node, Koichi

2018-03-23

Conditions such as hypertension and renal allograft rejection are accompanied by chronic, agonist-independent, signalling by angiotensin II AT 1 receptors. The current treatment paradigm for these diseases entails the preferred use of inverse agonist AT 1 receptor blockers (ARBs). However, variability in the inverse agonist activities of common biphenyl-tetrazole ARBs for the active state of AT 1 receptors often leads to treatment failure. Therefore, characterization of robust inverse agonist ARBs for the active state of AT 1 receptors is necessary. To identify the robust inverse agonist for active state of AT 1 receptors and its molecular mechanism, we performed site-directed mutagenesis, competition binding assay, inositol phosphate production assay and molecular modelling for both ground-state wild-type AT 1 receptors and active-state N111G mutant AT 1 receptors. Although candesartan and telmisartan exhibited weaker inverse agonist activity for N111G- compared with WT-AT 1 receptors, only eprosartan exhibited robust inverse agonist activity for both N111G- and WT- AT 1 receptors. Specific ligand-receptor contacts for candesartan and telmisartan are altered in the active-state N111G- AT 1 receptors compared with the ground-state WT-AT 1 receptors, suggesting an explanation of their attenuated inverse agonist activity for the active state of AT 1 receptors. In contrast, interactions between eprosartan and N111G-AT 1 receptors were not significantly altered, and the inverse agonist activity of eprosartan was robust. Eprosartan may be a better therapeutic option than other ARBs. Comparative studies investigating eprosartan and other ARBs for the treatment of diseases caused by chronic, agonist-independent, AT 1 receptor activation are warranted. © 2018 The British Pharmacological Society.

Α-amino-β-fluorocyclopropanecarboxylic acids as a new tool for drug development: synthesis of glutamic acid analogs and agonist activity towards metabotropic glutamate receptor 4.

Science.gov (United States)

Lemonnier, Gérald; Lion, Cédric; Quirion, Jean-Charles; Pin, Jean-Philippe; Goudet, Cyril; Jubault, Philippe

2012-08-01

Herein we describe the diastereoselective synthesis of glutamic acid analogs and the evaluation of their agonist activity towards metabotropic glutamate receptor subtype 4 (mGluR4). These analogs are based on a monofluorinated cyclopropane core substituted with an α-aminoacid function. The potential of this new building block as a tool for the development of a novel class of drugs is demonstrated with racemic analog 11a that displayed the best agonist activity with an EC50 of 340 nM. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

DEFF Research Database (Denmark)

Leonhardt, Julia; Villela, Daniel C.; Teichmann, Anke

2017-01-01

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may in...

Slow receptor dissociation kinetics differentiate macitentan from other endothelin receptor antagonists in pulmonary arterial smooth muscle cells.

Directory of Open Access Journals (Sweden)

John Gatfield

Full Text Available Two endothelin receptor antagonists (ERAs, bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH, a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC. The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP(1 assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K(b values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt(1/2 compared to bosentan and ambrisentan (ROt(1/2:17 minutes versus 70 seconds and 40 seconds, respectively. Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP(1 assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1 concentrations. However, prolongation of the ET-1 stimulation time beyond ROt(1/2 rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive

AMP Is an Adenosine A1 Receptor Agonist*

Science.gov (United States)

Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

2012-01-01

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

Discovery of a Series of Imidazo[4,5-b]pyridines with Dual Activity at Angiotensin II Type 1 Receptor and Peroxisome Proliferator-Activated Receptor-[gamma

Energy Technology Data Exchange (ETDEWEB)

Casimiro-Garcia, Agustin; Filzen, Gary F.; Flynn, Declan; Bigge, Christopher F.; Chen, Jing; Davis, Jo Ann; Dudley, Danette A.; Edmunds, Jeremy J.; Esmaeil, Nadia; Geyer, Andrew; Heemstra, Ronald J.; Jalaie, Mehran; Ohren, Jeffrey F.; Ostroski, Robert; Ellis, Teresa; Schaum, Robert P.; Stoner, Chad (Pfizer)

2013-03-07

Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPAR{gamma} confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPAR{gamma} activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC{sub 50} = 1.6 nM) with partial PPAR{gamma} agonism (EC{sub 50} = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.

Peroxisome proliferators-activated receptor (PPAR) regulation in cardiac metabolism and disease

NARCIS (Netherlands)

el Azzouzi, H.

2009-01-01

Peroxisome proliferators-activated receptors (PPARs) are members of the nuclear receptor family of ligand activated transcription factors and consist of the three isoforms, PPAR, PPAR/ and PPAR. Considerable evidence has established the importance of PPARs in myocardial lipid homeostasis and

CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein-Coupled Receptor CXCR2.

Science.gov (United States)

Boro, Monoranjan; Balaji, Kithiganahalli Narayanaswamy

2017-09-01

Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein-coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis -induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase C μ-dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C μ either by small interfering RNA-mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1β. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed therapeutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1β. Copyright © 2017 by The American Association of Immunologists, Inc.

The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study.

Science.gov (United States)

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

1999-11-19

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.]. The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.

Blocking Synaptic Removal of GluA2-Containing AMPA Receptors Prevents the Natural Forgetting of Long-Term Memories.

Science.gov (United States)

Migues, Paola Virginia; Liu, Lidong; Archbold, Georgina E B; Einarsson, Einar Ö; Wong, Jacinda; Bonasia, Kyra; Ko, Seung Hyun; Wang, Yu Tian; Hardt, Oliver

2016-03-23

The neurobiological processes underpinning the natural forgetting of long-term memories are poorly understood. Based on the critical role of GluA2-containing AMPA receptors (GluA2/AMPARs) in long-term memory persistence, we tested in rats whether their synaptic removal underpins time-dependent memory loss. We found that blocking GluA2/AMPAR removal with the interference peptides GluA23Y or G2CT in the dorsal hippocampus during a memory retention interval prevented the normal forgetting of established, long-term object location memories, but did not affect their acquisition. The same intervention also preserved associative memories of food-reward conditioned place preference that would otherwise be lost over time. We then explored whether this forgetting process could play a part in behavioral phenomena involving time-dependent memory change. We found that infusing GluA23Y into the dorsal hippocampus during a 2 week retention interval blocked generalization of contextual fear expression, whereas infusing it into the infralimbic cortex after extinction of auditory fear prevented spontaneous recovery of the conditioned response. Exploring possible physiological mechanisms that could be involved in this form of memory decay, we found that bath application of GluA23Y prevented depotentiation, but not induction of long-term potentiation, in a hippocampal slice preparation. Together, these findings suggest that a decay-like forgetting process that involves the synaptic removal of GluA2/AMPARs erases consolidated long-term memories in the hippocampus and other brain structures over time. This well regulated forgetting process may critically contribute to establishing adaptive behavior, whereas its dysregulation could promote the decline of memory and cognition in neuropathological disorders. The neurobiological mechanisms involved in the natural forgetting of long-term memory and its possible functions are not fully understood. Based on our previous work describing the

No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation.

Science.gov (United States)

Pfistershammer, Katharina; Klauser, Christoph; Pickl, Winfried F; Stöckl, Johannes; Leitner, Judith; Zlabinger, Gerhard; Majdic, Otto; Steinberger, Peter

2006-05-01

The B7 family member programmed-death-1-ligand 2 (PD-L2/B7-DC) is a ligand for programmed-death-receptor 1 (PD-1), a receptor involved in negative regulation of T cell activation. Several independent studies have reported that PD-L2, however, can also potently costimulate murine T cells via an additional yet unidentified receptor. In this study, we evaluated the contribution of PD-L2 to the activation of human T cells using a novel system of engineered T cell stimulators that expresses membrane-bound anti-CD3 antibodies. Analyzing early activation markers, cytokine production and proliferation, we found PD-L2 to consistently inhibit T cell activation. PD-L2 inhibition affected CD4+ and CD8+ T cells and was not abrogated by costimulation via CD28. Blocking PD-1 reverted the inhibitory effect of PD-L2, demonstrating involvement of this pathway. In human T cells, we found no evidence for any of the costimulatory effects described for PD-L2 in murine systems. In line with our functional data that do not point to stimulatory PD-L2-ligands, we show that binding of PD-L2-immunoglobulin to activated human T cells is abrogated by PD-1 antibodies. Our results demonstrate that PD-L2 negatively regulates human T cell activation and thus might be a candidate molecule for immunotherapeutic approaches aimed to attenuate pathological immune responses.

Molecular vibration-activity relationship in the agonism of adenosine receptors.

Science.gov (United States)

Chee, Hyun Keun; Oh, S June

2013-12-01

The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands.

Phasic dopamine release drives rapid activation of striatal D2-receptors

Science.gov (United States)

Marcott, Pamela F; Mamaligas, Aphroditi A; Ford, Christopher P

2014-01-01

Summary Striatal dopamine transmission underlies numerous goal-directed behaviors. Medium spiny neurons (MSNs) are a major target of dopamine in the striatum. However, as dopamine does not directly evoke a synaptic event in MSNs, the time course of dopamine signaling in these cells remains unclear. To examine how dopamine release activates D2-receptors on MSNs, G-protein activated inwardly rectifying potassium (GIRK2; Kir 3.2) channels were virally overexpressed in the striatum and the resulting outward currents were used as a sensor of D2-receptor activation. Electrical and optogenetic stimulation of dopamine terminals evoked robust D2-receptor inhibitory post-synaptic currents (IPSCs) in GIRK2-expressing MSNs that occurred in under a second. Evoked D2-IPSCs could be driven by repetitive stimulation and were not occluded by background dopamine tone. Together, the results indicate that D2-receptors on MSNs exhibit functional low affinity and suggest that striatal D2-receptors can encode both tonic and phasic dopamine signals. PMID:25242218

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

NARCIS (Netherlands)

Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

1991-01-01

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

Liver X receptor and peroxisome proliferator-activated receptor as integrators of lipid homeostasis and immunity.

Science.gov (United States)

Kidani, Yoko; Bensinger, Steven J

2012-09-01

Lipid metabolism has emerged as an important modulator of innate and adaptive immune cell fate and function. The lipid-activated transcription factors peroxisome proliferator-activated receptor (PPAR) α, β/δ, γ and liver X receptor (LXR) are members of the nuclear receptor superfamily that have a well-defined role in regulating lipid homeostasis and metabolic diseases. Accumulated evidence over the last decade indicates that PPAR and LXR signaling also influence multiple facets of inflammation and immunity, thereby providing important crosstalk between metabolism and immune system. Herein, we provide a brief introduction to LXR and PPAR biology and review recent discoveries highlighting the importance of PPAR and LXR signaling in the modulation of normal and pathologic states of immunity. We also examine advances in our mechanistic understanding of how nuclear receptors impact immune system function and homeostasis. Finally, we discuss whether LXRs and PPARs could be pharmacologically manipulated to provide novel therapeutic approaches for modulation of the immune system under pathologic inflammation or in the context of allergic and autoimmune disease. © 2012 John Wiley & Sons A/S.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

2016-01-29

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

International Nuclear Information System (INIS)

Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue; Tsuchihashi, Kenji; Ueda, Shiho; Masuko, Takashi; Saya, Hideyuki; Nagano, Osamu

2016-01-01

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

NARCIS (Netherlands)

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Oude Elferink, Ronald P. J.; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary

Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

International Nuclear Information System (INIS)

Diermeier, Simone; Horvath, Gabor; Knuechel-Clarke, Ruth; Hofstaedter, Ferdinand; Szoellosi, Janos; Brockhoff, Gero

2005-01-01

Background: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. Methods: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. Results: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. Conclusion: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and

Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor

Energy Technology Data Exchange (ETDEWEB)

Cubellis, M.V.; Andreasen, P.; Ragno, P.; Mayer, M.; Dano, K.; Blasi, F. (Univ. of Copenhagen (Denmark))

1989-07-01

Urokinase plasminogen activator (uPA) interacts with a surface receptor and with specific inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). The authors have now investigated whether PAI-1 can bind and inhibit receptor-bound uPA. Binding of {sup 125}I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-1 complexes, but not by PAI-1 alone. Preformed {sup 125}I-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAI-1 also binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptor-binding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition of PAI-1. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-1. The possibility that in adherent cells a different localization of PAI-1 and uPA leads to protection of uPA from PAI-1 is to be considered.

Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

Science.gov (United States)

Fischer, J A; Muff, R; Born, W

2002-08-01

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

Science.gov (United States)

Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

2005-03-01

Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

Science.gov (United States)

Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

2016-05-01

Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

NARCIS (Netherlands)

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Elferink, Ronald P. J. Oude; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPAR delta) is involved in regulation of energy homeostasis. Activation of PPAR delta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets

Directory of Open Access Journals (Sweden)

Marcinkiewicz C

2017-05-01

Full Text Available Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs functionalized with bitistatin (Bit, a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP. Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosis

Dopamine D2 receptor-mediated G-protein activation in rat striatum: functional autoradiography and influence of unilateral 6-hydroxydopamine lesions of the substantia nigra.

Science.gov (United States)

Newman-Tancredi, A; Cussac, D; Brocco, M; Rivet, J M; Chaput, C; Touzard, M; Pasteau, V; Millan, M J

2001-11-30

Unilateral 6-hydroxydopamine (6-OHDA) lesions of substantia nigra pars compacta (SNPC) neurons in rats induce behavioural hypersensitivity to dopaminergic agonists. However, the role of specific dopamine receptors is unclear, and potential alterations in their transduction mechanisms remain to be evaluated. The present study addressed these issues employing the dopaminergic agonist, quinelorane, which efficaciously stimulated G-protein activation (as assessed by [35S]GTPgammaS binding) at cloned hD2 (and hD3) receptors. At rat striatal membranes, dopamine stimulated [35S]GTPgammaS binding by 1.9-fold over basal, but its actions were only partially reversed by the selective D2/D3 receptor antagonist, raclopride, indicating the involvement of other receptor subtypes. In contrast, quinelorane-induced stimulation (48% of the effect of dopamine) was abolished by raclopride, and by the D2 receptor antagonist, L741,626. Further, novel antagonists selective for D3 and D4 receptors, S33084 and S18126, respectively, blocked the actions of quinelorane at concentrations corresponding to their affinities for D2 receptors. Quinelorane potently induced contralateral rotation in unilaterally 6-OHDA-lesioned rats, an effect abolished by raclopride and L741,626, but not by D3 and D4 receptor-selective doses of S33084 and S18126, respectively. In functional ([35S]GTPgammaS) autoradiography experiments, quinelorane stimulated G-protein activation in caudate putamen and, to a lesser extent, in nucleus accumbens and cingulate cortex of naive rats. In unilaterally SNPC-lesioned rats, quinelorane-induced G-protein activation in the caudate putamen on the non-lesioned side was similar to that seen in naive animals (approximately 50% stimulation), but significantly greater on the lesioned side (approximately 80%). This increase was both pharmacologically and regionally specific since it was reversed by raclopride, and was not observed in nucleus accumbens or cingulate cortex. In conclusion

Regulation of Liver Energy Balance by the Nuclear Receptors Farnesoid X Receptor and Peroxisome Proliferator Activated Receptor α.

Science.gov (United States)

Kim, Kang Ho; Moore, David D

2017-01-01

The liver undergoes major changes in substrate utilization and metabolic output over the daily feeding and fasting cycle. These changes occur acutely in response to hormones such as insulin and glucagon, with rapid changes in signaling pathways mediated by protein phosphorylation and other post-translational modifications. They are also reflected in chronic alterations in gene expression in response to nutrient-sensitive transcription factors. Among these, the nuclear receptors farnesoid X receptor (FXR) and peroxisome proliferator activated receptor α (PPARα) provide an intriguing, coordinated response to maintain energy balance in the liver. FXR is activated in the fed state by bile acids returning to the liver, while PPARα is activated in the fasted state in response to the free fatty acids produced by adipocyte lipolysis or possibly other signals. Key Messages: Previous studies indicate that FXR and PPARα have opposing effects on each other's primary targets in key metabolic pathways including gluconeogenesis. Our more recent work shows that these 2 nuclear receptors coordinately regulate autophagy: FXR suppresses this pathway of nutrient and energy recovery, while PPARα activates it. Another recent study indicates that FXR activates the complement and coagulation pathway, while earlier studies identify this as a negative target of PPARα. Since secretion is a very energy- and nutrient-intensive process for hepatocytes, it is possible that FXR licenses it in the nutrient-rich fed state, while PPARα represses it to spare resources in the fasted state. Energy balance is a potential connection linking FXR and PPARα regulation of autophagy and secretion, 2 seemingly unrelated aspects of hepatocyte function. FXR and PPARα act coordinately to promote energy balance and homeostasis in the liver by regulating autophagy and potentially protein secretion. It is quite likely that their impact extends to additional pathways relevant to hepatic energy balance, and

Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons

Directory of Open Access Journals (Sweden)

Huang Li-Yen

2007-08-01

Full Text Available Abstract Prostaglandin E2 (PGE2 is a well-known inflammatory mediator that enhances the excitability of DRG neurons. Homomeric P2X3 and heteromeric P2X2/3 receptors are abundantly expressed in dorsal root ganglia (DRG neurons and participate in the transmission of nociceptive signals. The interaction between PGE2 and P2X3 receptors has not been well delineated. We studied the actions of PGE2 on ATP-activated currents in dissociated DRG neurons under voltage-clamp conditions. PGE2 had no effects on P2X2/3 receptor-mediated responses, but significantly potentiated fast-inactivating ATP currents mediated by homomeric P2X3 receptors. PGE2 exerted its action by activating EP3 receptors. To study the mechanism underlying the action of PGE2, we found that the adenylyl cyclase activator, forskolin and the membrane-permeable cAMP analogue, 8-Br-cAMP increased ATP currents, mimicking the effect of PGE2. In addition, forskolin occluded the enhancement produced by PGE2. The protein kinase A (PKA inhibitors, H89 and PKA-I blocked the PGE2 effect. In contrast, the PKC inhibitor, bisindolymaleimide (Bis did not change the potentiating action of PGE2. We further showed that PGE2 enhanced α,β-meATP-induced allodynia and hyperalgesia and the enhancement was blocked by H89. These observations suggest that PGE2 binds to EP3 receptors, resulting in the activation of cAMP/PKA signaling pathway and leading to an enhancement of P2X3 homomeric receptor-mediated ATP responses in DRG neurons.

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor

International Nuclear Information System (INIS)

Morrison, W.J.; Dhar, A.; Shukla, S.D.

1989-01-01

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF stimulated incorporation of 32 P into proteins and caused [ 3 H]InsP 3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [ 3 H]InsP 3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [ 3 H]InsP 3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF

Pindolol antagonises G-protein activation at both pre- and postsynaptic serotonin 5-HT1A receptors: a.

Science.gov (United States)

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

2001-04-01

The arylalkylamine, pindolol, may potentiate the clinical actions of antidepressant agents. Although it is thought to act via blockade of 5-HT1A autoreceptors, its efficacy at these sites remains controversial. Herein, we evaluated the actions of pindolol at 5-HT1A autoreceptors and specific populations of postsynaptic 5-HT1A receptors employing [35S]GTPgammaS autoradiography, a measure of receptor-mediated G-protein activation. Both 8-OH-DPAT (1 microM) and 5-HT (10 microM) elicited a pronounced increase in [35S]GTPyS binding in the dorsal raphe nucleus, which contains serotonergic cell bodies bearing 5-HT1A autoreceptors. Pindolol abolished their actions. In the dentate gyrus, lateral septum and entorhinal cortex, structures enriched in postsynaptic 5-HT1A receptors, 8-OH-DPAT (1 microM) and 5-HT (10 microM) also elicited a marked increase in [35S]GTPgammaS binding which was likewise blocked by pindolol. The antagonism of 5-HT-induced [35S]GTPgammaS labelling in the dentate gyrus was shown to be concentration-dependent, yielding a pIC50 of 5.82. Pindolol did not, itself, affect [35S]GTPgammaS binding in any brain region examined. In conclusion, these data suggest that, as characterised by [35S]GTPgammaS autoradiography, and compared with 5-HT and 8-OH-DPAT, pindolol possesses low efficacy at both pre- and postsynaptic 5-HT1A receptors.

["Habitual" left branch block alternating with 2 "disguised" bracnch block].

Science.gov (United States)

Lévy, S; Jullien, G; Mathieu, P; Mostefa, S; Gérard, R

1976-10-01

Two cases of alternating left bundle branch block and "masquerading block" (with left bundle branch morphology in the stnadard leads and right bundle branch block morphology in the precordial leads) were studied by serial tracings and his bundle electrocardiography. In case 1 "the masquerading" block was associated with a first degree AV block related to a prolongation of HV interval. This case is to our knowledge the first cas of alternating bundle branch block in which his bundle activity was recorded in man. In case 2, the patient had atrial fibrilation and His bundle recordings were performed while differents degrees of left bundle branch block were present: The mechanism of the alternation and the concept of "masquerading" block are discussed. It is suggested that this type of block represents a right bundle branch block associated with severe lesions of the "left system".

Infusion of adrenergic receptor agonists and antagonists into the locus coeruleus and ventricular system of the brain. Effects on swim-motivated and spontaneous motor activity.

Science.gov (United States)

Weiss, J M; Simson, P G; Hoffman, L J; Ambrose, M J; Cooper, S; Webster, A

1986-04-01

These studies examined how pharmacological stimulation and blockade of alpha receptors would affect active motor behavior in rats. In experiment I, alpha-2 receptor antagonists (piperoxane, yohimbine) and agonists [clonidine, norepinephrine (NE)] were infused into various locations in the ventricular system of the brain, including the locus coeruleus region, and motor activity was measured. Activity was measured principally in a swim test but spontaneous (ambulatory) activity was also recorded while drugs were being infused. When infused into the locus coeruleus region, small doses of the antagonists piperoxane and yohimbine depressed activity in the swim test while infusion of the agonists clonidine and NE had the opposite effect of stimulating activity. These effects were highly specific to the region of the locus coeruleus, since infusions of these drugs into other nearby locations in the ventricular system or use of larger doses had different, often opposite effects. This was especially true of clonidine and NE which profoundly depressed activity when infused posterior to the locus coeruleus, particularly over the dorsal vagal complex. Infusion of small doses of these drugs into the lateral ventricle had effects similar to infusion into the locus coeruleus region, though less pronounced. Changes in spontaneous motor activity were also observed, but this measure differentiated the groups less well than did the swim test. In experiment II, the predominantly postsynaptic receptor agonists isoproterenol (beta agonist) and phenylephrine (alpha-1 agonist) were infused into the ventricular system. Since infusions of piperoxane and yohimbine into the locus coeruleus that decreased activity in experiment I increase the release of NE by blocking alpha-2 inhibitory receptors on cell bodies and dendrites of the locus coeruleus, experiment II tested whether ventricular infusion of predominantly postsynaptic receptor agonists would also decrease activity in the swim test

Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

Directory of Open Access Journals (Sweden)

Hyun Keun Chee

2013-12-01

Full Text Available The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands.

Peroxisome Proliferator-Activated Receptor Alpha Target Genes

Directory of Open Access Journals (Sweden)

Maryam Rakhshandehroo

2010-01-01

Full Text Available The peroxisome proliferator-activated receptor alpha (PPARα is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

Science.gov (United States)

Collin, Caitlin; Hauser, Frank; Gonzalez de Valdivia, Ernesto; de Valdivia, Ernesto Gonzalez; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M M; Khan, Zaid; Hansen, Niels O; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin R; Grimmelikhuijzen, Cornelis J P

2013-09-01

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

Blocking epithelial-to-mesenchymal transition in glioblastoma with a sextet of repurposed drugs: the EIS regimen.

Science.gov (United States)

Kast, Richard E; Skuli, Nicolas; Karpel-Massler, Georg; Frosina, Guido; Ryken, Timothy; Halatsch, Marc-Eric

2017-09-22

This paper outlines a treatment protocol to run alongside of standard current treatment of glioblastoma- resection, temozolomide and radiation. The epithelial to mesenchymal transition (EMT) inhibiting sextet, EIS Regimen, uses the ancillary attributes of six older medicines to impede EMT during glioblastoma. EMT is an actively motile, therapy-resisting, low proliferation, transient state that is an integral feature of cancers' lethality generally and of glioblastoma specifically. It is believed to be during the EMT state that glioblastoma's centrifugal migration occurs. EMT is also a feature of untreated glioblastoma but is enhanced by chemotherapy, by radiation and by surgical trauma. EIS Regimen uses the antifungal drug itraconazole to block Hedgehog signaling, the antidiabetes drug metformin to block AMP kinase (AMPK), the analgesic drug naproxen to block Rac1, the anti-fibrosis drug pirfenidone to block transforming growth factor-beta (TGF-beta), the psychiatric drug quetiapine to block receptor activator NFkB ligand (RANKL) and the antibiotic rifampin to block Wnt- all by their previously established ancillary attributes. All these systems have been identified as triggers of EMT and worthy targets to inhibit. The EIS Regimen drugs have a good safety profile when used individually. They are not expected to have any new side effects when combined. Further studies of the EIS Regimen are needed.

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

DEFF Research Database (Denmark)

Mollerup, Jens; Jørgensen, Sune T.; Hougaard, Charlotte

2001-01-01

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretrea...... by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity....

One feature of the activated southern Ordos block: the Ziwuling small earthquake cluster

Directory of Open Access Journals (Sweden)

Li Yuhang

2014-08-01

Full Text Available Small earthquakes (Ms > 2.0 have been recorded from 1970 to the present day and reveal a significant difference in seismicity between the stable Ordos block and its active surrounding area. The southern Ordos block is a conspicuous small earthquake belt clustered and isolated along the NNW direction and extends to the inner stable Ordos block; no active fault can match this small earthquake cluster. In this paper, we analyze the dynamic mechanism of this small earthquake cluster based on the GPS velocity field (from 1999 to 2007, which are mainly from Crustal Movement Observation Network of China (CMONOC with respect to the north and south China blocks. The principal direction of strain rate field, the expansion ratefield, the maximum shear strain rate, and the rotation rate were constrained using the GPS velocity field. The results show that the velocity field, which is bounded by the small earthquake cluster from Tongchuan to Weinan, differs from the strain rate field, and the crustal deformation is left-lateral shear. This left-lateral shear belt not only spatially coincides with the Neo-tectonic belt in the Weihe Basin but also with the NNW small earthquake cluster (the Ziwuling small earthquake cluster. Based on these studies, we speculate that the NNW small earthquake cluster is caused by left-lateral shear slip, which is prone to strain accumulation. When the strain releases along the weak zone of structure, small earthquakes diffuse within its upper crust. The maximum principal compression strees direction changed from NE-SW to NEE-SWW, and the former reverse faults in the southwestern margin of the Ordos block became a left-lateral strike slip due to readjustment of the tectonic strees field after the middle Pleistocene. The NNW Neo-tectonic belt in the Weihe Basin, the different movement character of the inner Weihe Basin (which was demonstrated through GPS measurements and the small earthquake cluster belt reflect the activated

Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

Science.gov (United States)

Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

2015-06-01

Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

Activation of synaptic and extrasynaptic glycine receptors by taurine in preoptic hypothalamic neurons.

Science.gov (United States)

Bhattarai, Janardhan Prasad; Park, Soo Joung; Chun, Sang Woo; Cho, Dong Hyu; Han, Seong Kyu

2015-11-03

Taurine is an essential amino-sulfonic acid having a fundamental function in the brain, participating in both cell volume regulation and neurotransmission. Using a whole cell voltage patch clamp technique, the taurine-activated neurotransmitter receptors in the preoptic hypothalamic area (PHA) neurons were investigated. In the first set of experiments, different concentrations of taurine were applied on PHA neurons. Taurine-induced responses were concentration-dependent. Taurine-induced currents were action potential-independent and sensitive to strychnine, suggesting the involvement of glycine receptors. In addition, taurine activated not only α-homomeric, but also αβ-heteromeric glycine receptors in PHA neurons. Interestingly, a low concentration of taurine (0.5mM) activated glycine receptors, whereas a higher concentration (3mM) activated both glycine and gamma-aminobutyric acid A (GABAA) receptors in PHA neurons. These results suggest that PHA neurons are influenced by taurine and respond via glycine and GABAA receptors. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

Energy Technology Data Exchange (ETDEWEB)

Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

2014-09-15

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

Identification of an Activating Chicken Ig-like Receptor Recognizing Avian Influenza Viruses

NARCIS (Netherlands)

Jansen, Christine A; van Haarlem, Daphne A; Sperling, Beatrice; van Kooten, Peter J; de Vries, Erik; Viertlboeck, Birgit C; Vervelde, Lonneke; Göbel, Thomas W

2016-01-01

Chicken Ig-like receptors (CHIRs) represent a multigene family encoded by the leukocyte receptor complex that encodes a variety of receptors that are subdivided into activating CHIR-A, inhibitory CHIR-B, and bifunctional CHIR-AB. Apart from CHIR-AB, which functions as an Fc receptor, CHIR ligands

Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association

Directory of Open Access Journals (Sweden)

Filippo eConti

2015-01-01

Full Text Available To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS, avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with Escherichia coli LPS or with the LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid activating macrophages by the disruption of the TLR-2 and TLR-4 association.

Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

Directory of Open Access Journals (Sweden)

Mahmoud M. Mansour

2008-01-01

Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

Directory of Open Access Journals (Sweden)

Gordon Richard

2012-04-01

Full Text Available Abstract Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1 were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ, an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (siRNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/− mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

Science.gov (United States)

Bele, Tanja; Fabbretti, Elsa

2016-08-01

P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

Anti-tumor Activity of Toll-Like Receptor 7 Agonists

Directory of Open Access Journals (Sweden)

Huju Chi

2017-05-01

Full Text Available Toll-like receptors (TLRs are a class of pattern recognition receptors that play a bridging role in innate immunity and adaptive immunity. The activated TLRs not only induce inflammatory responses, but also elicit the development of antigen specific immunity. TLR7, a member of TLR family, is an intracellular receptor expressed on the membrane of endosomes. TLR7 can be triggered not only by ssRNA during viral infections, but also by immune modifiers that share a similar structure to nucleosides. Its powerful immune stimulatory action can be potentially used in the anti-tumor therapy. This article reviewed the anti-tumor activity and mechanism of TLR7 agonists that are frequently applied in preclinical and clinical investigations, and mainly focused on small synthetic molecules, including imiquimod, resiquimod, gardiquimod, and 852A, etc.

Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

Science.gov (United States)

Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

2015-10-01

Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.