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Sample records for a2b receptor subtype

  1. A(2B) receptors mediate antimitogenesis in vascular smooth muscle cells.

    PubMed

    Dubey, R K; Gillespie, D G; Shue, H; Jackson, E K

    2000-01-01

    Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis

  2. The resurgence of A2B adenosine receptor signaling

    PubMed Central

    Aherne, Carol M.; Kewley, Emily M.; Eltzschig, Holger K.

    2010-01-01

    Since its discovery as a low-affinity adenosine receptor (AR), the A2B receptor (A2BAR), has proven enigmatic in its function. The previous discovery of the A2AAR, which shares many similarities with the A2BAR but demonstrates significantly greater affinity for its endogenous ligand, led to the original perception that the A2BAR was not of substantial physiologic relevance. In addition, lack of specific pharmacological agents targeting the A2BAR made its initial characterization challenging. However, the importance of this receptor was reconsidered when it was observed that the A2BAR is highly transcriptionally regulated by factors implicated in inflammatory hypoxia. Moreover, the notion that during ischemia or inflammation extracellular adenosine is dramatically elevated to levels sufficient for A2BAR activation, indicated that A2BAR signaling may be important to dampen inflammation particularly during tissue hypoxia. In addition, the recent advent of techniques for murine genetic manipulation along with development of pharmacological agents with enhanced A2BAR specificity has provided invaluable tools for focused studies on the explicit role of A2BAR signaling in different disease models. Currently, studies performed with combined genetic and pharmacological approaches have demonstrated that A2BAR signaling plays a tissue protective role in many models of acute diseases e.g. myocardial ischemia, or acute lung injury. These studies indicate that the A2BAR is expressed on a wide variety of cell types and exerts tissue/cell specific effects. This is an important consideration for future studies where tissue or cell type specific targeting of the A2BAR may be used as therapeutic approach. PMID:20546702

  3. 1-, 3- and 8-substituted-9-deazaxanthines as potent and selective antagonists at the human A2B adenosine receptor.

    PubMed

    Stefanachi, Angela; Brea, Jose Manuel; Cadavid, Maria Isabel; Centeno, Nuria B; Esteve, Cristina; Loza, Maria Isabel; Martinez, Ana; Nieto, Rosa; Raviña, Enrique; Sanz, Ferran; Segarra, Victor; Sotelo, Eddy; Vidal, Bernat; Carotti, Angelo

    2008-03-15

    A large series of piperazin-, piperidin- and tetrahydroisoquinolinamides of 4-(1,3-dialkyl-9-deazaxanthin-8-yl)phenoxyacetic acid were prepared through conventional or multiple parallel syntheses and evaluated for their binding affinity at the recombinant human adenosine receptors, chiefly at the hA(2B) and hA(2A) receptor subtypes. Several ligands endowed with high binding affinity at hA(2B) receptors, excellent selectivity over hA(2A) and hA(3) and a significant, but lower, selectivity over hA(1) were identified. Among them, piperazinamide derivatives 23 and 52, and piperidinamide derivative 69 proved highly potent at hA(2B) (K(i)=11, 2 and 5.5 nM, respectively) and selective towards hA(2A) (hA(2A)/hA(2B) SI=912, 159 and 630, respectively), hA(3) (hA(3)/hA(2B) SI=>100, 3090 and >180, respectively) and hA(1) (hA(1)/hA(2B) SI=>100, 44 and 120, respectively), SI being the selectivity index. A number of selected ligands tested in functional assays in vitro showed very interesting antagonist activities and efficacies at both A(2A) and A(2B) receptor subtypes, with pA(2) values close to the corresponding pK(i)s. Structure-affinity and structure-selectivity relationships suggested that the binding potency at the hA(2B) receptor may be increased by lipophilic substituents at the N4-position of piperazinamides and that an ortho-methoxy substituent at the 8-phenyl ring and alkyl groups at N1 larger than the ones at N3, in the 9-deazaxanthine ring, may strongly enhance the hA(2A)/hA(2B) SI. PMID:18226909

  4. Discovery and optimization of potent and selective functional antagonists of the human adenosine A2B receptor.

    PubMed

    Bedford, Simon T; Benwell, Karen R; Brooks, Teresa; Chen, Ijen; Comer, Mike; Dugdale, Sarah; Haymes, Tim; Jordan, Allan M; Kennett, Guy A; Knight, Anthony R; Klenke, Burkhard; LeStrat, Loic; Merrett, Angela; Misra, Anil; Lightowler, Sean; Padfield, Anthony; Poullennec, Karine; Reece, Mark; Simmonite, Heather; Wong, Melanie; Yule, Ian A

    2009-10-15

    We herein report the discovery of a novel class of antagonists of the human adenosine A2B receptor. This low molecular weight scaffold has been optimized to offer derivatives with potential utility for the alleviation of conditions associated with this receptor subtype, such as nociception, diabetes, asthma and COPD. Furthermore, preliminary pharmacokinetic analysis has revealed compounds with profiles suitable for either inhaled or systemic routes of administration.

  5. 3D-pharmacophore models for selective A2A and A2B adenosine receptor antagonists.

    PubMed

    Wei, Jing; Wang, Songqing; Gao, Shaofen; Dai, Xuedong; Gao, Qingzhi

    2007-01-01

    Three-dimensional pharmacophore models were generated for A2A and A2B adenosine receptors (ARs) based on highly selective A2A and A2B antagonists using the Catalyst program. The best pharmacophore model for selective A2A antagonists (Hypo-A2A) was obtained through a careful validation process. Four features contained in Hypo-A2A (one ring aromatic feature (R), one positively ionizable feature (P), one hydrogen bond acceptor lipid feature (L), and one hydrophobic feature (H)) seem to be essential for antagonists in terms of binding activity and A2A AR selectivity. The best pharmacophore model for selective A2B antagonists (Hypo-A2B) was elaborated by modifying the Catalyst common features (HipHop) hypotheses generated from the selective A2B antagonists training set. Hypo-A2B also consists of four features: one ring aromatic feature (R), one hydrophobic aliphatic feature (Z), and two hydrogen bond acceptor lipid features (L). All features play an important role in A2B AR binding affinity and are essential for A2B selectivity. Both A2A and A2B pharmacophore models have been validated toward a wide set of test molecules containing structurally diverse selective antagonists of all AR subtypes. They are capable of identifying correspondingly high potent antagonists and differentiating antagonists between subtypes. The results of our study will act as a valuable tool for retrieving structurally diverse compounds with desired biological activities and designing novel selective adenosine receptor ligands. PMID:17330954

  6. 3D-pharmacophore models for selective A2A and A2B adenosine receptor antagonists.

    PubMed

    Wei, Jing; Wang, Songqing; Gao, Shaofen; Dai, Xuedong; Gao, Qingzhi

    2007-01-01

    Three-dimensional pharmacophore models were generated for A2A and A2B adenosine receptors (ARs) based on highly selective A2A and A2B antagonists using the Catalyst program. The best pharmacophore model for selective A2A antagonists (Hypo-A2A) was obtained through a careful validation process. Four features contained in Hypo-A2A (one ring aromatic feature (R), one positively ionizable feature (P), one hydrogen bond acceptor lipid feature (L), and one hydrophobic feature (H)) seem to be essential for antagonists in terms of binding activity and A2A AR selectivity. The best pharmacophore model for selective A2B antagonists (Hypo-A2B) was elaborated by modifying the Catalyst common features (HipHop) hypotheses generated from the selective A2B antagonists training set. Hypo-A2B also consists of four features: one ring aromatic feature (R), one hydrophobic aliphatic feature (Z), and two hydrogen bond acceptor lipid features (L). All features play an important role in A2B AR binding affinity and are essential for A2B selectivity. Both A2A and A2B pharmacophore models have been validated toward a wide set of test molecules containing structurally diverse selective antagonists of all AR subtypes. They are capable of identifying correspondingly high potent antagonists and differentiating antagonists between subtypes. The results of our study will act as a valuable tool for retrieving structurally diverse compounds with desired biological activities and designing novel selective adenosine receptor ligands.

  7. Contribution of Adenosine A2B Receptors in Clostridium difficile Intoxication and Infection

    PubMed Central

    Li, Yuesheng; Calabrese, Gina M.; Freire, Rosemayre S.; Zaja-Milatovic, Snjezana; van Opstal, Edward; Figler, Robert A.; Linden, Joel; Guerrant, Richard L.

    2012-01-01

    Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A2B adenosine receptors (A2BARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A2BARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A2BARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A1, A2A, A2B, and A3) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A2BAR−/− mice were treated with TcdA, with or without the selective A2BAR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A2BAR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A2BAR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A2BARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A2BARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A2BARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A2BAR activation may be a potential strategy to limit morbidity and mortality from CDI. PMID:23045479

  8. In vivo adenosine A(2B) receptor desensitization in guinea-pig airway smooth muscle: implications for asthma.

    PubMed

    Breschi, Maria Cristina; Blandizzi, Corrado; Fogli, Stefano; Martinelli, Cinzia; Adinolfi, Barbara; Calderone, Vincenzo; Camici, Marcella; Martinotti, Enrica; Nieri, Paola

    2007-12-01

    This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

  9. Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist

    PubMed Central

    2010-01-01

    The structure−activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels. PMID:24900298

  10. Discovery of Potent and Highly Selective A2B Adenosine Receptor Antagonist Chemotypes.

    PubMed

    El Maatougui, Abdelaziz; Azuaje, Jhonny; González-Gómez, Manuel; Miguez, Gabriel; Crespo, Abel; Carbajales, Carlos; Escalante, Luz; García-Mera, Xerardo; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2016-03-10

    Three novel families of A2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2(1H)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A2B ligand that exhibits complete selectivity toward A1, A2A, and A3 receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A2A receptor.

  11. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease.

    PubMed

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K; Blackwell, Timothy S; Xia, Yang; Johnston, Richard A; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R

    2012-06-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A(2B)R) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A(2B)R or treatment with the A(2B)R antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A(2B)R attenuated vascular remodeling and hypertension in our model. Furthermore, direct A(2B)R activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A(2B)R antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.

  12. Probing biased/partial agonism at the G protein-coupled A(2B) adenosine receptor.

    PubMed

    Gao, Zhan-Guo; Balasubramanian, Ramachandran; Kiselev, Evgeny; Wei, Qiang; Jacobson, Kenneth A

    2014-08-01

    G protein-coupled A(2B) adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A(2B)AR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A(2B)AR. In cAMP accumulation assays, both 5'-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5'-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A(2B)AR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A(2B)AR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N⁶-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A(2B)AR antagonist in MIN-6 mouse pancreatic β cells expressing low A(2B)AR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target.

  13. Targeting the A2B adenosine receptor during gastrointestinal ischemia and inflammation

    PubMed Central

    Eltzschig, Holger K; Rivera-Nieves, Jesus; Colgan, Sean P

    2014-01-01

    Extracellular adenosine functions as an endogenous distress signal via activation of four distinct adenosine receptors (A1, A2A, A2B and A3). Conditions of limited oxygen availability or acute inflammation lead to elevated levels of extracellular adenosine and enhanced signaling events. This relates to a combination of four mechanisms: i) increased production of adenosine via extracellular phosphohydrolysis of precursor molecules (particularly ATP and ADP); ii) increased expression and signaling via hypoxia-induced adenosine receptors, particularly the A2B adenosine receptor; iii) attenuated uptake from the extracellular towards the intracellular compartment; and iv) attenuated intracellular metabolism. Due to their large surface area, mucosal organs are particularly prone to hypoxia and ischemia associated inflammation. Therefore, it is not surprising that adenosine production and signaling plays a central role in attenuating tissue inflammation and injury during intestinal ischemia or inflammation. In fact, recent studies combining pharmacological and genetic approaches demonstrated that adenosine signaling via the A2B adenosine receptor dampens mucosal inflammation and tissue injury during intestinal ischemia or experimental colitis. This review outlines basic principles of extracellular adenosine production, signaling, uptake and metabolism. In addition, we discuss the role of this pathway in dampening hypoxia-elicited inflammation, specifically in the setting of intestinal ischemia and inflammation. PMID:19769545

  14. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 1. Expression of receptor mRNA in four mouse hematopoietic precursor cells.

    PubMed

    Streitová, D; Sefc, L; Savvulidi, F; Pospísil, M; Holá, J; Hofer, M

    2010-01-01

    Four mouse bone marrow or thymus cell populations, namely granulopoietic/monocytopoietic, erythropoietic, B-lymphopoietic, and T-lymphopoietic precursor cells have been assayed by RT-PCR technique for the presence and relative amounts of adenosine A(1), A(2a), A(2b), and A(3) receptor mRNA. It has been found that (i) all four populations studied express all four adenosine receptor subtypes, (ii) the A(1), receptor is the least expressed in all populations studied, (iii) the A(3) receptor is markedly expressed in the populations of granulopoietic/monocytopoietic and erythropoietic cells, (iv) the A(2a) receptor is markedly expressed in the populations of B-lymphopoietic and T-lymphopoietic cells, and v) the A(2b) receptor does not predominate in any of the precursor cells studied. Our data offer a new possibility for the assessment of the readiness of these cells to respond, by receptor-mediated mechanisms, to adenosine or its analogs present in the tissues as a result of endogenous processes and/or following their administration.

  15. The impact of adenosine and A(2B) receptors on glucose homoeostasis.

    PubMed

    Rüsing, D; Müller, C E; Verspohl, E J

    2006-12-01

    Adenosine and adenosine receptor antagonists are involved in glucose homoeostasis. The participating receptors are not known, mainly due to a lack of specific agonists and antagonists, but are reasonable targets for anti-diabetic therapy. The stable, albeit nonselective, adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) (10 microM) reduced glucose-stimulated insulin release from INS-1 cells. This was mimicked by A(1)-(CHA), A(2A)-(CGS-21680) and A(3)-receptor agonists (Cl-IB-MECA). Two newly synthesized A(2B)-receptor antagonists, PSB-53 and PSB-1115, counteracted the inhibitory effect of NECA. These in-vitro effects were mirrored by in-vivo data with respect to CHA, CGS and Cl-IB-MECA. Distinct concentrations of either PSB-53 or PSB-1115 reversed the decrease in plasma insulin induced by NECA. This was not mimicked by a corresponding change in blood glucose. The effect of PSB-1115 was also obvious in diabetic GotoKakizaki rats: plasma insulin was increased whereas blood glucose was unchanged. During most experiments the effects on blood glucose were not impressive probably because of the physiologically necessary homoeostasis. The adenosine levels were not different in normal Wistar rats and in diabetic GotoKakzaki rats. Altogether the A(2B)-receptor antagonists showed an anti-diabetic potential mainly by increasing plasma insulin levels under conditions when the adenosine tonus was elevated in-vivo and increased insulin release in-vitro.

  16. Adenosine A2B Receptor: From Cell Biology to Human Diseases.

    PubMed

    Sun, Ying; Huang, Pingbo

    2016-01-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR's functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases, and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases. PMID:27606311

  17. Adenosine A2B receptor: from cell biology to human diseases

    NASA Astrophysics Data System (ADS)

    Sun, Ying; Huang, Pingbo

    2016-08-01

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

  18. Adenosine A2B Receptor: From Cell Biology to Human Diseases

    PubMed Central

    Sun, Ying; Huang, Pingbo

    2016-01-01

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

  19. Adenosine A2B Receptor: From Cell Biology to Human Diseases

    PubMed Central

    Sun, Ying; Huang, Pingbo

    2016-01-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR's functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases, and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases. PMID:27606311

  20. A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

    PubMed

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

    2015-01-01

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

  1. A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

    PubMed

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

    2015-01-01

    Diabetes is an important risk factor for erectile dysfunction (ED). Recent studies have indicated that A2B adenosine receptor (ADORA2B) signaling is essential for penile erection. Thus, we hypothesize that diabetic ED may be attributed to impaired A2B adenosine signaling. To test this hypothesis, we generated diabetic rats by injecting streptozocin as animal model. After 12 weeks, immunohistochemistry staining was used to localize the expression of ADORA2B. Western Blot and quantitative PCR were employed to determine ADORA2B expression level. Intracavernosal pressure (ICP) measurement was used to evaluate erectile function. Diabetic rats received a single intravenous injection of BAY 60-6583, an ADORA2B agonist, or vehicle solution, at 60 min before the ICP measurement. The results showed that ADORA2B expressed in the nerve bundle, smooth muscle, and endothelium in penile tissue of control mice. Western Blot and quantitative PCR results indicated that the expression levels of ADORA2B protein and mRNA were significantly reduced in penile tissues of diabetic rats. Functional studies showed that the erectile response induced by electrical stimulation was remarkably decreased in diabetic rats, compared with age-matched control rats. However, at 60 min after BAY 60-6583 treatment, the erectile function was improved in diabetic rats, suggesting that enhancement of ADORA2B signaling may improve erectile function in diabetic ED. This preclinical study has revealed a previously unrecognized therapeutic possibility of BAY 60-6583 as an effective and mechanism-based drug to treat diabetic ED. In conclusion, we propose that impaired A2B adenosine signaling is one of the pathological mechanisms of diabetic ED. PMID:26447087

  2. NMDA receptor surface mobility depends on NR2A-2B subunits

    PubMed Central

    Groc, Laurent; Heine, Martin; Cousins, Sarah L.; Stephenson, F. Anne; Lounis, Brahim; Cognet, Laurent; Choquet, Daniel

    2006-01-01

    The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking. PMID:17124177

  3. The Quintiles Prize Lecture 2004. The identification of the adenosine A2B receptor as a novel therapeutic target in asthma.

    PubMed

    Holgate, Stephen T

    2005-08-01

    Adenosine is a powerful bronchoconstrictor of asthmatic, but not normal, airways. In vitro studies on isolated human mast cells and basophils revealed that adenosine and selective analogues augmented inflammatory mediator release from mast cells by stimulating A(2) receptors. Pharmacological blockade of mast cell mediator release in vivo also attenuated adenosine-induced bronchoconstriction, as did theophylline, by adenosine A(2) receptor antagonism. Further in vitro studies revealed that the asthmatic response to adenosine is likely to be mediated via the A(2B) subtype which is selectively antagonised by enprofylline. Studies in animal models, especially mice, have shown a close synergistic interaction between adenosine, Th2 and airway remodelling responses. The recent description of A(2B) receptors on human airway smooth muscle cells that mediate cytokine and chemokine release and induce differentiation of fibroblasts into myofibroblasts strengthens the view that adenosine maybe more than an inflammatory mediator in asthma but also participates in airway wall remodelling in this disease. These data have provided a firm basis for developing adenosine A(2B) receptor antagonists as a new therapeutic approach to this disease.

  4. The Quintiles Prize Lecture 2004: The identification of the adenosine A2B receptor as a novel therapeutic target in asthma

    PubMed Central

    Holgate, Stephen T

    2005-01-01

    Adenosine is a powerful bronchoconstrictor of asthmatic, but not normal, airways. In vitro studies on isolated human mast cells and basophils revealed that adenosine and selective analogues augmented inflammatory mediator release from mast cells by stimulating A2 receptors. Pharmacological blockade of mast cell mediator release in vivo also attenuated adenosine-induced bronchoconstriction, as did theophylline, by adenosine A2 receptor antagonism. Further in vitro studies revealed that the asthmatic response to adenosine is likely to be mediated via the A2B subtype which is selectively antagonised by enprofylline. Studies in animal models, especially mice, have shown a close synergistic interaction between adenosine, Th2 and airway remodelling responses. The recent description of A2B receptors on human airway smooth muscle cells that mediate cytokine and chemokine release and induce differentiation of fibroblasts into myofibroblasts strengthens the view that adenosine maybe more than an inflammatory mediator in asthma but also participates in airway wall remodelling in this disease. These data have provided a firm basis for developing adenosine A2B receptor antagonists as a new therapeutic approach to this disease. PMID:15980878

  5. The Quintiles Prize Lecture 2004. The identification of the adenosine A2B receptor as a novel therapeutic target in asthma.

    PubMed

    Holgate, Stephen T

    2005-08-01

    Adenosine is a powerful bronchoconstrictor of asthmatic, but not normal, airways. In vitro studies on isolated human mast cells and basophils revealed that adenosine and selective analogues augmented inflammatory mediator release from mast cells by stimulating A(2) receptors. Pharmacological blockade of mast cell mediator release in vivo also attenuated adenosine-induced bronchoconstriction, as did theophylline, by adenosine A(2) receptor antagonism. Further in vitro studies revealed that the asthmatic response to adenosine is likely to be mediated via the A(2B) subtype which is selectively antagonised by enprofylline. Studies in animal models, especially mice, have shown a close synergistic interaction between adenosine, Th2 and airway remodelling responses. The recent description of A(2B) receptors on human airway smooth muscle cells that mediate cytokine and chemokine release and induce differentiation of fibroblasts into myofibroblasts strengthens the view that adenosine maybe more than an inflammatory mediator in asthma but also participates in airway wall remodelling in this disease. These data have provided a firm basis for developing adenosine A(2B) receptor antagonists as a new therapeutic approach to this disease. PMID:15980878

  6. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease

    PubMed Central

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D.; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K.; Blackwell, Timothy S.; Xia, Yang; Johnston, Richard A.; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R.

    2012-01-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A2BR) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A2BR or treatment with the A2BR antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A2BR attenuated vascular remodeling and hypertension in our model. Furthermore, direct A2BR activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A2BR antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.—Karmouty-Quintana, H., Zhong, H., Acero, L., Weng, T., Melicoff, E., West, J. D., Hemnes, A., Grenz, A., Eltzschig, H. K., Blackwell, T. S., Xia, Y., Johnston, R. A., Zeng, D., Belardinelli, L., Blackburn, M. R. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease. PMID:22415303

  7. IFN-γ Prevents Adenosine Receptor (A2bR) Upregulation To Sustain the Macrophage Activation Response.

    PubMed

    Cohen, Heather B; Ward, Amanda; Hamidzadeh, Kajal; Ravid, Katya; Mosser, David M

    2015-10-15

    The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. In this study, we demonstrate that, following TLR stimulation, macrophages upregulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This upregulation of A2bR leads to the induction of macrophages with an immunoregulatory phenotype and the downregulation of inflammation. IFN-γ priming of macrophages selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and to prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNF-α and IL-12 in response to TLR ligation. The pharmacologic inhibition or the genetic deletion of the A2bR results in a hyperinflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the antimicrobial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated. PMID:26355158

  8. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 2. Expression of receptor mRNA in resting and lipopolysaccharide-activated mouse RAW 264.7 macrophages.

    PubMed

    Streitová, D; Hofer, M; Holá, J; Vacek, A; Pospísil, M

    2010-01-01

    Expression of mRNA for adenosine receptor subtypes A(1), A(2a), A(2b), and A(3) in normal and lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages has been investigated using the method of quantitative real-time polymerase chain reaction. The results have shown a very low, unquantifiable expression of adenosine A(1) receptor mRNA in both normal and LPS-activated macrophages. The other three adenosine receptor mRNAs have been found to be expressed at various but always quantifiable levels. Activation of the macrophages by LPS induced upregulation of the expression of adenosine receptor A(2a) and A(2b) mRNA, whereas the expression of adenosine receptor A(3) mRNA was downregulated. Unstimulated macrophages exhibited a high expression of the A(2b) adenosine receptor mRNA. The findings are discussed from the point of view of the antiinflammatory and hematopoiesis-stimulating roles of the adenosine receptor signaling.

  9. Identification of angiotensin II receptor subtypes

    SciTech Connect

    Chiu, A.T.; Herblin, W.F.; McCall, D.E.; Ardecky, R.J.; Carini, D.J.; Duncia, J.V.; Pease, L.J.; Wong, P.C.; Wexler, R.R.; Johnson, A.L.; )

    1989-11-30

    We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.

  10. Structure Based Prediction of Subtype-Selectivity for Adenosine Receptor Antagonists

    PubMed Central

    Katritch, Vsevolod; Kufareva, Irina; Abagyan, Ruben

    2010-01-01

    One of the major hurdles in the development of safe and effective drugs targeting G-protein coupled receptors (GPCRs) is finding ligands that are highly selective for a specific receptor subtype. Structural understanding of subtype-specific binding pocket variations and ligand-receptor interactions may greatly facilitate design of selective ligands. To gain insights into the structural basis of ligand subtype selectivity within the family of adenosine receptors (AR: A1, A2A, A2B, and A3) we generated 3D models of all four subtypes using the recently determined crystal structure of the AA2AR as a template, and employing the methodology of ligand-guided receptor optimization for refinement. This approach produced 3D conformational models of AR subtypes that effectively explain binding modes and subtype selectivity for a diverse set of known AR antagonists. Analysis of the subtype-specific ligand-receptor interactions allowed identification of the major determinants of ligand selectivity, which may facilitate discovery of more efficient drug candidates. PMID:20637786

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  13. Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

    PubMed Central

    Du, Xiaolong; Ou, Xuehai; Song, Tao; Zhang, Wentao; Cong, Fei; Zhang, Shihui

    2015-01-01

    Angiogenesis is critical to wound repair due to its role in providing oxygen and nutrients that are required to support the growth and function of reparative cells in damaged tissues. Adenosine receptors are claimed to be of paramount importance in driving wound angiogenesis by inducing VEGF. However, the underlying mechanisms for the regulation of adenosine receptors in VEGF as well as eNOS remain poorly understood. In the present study, we found that adenosine and the non-selective adenosine receptor agonists (NECA) induced tube formation in HMEC-1 in a dose-dependent manner. Adenosine or NECA (10 µmol/L) significantly augmented the number and length of the segments in comparison with the control. Simultaneously, VEGF and eNOS were significantly upregulated following the administration of 10 µmol/L NECA, while they were suppressed after A2B AR genetic silencing and pharmacological inhibition by MRS1754. In addition, VEGF expression and eNOS bioavailability elimination significantly reduced the formation of capillary-like structures. Furthermore, the activation of A2B AR by NECA significantly increased the intracellular cAMP levels and concomitant CREB phosphorylation, eventually leading to the production of VEGF in HMEC-1. However, the activated PKA-CREB pathway seemed to be invalidated in the induction of eNOS. Moreover, we found that the elicited PI3K/AKT signaling in response to the induction of NECA assisted in regulating eNOS but failed to impact on VEGF generation. In conclusion, the A2B AR activation-driven angiogenesis via cAMP-PKA-CREB mediated VEGF production and PI3K/AKT-dependent upregulation of eNOS in HMEC-1. PMID:25966978

  14. Novel role of hnRNP-A2/B1 in modulating aryl hydrocarbon receptor ligand sensitivity.

    PubMed

    Cho, See-Wun; Suzuki, Ken-ichi; Miura, Yoshiaki; Miyazaki, Tatsuhiko; Nose, Masato; Iwata, Hisato; Kim, Eun-Young

    2015-11-01

    The aryl hydrocarbon receptor (AHR) is responsible for susceptibility to its ligand-dependent responses. However, the effect of non-AHR factors is less clear. To explore the non-AHR factors, we used two mouse strains with different AHR genetic variants, namely C3H/lpr and MRL/lpr strains with Ala and Val as the 375th amino acid residue, respectively. To assess the contribution of AHR alone, COS-7 cells transiently expressing AHR from each strain were treated with 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and xenobiotic-responsive element (XRE)-driven reporter gene activities were measured. FICZ-EC50 values for the C3H/lpr and MRL/lpr AHR-mediated transactivation were 0.023 and 0.046 nM, respectively, indicating a similar susceptibility in both AHR genotypes. In contrast, C3H/lpr AHR was fourfold more sensitive to TCDD than MRL/lpr AHR. By a pull-down assay using a XRE-containing PCR product as bait and the hepatic nuclear extracts of both FICZ-treated mouse strains, we identified two interacting proteins as heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2) and its splicing variant (hnRNP-A2b). Immunoprecipitation assays demonstrated the AHR interaction with hnRNP-A2/B1. When hnRNP-A2 was co-expressed with the MRL/lpr or C3H/lpr AHR in COS-7, FICZ treatment decreased EC50 to about threefold in both AHR genotypes, compared with EC50 in AHR alone. Similarly, hnRNP-A2b co-expression also lowered the FICZ-EC50 values. In TCDD-treated COS-7, responses depended on the AHR genotype; while no change in TCDD-EC50 was observed for C3H/lpr AHR when hnRNP-A2 was co-expressed, the value was reduced to nearly tenfold for MRL/lpr AHR. Co-transfection with hnRNP-A2b attenuated the AHR sensitivity to TCDD. In conclusion, the hnRNP-A2/B1 interacting with AHR may be a modulator of the AHR ligand sensitivity.

  15. The Reno-Vascular A2B Adenosine Receptor Protects the Kidney from Ischemia

    PubMed Central

    Grenz, Almut; Osswald, Hartmut; Eckle, Tobias; Yang, Dan; Zhang, Hua; Tran, Zung Vu; Klingel, Karin; Ravid, Katya; Eltzschig, Holger K

    2008-01-01

    Background Acute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP). Methods and Findings For this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1−/−, A2A−/−, or A3AR−/− mice. In contrast, protection from ischemia was abolished in A2BAR−/− mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60–6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs. Conclusions These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia. PMID:18578565

  16. Pharmacologic specificity of alpha-2 adrenergic receptor subtypes

    SciTech Connect

    Petrash, A.; Bylund, D.

    1986-03-01

    The authors have defined alpha-2 adrenergic receptor subtypes in human and rat tissues using prazosin as a subtype selective drug. Prazosin has a lower affinity (250 nM) at alpha-2A receptor and a higher affinity (5 nM) at alpha-2B receptors. In order to determine if other adrenergic drugs are selective for one or the other subtypes, the authors performed (/sup 3/H)yohimbine inhibition experiments with various adrenergic drugs in tissues containing alpha-2A, alpha-2B or both subtypes. Oxymetazoline, WB4101 and yohimbine were found to be 80-, 20- and 10-fold more potent at alpha-2A receptors than at alpha-2B receptors. Phentolamine, adazoxan, (+)- and (-)-mianserin, clonidine, (+)-butaclamol, (-)- and (+)-norepinephrine, epinephrine, dopamine and thioridazine were found to have equal affinities for the two subtypes. These results further validate the subdivision of alpha-2 adrenergic receptors into alpha-2A and alpha-2B subtypes.

  17. Molecular biology of somatostatin receptor subtypes.

    PubMed

    Patel, Y C; Greenwood, M; Panetta, R; Hukovic, N; Grigorakis, S; Robertson, L A; Srikant, C B

    1996-08-01

    Somatostatin (SRIF) receptors (ssts) comprise a family of heptahelical membrane proteins encoded by five related genes that map to separate chromosomes and which, with the exception of sst1, are intronless. The ssts1-4 display weak selectivity for SRIF-14 binding, whereas sst5 is SRIF-28-selective. Based on structural similarity and reactivity for octapeptide and hexapeptide sst analogs, ssts2,3 and sst5 belong to a similar sst subclass; ssts1-4 react poorly with these analogs and belong to a separate subclass. All five ssts are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin-sensitive guanosine triphosphate (GTP)-binding proteins. mRNA for ssts1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective and tissue- and species-specific. All pituitary cell subsets express sst2 and sst5, with sst5 being more abundant. Individual pituitary cells coexpress multiple sst subtypes. The binding pocket for SRIF-14 ligand lies deep within the membrane in transmembrane domains (TMDs) 3 to 7. Except for extracellular loop 2, it does not involve the other exofacial structures. Human (h)sst2A and hsst5 undergo agonist-mediated desensitization, associated with receptor internalization. The C-tail segment of hsst5 displays positive molecular internalization signals. The ssts inhibit the growth of tumor cells directly, through blockade of mitogenic signaling leading to growth arrest and through induction of apoptosis. This process is associated with translocation of phosphotyrosine phosphatase (PTP) 1C from the cytosol to the membrane.

  18. Discovery of 3,4-Dihydropyrimidin-2(1H)-ones As a Novel Class of Potent and Selective A2B Adenosine Receptor Antagonists.

    PubMed

    Crespo, Abel; El Maatougui, Abdelaziz; Biagini, Pierfrancesco; Azuaje, Jhonny; Coelho, Alberto; Brea, José; Loza, María Isabel; Cadavid, María Isabel; García-Mera, Xerardo; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2013-11-14

    We describe the discovery and optimization of 3,4-dihydropyrimidin-2(1H)-ones as a novel family of (nonxanthine) A2B receptor antagonists that exhibit an unusually high selectivity profile. The Biginelli-based hit optimization process enabled a thoughtful exploration of the structure-activity and structure-selectivity relationships for this chemotype, enabling the identification of ligands that combine structural simplicity with excellent hA2B AdoR affinity and remarkable selectivity profiles.

  19. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling

    PubMed Central

    Wen, Jiaming; Jiang, Xianzhen; Dai, Yingbo; Zhang, Yujin; Tang, Yuxin; Sun, Hong; Mi, Tiejuan; Phatarpekar, Prasad V.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2010-01-01

    offer a potential target for prevention and treatment of penile fibrosis, a dangerous complication seen in priapism.—Wen, J., Jiang, X., Dai, Y., Zhang, Y., Tang, Y., Sun, H., Mi, T., Phatarpekar, P. V., Kellems, R. E., Blackburn, M. R., Xia, Y. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling. PMID:19858092

  20. Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like cells to myocardial scar formation.

    PubMed

    Ryzhov, Sergey; Sung, Bong Hwan; Zhang, Qinkun; Weaver, Alissa; Gumina, Richard J; Biaggioni, Italo; Feoktistov, Igor

    2014-09-01

    Adenosine levels increase in ischemic hearts and contribute to the modulation of that pathological environment. We previously showed that A2B adenosine receptors on mouse cardiac Sca1(+)CD31(-) mesenchymal stromal cells upregulate secretion of paracrine factors that may contribute to the improvement in cardiac recovery seen when these cells are transplanted in infarcted hearts. In this study, we tested the hypothesis that A2B receptor signaling regulates the transition of Sca1(+)CD31(-) cells, which occurs after myocardial injury, into a myofibroblast phenotype that promotes myocardial repair and remodeling. In vitro, TGFβ1 induced the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and increased collagen I generation in Sca1(+)CD31(-) cells. Stimulation of A2B receptors attenuated TGFβ1-induced collagen I secretion but had no effect on αSMA expression. In vivo, myocardial infarction resulted in a rapid increase in the numbers of αSMA-positive cardiac stromal cells by day 5 followed by a gradual decline. Genetic deletion of A2B receptors had no effect on the initial accumulation of αSMA-expressing stromal cells but hastened their subsequent decline; the numbers of αSMA-positive cells including Sca1(+)CD31(-) cells remained significantly higher in wild type compared with A2B knockout hearts. Thus, our study revealed a significant contribution of cardiac Sca1(+)CD31(-) cells to the accumulation of αSMA-expressing cells after infarction and implicated A2B receptor signaling in regulation of myocardial repair and remodeling by delaying deactivation of these cells. It is plausible that this phenomenon may contribute to the beneficial effects of transplantation of these cells to the injured heart.

  1. Role of A2B Adenosine Receptors in Regulation of Paracrine Functions of Stem Cell Antigen 1-Positive Cardiac Stromal Cells

    PubMed Central

    Ryzhov, Sergey; Goldstein, Anna E.; Novitskiy, Sergey V.; Blackburn, Michael R.; Biaggioni, Italo

    2012-01-01

    The existence of multipotent cardiac stromal cells expressing stem cell antigen (Sca)-1 has been reported, and their proangiogenic properties have been demonstrated in myocardial infarction models. In this study, we tested the hypothesis that stimulation of adenosine receptors on cardiac Sca-1+ cells up-regulates their secretion of proangiogenic factors. We found that Sca-1 is expressed in subsets of mouse cardiac stromal CD31− and endothelial CD31+ cells. The population of Sca-1+CD31+ endothelial cells was significantly reduced, whereas the population of Sca-1+CD31− stromal cells was increased 1 week after myocardial infarction, indicating their relative functional importance in this pathophysiological process. An increase in adenosine levels in adenosine deaminase-deficient mice in vivo significantly augmented vascular endothelial growth factor (VEGF) production in cardiac Sca-1+CD31− stromal cells but not in Sca-1+CD31+ endothelial cells. We found that mouse cardiac Sca-1+CD31− stromal cells predominantly express mRNA encoding A2B adenosine receptors. Stimulation of adenosine receptors significantly increased interleukin (IL)-6, CXCL1 (a mouse ortholog of human IL-8), and VEGF release from these cells. Using conditionally immortalized Sca-1+CD31− stromal cells obtained from wild-type and A2B receptor knockout mouse hearts, we demonstrated that A2B receptors are essential for adenosine-dependent up-regulation of their paracrine functions. We found that the human heart also harbors a population of stromal cells similar to the mouse cardiac Sca-1+CD31− stromal cells that increase release of IL-6, IL-8, and VEGF in response to A2B receptor stimulation. Thus, our study identified A2B adenosine receptors on cardiac stromal cells as potential targets for up-regulation of proangiogenic factors in the ischemic heart. PMID:22431204

  2. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  3. Homology modelling of the human adenosine A2B receptor based on X-ray structures of bovine rhodopsin, the beta2-adrenergic receptor and the human adenosine A2A receptor.

    PubMed

    Sherbiny, Farag F; Schiedel, Anke C; Maass, Astrid; Müller, Christa E

    2009-11-01

    A three-dimensional model of the human adenosine A2B receptor was generated by means of homology modelling, using the crystal structures of bovine rhodopsin, the beta2-adrenergic receptor, and the human adenosine A2A receptor as templates. In order to compare the three resulting models, the binding modes of the adenosine A2B receptor antagonists theophylline, ZM241385, MRS1706, and PSB601 were investigated. The A2A-based model was much better able to stabilize the ligands in the binding site than the other models reflecting the high degree of similarity between A2A and A2B receptors: while the A2B receptor shares about 21% of the residues with rhodopsin, and 31% with the beta2-adrenergic receptor, it is 56% identical to the adenosine A2A receptor. The A2A-based model was used for further studies. The model included the transmembrane domains, the extracellular and the intracellular hydrophilic loops as well as the terminal domains. In order to validate the usefulness of this model, a docking analysis of several selective and nonselective agonists and antagonists was carried out including a study of binding affinities and selectivities of these ligands with respect to the adenosine A2A and A2B receptors. A common binding site is proposed for antagonists and agonists based on homology modelling combined with site-directed mutagenesis and a comparison between experimental and calculated affinity data. The new, validated A2B receptor model may serve as a basis for developing more potent and selective drugs.

  4. Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels

    SciTech Connect

    Garcia-Villalon, A.L.; Krause, D.N.; Ehlert, F.J.; Duckles, S.P. )

    1991-07-01

    The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 (11-2-((2-(diethylaminomethyl)- 1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)benzodiazepine-6-one), hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of (3H)quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of (3H)-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.

  5. Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes

    PubMed Central

    Rudolph, Uwe; Knoflach, Frédéric

    2012-01-01

    GABAA receptors are a family of ligand-gated ion channels which are essential for the regulation of central nervous system function. Benzodiazepines – which target GABAA receptors containing the α1, α2, α3, or α5 subunits non-selectively – have been in clinical use for decades and are still among the most widely prescribed drugs for the treatment of insomnia and anxiety disorders. However, their use is limited by side effects and the risk of drug dependence. In the past decade, the identification of separable key functions of GABAA receptor subtypes suggests that receptor subtype-selective compounds could overcome the limitations of classical benzodiazepines and, furthermore, might be valuable for novel indications, such as analgesia, depression, schizophrenia, cognitive enhancement and stroke. PMID:21799515

  6. Oestrogen compromises the facilitatory effect of chronic nicotine on adenosine A2B receptor-K(+) channel-mediated renal vasodilation.

    PubMed

    El-Mas, Mahmoud M; El-Gowilly, Sahar M; Elsalakawy, Lamia K; El-Gowelli, Hanan M

    2014-08-01

    We have shown previously that the renal vasodilatory action of the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) in female rats is mediated via preferential activation of adenosine A2B receptor (A2B R)-K(+) channel signalling. In the present study, we tested the hypothesis that the renal vasodilatory effect of NECA and its A2B R/K(+) channel specificities are altered by chronic nicotine administration. The oestrogenic modulation of the nicotine-NECA renovascular interaction was also evaluated by determining the effect of ovariectomy (OVX) and oestrogen replacement (OVXE2) on the evoked responses. In isolated phenylephrine-preconstricted perfused kidneys obtained from sham-operated rats, vasodilation in response to cumulative bolus injections of NECA (1.6-50 nmol) or papaverine (1-243 nmol) were not affected by nicotine (1-8 mg/kg per day, i.p., 2 weeks). However, vasodilator responses to NECA, but not papaverine, were reduced in kidneys of OVX rats and restored to near-sham values after E2 replacement. Further, nicotine increased NECA-induced vasodilation in perfused kidneys from OVX rats, but failed to do so in OVXE2 preparations. The enhanced NECA responsiveness in nicotine-treated OVX preparations was abolished after infusion (into isolated kidneys) of 10 μmol/L alloxazine (A2B R antagonist) or BaCl2 plus glibenclamide (blockers of inward rectifier and ATP-sensitive K(+) channels, respectively). Vasodilator responses to 0.05-1.6 μmol minoxidil (a K(+) channel opener) were increased by nicotine in OVX, but not OVXE2, preparations and this increase was abolished after infusion of BaCl2  + glibenclamide. Together, the data suggest that chronic nicotine enhances A2B R/K(+) channel-mediated renal vasodilation in oestrogen-depleted rats. PMID:24827542

  7. Multiple estrogen receptor subtypes influence ingestive behavior in female rodents.

    PubMed

    Santollo, Jessica; Daniels, Derek

    2015-12-01

    Postmenopausal women are at an increased risk of obesity and cardiovascular-related diseases. This is attributable, at least in part, to loss of the ovarian hormone estradiol, which inhibits food and fluid intake in humans and laboratory animal models. Although the hypophagic and anti-dipsogenic effects of estradiol have been well documented for decades, the precise mechanisms underlying these effects are not fully understood. An obvious step toward addressing this open question is identifying which estrogen receptor subtypes are involved and what intracellular processes are involved. This question, however, is complicated not only by the variety of estrogen receptor subtypes that exist, but also because many subtypes have multiple locations of action (i.e. in the nucleus or in the plasma membrane). This review will highlight our current understanding of the roles that specific estrogen receptor subtypes play in mediating estradiol's anorexigenic and anti-dipsogenic effects along with highlighting the many open questions that remain. This review will also describe recent work being performed by our laboratory aimed at answering these open questions.

  8. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    PubMed

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  9. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

    PubMed Central

    Watson, Michael J.; Lee, Shernita L.; Marklew, Abigail J.; Gilmore, Rodney C.; Gentzsch, Martina; Sassano, Maria F.; Gray, Michael A.; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  10. Characterization of muscarinic receptor subtypes in human tissues

    SciTech Connect

    Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

    1988-01-01

    The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with (/sup 3/H)Pirenzepine and (/sup 3/H)N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M/sub 1/ neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M/sub 1/, the cardiac M/sub 2/ and the glandular M/sub 3/.

  11. Remifentanil-induced preconditioning has cross-talk with A1 and A2B adenosine receptors in ischemic-reperfused rat heart.

    PubMed

    Lee, Yong-Cheol; Jung, Jiyoon; Park, Sang-Jin

    2016-01-01

    The purpose of this study was to determine whether there is a cross-talk between opioid receptors (OPRs) and adenosine receptors (ADRs) in remifentanil preconditioning (R-Pre) and, if so, to investigate the types of ADRs involved in the cross-talk. Isolated rat hearts received 30 min of regional ischemia followed by 2 hr of reperfusion. OPR and ADR antagonists were perfused from 10 min before R-Pre until the end of R-Pre. The heart rate, left ventricular developed pressure (LVDP),velocity of contraction (+dP/dtmax), and coronary flow (CF) were recorded. The area at risk and area of necrosis were measured. After reperfusion, the LVDP, +dP/dtmax,and CF showed a significant increase in the R-Pre group compared with the control group (no intervention before or after regional ischemia). These increases in the R-Pre group were blocked by naloxone, a nonspecific ADR antagonist, an A1 ADR antagonist, and an A2B ADR antagonist. The infarct size was reduced significantly in the R-Pre group compared with the control group. The infarct-reducing effect in the R-Pre group was blocked by naloxone, the nonspecific ADR antagonist, the A1 ADR antagonist, and the A2B ADR antagonist. The results of this study demonstrate that there is cross-talk between ADRs and OPRs in R-Pre and that A1 ADR and A2B ADR appear to be involved in the cross-talk. PMID:26773185

  12. 5-Hydroxytryptamine Receptor Subtypes and their Modulators with Therapeutic Potentials

    PubMed Central

    Pithadia, Anand B.; Jain, Sunita M.

    2009-01-01

    5-hydroxytryptamine (5-HT) has become one of the most investigated and complex biogenic amines. The main receptors and their subtypes, e.g., 5-HTI (5-HT1A, 5-HT1B, 5-HTID, 5-HTIE and 5-HT1F), 5-HT2 (5-HT2A, 5-HT2B and 5-HT2C), 5-HT3, 5-HT4, 5-HT5 (5-HT5A, 5-HT5B), 5-HT6 and 5-HT7 have been identified. Specific drugs which are capable of either selectively stimulating or inhibiting these receptor subtypes are being designed. This has generated therapeutic potentials of 5-HT receptor modulators in a variety of disease conditions. Conditions where 5-HT receptor modulators have established their use with distinct efficacy and advantages include migraine, anxiety, psychosis, obesity and cancer therapy-induced vomiting by cytotoxic drugs and radiation. Discovery of 5-HT, its biosynthesis, metabolism, physiological role and the potential of 5-HT receptor modulators in various nervous, cardiovascular and gastrointestinal tract disorders, bone growth and micturition have been discussed in this article. Keywords 5-hydroxytryptamine (5-HT) receptors; Modulators; Biogenic amines PMID:22505971

  13. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression

    PubMed Central

    Ning, Chen; Wen, Jiaming; Zhang, Yujin; Dai, Yingbo; Wang, Wei; Zhang, Weiru; Qi, Lin; Grenz, Almut; Eltzschig, Holger K.; Blackburn, Michael R.; Kellems, Rodney E.; Xia, Yang

    2014-01-01

    Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A2B adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA−/− and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.—Ning, C., Wen, J., Zhang, Y., Dai, Y., Wang, W., Zhang, W., Qi, L., Grenz, A., Eltzschig, H. K., Blackburn, M. R., Kellems, R. E., Xia, Y. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression. PMID:24614760

  14. Angiotensin II receptor subtypes in rat renal preglomerular vessels.

    PubMed

    De León, H; Garcia, R

    1992-01-01

    A simple technique to isolate rat renal preglomerular vessels is described. Kidneys were pressed against a 0.3 mm stainless steel grid. The whole vascular tree, including the interlobar, arcuate, and interlobular arteries, as well as the afferent arterioles, remained on the grid surface from where they were recovered. Extensive washing yielded a highly pure preparation of renal microvessels. Radioligand binding experiments were performed to characterize 125I-[Sar1,Ile8]-ANG II binding sites in preglomerular microvessel membranes. Equilibrium saturation binding experiments revealed the presence of one group of high affinity receptors (Kd = 1.22 +/- 0.171 nM; Bmax = 209 +/- 14 fmol/mg protein). Competitive inhibition experiments with two highly specific nonpeptide ANG II antagonists, losartan (DuP 753), which is specific for the AT1 receptor subtype, and PD123319, which is specific for the AT2 subtype, demonstrated that the large majority of, if not all, ANG II receptors in rat renal preglomerular vessels correspond to the AT1 subtype. PMID:1299411

  15. Endothelin Receptor Subtype Distribution Predisposes Coronary Arteries to Damage

    PubMed Central

    Louden, Calvert S.; Nambi, Ponnal; Pullen, Mark A.; Thomas, Roberta A.; Tierney, Lauren A.; Solleveld, Henk A.; Schwartz, Lester W.

    2000-01-01

    Several vasoactive drugs that lower blood pressure and increase heart rate induce regional cardiotoxicity in the dog, most frequently of right coronary arteries and right atrium. The basis for this selective damage is thought to result from local changes in vascular tone and blood flow. Administration of an endothelin receptor antagonist (ETRA, SB 209670) to dogs induced damage most frequent and severe in the right coronary artery and right atrium. Because site predisposition may correlate with distribution of vasoactive receptors, the objectives of this study were to map endothelin (ET) receptor distribution and density within regions of dog heart using both gene (mRNA) and protein expression endpoints for dog ETA and ETB receptors, and, additionally, correlate ET receptor subtype density with regional cardiac blood flow. A 10- to 15-mmHg reduction in mean arterial pressure with a concomitant increase in heart rate (10–20%), a six- and twofold increase in regional blood flow to the right and left atrium, respectively, and acute hemorrhage, medial necrosis, and inflammation were observed in the right coronary arteries and arteries of the right atrium after ETRA infusion for 5 days. Radioligand protein binding to quantify both ET receptors in normal dog heart indicated a twofold greater density of ET receptors in atrial regions versus ventricular regions. Importantly, ET receptor density in coronary arteries was markedly (about five- to sixfold) increased above that in atrial or ventricular tissues. ET receptor subtype characterization indicated ETB receptors were three times more prevalent in right coronary arteries compared to left coronary arteries and in situ hybridization confirmed localization of ETB in vascular smooth muscle. ETA receptor density was comparable in right and left coronary arteries. Quantitative real-time polymerase chain reaction for ETA and ETB receptor mRNA transcripts supported the site prevalence for message distribution. Consequently

  16. Role of adenosine receptor subtypes in methamphetamine reward and reinforcement.

    PubMed

    Kavanagh, Kevin A; Schreiner, Drew C; Levis, Sophia C; O'Neill, Casey E; Bachtell, Ryan K

    2015-02-01

    The neurobiology of methamphetamine (MA) remains largely unknown despite its high abuse liability. The present series of studies explored the role of adenosine receptors on MA reward and reinforcement and identified alterations in the expression of adenosine receptors in dopamine terminal areas following MA administration in rats. We tested whether stimulating adenosine A1 or A2A receptor subtypes would influence MA-induced place preference or MA self-administration on fixed and progressive ratio schedules in male Sprague-Dawley rats. Stimulation of either adenosine A1 or A2A receptors significantly reduced the development of MA-induced place preference. Stimulating adenosine A1, but not A2A, receptors reduced MA self-administration responding. We next tested whether repeated experimenter-delivered MA administration would alter the expression of adenosine receptors in the striatal areas using immunoblotting. We observed no change in the expression of adenosine receptors. Lastly, rats were trained to self-administer MA or saline for 14 days and we detected changes in adenosine A1 and A2A receptor expression using immunoblotting. MA self-administration significantly increased adenosine A1 in the nucleus accumbens shell, caudate-putamen and prefrontal cortex. MA self-administration significantly decreased adenosine A2A receptor expression in the nucleus accumbens shell, but increased A2A receptor expression in the amygdala. These findings demonstrate that MA self-administration produces selective alterations in adenosine receptor expression in the nucleus accumbens shell and that stimulation of adenosine receptors reduces several behavioral indices of MA addiction. Together, these studies shed light onto the neurobiological alterations incurred through chronic MA use that may aid in the development of treatments for MA addiction.

  17. Low dose acute alcohol effects on GABAA receptor subtypes

    PubMed Central

    Wallner, Martin; Hanchar, H. Jacob; Olsen, Richard W.

    2010-01-01

    GABAA receptors (GABAARs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABAAR subtypes, nonsynaptic GABAARs have recently emerged as important regulators of neuronal excitability. While high doses (≥100 mM) of ethanol have been reported to enhance activity of most GABAAR subtypes, most abundant synaptic GABAARs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (<30 mM). However, extrasynaptic δ and β3 subunit-containing GABAARs, associated in the brain with α4or α6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar α6 subunit (α6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant α6β3δ receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on α4/6/β3δ receptors, without reducing GABA-induced currents. In binding assays α4β3δ GABAARs bind [3H] Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513’s block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABAAR subtypes. PMID:16814864

  18. Photoaffinity labeling of the somatostatin receptor: identification of molecular subtypes.

    PubMed

    Srikant, C B; Murthy, K K; Escher, E E; Patel, Y C

    1992-05-01

    Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and may exhibit subtypes selective for SS-14 and SS-28. Whether this heterogeneity can be explained by separate molecular forms of the receptor protein is unclear. In the present study, we have developed a novel photosensitive azido derivative of the octapeptide SS analog Tyr3 SMS (EE 581) and used it as a photoaffinity probe to characterize the molecular components of the SS receptor in five receptor positive tissues (normal rat brain, pituitary, pancreas, and adrenal cortex, and mouse AtT-20 pituitary tumor cells). [125I]EE-581 labeled specific high affinity binding sites in all these tissues (Kd range 1.3-1.67 nM). Photoaffinity labeled membrane SS receptors were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Three specifically labeled SS receptor proteins of 80 kilodaltons (kDa), 58 kDa, and 32 kDa were identified and exhibited a tissue-specific distribution. The 58 kDa species was the exclusive form in pancreas, adrenal cortex, and AtT-20 cells and the dominant form in brain. The 32 kDa receptor protein was expressed as a minor form (ratio of 58 kDa:32 kDa 3:1), exclusively in brain. The 80 kDa receptor was found only in the pituitary where it occurred as the sole SS receptor species. Competition experiments showed that the 58 kDa and 32 kDa receptor proteins in brain reacted with SS-14 greater than SS-28; in contrast, the 58 kDa protein in AtT-20 cells bound SS-28 greater than SS-14 suggesting the existence of distinct subtypes of the 58 kDa receptor in these two tissues. These data represent the first systematic evaluation of the molecular forms of SS receptor proteins by photoaffinity labeling in different target tissues and provide direct evidence for molecular heterogeneity and SS-14/SS-28 selectivity; a major 58 kDa protein present in most tissues, an additional 32 kDa protein uniquely expressed in brain, and an

  19. MicroRNA signatures predict dysregulated vitamin D receptor and calcium pathways status in limb girdle muscle dystrophies (LGMD) 2A/2B.

    PubMed

    Aguennouz, M; Lo Giudice, C; Licata, N; Rodolico, C; Musumeci, O; Fanin, M; Migliorato, A; Ragusa, M; Macaione, V; Di Giorgio, R M; Angelini, C; Toscano, A

    2016-08-01

    miRNA expression profile and predicted pathways involved in selected limb-girdle muscular dystrophy (LGMD)2A/2B patients were investigated. A total of 187 miRNAs were dysregulated in all patients, with six miRNAs showing opposite regulation in LGMD2A versus LGMD2B patients. Silico analysis evidence: (1) a cluster of the dysregulated miRNAs resulted primarily involved in inflammation and calcium metabolism, and (2) two genes predicted as controlled by calcium-assigned miRNAs (Vitamin D Receptor gene and Guanine Nucleotide Binding protein beta polypeptide 1gene) showed an evident upregulation in LGMD2B patients, in accordance with miRNA levels. Our data support alterations in calcium pathway status in LGMD 2A/B, suggesting myofibre calcium imbalance as a potential therapeutic target. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27558075

  20. Functional subtyping of muscarinic receptors on canine esophageal mucosa.

    PubMed

    Lad, R; Donoff, B; Rangachari, P K

    1991-09-01

    Serosal addition of muscarinic agonists elicited rapid changes in electrical parameters across the isolated canine esophageal epithelium set up in vitro. Both carbachol and the M1-selective agonist, McNeil A343 (McN), increased transmucosal potential differences (PDs), decreased transmucosal resistances (R), and increased short-circuit currents (Isc). Carbachol was more potent and more effective than McN. Muscarinic antagonists were used to define the muscarinic receptor involved. The pA2 values obtained with Schild plots were as follows: atropine 9.14, 4-DAMP 8.98, AFDX-116 6.71, and pirenzepine 7.12. Low concentrations of pirenzepine (10(-8) M), produced a rightward shift in the dose-response curve to McN, without inhibiting responses to carbachol. Thus the receptor subtype is clearly not an M2. As in other glandular systems, M3 receptors are present. Whether M1 receptors also exist requires better definition of receptor densities-reserves in this tissue. Carbachol induced net secretion of Na and Cl and converted a predominantly absorptive tissue to a secretory one. PMID:1716057

  1. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation

    PubMed Central

    Wen, Jiaming; Grenz, Almut; Zhang, Yujin; Dai, Yingbo; Kellems, Rodney E.; Blackburn, Michael R.; Eltzschig, Holger K.; Xia, Yang

    2011-01-01

    Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5′-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A2B receptor (A2BR) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A2BR signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A2BR in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A2BR activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.—Wen, J., Grenz, A., Zhang, Y., Dai, Y., Kellems, R. E., Blackburn, M. R., Eltzschig, H. K., Xia, Y. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation. PMID

  2. Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C.

    PubMed

    Riemenschneider, Mona; Cashin, Kieran Y; Budeus, Bettina; Sierra, Saleta; Shirvani-Dastgerdi, Elham; Bayanolhagh, Saeed; Kaiser, Rolf; Gorry, Paul R; Heider, Dominik

    2016-01-01

    Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients. PMID:27126912

  3. Adrenergic receptor subtypes in the cerebral circulation of newborn piglets

    SciTech Connect

    Wagerle, L.C.; Delivoria-Papadopoulos, M.

    1987-06-01

    The purpose of this study was to identify the ..cap alpha..-adrenergic receptor subtype mediating cerebral vasoconstriction during sympathetic nerve stimulation in the newborn piglet. The effect of ..cap alpha../sub 1/- and ..cap alpha../sub 2/-antagonists prazosin and yohimbine on the cerebrovascular response to unilateral electrical stimulation (15 Hz, 15 V) of the superior cervical sympathetic trunk was studied in 25 newborn piglets. Regional cerebral blood flow was measured with tracer microspheres. Sympathetic stimulation decreased blood flow to the ipsilateral cerebrum hippocampus, choroid plexus, and masseter muscle. ..cap alpha../sub 1/-Adrenergic receptor blockade with prazosin inhibited the sympathetic vasoconstriction in the cerebrum, hippocampus, and masseter muscle and abolished it in the choroid plexus. ..cap alpha../sub s/-Adrenergic receptor blockade with yohimbine had no effect. Following the higher dose of yohimbine, however, blood flow to all brain regions was increased by approximately two-fold, possibly due to enhanced cerebral metabolism. These data demonstrate that vascular ..cap alpha../sub 1/-adrenergic receptors mediate vasoconstriction to neuroadrenergic stimulation in cerebral resistance vessels in the newborn piglet.

  4. Molecular and cellular analysis of human histamine receptor subtypes

    PubMed Central

    Seifert, Roland; Strasser, Andrea; Schneider, Erich H.; Neumann, Detlef; Dove, Stefan; Buschauer, Armin

    2013-01-01

    The human histamine receptors hH1R and hH2R constitute important drug targets, and hH3R and hH4R have substantial potential in this area. Considering the species-specificity of pharmacology of HxR orthologs, it is important to analyze hHxRs. Here,we summarize current knowledge of hHxRs endogenously expressed in human cells and hHxRs recombinantly expressed in mammalian and insect cells. We present the advantages and disadvantages of the various systems. We also discuss problems associated with the use of hHxR antibodies, an issue of general relevance for G-protein-coupled receptors (GPCRs). There is much greater overlap in activity of ‘selective’ ligands for other hHxRs than the cognate receptor subtype than generally appreciated. Studies with native and recombinant systems support the concept of ligand-specific receptor conformations, encompassing agonists and antagonists. It is emerging that for characterization of hHxR ligands, one cannot rely on a single test system and a single parameter. Rather, multiple systems and parameters have to be studied. Although such studies are time-consuming and expensive, ultimately, they will increase drug safety and efficacy. PMID:23254267

  5. From molecular phylogeny towards differentiating pharmacology for NMDA receptor subtypes.

    PubMed

    Platt, Randall J; Curtice, Kigen J; Twede, Vernon D; Watkins, Maren; Gruszczyński, Paweł; Bulaj, Grzegorz; Horvath, Martin P; Olivera, Baldomero M

    2014-04-01

    In order to decode the roles that N-methyl-D-aspartate (NMDA) receptors play in excitatory neurotransmission, synaptic plasticity, and neuropathologies, there is need for ligands that differ in their subtype selectivity. The conantokin family of Conus peptides is the only group of peptidic natural products known to target NMDA receptors. Using a search that was guided by phylogeny, we identified new conantokins from the marine snail Conus bocki that complement the current repertoire of NMDA receptor pharmacology. Channel currents measured in Xenopus oocytes demonstrate conantokins conBk-A, conBk-B, and conBk-C have highest potencies for NR2D containing receptors, in contrast to previously characterized conantokins that preferentially block NR2B containing NMDA receptors. Conantokins are rich in γ-carboxyglutamate, typically 17-34 residues, and adopt helical structure in a calcium-dependent manner. As judged by CD spectroscopy, conBk-C adopts significant helical structure in a calcium ion-dependent manner, while calcium, on its own, appears insufficient to stabilize helical conformations of conBk-A or conBk-B. Molecular dynamics simulations help explain the differences in calcium-stabilized structures. Two-dimensional NMR spectroscopy shows that the 9-residue conBk-B is relatively unstructured but forms a helix in the presence of TFE and calcium ions that is similar to other conantokin structures. These newly discovered conantokins hold promise that further exploration of small peptidic antagonists will lead to a set of pharmacological tools that can be used to characterize the role of NMDA receptors in nervous system function and disease.

  6. A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Bustamante, Salvador; García-Sacristán, Albino; Orensanz, Luis M

    1999-01-01

    or NO, through activation of A2B-receptors located in the smooth muscle. This relaxation may modulate the ureteral NANC excitatory neurotransmission through a postsynaptic mechanism. PMID:10193777

  7. Propofol Restores Transient Receptor Potential Vanilloid Receptor Subtype-1 Sensitivity via Activation of Transient Receptor Potential Ankyrin Receptor Subtype-1 in Sensory Neurons

    PubMed Central

    Zhang, Hongyu; Wickley, Peter J.; Sinha, Sayantani; Bratz, Ian N.; Damron, Derek S.

    2011-01-01

    Background Crosstalk between peripheral nociceptors belonging to the transient receptor potential vanilloid receptor subtype-1 (TRPV1) and ankyrin subtype-1 (TRPA1) family has recently been demonstrated. Moreover, the intravenous anesthetic propofol has been shown to directly activate TRPA1 receptors, and indirectly restore sensitivity of TRPV1 receptors in dorsal root ganglion (DRG) sensory neurons. Our objective was to determine the extent to which TRPA1 activation is involved in mediating the propofol-induced restoration of TRPV1 sensitivity. Methods Mouse DRG neurons were isolated by enzymatic dissociation and grown for 24 h. F-11 cells were transfected with complementary DNA for both TRPV1 and TRPA1 or TRPV1 only. Intracellular Ca2+ concentration was measured in individual cells via fluorescence microscopy. Following TRPV1 de-sensitization with capsaicin (100 nM), cells were treated with propofol (1, 5 and 10 μM) alone, propofol in the presence of the TRPA1 antagonist, HC-030031 (0.5 μM) or the TRPA1 agonist, Allyl isothiocyanate (AITC, 100 μM) and capsaicin was then reapplied. Results In DRG neurons that contain both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in DRG neurons containing only TRPV1 receptors, exposure to propofol or AITC following de-sensitization did not restore capsaicin-induced TRPV1 sensitivity. Similarly, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in F-11 cells transfected with TRPV1 only, neither propofol nor AITC were capable of restoring TRPV1 sensitivity. Conclusions These data demonstrate that propofol restores TRPV1 sensitivity in primary DRG neurons and in cultured F-11 cells transfected with both the TRPV1 and TRPA1 receptors via a TRPA1-dependent process. Propofol’s effects on sensory neurons may be clinically important and contribute to peripheral sensitization to nociceptive stimuli in traumatized tissue. PMID:21364461

  8. Selective labeling and localization of the M4 (m4) muscarinic receptor subtype.

    PubMed

    Ferrari-Dileo, G; Waelbroeck, M; Mash, D C; Flynn, D D

    1994-12-01

    We report here a novel strategy for the selective labeling and localization of the M4 (m4) muscarinic receptor subtype, based on the distinct kinetics of the muscarinic antagonists dexetimide and N-methylscopolamine (NMS) and on the selectivity profile of guanylpirenzepine and AF-DX 116 for the m1-m5 muscarinic receptor subtypes expressed in CHO-K1 cells. Incubation with 10 nM dexetimide, a nonselective antagonist, resulted in > 90% occupancy of each of the m1-m5 receptor subtypes. The relatively rapid rates of dexetimide dissociation from the m1, m2, and m4 receptor subtypes (t1/2 values of < 12.5 min) and the slower rates of dexetimide dissociation from the m3 and m5 receptor subtypes (t1/2 values of 65 and 75 min, respectively) favored the labeling of the m1, m2, and m4 receptor subtypes with short incubations with [3H]NMS. Inclusion of 200 nM guanylpirenzepine and 250 nM AF-DX 116 prevented the binding of [3H]NMS to the majority of the m1 and m2 receptor subtypes, respectively, resulting in primary labeling of the m4 receptor subtype. Brief dissociation of the radioligand in the presence of 1 microM atropine improved the ratio of m4 to m2 labeling by selectively removing [3H]NMS from the m2 subtype. Under these conditions, the ratio of [3H]NMS binding to the m4 versus m1, m2, m3, and m5 receptor subtypes was 4:1. In vitro autoradiography combined with these m4-selective labeling conditions demonstrated that the M4 (m4) receptor subtype was localized to the primary visual area (V1, area 17, occipital cortex) and the basal ganglia, a distribution distinct from that demonstrated for the M1 (m1), M2 (m2), and M3 (m3) receptor subtypes. These results demonstrate that a combination of the distinct kinetics of dexetimide and NMS and the receptor subtype selectivity of guanylpirenzepine and AF-DX 116 provides a valuable labeling strategy to examine the distribution and localization of the M4 (m4) muscarinic receptor subtype in brain, peripheral tissues, and cell lines

  9. Multiple GABAA receptor subtypes regulate hippocampal ripple oscillations.

    PubMed

    Ponomarenko, A A; Korotkova, T M; Sergeeva, O A; Haas, H L

    2004-10-01

    High-frequency oscillations (140-200 Hz) were recorded in behaving rats from the CA1 area of the hippocampus. As generation of these synchronous patterns is assumed to depend on coordinated interneuronal inhibition, we studied the interference of benzodiazepines with the fine structure and occurrence of ripple oscillations. The nonselective GABAA receptor alpha-subunit agonist, diazepam, lowered the frequency of ripple oscillations and reduced their occurrence, amplitude and duration. Zolpidem, an alpha1-subunit selective benzodiazepine elevated ripple duration but acted similar to diazepam in other respects. The nonselective alpha-subunit benzodiazepine antagonist, flumazenil, reduced ripple numbers, amplitude and duration. Wavelet based analysis of the dynamics of intraripple frequency revealed a dramatic decay within a ripple. Only diazepam (1 mg/kg) accelerated this intraripple frequency accommodation. The effects were not due to increased behavioural activity and alertness as evident from vigilance state control. The results suggest a differential role of GABAA receptor subtype specific inhibitory mechanisms in the mediation and fine-tuning of the network synchronization during approximately 200 Hz hippocampal oscillations.

  10. Differential interactions of indolealkylamines with 5-hydroxytryptamine receptor subtypes.

    PubMed

    McKenna, D J; Repke, D B; Lo, L; Peroutka, S J

    1990-03-01

    Affinities of drugs for 21 indolealkylamine derivatives, some with putative hallucinogenic activity, were determined at 5-HT1A, 5-HT2A and 5-HT2B recognition sites, using radioligand competition studies. Nearly all of the derivatives displayed greatest potency for the 5-HT2A receptor, labelled by [125I]R-(-)DOI in the cortex of the rat. Most derivatives displayed 2-10 times lower affinity at the HT2B receptor labelled by [3H]ketanserin in bovine cortex. Derivatives lacking ring substituents displayed lower affinities for all of the recognition sites, compared to derivatives substituted in the 4- or 5-position of the indole ring. The 4-hydroxylated derivatives displayed 25-380-fold selectivity for the 5-HT2A site, vs the 5-HT1A site, while the 5-substituted derivatives displayed approximately equal potency at the 5-HT1A and 5-HT2A sites. Affinity of all the compounds at the 5-HT2B site was greater than 300 nM. The 6-substituted derivatives displayed greater than micromolar affinities for all of the 5-HT recognition sites examined. The size of the N,N-dialkyl substituent was a secondary determinant of affinity, with groups larger than N,N-diisopropyl resulting in a marked reduction in affinity at both the 5-HT2A and 5-HT1A recognition sites. This study demonstrated that hallucinogenic 4-hydroxy-indolealkylamines, like psychotomimetic phenylisopropylamines, bind potently and selectively to the 5-HT2A recognition site, labelled by [125I]R-(-)DOI. This provides further evidence indicating that this recently described subtype of the 5-HT2 receptor may partially mediate the action of hallucinogenic agents.

  11. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

    PubMed Central

    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.

    2016-01-01

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  12. Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

    PubMed

    Katz, Jonathan L; Hiranita, Takato; Kopajtic, Theresa A; Rice, Kenner C; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H; McCurdy, Christopher R

    2016-07-01

    The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

  13. Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists

    PubMed Central

    Hiranita, Takato; Kopajtic, Theresa A.; Rice, Kenner C.; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H.; McCurdy, Christopher R.

    2016-01-01

    The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

  14. Classification of Dopamine Receptor Genes in Vertebrates: Nine Subtypes in Osteichthyes.

    PubMed

    Yamamoto, Kei; Fontaine, Romain; Pasqualini, Catherine; Vernier, Philippe

    2015-01-01

    Dopamine neurotransmission regulates various brain functions, and its regulatory roles are mediated by two families of G protein-coupled receptors: the D1 and D2 receptor families. In mammals, the D1 family comprises two receptor subtypes (D1 and D5), while the D2 family comprises three receptor subtypes (D2, D3 and D4). Phylogenetic analyses of dopamine receptor genes strongly suggest that the common ancestor of Osteichthyes (bony jawed vertebrates) possessed four subtypes in the D1 family and five subtypes in the D2 family. Mammals have secondarily lost almost half of the ancestral dopamine receptor genes, whereas nonmammalian species kept many of them. Although the mammalian situation is an exception among Osteichthyes, the current classification and characterization of dopamine receptors are based on mammalian features, which have led to confusion in the identification of dopamine receptor subtypes in nonmammalian species. Here we begin by reviewing the history of the discovery of dopamine receptors in vertebrates. The recent genome sequencing of coelacanth, gar and elephant shark led to the proposal of a refined scenario of evolution of dopamine receptor genes. We also discuss a current problem of nomenclature of dopamine receptors. Following the official nomenclature of mammalian dopamine receptors from D1 to D5, we propose to name newly identified receptor subtypes from D6 to D9 in order to facilitate the use of an identical name for orthologous genes among different species. To promote a nomenclature change which allows distinguishing the two dopamine receptor families, a nomenclature consortium is needed. This comparative perspective is crucial to correctly interpret data obtained in animal studies on dopamine-related brain disorders, and more fundamentally, to understand the characteristics of dopamine neurotransmission in vertebrates. PMID:26613258

  15. Affinities of brompheniramine, chlorpheniramine, and terfenadine at the five human muscarinic cholinergic receptor subtypes.

    PubMed

    Yasuda, S U; Yasuda, R P

    1999-04-01

    Anticholinergic effects are presumed to be the mechanism for the efficacy of chlorpheniramine in symptomatic relief of the common cold. Terfenadine, a second-generation antihistamine, reportedly lacks anticholinergic side effects. We evaluated affinities of two commonly used over-the-counter antihistamines, brompheniramine and chlorpheniramine, as well as terfenadine in comparison with atropine at the five human muscarinic cholinergic receptor subtypes using CHO cells stably transfected with the individual subtypes. Atropine was more potent than all three drugs at m1-m5 (p<0.01). No significant difference was observed between chlorpheniramine and brompheniramine. Atropine, brompheniramine, and chlorpheniramine could not discriminate between m1-m5. Terfenadine demonstrated subtype selectivity at m3. In vitro comparisons in human muscarinic receptor subtypes could potentially be used to predict clinical anticholinergic effects of antihistamines and to target receptor-specific effects of such agents.

  16. Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung

    SciTech Connect

    Mak, J.C.; Barnes, P.J. )

    1990-06-01

    Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using (3H)(-)quinuclidinyl benzilate (( 3H)QNB) and selective muscarinic antagonists. (3H)QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with (3H)pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies.

  17. Muscarinic receptor subtypes as potential targets to modulate oligodendrocyte progenitor survival, proliferation, and differentiation.

    PubMed

    De Angelis, Federica; Bernardo, Antonietta; Magnaghi, Valerio; Minghetti, Luisa; Tata, Ada Maria

    2012-05-01

    Acetylcholine (ACh) is a major neurotransmitter but also an important signaling molecule in neuron-glia interactions. Expression of ACh receptors has been reported in several glial cell populations, including oligodendrocytes (OLs). Nonetheless, the characterization of muscarinic receptors in these cells, as well as the description of the cholinergic effects at different stages of OL development, is still incomplete. In this study, we characterized the pattern of expression of muscarinic receptor subtypes in primary cultures of rat oligodendrocyte progenitor cells (OPC) and mature OLs, at both mRNA and protein levels. We found that muscarinic receptor expression is developmentally regulated. M1, M3, and M4 receptors were the main subtypes expressed in OPC, whereas all receptor subtypes were expressed at low levels in mature OLs. Exposure of OPC to muscarine enhanced cell proliferation, an effect mainly due to M1, M3, and M4 receptor subtypes as demonstrated by pharmacological competition with selective antagonists. Conversely, M2 receptor activation impaired OPC survival. In line with the mitogenic activity, muscarinic receptor activation increased the expression of platelet derived growth factor receptor α. Muscarine stimulation increased CX32 and myelin basic protein expression, left unaffected that of myelin proteolipid protein (PLP), and decreased member of the family of epidermal growth factor receptor (EGFR) ErbB3/ErbB4 receptor expression indicating a predominant role of muscarinic receptors in OPC. These findings suggest that ACh may contribute to the maintenance of an immature proliferating progenitor pool and impair the progression toward mature stage. This hypothesis is further supported by increased expression of Notch-1 in OL on muscarinic activation.

  18. Phosphoinositide system-linked serotonin receptor subtypes and their pharmacological properties and clinical correlates.

    PubMed Central

    Pandey, S C; Davis, J M; Pandey, G N

    1995-01-01

    Serotonergic neurotransmission represents a complex mechanism involving pre- and post-synaptic events and distinct 5-HT receptor subtypes. Serotonin (5-HT) receptors have been classified into several categories, and they are termed as 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 type receptors. 5-HT1 receptors have been further subdivided into 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F. 5-HT2 receptors have been divided into 5-HT2A, 5-HT2B and 5-HT2C receptors. All 5-HT2 receptor subtypes are linked to the multifunctional phosphoinositide (PI) signalling system. 5-HT3 receptors are considered ion-gated receptors and are also linked to the PI signalling system by an unknown mechanism. The 5-HT2A receptor subtype is the most widely studied of the 5-HT receptors in psychiatric disorders (for example, suicide, depression and schizophrenia) as well as in relation to the mechanism of action of antidepressant drugs. The roles of 5-HT2C and 5-HT3 receptors in psychiatric disorders are less clear. These 5-HT receptors also play an important role in alcoholism. It has been shown that 5-HT2A, 5-HT2C and 5-HT3 antagonists cause attenuation of alcohol intake in animals and humans. However, the exact mechanisms are unknown. The recent cloning of the cDNAs for 5-HT2A, 5-HT2C and 5-HT3 receptors provides the opportunity to explore the molecular mechanisms responsible for the alterations in these receptors during illness as well as pharmacotherapy. This review article will focus on the current research into the pharmacological properties, molecular biology, and clinical correlates of 5-HT2A, 5-HT2C and 5-HT3 receptors. PMID:7786883

  19. Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 1 (EP₁) in zebrafish.

    PubMed

    Kwok, Amy H Y; Wang, Yajun; Leung, Frederick C

    2012-09-01

    Prostaglandins E (PGE) and F (PGF) mediate diverse physiological functions via their cell surface receptors - prostaglandin E receptor (EP) subtypes 1, 2, 3 and 4 (EP(1); EP(2); EP(3); EP(4)) and F receptor (FP). In teleost fishes, PGE was implicated in gill epithelium ion transport, while both PGE and PGF were involved in oocyte maturation, follicular rupture and coordination of reproductive behaviors. However, little is known about the mechanisms behind their actions. In present study, we first identified the full-length ORF cDNA clones of three zebrafish prostaglandin E receptor subtype 1 (zEP(1)) isoforms - zEP(1a), zEP(1b) and zEP(1c) - and FP (zFP) from adult ovary. RT-PCR showed that zEP(1a), zEP(1b) and zFP are widely expressed in adult tissues, while zEP(1c) mRNA expression is mainly confined in brain and kidney. Using a pGL3-NFAT-RE luciferase reporter system, both zEP(1a) and zEP(1b) expressed in DF-1 cells were shown to be activated by PGE(2) potently while zEP(1c) and zFP were activated by PGF(2a) effectively, suggesting that the four receptors are functionally coupled to intracellular Ca(2+)-signaling pathway. Furthermore, EP1a and EP1b, but not EP1c were suggested to couple to cAMP-PKA signaling pathway using a pGL3-CRE luciferase reporter assay. Although zEP(1c) might originate as a paralog to zEP(1a) and zEP(1b), its functional coupling to PGF(2α) instead of PGE(2) suggested that zEP(1) isoforms might have sub-functionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Characterization of these receptors undoubtedly furthered our understanding on the diverse yet highly target-specific responses of prostaglandins in teleosts. PMID:22617193

  20. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  1. Expression of purinergic P2X receptor subtypes 1, 2, 3 and 7 in equine laminitis.

    PubMed

    Zamboulis, Danae E; Senior, Mark; Clegg, Peter D; Milner, Peter I

    2013-11-01

    Tissue sensitisation and chronic pain have been described in chronic-active laminitis in the horse, making treatment of such cases difficult. Purinergic P2X receptors are linked to chronic pain and inflammation. The aim of this study was to examine the expression of purinergic P2X receptor subtypes 1, 2, 3 and 7 in the hoof, palmar digital vessels and nerve, dorsal root ganglia and spinal cord in horses with chronic-active laminitis (n=5) compared to non-laminitic horses (n=5). Immunohistochemical analysis was performed on tissue sections using antibodies against P2X receptor subtypes 1-3 and 7. In horses with laminitis, there was a reduction in the thickness of the tunica media layer of the palmar digital vein as a proportion of the whole vessel diameter (0.48±0.05) compared to the non-laminitic group (0.57±0.04; P=0.02). P2X receptor subtype 3 was expressed in the smooth muscle layer (tunica media) of the palmar digital artery of horses with laminitis, but was absent in horses without laminitis. There was strong expression of P2X receptor subtype 7 in the proliferating, partially keratinised, epidermal cells of the secondary epidermal lamellae in the hooves of horses with laminitis, but no immunopositivity in horses without laminitis.

  2. Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens.

    PubMed

    Tohyama, Saki; Miyagawa, Shinichi; Lange, Anke; Ogino, Yukiko; Mizutani, Takeshi; Tatarazako, Norihisa; Katsu, Yoshinao; Ihara, Masaru; Tanaka, Hiroaki; Ishibashi, Hiroshi; Kobayashi, Tohru; Tyler, Charles R; Iguchi, Taisen

    2015-06-16

    Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research. PMID:26032098

  3. Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens.

    PubMed

    Tohyama, Saki; Miyagawa, Shinichi; Lange, Anke; Ogino, Yukiko; Mizutani, Takeshi; Tatarazako, Norihisa; Katsu, Yoshinao; Ihara, Masaru; Tanaka, Hiroaki; Ishibashi, Hiroshi; Kobayashi, Tohru; Tyler, Charles R; Iguchi, Taisen

    2015-06-16

    Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

  4. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    NASA Astrophysics Data System (ADS)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  5. Distribution and effects of the muscarinic receptor subtypes in the primary visual cortex

    PubMed Central

    Groleau, Marianne; Kang, Jun Il; Huppé-Gourgues, Frédéric; Vaucher, Elvire

    2015-01-01

    Muscarinic cholinergic receptors modulate the activity and plasticity of the visual cortex. Muscarinic receptors are divided into five subtypes that are not homogeneously distributed throughout the cortical layers and cells types. This distribution results in complex action of the muscarinic receptors in the integration of visual stimuli. Selective activation of the different subtypes can either strengthen or weaken cortical connectivity (e.g., thalamocortical vs. corticocortical), i.e., it can influence the processing of certain stimuli over others. Moreover, muscarinic receptors differentially modulate some functional properties of neurons during experience-dependent activity and cognitive processes and they contribute to the fine-tuning of visual processing. These functions are involved in the mechanisms of attention, maturation and learning in the visual cortex. This minireview describes the anatomo-functional aspects of muscarinic modulation of the primary visual cortex’s (V1) microcircuitry. PMID:26150786

  6. Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

    PubMed

    Monnerie, H; Hsu, F-C; Coulter, D A; Le Roux, P D

    2010-12-29

    The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered

  7. The N-terminal domain of GluR6-subtype glutamate receptor ion channels

    SciTech Connect

    Kumar, Janesh; Schuck, Peter; Jin, Rongsheng; Mayer, Mark L.

    2009-09-25

    The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.

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

    PubMed

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

    1992-12-01

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

  9. The role of serotonin receptor subtypes in treating depression: a review of animal studies

    PubMed Central

    Carr, Gregory V.

    2012-01-01

    Rationale Serotonin reuptake inhibitors (SSRIs) are effective in treating depression. Given the existence of different families and subtypes of 5-HT receptors, multiple 5-HT receptors may be involved in the antidepressant-like behavioral effects of SSRIs. Objective Behavioral pharmacology studies investigating the role of 5-HT receptor subtypes in producing or blocking the effects of SSRIs were reviewed. Results Few animal behavior tests were available to support the original development of SSRIs. Since their development, a number of behavioral tests and models of depression have been developed that are sensitive to the effects of SSRIs, as well as to other types of antidepressant treatments. The rationale for the development and use of these tests is reviewed. Behavioral effects similar to those of SSRIs (antidepressant-like) have been produced by agonists at 5-HT1A, 5-HT1B, 5-HT2C, 5-HT4, and 5-HT6 receptors. Also, antagonists at 5-HT2A, 5-HT2C, 5-HT3, 5- HT6, and 5-HT7 receptors have been reported to produce antidepressant-like responses. Although it seems paradoxical that both agonists and antagonists at particular 5-HT receptors can produce antidepressant-like effects, they probably involve diverse neurochemical mechanisms. The behavioral effects of SSRIs and other antidepressants may also be augmented when 5-HT receptor agonists or antagonists are given in combination. Conclusions The involvement of 5-HT receptors in the antidepressant-like effects of SSRIs is complex and involves the orchestration of stimulation and blockade at different 5-HT receptor subtypes. Individual 5-HT receptors provide opportunities for the development of a newer generation of antidepressants that may be more beneficial and effective than SSRIs. PMID:21107537

  10. Expression of two alpha 2-adrenergic receptor subtypes in human placenta: evidence from direct binding studies.

    PubMed

    Falkay, G; Kovács, L

    1994-09-01

    Adrenergic receptors may play an important role for mediating a variety of metabolic and haemodynamic effects of catecholamines including placental blood flow. The alpha-adrenergic receptors of the human placenta were characterized in vitro by the use of [3H]rauwolscine and [3H]prazosin as radioligands. Saturation experiments would suggest that the alpha-adrenoceptors in the human placenta are alpha 2. Comparative binding studies were performed, using recently synthesized compounds (Beecham Pharmaceuticals, UK) selective for alpha 2A (BRL-44408) and alpha 2B (BRL-41992) subtypes. The results indicate that human placenta contains at least two pharmacologically distinct alpha 2-adrenoceptor subtypes with approximately 60 per cent alpha 2A and 40 per cent alpha 2B receptors. In contrast with the pattern of increasing beta-adrenoceptor density, the concentration of alpha 2-adrenoceptors in term placentae is significantly lower than in placentae from the first trimester.

  11. Amiloride inhibition of gamma-aminobutyric acid(A) receptors depends upon the alpha subunit subtype.

    PubMed

    Fisher, Janet L

    2002-06-01

    gamma-Aminobutyric acid(A) (GABA(A)) receptors (GABARs) are responsible for most fast inhibitory neurotransmission in the mammalian brain. The GABARs contain several allosteric modulatory sites, many of which are useful clinically. The activity of most of these modulators depends upon the subunit composition of the receptor. The diuretic amiloride was previously reported to inhibit GABARs in frog sensory neurons. We measured its effects on recombinant GABARs to determine its mechanism of action at mammalian receptors and to examine the effect of subunit composition. Amiloride acted primarily as a competitive antagonist, reducing the sensitivity of the receptor to GABA without affecting the maximal current amplitude. Receptors containing an alpha6 subunit were about 10-fold more sensitive to amiloride than those containing other alpha subunits. In contrast, the identity of the beta or gamma subtype had little effect on amiloride sensitivity. Although several other modulators have specific effects at alpha6-containing receptors, amiloride is the first inhibitor to be reported with no additional dependence on the identity of the beta or gamma subunit. Therefore, it probably represents a unique modulatory site on the GABAR, which could be useful for developing drugs targeting these receptors. The selective activity of amiloride could also be helpful for isolating the contribution of receptors composed of alpha6 subtypes in heterogeneous native GABAR populations.

  12. Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 3 (EP3) in chickens.

    PubMed

    Kwok, Amy H Y; Wang, Yajun; Leung, Frederick C

    2012-10-01

    Prostaglandin E and F regulate diverse physiological functions including gastrointestinal motility, fever induction and reproduction. This multitude of biological effects is mediated via their four E receptor subtypes (EP(1), EP(2), EP(3) and EP(4)) and F receptor (FP), respectively. Majority of these studies was performed in mammalian species, while investigations on their roles were impeded by inadequate information on their receptors in avian species. In present study, full-length cDNAs of chicken EP(3) (cEP(3)) and two isoforms of FP - cFPa and cFPb - were cloned from adult hen ovary. The putative cEP(3) and cFPa share high amino acid sequence identity with their respective orthologs, while the predicted cFPb is a novel middle-truncated splice variant which lacks 107 amino acids between transmembrane domains 4 and 6. RT-PCR showed that cEP(3), cFPa and cFPb are widely expressed in adult tissues examined, including ovary and oviduct. Using a pGL3-CRE luciferase reporter system, cEP(3)-expressing DF1 cells inhibited forskolin-induced luciferase activity (EC(50): <1.9 pM) upon PGE(2) treatment, suggesting that cEP(3) may functionally couple to Gi protein. Upon PGF(2α) addition, cFPa was shown to potentially couple to intracellular Ca(2+)-signaling pathway by pGL3-NFAT-RE reporter assay (EC(50): 2.9 nM), while cFPb showed no response. Using a pGL4-SRE reporter system, both cEP(3) and cFPa exhibited potential MAPK activation by PGE(2) and PGF(2α) at EC(50) 0.34 and 13 nM, respectively. Molecular characterization of these receptors paved the road to the better understanding of PGE(2) and PGF(2α) roles in avian physiology and comparative endocrinology studies. PMID:22885557

  13. Ligand binding properties of muscarinic acetylcholine receptor subtypes (m1-m5) expressed in baculovirus-infected insect cells.

    PubMed

    Dong, G Z; Kameyama, K; Rinken, A; Haga, T

    1995-07-01

    Five subtypes of muscarinic acetylcholine receptors (m1-m5) have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system. Up to 6 nmol of muscarinic acetylcholine receptors were produced by 1 liter culture; 0.3 to 0.6 (human m1), 3 to 6 (human m2), 2 to 4 (rat m3), 1 to 2 (rat m4) and 0.5 to 1 (human m5) nmol. Pirenzepine, AF-DX116 and hexahidrosiladifenidol showed the highest affinity for the m1, m2 and m3 subtype, respectively, indicating that these receptors expressed in Sf9 cells retain the same substrate specificity as those in mammalian tissues or cultured cells. Among 32 kinds of muscarinic ligands examined in the present studies, prifinium was found to have the highest affinity for the m4 subtype, and pilocarpine, oxotremorine, McN-A343 and promethazine the highest affinity for the m5 subtype, although the differences in the affinities among the five subtypes were less than 10-fold. Alcuronium increased the binding of [3H]N-methylscopalamine to the m2 subtype, but not the m1, m4 and m5 subtypes and only slightly to the m3 subtype. Similar but smaller effects of fangchinoline and tetrandrine were found for [3H]N-methylscopalamine binding to only the m3 subtype. These effects may also be useful for the discrimination of individual subtypes. PMID:7616422

  14. Regulation of subtypes of beta-adrenergic receptors in rat brain following treatment with 6-hydroxydopamine

    SciTech Connect

    Johnson, E.W.; Wolfe, B.B.; Molinoff, P.B.

    1989-07-01

    The technique of quantitative autoradiography has been used to localize changes in the densities of subtypes of beta-adrenergic receptors in rat brain following treatment with 6-hydroxydopamine. Previously reported increases in the density of beta 1-adrenergic receptors in the cerebral cortex were confirmed. The anatomical resolution of autoradiography made it possible to detect changes in the density of beta 2-adrenergic receptors in the cortex and in a number of other brain regions. The density of beta 1-adrenergic receptors increased from 30 to 50% depending on the region of the cortex being examined. The increase in the somatomotor cortex was greater than that in the frontal or occipital cortex. The increase in the density of beta 2-adrenergic receptors in the cortex was not as widespread as that of beta 1-adrenergic receptors and occurred primarily in frontal cortex, where the density of receptors increased by 40%. The densities of both beta 1- and beta 2-adrenergic receptors increased in a number of forebrain, thalamic, and midbrain structures. Selective changes in the density of beta 1-adrenergic receptors were observed in the superficial gray layer of the superior colliculus and in the amygdala. The density of beta 2-adrenergic receptors increased in the caudate-putamen, the substantia nigra, and the lateral and central nuclei of the thalamus, whereas the density of beta 1-adrenergic receptors did not change in these regions. The densities of both subtypes of beta-adrenergic receptors increased in the hippocampus, the cerebellum, the lateral posterior nucleus of the thalamus, and the dorsal lateral geniculate.

  15. The type I interleukin-1 receptor mediates fever in the rat as shown by interleukin-1 receptor subtype selective ligands.

    PubMed

    Malinowsky, D; Chai, Z; Bristulf, J; Simoncsits, A; Bartfai, T

    1995-12-01

    The interleukin-1 (IL-1) system possesses two distinct receptors (type I and type II) which, together with the accessory protein, mediate a multitude of responses to IL-1 alpha and IL-1 beta, including fever. So far, no receptor subtype-specific ligands have been described. Since both types of IL-1 receptors occur in the thermoregulatory areas it was unclear which IL-1 receptor type mediates fever. We report here that for a series of deletion mutants of human recombinant IL-1 beta (hrIL-1 beta), the affinity of these ligands for the type I IL-1 receptor correlates with their efficacy to evoke the fever response (hrIL-1 beta > des-SND52-54 > des-QGE48-50 > des-I56). Thus, the results suggest that agonist occupancy of the type I IL-1 receptor is essential for IL-1 beta-mediated fever.

  16. Pharmacology and therapeutic applications of A3 receptor subtype.

    PubMed

    Fishman, Pnina; Bar-Yehuda, Sara

    2003-01-01

    The present study summarizes the biological effects elicit upon A(3) adenosine receptor (A(3)AR) activation in normal and tumor cells. Anti-inflamatory response is mediated upon A(3)AR activation in neutrophils, eosinophils and macrophages via direct effect on cell degranulation or the production of anti-inflamatory cytokines. In basophils, which highly express A(3)AR, degranulation and mediator release upon receptor activation lead to pro-inflammatory effects resulting in bronchospasm and asthma. In other normal cells such as cardiomyocytes, neuronal cells and bone marrow cells A(1)AR activation induces cytoprotective effects in vitro. In vivo, A(3)AR agonists act as cardio- and neuroprotective agents and attenuate ischemic damage. Furthermore, agonists to A(3)AR induce granulocyte colony stimulating factor (G-CSF) production and myeloprotective effect in chemotherapy treated mice. Interestingly, A(3)AR agonists inhibit tumor cell growth both in vitro and in vivo through a cytostatic effect mediated via the de-regulation of the Wnt signaling pathway. The variety of activities elicit by A(3)AR agonists suggest their potential use as therapeutic agents in inflammation, brain/cardiac ischemia and cancer. Antagonists to A(3)AR may be implemented to the therapy of asthma and additional allergic conditions.

  17. Switching of chemoattractant receptors programs development and morphogenesis in Dictyostelium: receptor subtypes activate common responses at different agonist concentrations.

    PubMed

    Kim, J Y; Borleis, J A; Devreotes, P N

    1998-05-01

    One of the common functional features among G-protein coupled receptors is the occurrence of multiple subtypes involved in similar signal transduction events. The cAMP chemoattractant receptor family of Dictyostelium discoideum is composed of four receptors (cAR1-cAR4), which are expressed sequentially throughout the developmental transition from a unicellular to a multicellular organism. The receptors differ in affinity for cAMP and in the sequences of their C-terminal domains. In this study, we constitutively expressed cAR1, cAR2, and cAR3 as well as a series of chimeric and mutant receptors and assessed the capacity of each to mediate chemotaxis, activation of adenylyl cyclase and actin polymerization, and rescue the developmental defect of car1-/car3- cells. We found that various receptors and mutants sense different concentration ranges of cAMP but all can mediate identical responses during the aggregation stage of development. The responses displayed very similar kinetics, suggesting no major differences in regulatory properties attributable to the C-terminal domains. We speculate that switching of receptor subtypes during development enables the organism to respond to the changing concentrations of the chemoattractant and thereby program morphogenesis appropriately. PMID:9578623

  18. NPY receptor subtype specification for behavioral adaptive strategies during limited food access.

    PubMed

    Pjetri, E; Adan, R A; Herzog, H; de Haas, R; Oppelaar, H; Spierenburg, H A; Olivier, B; Kas, M J

    2012-02-01

    The neuropeptide Y (NPY) system in the brain regulates a wide variety of behavioral, metabolic and hormonal homeostatic processes required for energy balance control. During times of limited food availability, NPY promotes behavioral hyperactivity necessary to explore and prepare for novel food resources. As NPY can act via 5 different receptor subtypes, we investigated the path through which NPY affects different behavioral components relevant for adaptation to such conditions. We tested NPY Y1 and Y2 receptor knockout mice and their wild-type littermate controls in a daily scheduled limited food access paradigm with unlimited access to running wheel. Here we show that NPY Y1 receptor deficient mice lack the expression of appetitive behavior and that NPY Y2 receptors control the level of hyperactive behavior under these conditions. Thus, receptor specificity determines the differential expression of NPY-mediated behavioral adaptations to overcome a negative energy status.

  19. Metabotropic glutamate receptor subtype 5: molecular pharmacology, allosteric modulation and stimulus bias.

    PubMed

    Sengmany, K; Gregory, K J

    2016-10-01

    The metabotropic glutamate receptor subtype 5 (mGlu5 ) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR-based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine-tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus-bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  20. Ligand Binding and Subtype Selectivity of the Human A2A Adenosine Receptor

    PubMed Central

    Jaakola, Veli-Pekka; Lane, J. Robert; Lin, Judy Y.; Katritch, Vsevolod; IJzerman, Adriaan P.; Stevens, Raymond C.

    2010-01-01

    The crystal structure of the human A2A adenosine receptor bound to the A2A receptor-specific antagonist, ZM241385, was recently determined at 2.6-Å resolution. Surprisingly, the antagonist binds in an extended conformation, perpendicular to the plane of the membrane, and indicates a number of interactions unidentified before in ZM241385 recognition. To further understand the selectivity of ZM241385 for the human A2A adenosine receptor, we examined the effect of mutating amino acid residues within the binding cavity likely to have key interactions and that have not been previously examined. Mutation of Phe-168 to Ala abolishes both agonist and antagonist binding as well as receptor activity, whereas mutation of this residue to Trp or Tyr had only moderate effects. The Met-177 → Ala mutation impeded antagonist but not agonist binding. Finally, the Leu-249 → Ala mutant showed neither agonist nor antagonist binding affinity. From our results and previously published mutagenesis data, we conclude that conserved residues Phe-168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a central role in coordinating the bicyclic core present in both agonists and antagonists. By combining the analysis of the mutagenesis data with a comparison of the sequences of different adenosine receptor subtypes from different species, we predict that the interactions that determine subtype selectivity reside in the more divergent “upper” region of the binding cavity while the “lower” part of the binding cavity is conserved across adenosine receptor subtypes. PMID:20147292

  1. US Incidence of Breast Cancer Subtypes Defined by Joint Hormone Receptor and HER2 Status

    PubMed Central

    Altekruse, Sean F.; Li, Christopher I.; Chen, Vivien W.; Clarke, Christina A.; Ries, Lynn A. G.; Cronin, Kathleen A.

    2014-01-01

    Background In 2010, Surveillance, Epidemiology, and End Results (SEER) registries began collecting human epidermal growth factor 2 (HER2) receptor status for breast cancer cases. Methods Breast cancer subtypes defined by joint hormone receptor (HR; estrogen receptor [ER] and progesterone receptor [PR]) and HER2 status were assessed across the 28% of the US population that is covered by SEER registries. Age-specific incidence rates by subtype were calculated for non-Hispanic (NH) white, NH black, NH Asian Pacific Islander (API), and Hispanic women. Joint HR/HER2 status distributions by age, race/ethnicity, county-level poverty, registry, stage, Bloom–Richardson grade, tumor size, and nodal status were evaluated using multivariable adjusted polytomous logistic regression. All statistical tests were two-sided. Results Among case patients with known HR/HER2 status, 36810 (72.7%) were found to be HR+/HER2−, 6193 (12.2%) were triple-negative (HR−/HER2−), 5240 (10.3%) were HR+/HER2+, and 2328 (4.6%) were HR−/HER2+; 6912 (12%) had unknown HR/HER2 status. NH white women had the highest incidence rate of the HR+/HER2− subtype, and NH black women had the highest rate of the triple-negative subtype. Compared with women with the HR+/HER2− subtype, triple-negative patients were more likely to be NH black and Hispanic; HR+/HER2+ patients were more likely to be NH API; and HR−/HER2+ patients were more likely to be NH black, NH API, and Hispanic. Patients with triple-negative, HR+/HER2+, and HR−/HER2+ breast cancer were 10% to 30% less likely to be diagnosed at older ages compared with HR+/HER2− patients and 6.4-fold to 20.0-fold more likely to present with high-grade disease. Conclusions In the future, SEER data can be used to monitor clinical outcomes in women diagnosed with different molecular subtypes of breast cancer for a large portion (approximately 28%) of the US population. PMID:24777111

  2. Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart.

    PubMed

    Malloy, Cole A; Ritter, Kyle; Robinson, Jonathan; English, Connor; Cooper, Robin L

    2016-01-01

    The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart's pacemaker activity.

  3. Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

    PubMed Central

    Liggett, S B; Freedman, N J; Schwinn, D A; Lefkowitz, R J

    1993-01-01

    The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In

  4. Are so many adrenergic receptor subtypes really present in domestic animal tissues? A pharmacological perspective.

    PubMed

    Badino, P; Odore, R; Re, G

    2005-09-01

    Adrenergic receptors (ARs) are the cellular membrane binding sites through which natural catecholamines and sympathomimetic drugs exert their physiological and pharmacological effects. In recent decades, studies to clarify the distribution and function of ARs have been performed mostly on cultured cells, laboratory animals and human target tissues, but little is known about these aspects in domestic animals. This review focuses on AR structure, classification and signalling pathways and on AR subtype distribution in target tissues of some domestic animals, namely dogs, horses and bovines. In these species, different alpha- and beta-AR subtypes have been characterized and the functions controlled by the adrenergic systems have been studied. In the dog, the role played by the adrenergic system in the pathogenesis of cardiovascular disorders and in the modulation of canine aggression has roused particular interest. In dogs affected by dilated cardiomyopathy a significant down-regulation of beta-ARs has been observed both in the heart and circulating lymphocytes. This finding confirms the involvement of the adrenergic system in the pathogenesis and progression of the disorder and suggests new therapeutic strategies. In the horse, AR distribution has been studied in the cardiac, respiratory and gastrointestinal systems as well as in digital veins and arteries. The cardiac beta-ARs in healthy horses seem to be predominantly represented by the beta(1) subtype. In this species, heart failure may increase the expression of the beta(2) subtype, rather than causing AR down-regulation. Different beta- and alpha-AR subtypes have been characterized in the smooth muscle of equine ileum. The sympathetic relaxation of equine ileum smooth muscle seems to depend mainly on beta(3)-AR subtype activation, with minor involvement of the beta(2) subtype. In the respiratory tract, regional differences have been evidenced in the functionality of beta-AR subtype. The beta(2) subtype

  5. Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

    PubMed Central

    Kirsch, Glenn E.; Fedorov, Nikolai B.; Kuryshev, Yuri A.; Liu, Zhiqi; Orr, Michael S.

    2016-01-01

    Abstract The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity. PMID:27505073

  6. Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors.

    PubMed

    Kirsch, Glenn E; Fedorov, Nikolai B; Kuryshev, Yuri A; Liu, Zhiqi; Armstrong, Lucas C; Orr, Michael S

    2016-08-01

    The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity. PMID:27505073

  7. Effect of the alpha subunit subtype on the macroscopic kinetic properties of recombinant GABA(A) receptors.

    PubMed

    Picton, Amber J; Fisher, Janet L

    2007-08-24

    The GABA(A) receptors (GABARs) are chloride-permeable ligand-gated ion channels responsible for fast inhibitory neurotransmission. These receptors are structurally heterogeneous, and in mammals can be formed from a combination of sixteen different subunit subtypes. Much of this variety comes from the six different alpha subunit subtypes. All neuronal GABARs contain an alpha subunit, and the identity of the alpha subtype affects the pharmacological properties of the receptors. The expression of each of the different alpha subtypes is regulated developmentally and regionally and changes with both normal physiological processes such development and synaptic plasticity, and pathological conditions such as epilepsy. In order to understand the functional significance of this structural heterogeneity, we examined the effect of the alpha subtype on the receptor's response to GABA. Each of the six alpha subtypes was transiently co-expressed with the beta3 and gamma2L subunits in mammalian cells. The sensitivity to GABA was measured with whole-cell recordings. We also determined the activation, deactivation, desensitization, and recovery kinetics for the six isoforms using rapid application recordings from excised macropatches. We found unique characteristics associated with each alpha subunit subtype. These properties would be expected to influence the post-synaptic response to GABA, creating functional diversity among neurons expressing different alpha subunits.

  8. Somatostatin receptor subtypes in neuroendocrine tumor cell lines and tumor tissues.

    PubMed

    Jonas, S; John, M; Boese-Landgraf, J; Häring, R; Prevost, G; Thomas, F; Rosewicz, S; Riecken, E O; Wiedenmann, B; Neuhaus, P

    1995-01-01

    Somatostatin receptor scintigraphy (SRS) is positive in approximately 80% of all patients who have been found to have neuroendocrine (NE) gastroenteropancreatic (GEP) tumors. The reasons for negative results are unclear. The aim of the present study was identification of the specific somatostatin receptor (SSTR) subtypes that are responsible for the in vivo binding of the widely used somatostatin (SST) analogues octreotide and lanreotide in human neuroendocrine gastroenteropancreatic tumors. Ten patients were subjected to SRS with radiolabeled octreotide. Following surgical resection, tumor tissues were analyzed for SSTR subtype mRNA expression by the reverse transcription-polymerase chain reaction (RT-PCR). In addition, SSTR subtype transcripts were investigated by Northern blot analysis and RT-PCR in neuroendocrine tumor cell lines. Expression of SSTR at the protein level was studied by chemical cross-linking experiments. Three patients were negative by SRS. However, RT-PCR revealed most prominently SSTR 2 expression in all tumor specimens. In addition, all tumor tissues analyzed by chemical crosslinking exhibited SST-14 binding sites, indicating that at least some NE tumors were false-negative on SRS.

  9. Identification of the ETA receptor subtype that mediates endothelin induced autocrine proliferation of normal human keratinocytes.

    PubMed

    Bagnato, A; Venuti, A; Di Castro, V; Marcante, M L

    1995-04-01

    Endothelin-1 has a wide range of pharmacological effects in various tissues and acts as autocrine/paracrine factor. The potential of ET-1 to function as an autocrine growth factor was evaluated in normal human keratinocytes. Radioligand binding studies showed that 125I-ET-1 bound to a single class of high-affinity-binding sites on the surface of the cells. The dissociation constant was 0.045 nM with receptor numbers of 1700 sites/cell. Treatment with serum caused increases in expression of binding sites (3500 sites/cell), with no change in binding affinity. ET-1 stimulated thymidine incorporation in these cells that expressed ET receptors. An ET antagonist selective for the ETA receptor subtype (BQ 123) inhibited DNA synthesis stimulated by ET-1 and reduced the basal growth rate of unstimulated cells. These data suggest that the ET-1 induced DNA synthesis is mediated by ETA receptor subtype and that endogenously produced ET-1 promotes the autocrine proliferation of keratinocytes.

  10. Mg2+ imparts NMDA receptor subtype selectivity to the Alzheimer's drug memantine.

    PubMed

    Kotermanski, Shawn E; Johnson, Jon W

    2009-03-01

    N-methyl-D-aspartate receptors (NMDARs) mediate interneuronal communication and are broadly involved in nervous system physiology and pathology (Dingledine et al., 1999). Memantine, a drug that blocks the ion channel formed by NMDARs, is a widely prescribed treatment of Alzheimer's disease (Schmitt, 2005; Lipton, 2006; Parsons et al., 2007). Research on memantine's mechanism of action has focused on the NMDAR subtypes most highly expressed in adult cerebral cortex, NR1/2A and NR1/2B receptors (Cull-Candy and Leszkiewicz, 2004), and has largely ignored interactions with extracellular Mg(2+) (Mg(2+)(o)). Mg(2+)(o) is an endogenous NMDAR channel blocker that binds near memantine's binding site (Kashiwagi et al., 2002; Chen and Lipton, 2005). We report that a physiological concentration (1 mM) of Mg(2+)(o) decreased memantine inhibition of NR1/2A and NR1/2B receptors nearly 20-fold at a membrane voltage near rest. In contrast, memantine inhibition of the other principal NMDAR subtypes, NR1/2C and NR1/2D receptors, was decreased only approximately 3-fold. As a result, therapeutic memantine concentrations should have negligible effects on NR1/2A or NR1/2B receptor activity but pronounced effects on NR1/2C and NR1/2D receptors. Quantitative modeling showed that the voltage dependence of memantine inhibition also is altered by 1 mM Mg(2+)(o). We report similar results with the NMDAR channel blocker ketamine, a drug used to model schizophrenia (Krystal et al., 2003). These results suggest that currently hypothesized mechanisms of memantine and ketamine action should be reconsidered and that NR1/2C and/or NR1/2D receptors play a more important role in cortical physiology and pathology than previously appreciated.

  11. Monovalent cation and amiloride analog modulation of adrenergic ligand binding to the unglycosylated alpha 2B-adrenergic receptor subtype

    SciTech Connect

    Wilson, A.L.; Seibert, K.; Brandon, S.; Cragoe, E.J. Jr.; Limbird, L.E. )

    1991-04-01

    The unglycosylated alpha 2B subtype of the alpha 2-adrenergic receptor found in NG-108-15 cells possesses allosteric regulation of adrenergic ligand binding by monovalent cations and 5-amino-substituted amiloride analogs. These findings demonstrate that allosteric modulation of adrenergic ligand binding is not a property unique to the alpha 2A subtype. The observation that amiloride analogs as well as monovalent cations can modulate adrenergic ligand binding to the nonglycosylated alpha 2B subtype indicates that charge shielding due to carbohydrate moieties does not play a role in this allosteric modulation but, rather, these regulatory effects result from interactions of cations and amiloride analogs with the protein moiety of the receptor. Furthermore, the observation that both alpha 2A and alpha 2B receptor subtypes are modulated by amiloride analogs suggests that structural domains that are conserved between the two are likely to be involved in this allosteric modulation.

  12. Common genetic variation in adiponectin, leptin, and leptin receptor and association with breast cancer subtypes.

    PubMed

    Nyante, Sarah J; Gammon, Marilie D; Kaufman, Jay S; Bensen, Jeannette T; Lin, Dan Yu; Barnholtz-Sloan, Jill S; Hu, Yijuan; He, Qianchuan; Luo, Jingchun; Millikan, Robert C

    2011-09-01

    Adipocytokines are produced by visceral fat, and levels may be associated with breast cancer risk. We investigated whether single nucleotide polymorphisms (SNPs) in adipocytokine genes adiponectin (ADIPOQ), leptin (LEP), and the leptin receptor (LEPR) were associated with basal-like or luminal A breast cancer subtypes. 104 candidate and tag SNPs were genotyped in 1776 of 2022 controls and 1972 (200 basal-like, 679 luminal A) of 2311 cases from the Carolina Breast Cancer Study (CBCS), a population-based case-control study of whites and African Americans. Breast cancer molecular subtypes were determined by immunohistochemistry. Genotype odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. Haplotype ORs and 95% CIs were estimated using Hapstat. Interactions with waist-hip ratio were evaluated using a multiplicative interaction term. Ancestry was estimated from 144 ancestry informative markers (AIMs), and included in models to control for population stratification. Candidate SNPs LEPR K109R (rs1137100) and LEPR Q223R (rs1137101) were positively associated with luminal A breast cancer, whereas ADIPOQ +45 T/G (rs2241766), ADIPOQ +276 G/T (rs1501299), and LEPR K656N (rs8129183) were not associated with either subtype. Few patterns were observed among tag SNPs, with the exception of 3 LEPR SNPs (rs17412175, rs9436746, and rs9436748) that were in moderate LD and inversely associated with basal-like breast cancer. However, no SNP associations were statistically significant after adjustment for multiple comparisons. Haplotypes in LEP and LEPR were associated with both basal-like and luminal A subtypes. There was no evidence of interaction with waist-hip ratio. Data suggest associations between LEPR candidate SNPs and luminal A breast cancer in the CBCS and LEPR intron 2 tag SNPs and basal-like breast cancer. Replication in additional studies where breast cancer subtypes have been defined is necessary to confirm these

  13. Common genetic variation in adiponectin, leptin, and leptin receptor and association with breast cancer subtypes

    PubMed Central

    Nyante, Sarah J.; Gammon, Marilie D.; Kaufman, Jay S.; Bensen, Jeannette T.; Lin, Dan Yu; Barnholtz-Sloan, Jill S.; Hu, Yijuan; He, Qianchuan; Luo, Jingchun; Millikan, Robert C.

    2012-01-01

    Adipocytokines are produced by visceral fat, and levels may be associated with breast cancer risk. We investigated whether single nucleotide polymorphisms (SNPs) in adipocytokine genes adiponectin (ADIPOQ), leptin (LEP), and the leptin receptor (LEPR) were associated with basal-like or luminal A breast cancer subtypes. 104 candidate and tag SNPs were genotyped in 1776 of 2022 controls and 1972 (200 basal-like, 679 luminal A) of 2311 cases from the Carolina Breast Cancer Study (CBCS), a population-based case–control study of whites and African Americans. Breast cancer molecular subtypes were determined by immunohistochemistry. Genotype odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. Haplotype ORs and 95% CIs were estimated using Hapstat. Interactions with waist-hip ratio were evaluated using a multiplicative interaction term. Ancestry was estimated from 144 ancestry informative markers (AIMs), and included in models to control for population stratification. Candidate SNPs LEPR K109R (rs1137100) and LEPR Q223R (rs1137101) were positively associated with luminal A breast cancer, whereas ADIPOQ +45 T/G (rs2241766), ADIPOQ +276 G/T (rs1501299), and LEPR K656N (rs8129183) were not associated with either subtype. Few patterns were observed among tag SNPs, with the exception of 3 LEPR SNPs (rs17412175, rs9436746, and rs9436748) that were in moderate LD and inversely associated with basal-like breast cancer. However, no SNP associations were statistically significant after adjustment for multiple comparisons. Haplotypes in LEP and LEPR were associated with both basal-like and luminal A subtypes. There was no evidence of interaction with waist-hip ratio. Data suggest associations between LEPR candidate SNPs and luminal A breast cancer in the CBCS and LEPR intron 2 tag SNPs and basal-like breast cancer. Replication in additional studies where breast cancer subtypes have been defined is necessary to confirm these

  14. Molecular subtype profiling of invasive breast cancers weakly positive for estrogen receptor.

    PubMed

    Sheffield, Brandon S; Kos, Zuzana; Asleh-Aburaya, Karama; Wang, Xiu Qing; Leung, Samuel; Gao, Dongxia; Won, Jennifer; Chow, Christine; Rachamadugu, Rakesh; Stijleman, Inge; Wolber, Robert; Gilks, C Blake; Myles, Nickolas; Thomson, Tom; Hayes, Malcolm M; Bernard, Philip S; Nielsen, Torsten O; Chia, Stephen K L

    2016-02-01

    The estrogen receptor (ER) is a key predictive biomarker in the treatment of breast cancer. There is uncertainty regarding the use of hormonal therapy in the setting of weakly positive ER by immunohistochemistry (IHC). We report intrinsic subtype classification on a cohort of ER weakly positive early-stage breast cancers. Consecutive cases of breast cancer treated by primary surgical resection were retrospectively identified from 4 centers that engage in routine external proficiency testing for breast biomarkers. ER-negative (Allred 0 and 2) and ER weakly positive (Allred 3-5) cases were included. Gene expression profiling was performed using qRT-PCR. Intrinsic subtype prediction was made based upon the PAM50 gene expression signature. 148 cases were included in the series: 60 cases originally diagnosed as ER weakly positive and 88 ER negative. Of the cases originally assessed as ER weakly positive, only 6 (10 %) were confirmed to be of luminal subtype by gene expression profiling; the remaining 90 % of cases were classified as basal-like or HER2-enriched subtypes. This was not significantly different than the fraction of luminal cases identified in the IHC ER-negative cohort (5 (5 %) luminal, 83(95 %) non-luminal). Recurrence-free, and overall, survival rates were similar in both groups (p = 0.4 and 0.5, respectively) despite adjuvant hormonal therapy prescribed in the majority (59 %) of weakly positive ER cases. Weak ER expression by IHC is a poor correlate of luminal subtype in invasive breast cancer. In the setting of highly sensitive and robust IHC methodology, cutoffs for ER status determination and subsequent systemic therapy should be revisited. PMID:26846986

  15. Alterations of muscarinic receptor subtypes in pathways relating to memory: Effects of lesions and transplants

    SciTech Connect

    Dawson, V.L.

    1989-01-01

    Muscarinic cholinergic receptors have been classified pharmacologically into two distinct populations designated muscarinic type-one (M-1) and mscarinic type-two (M-2). The semiquantitative technique of receptor autoradiography was used to examine the anatomical and cellular distribution, and densities of M-1 and M-2 receptors in the rate brain. Muscarinic receptors were labeled with the classical antagonist ({sup 3}H)quinuclidinyl benzilate (QNB). Differentiation of the muscarinic subtypes was accomplished by competition studies of ({sup 3}H)QNB against the relatively selective M-1 antagonist pirenzepine (PZ), and the relatively selective M-2 antagonist, AFDX-116. In addition, M-1 and M-2 receptors were directly labeled with ({sup 3}H)PZ and ({sup 3}H)AFDX-116, respectively. Cholinergic pathways from the large cholinergic neurons in the nucleus basalis magnocellularis (NBM) to the cortex and from the medial septum (MS) to the hippocampus were examined by lesioning with the selective cholinergic neurotoxin, AF64A. Bilateral cerebral cortical infarction was performed in order to analyze potential changes in muscarinic receptor populations in subcortical structures that are sensitive to cortical infarction. Finally, the response of muscarinic receptors to fetal septodiagonal band transplants in the deafferentated hippocampus was examined.

  16. The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

    PubMed

    Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V

    2012-09-01

    The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

  17. Quantitative method of analyzing the interaction of slightly selective radioligands with multiple receptor subtypes

    SciTech Connect

    McGonigle, P.; Neve, K.A.; Molinoff, P.B.

    1986-10-01

    Subclasses of receptors exist for most neurotransmitters. Frequently, two subtypes of receptors coexist in the same tissue and, in some cases, they mediate the same physiological response. In tissues with two classes of binding sites for a given hormone, an estimate of the proportion of each class of binding sites is obtained by inhibiting the binding of a single concentration of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of receptors will only be obtained, however, if the radioligand is entirely nonselective. Selectivity of just 2- to 3-fold can markedly influence the results of subtype analysis. The conclusion that a radioligand is nonselective is usually based on the results of a saturation binding curve. If Scatchard analysis results in a linear plot, the radioligand is nonselective. Scatchard analysis cannot distinguish between a radioligand that is nonselective and one that is slightly selective. The use of a slightly selective radioligand can lead to errors of 50% or more, depending on the concentration of the radioligand relative to the Kd values of the two classes of sites. A new method has been developed that can be used to quantitate 2- to 3-fold differences in the affinity of two distinct classes of binding sites for a radioligand. This approach requires that a series of inhibition experiments with a selective unlabeled ligand be performed in the presence of increasing concentrations of the radioligand. Analysis of the resulting inhibition curves, utilizing the mathematical modeling program MLAB on the PROPHET system, yields accurate estimates of the density of each class of receptor as well as the affinity of each receptor for the labeled and unlabeled ligands. This approach was used to determine whether /sup 125/I-iodopindolol shows selectivity for beta 1- or beta 2-adrenergic receptors.

  18. α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration.

    PubMed

    Marks, Michael J; Grady, Sharon R; Salminen, Outi; Paley, Miranda A; Wageman, Charles R; McIntosh, J Michael; Whiteaker, Paul

    2014-07-01

    Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where *indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*-nAChR are down-regulated following chronic nicotine exposure (unlike other subtypes that have been investigated - most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose-responses and quantitative ligand-binding autoradiography were used to define nicotine sensitivity of changes in α4β2*-nAChR and α6β2*-nAChR expression. α6β2*-nAChR down-regulation by chronic nicotine exposure in dopaminergic and optic-tract nuclei was ≈three-fold more sensitive than up-regulation of α4β2*-nAChR. In contrast, nAChR-mediated [(3) H]-dopamine release from dopamine-terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, whereas dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR-mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [(3) H]-DA release are primarily owing to changes in nAChR, rather than in dopaminergic, function. This study examined dose-response relationships for murine α6β2*-nicotinic acetylcholine receptor (nAChR) down-regulation by chronic nicotine treatment. The ID50 value for α6β2* down-regulation (35 nM) is ≈ 3x lower than the ED50 value for α4β2* nAChR up-regulation (95 nM), both well within the range reached by human smokers. Chronic nicotine treatment altered α6β2*- and α4

  19. Antihypertensive effects of selective prostaglandin E2 receptor subtype 1 targeting

    PubMed Central

    Guan, Youfei; Zhang, Yahua; Wu, Jing; Qi, Zhonghua; Yang, Guangrui; Dou, Dou; Gao, Yuansheng; Chen, Lihong; Zhang, Xiaoyan; Davis, Linda S.; Wei, Mingfeng; Fan, Xuefeng; Carmosino, Monica; Hao, Chuanming; Imig, John D.; Breyer, Richard M.; Breyer, Matthew D.

    2007-01-01

    Clinical use of prostaglandin synthase–inhibiting NSAIDs is associated with the development of hypertension; however, the cardiovascular effects of antagonists for individual prostaglandin receptors remain uncharacterized. The present studies were aimed at elucidating the role of prostaglandin E2 (PGE2) E-prostanoid receptor subtype 1 (EP1) in regulating blood pressure. Oral administration of the EP1 receptor antagonist SC51322 reduced blood pressure in spontaneously hypertensive rats. To define whether this antihypertensive effect was caused by EP1 receptor inhibition, an EP1-null mouse was generated using a “hit-and-run” strategy that disrupted the gene encoding EP1 but spared expression of protein kinase N (PKN) encoded at the EP1 locus on the antiparallel DNA strand. Selective genetic disruption of the EP1 receptor blunted the acute pressor response to Ang II and reduced chronic Ang II–driven hypertension. SC51322 blunted the constricting effect of Ang II on in vitro–perfused preglomerular renal arterioles and mesenteric arteriolar rings. Similarly, the pressor response to EP1-selective agonists sulprostone and 17-phenyltrinor PGE2 were blunted by SC51322 and in EP1-null mice. These data support the possibility of targeting the EP1 receptor for antihypertensive therapy. PMID:17710229

  20. Type 1 angiotensin II receptor subtypes in kidney of normal and salt-sensitive hypertensive rats.

    PubMed

    Bouby, N; Bankir, L; Llorens-Cortes, C

    1996-03-01

    We studied the localization and regulation of the two type 1 angiotensin II receptor subtypes AT(1A) and AT(1B) in different renal zones of the rat kidney by a reverse transcription-polymerase chain reaction amplification method. The yield of the reaction was quantified with an internal standard that was a 63-bp deleted mutant cRNA of the AT(1A) receptor. In kidneys of male Sprague-Dawley rats (n=4), the levels of AT(1A) and AT(1B) receptor mRNAs were highest in the inner stripe of the outer medulla, lowest in the inner medulla, and intermediate in the cortex and outer stripe of the outer medulla. Results (mean+/-SE) expressed in 10(5) molecules per microgram total RNA were for cortex outer stripe, inner stripe, and inner medulla, respectively, 171 +/- 15, 152 +/- 27, 322 +/- 10, and 73 +/- 3 for AT(1A), and 35 +/- 9, 26 +/- 1, 71 +/- 10, and 53 +/- 11 for AT(1B). In sabra rats sensitive (n=6) or resistant (n=6) to salt-induced hypertension and maintained on a normal salt diet, the percentage and level of each receptor subtype mRNA in cortex and outer stripe were similar in the two strains and comparable to those observed in Sprague-Dawley rats. However, AT(1A) of the inner stripe was significantly decreased in salt-resistant compared with salt-sensitive rats (166 +/- 28 and 318 +/- 58 10(5) molecules per microgram total RNA, respectively). These modifications were organ specific because no difference in the level of the receptor mRNAs was observed in the liver of the two Sabra rat strains, whereas a twofold increase in AT(1A) mRNA level but not in AT(1B) mRNA level was apparent in adrenal and in one renal zone, the inner stripe of the outer medulla, of hypertension-prone Sabra rats.

  1. Estrogen receptor subtypes selectively mediate female mouse reproductive abnormalities induced by neonatal exposure to estrogenic chemicals.

    PubMed

    Nakamura, Takeshi; Katsu, Yoshinao; Watanabe, Hajime; Iguchi, Taisen

    2008-11-20

    Perinatal exposure to estrogens such as diethylstilbestrol (DES), and to estrogenic chemicals, induces persistent anovulation caused by alteration of hypothalamic-pituitary-gonadal (HPG) axis, polyovular follicles, uterine abnormalities and persistent vaginal changes in mice. Most activities of estrogenic chemicals are mediated through estrogen receptor alpha (ERalpha) and/or ERbeta. However, little was known about the relative contribution of the individual ER subtypes in induction of abnormalities. We tested the effects of neonatal exposure to ER selective ligands and DES on female mice. Transactivation assays using mouse ERalpha and ERbeta showed that 10(-10)M DES activated both ER subtypes and that the ERalpha agonist (propyl pyrazole triol, PPT) and the ERbeta agonist (diarylpropionitrile, DPN) selectively activated their respective ERs at 10(-9)M. Neonatal female mice were injected subcutaneously with DES, PPT or DPN and the animals were examined at 13 and 15 weeks of age, respectively. Persistent estrous smears and anovulation were induced in all mice by 0.025-2.5 microg DES and 2.5-25 microg PPT, but not by DPN, suggesting that the observed anovulation was primarily mediated through ERalpha. Disorganization of uterine musculature and ovary-independent vaginal epithelial cell proliferation accompanied by persistent expression of EGF-related genes and interleukin-1-related genes were also mediated through ERalpha. In contrast, polyovular follicles were induced by neonatal treatment with both ERalpha and ERbeta ligands, suggesting that ovarian abnormalities are mediated through both ER subtypes.

  2. Structure-Based Evolution of Subtype-Selective Neurotensin Receptor Ligands

    PubMed Central

    Schaab, Carolin; Kling, Ralf Christian; Einsiedel, Jürgen; Hübner, Harald; Clark, Tim; Seebach, Dieter; Gmeiner, Peter

    2014-01-01

    Subtype-selective agonists of the neurotensin receptor NTS2 represent a promising option for the treatment of neuropathic pain, as NTS2 is involved in the mediation of μ-opioid-independent anti-nociceptive effects. Based on the crystal structure of the subtype NTS1 and previous structure–activity relationships (SARs) indicating a potential role for the sub-pocket around Tyr11 of NT(8–13) in subtype-specific ligand recognition, we have developed new NTS2-selective ligands. Starting from NT(8–13), we replaced the tyrosine unit by β2-amino acids (type 1), by heterocyclic tyrosine bioisosteres (type 2) and peptoid analogues (type 3). We were able to evolve an asymmetric synthesis of a 5-substituted azaindolylalanine and its application as a bioisostere of tyrosine capable of enhancing NTS2 selectivity. The S-configured test compound 2 a, [(S)-3-(pyrazolo[1,5-a]pyridine-5-yl)-propionyl11]NT(8–13), exhibits substantial NTS2 affinity (4.8 nm) and has a nearly 30-fold NTS2 selectivity over NTS1. The (R)-epimer 2 b showed lower NTS2 affinity but more than 600-fold selectivity over NTS1. PMID:25478316

  3. Structure-based evolution of subtype-selective neurotensin receptor ligands.

    PubMed

    Schaab, Carolin; Kling, Ralf Christian; Einsiedel, Jürgen; Hübner, Harald; Clark, Tim; Seebach, Dieter; Gmeiner, Peter

    2014-10-01

    Subtype-selective agonists of the neurotensin receptor NTS2 represent a promising option for the treatment of neuropathic pain, as NTS2 is involved in the mediation of μ-opioid-independent anti-nociceptive effects. Based on the crystal structure of the subtype NTS1 and previous structure-activity relationships (SARs) indicating a potential role for the sub-pocket around Tyr11 of NT(8-13) in subtype-specific ligand recognition, we have developed new NTS2-selective ligands. Starting from NT(8-13), we replaced the tyrosine unit by β(2)-amino acids (type 1), by heterocyclic tyrosine bioisosteres (type 2) and peptoid analogues (type 3). We were able to evolve an asymmetric synthesis of a 5-substituted azaindolylalanine and its application as a bioisostere of tyrosine capable of enhancing NTS2 selectivity. The S-configured test compound 2 a, [(S)-3-(pyrazolo[1,5-a]pyridine-5-yl)-propionyl(11)]NT(8-13), exhibits substantial NTS2 affinity (4.8 nm) and has a nearly 30-fold NTS2 selectivity over NTS1. The (R)-epimer 2 b showed lower NTS2 affinity but more than 600-fold selectivity over NTS1. PMID:25478316

  4. Alpha1-adrenoreceptor in human hippocampus: binding and receptor subtype mRNA expression.

    PubMed

    Szot, Patricia; White, Sylvia S; Greenup, J Lynne; Leverenz, James B; Peskind, Elaine R; Raskind, Murray A

    2005-10-01

    Alpha1-adrenoreceptors (AR), of which three subtypes exist (alpha1A-, alpha1B- and alpha1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, alpha1-ARs are found in the hippocampus where animal studies have shown the ability of alpha1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of alpha1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (alpha1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogeneously. Human alpha1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while alpha1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. alpha1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal alpha1-AR localization between rat and humans and further describe a difference in the localization of the alpha1A- and alpha1D-AR mRNA subtype between rats and humans. PMID:16039007

  5. Quantification of beta adrenergic receptor subtypes in beta-arrestin knockout mouse airways.

    PubMed

    Hegde, Akhil; Strachan, Ryan T; Walker, Julia K L

    2015-01-01

    In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference

  6. Expression of the Somatostatin Receptor Subtype 4 in Intact and Inflamed Pulmonary Tissues

    PubMed Central

    Varecza, Zoltán; Elekes, Krisztián; László, Terézia; Perkecz, Anikó; Pintér, Erika; Sándor, Zoltán; Szolcsányi, János; Keszthelyi, Dániel; Szabó, Árpád; Sándor, Katalin; Molnár, Tamás F.; Szántó, Zalán; Pongrácz, Judit E.; Helyes, Zsuzsanna

    2009-01-01

    Somatostatin released from capsaicin-sensitive sensory nerves of the lung during endotoxin-induced murine pneumonitis inhibits inflammation and hyperresponsiveness, presumably via somatostatin receptor subtype 4 (sst4). The goal of the present study was to identify sst4 receptors in mouse and human lungs and to reveal its inflammation-induced alterations with real-time quantitative PCR, Western blot, and immunohistochemistry. In non-inflamed mouse and human lungs, mRNA expression and immunolocalization of sst4 are very similar. They are present on bronchial epithelial, vascular endothelial, and smooth-muscle cells. The sst4 receptor protein in the mouse lung significantly increases 24 hr after intranasal endotoxin administration as well as in response to 3 months of whole-body cigarette smoke exposure, owing to the infiltrating sst4-positivite mononuclear cells and neutrophils. In the chronically inflamed human lung, the large number of activated macrophages markedly elevate sst4 mRNA levels, although there is no change in acute purulent pneumonia, in which granulocytes accumulate. Despite mouse granulocytes, human neutrophils do not show sst4 immunopositivity. We provide the first evidence for the expression, localization, and inflammation-induced alterations of sst4 receptors in murine and human lungs. Inasmuch as tissue distribution of this receptor is highly similar, extrapolation of murine experimental results to human conditions might be possible. (J Histochem Cytochem 57:1127–1137, 2009) PMID:19687471

  7. Oestradiol-induced synapse formation in the female hippocampus: roles of oestrogen receptor subtypes.

    PubMed

    Zhou, L; Fester, L; Haghshenas, S; de Vrese, X; von Hacht, R; Gloger, S; Brandt, N; Bader, M; Vollmer, G; Rune, G M

    2014-07-01

    During the oestrus cycle, varying spine synapse density correlates positively with varying local synthesis of oestradiol in the hippocampus. In this context, the roles of the oestrogen receptor (ER) subtypes ERα and β are not fully understood. In the present study, we used neonatal hippocampal slice cultures from female rats because these cultures synthesise oestradiol and express both receptor subtypes, and inhibition of oestradiol synthesis in these cultures results in spine synapse loss. Using electron microscopy, we tested the effects on spine synapse density in response to agonists of both ERα and ERβ. Application of agonists to the cultures had no effect. After inhibition of oestradiol synthesis, however, agonists of ERα induced spine synapse formation, whereas ERβ agonists led to a reduction in spine synapse density in the CA1 region of these cultures. Consistently, up-regulation of ERβ in the hippocampus of adult female aromatase-deficient mice is paralleled by hippocampus-specific spine synapse loss in this mutant. Finally, we found an increase in spine synapses in the adult female ERβ knockout mouse, but no effect in the adult female ERα knockout mouse. Our data suggest antagonistic roles of ERβ and ERα in spine synapse formation in the female hippocampus, which may contribute to oestrus cyclicity of spine synapse density in the hippocampus.

  8. Iterative experimental and virtual high-throughput screening identifies metabotropic glutamate receptor subtype 4 positive allosteric modulators

    PubMed Central

    Mueller, Ralf; Dawson, Eric S.; Niswender, Colleen M.; Butkiewicz, Mariusz; Hopkins, Corey R.; Weaver, C. David; Lindsley, Craig W.; Conn, P. Jeffrey; Meiler, Jens

    2013-01-01

    Activation of metabotropic glutamate receptor subtype 4 has been shown to be efficacious in rodent models of Parkinson’s disease. Artificial neural networks were trained based on a recently reported high throughput screen which identified 434 positive allosteric modulators of metabotropic glutamate receptor subtype 4 out of a set of approximately 155,000 compounds. A jury system containing three artificial neural networks achieved a theoretical enrichment of 15.4 when selecting the top 2% compounds of an independent test dataset. The model was used to screen an external commercial database of approximately 450,000 drug-like compounds. 1,100 predicted active small molecules were tested experimentally using two distinct assays of mGlu4 activity. This experiment yielded 67 positive allosteric modulators of metabotropic glutamate receptor subtype 4 that confirmed in both experimental systems. Compared to the 0.3% active compounds in the primary screen, this constituted an enrichment of 22 fold. PMID:22592386

  9. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  10. Multiple microvascular and astroglial 5-hydroxytryptamine receptor subtypes in human brain: molecular and pharmacologic characterization.

    PubMed

    Cohen, Z; Bouchelet, I; Olivier, A; Villemure, J G; Ball, R; Stanimirovic, D B; Hamel, E

    1999-08-01

    Physiologic and anatomic evidence suggest that 5-hydroxytryptamine (5-HT) neurons regulate local cerebral blood flow and blood-brain barrier permeability. To evaluate the possibility that some of these effects occur directly on the blood vessels, molecular and/or pharmacologic approaches were used to assess the presence of 5-HT receptors in human brain microvascular fractions, endothelial and smooth muscle cell cultures, as well as in astroglial cells which intimately associate with intraparenchymal blood vessels. Isolated microvessels and capillaries consistently expressed messages for the h5-HT1B, h5-HT1D, 5-HT1F, 5-HT2A but not 5-HT7 receptors. When their distribution within the vessel wall was studied in more detail, it was found that capillary endothelial cells exhibited mRNA for the h5-HT1D and for the 5-HT7 receptors whereas microvascular smooth muscle cells, in addition to h5-HT1D and 5-HT7, also showed polymerase chain reaction products for h5-HT1B receptors. Expression of 5-HT1F and 5-HT2A receptor mRNAs was never detected in any of the microvascular cell cultures. In contrast, messages for all 5-HT receptors tested were detected in human brain astrocytes with a predominance of the 5-HT2A and 5-HT7 subtypes. In all cultures, sumatriptan inhibited (35-58%, P < .05) the forskolin-stimulated production of cyclic AMP, an effect blocked by the 5-HT1B/1D receptor antagonists GR127935 and GR55562. In contrast, 5-carboxamidotryptamine induced strong increases (> or = 400%, P < .005) in basal cyclic AMP levels that were abolished by mesulergine, a nonselective 5-HT7 receptor antagonist. Only astroglial cells showed a ketanserin-sensitive increase (177%, P < .05) in IP3 formation when exposed to 5-HT. These results show that specific populations of functional 5-HT receptors are differentially distributed within the various cellular compartments of the human cortical microvascular bed, and that human brain astroglial cells are endowed with multiple 5-HT receptors

  11. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

    SciTech Connect

    Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L.

    2008-10-27

    NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

  12. Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

    PubMed

    Forkuo, Gloria S; Guthrie, Margaret L; Yuan, Nina Y; Nieman, Amanda N; Kodali, Revathi; Jahan, Rajwana; Stephen, Michael R; Yocum, Gene T; Treven, Marco; Poe, Michael M; Li, Guanguan; Yu, Olivia B; Hartzler, Benjamin D; Zahn, Nicolas M; Ernst, Margot; Emala, Charles W; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

    2016-06-01

    Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.

  13. Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments.

    PubMed

    Forkuo, Gloria S; Guthrie, Margaret L; Yuan, Nina Y; Nieman, Amanda N; Kodali, Revathi; Jahan, Rajwana; Stephen, Michael R; Yocum, Gene T; Treven, Marco; Poe, Michael M; Li, Guanguan; Yu, Olivia B; Hartzler, Benjamin D; Zahn, Nicolas M; Ernst, Margot; Emala, Charles W; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

    2016-06-01

    Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects. PMID:27120014

  14. Activation of extrasynaptic NMDA receptors induces a PKC-dependent switch in AMPA receptor subtypes in mouse cerebellar stellate cells.

    PubMed

    Sun, Lu; June Liu, Siqiong

    2007-09-01

    The repetitive activation of synaptic glutamate receptors can induce a lasting change in the number or subunit composition of synaptic AMPA receptors (AMPARs). However, NMDA receptors that are present extrasynaptically can also be activated by a burst of presynaptic activity, and thus may be involved in the induction of synaptic plasticity. Here we show that the physiological-like activation of extrasynaptic NMDARs induces a lasting change in the synaptic current, by changing the subunit composition of AMPARs at the parallel fibre-to-cerebellar stellate cell synapse. This extrasynaptic NMDAR-induced switch in synaptic AMPARs from GluR2-lacking (Ca(2+)-permeable) to GluR2-containing (Ca(2+)-impermeable) receptors requires the activation of protein kinase C (PKC). These results indicate that the activation of extrasynaptic NMDARs by glutamate spillover is an important mechanism that detects the pattern of afferent activity and subsequently exerts a remote regulation of AMPAR subtypes at the synapse via a PKC-dependent pathway.

  15. Multiple receptor subtypes mediate the effects of serotonin on rat subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Scrogin, K. E.; Johnson, A. K.; Schmid, H. A.

    1998-01-01

    The subfornical organ (SFO) receives significant serotonergic innervation. However, few reports have examined the functional effects of serotonin on SFO neurons. This study characterized the effects of serotonin on spontaneously firing SFO neurons in the rat brain slice. Of 31 neurons tested, 80% responded to serotonin (1-100 microM) with either an increase (n = 15) or decrease (n = 10) in spontaneous activity. Responses to serotonin were dose dependent and persisted after synaptic blockade. Excitatory responses could also be mimicked by the 5-hydroxytryptamine (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; 1-10 microM) and could be blocked by the 5-HT2A/2C-receptor antagonist LY-53,857 (10 microM). LY-53,857 unmasked inhibitory responses to serotonin in 56% of serotonin-excited cells tested. Serotonin-inhibited cells were also inhibited by the 5-HT1A-receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT; 1-10 microM; n = 7). The data indicate that SFO neurons are responsive to serotonin via postsynaptic activation of multiple receptor subtypes. The results suggest that excitatory responses to serotonin are mediated by 5-HT2A or 5-HT2C receptors and that inhibitory responses may be mediated by 5-HT1A receptors. In addition, similar percentages of serotonin-excited and -inhibited cells were also sensitive to ANG II. As such the functional relationship between serotonin and ANG II in the SFO remains unclear.

  16. Lynx1 and Aβ1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes.

    PubMed

    Thomsen, Morten S; Arvaniti, Maria; Jensen, Majbrit M; Shulepko, Mikhail A; Dolgikh, Dmitry A; Pinborg, Lars H; Härtig, Wolfgang; Lyukmanova, Ekaterina N; Mikkelsen, Jens D

    2016-10-01

    Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, β2, and β4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease. PMID:27460145

  17. Effect of estrogen receptor-subtype-specific ligands on fertility in adult male rats.

    PubMed

    Dumasia, Kushaan; Kumar, Anita; Kadam, Leena; Balasinor, N H

    2015-06-01

    Maintenance of normal male fertility relies on the process of spermatogenesis which is under complex endocrine control by mechanisms involving gonadotropin and steroid hormones. Although testosterone is the primary sex steroid in males, estrogen is locally produced in the testis and plays a very crucial role in male fertility. This is evident from presence of both the estrogen receptors alpha (ERα) and beta (ERβ) in the testis and their absence, as in the case of knockout mice models, leads to sterility. The present study was undertaken to understand individual roles of the two ERs in spermatogenesis and their direct contribution towards the maintenance of male fertility using receptor-subtype-specific ligands. Administration of ERα and β agonists to adult male rats for 60 days results in a significant decrease in fertility, mainly due to an increase in pre- and post-implantation loss and a concomitant decrease in litter size and sperm counts. Our results indicate that ERα is mainly involved in negative feedback regulation of gonadotropin hormones, whereas both ERs are involved in regulation of prolactin and testosterone production. Histological examinations of the testis reveal that ERβ could be involved in the process of spermiation since many failed spermatids were observed in stages IX-XI following ERβ agonist treatment. Our results indicate that overactivation of estrogen signaling through either of its receptors can have detrimental effects on the fertility parameters and that the two ERs have both overlapping and distinct roles in maintenance of male fertility.

  18. Effect of estrogen receptor-subtype-specific ligands on fertility in adult male rats.

    PubMed

    Dumasia, Kushaan; Kumar, Anita; Kadam, Leena; Balasinor, N H

    2015-06-01

    Maintenance of normal male fertility relies on the process of spermatogenesis which is under complex endocrine control by mechanisms involving gonadotropin and steroid hormones. Although testosterone is the primary sex steroid in males, estrogen is locally produced in the testis and plays a very crucial role in male fertility. This is evident from presence of both the estrogen receptors alpha (ERα) and beta (ERβ) in the testis and their absence, as in the case of knockout mice models, leads to sterility. The present study was undertaken to understand individual roles of the two ERs in spermatogenesis and their direct contribution towards the maintenance of male fertility using receptor-subtype-specific ligands. Administration of ERα and β agonists to adult male rats for 60 days results in a significant decrease in fertility, mainly due to an increase in pre- and post-implantation loss and a concomitant decrease in litter size and sperm counts. Our results indicate that ERα is mainly involved in negative feedback regulation of gonadotropin hormones, whereas both ERs are involved in regulation of prolactin and testosterone production. Histological examinations of the testis reveal that ERβ could be involved in the process of spermiation since many failed spermatids were observed in stages IX-XI following ERβ agonist treatment. Our results indicate that overactivation of estrogen signaling through either of its receptors can have detrimental effects on the fertility parameters and that the two ERs have both overlapping and distinct roles in maintenance of male fertility. PMID:25869617

  19. Subtype selective NMDA receptor antagonists induce recovery of synapses lost following exposure to HIV-1 Tat

    PubMed Central

    Shin, AH; Kim, HJ; Thayer, SA

    2012-01-01

    BACKGROUND AND PURPOSE Neurocognitive disorders afflict approximately 20% of HIV-infected patients. HIV-1-infected cells in the brain shed viral proteins such as transactivator of transcription (Tat). Tat elicits cell death and synapse loss via processes initiated by NMDA receptor activation but mediated by separate downstream signalling pathways. Subunit selective NMDA receptor antagonists may differentially modulate survival relative to synaptic changes. EXPERIMENTAL APPROACH Tat-evoked cell death was quantified by measuring propidium iodide uptake into rat hippocampal neurons in culture. The effects of Tat on synaptic changes were measured using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein. KEY RESULTS Dizocilpine, a non-competitive NMDA receptor antagonist, inhibited Tat-induced synapse loss, subsequent synapse recovery and Tat-induced cell death with comparable potencies. Memantine (10 µM) and ifenprodil (10 µM), which preferentially inhibit GluN2B-containing NMDA receptors, protected from Tat-induced cell death with no effect on synapse loss. Surprisingly, memantine and ifenprodil induced synapse recovery in the presence of Tat. In contrast, the GluN2A-prefering antagonist TCN201 prevented synapse loss and recovery with no effect on cell death. CONCLUSIONS AND IMPLICATIONS Synapse loss is a protective mechanism that enables the cell to cope with excess excitatory input. Thus, memantine and ifenprodil are promising neuroprotective drugs because they spare synaptic changes and promote survival. These GluN2B-preferring drugs induced recovery from Tat-evoked synapse loss, suggesting that synaptic pharmacology changed during the neurotoxic process. NMDA receptor subtypes differentially participate in the adaptation and death induced by excitotoxic insult. PMID:22142193

  20. Differentiation of muscarinic cholinergic receptor subtypes in human cortex and pons - Implications for anti-motion sickness therapy

    NASA Technical Reports Server (NTRS)

    Mccarthy, Bruce G.; Peroutka, Stephen J.

    1988-01-01

    Radioligand binding studies were used to analyze muscarinic cholinergic receptor subtypes in human cortex and pons. Muscarinic cholinergic receptors were labeled by H-3-quinuclidinyl benzilate (H-3-QNB). Scopolamine was equipotent in both brain regions and did not discriminate subtypes of H-3-QNB binding. By contrast, the M1 selective antagonist pirenzepine was approximately 33-fold more potent in human cortex than pons. Carbachol, a putative M2 selective agonist, was more than 100-fold more potent in human pons than cortex. These results demonstrate that the human pons contains a relatively large proportion of carbachol-sensitive muscarinic cholinergic receptors. Drugs targeted to this subpopulation of muscarinic cholinergic receptors may prove to be effective anti-motion sickness agents with less side effects than scopolamine.

  1. Receptor-Binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

    PubMed Central

    Gambaryan, Alexandra S.; Matrosovich, Tatyana Y.; Philipp, Jennifer; Munster, Vincent J.; Fouchier, Ron A. M.; Cattoli, Giovanni; Capua, Ilaria; Krauss, Scott L.; Webster, Robert G.; Banks, Jill; Bovin, Nicolai V.; Klenk, Hans-Dieter

    2012-01-01

    Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO3)GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO3)GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry. PMID:22345462

  2. Classification of M/sub 1/ and M/sub 2/ receptor subtypes in vivo by autoradiography using (/sup 125/I) (R,R) 4IQNB: Implications for imaging receptor subtypes

    SciTech Connect

    Gibson, R.E.; Moody, T.; Kzeszotarski, W.J.; Schneidau, T.S.; Jagoda, E.M.; Reba, R.C.

    1985-05-01

    (/sup 125/I) (R,R) 3-Quinuclidinyl 4-Iodobenzilate (4IQNB) is a high affinity radiotracer for the muscarinic acetylcholine receptor which exhibits differential kinetics of dissociation from the receptor subtypes, M/sub 1/ and M/sub 2/. The authors have determined the relative percentages of M/sub 1/ to M/sub 2/-receptor subtype in six structures of rat brain by equilibrium competition using the selective antagonist, QNX, and by analysis of the off-rate profiles for 4IQNB. The results are comparable and provide: (% M/sub 1/) caudate nucleus - 100%, hippocampus - 92%, cortex - 82%, thalamus - 6%, superior + inferior colliculi - 41%, and pons - 23%. To determine the relative proportions of M/sub 1/ to M/sub 2/ receptors in vivo we examined the distribution of 4IQNB at 2 h and 24 h by autoradiography. At 2 h, both M/sub 1/ and M/sub 2/ receptors will be labeled but at 24 h only the M/sub 1/ receptor will retain radiotracer. At 2 h, all structures of the brain are variably labeled with the cortex, hippocampus, caudate nucleus, olfactory nuclei, nucleus accumbens, pontine nuclei, and anteroventral thalamic nucleus (AV) most heavily labeled. At 24 h, both the pontine and AV, as well as the less heavily labeled hypothalamus, superior colliculus and mesencephalic nuclei, are devoid of radiotracer thus indicating predominantly M/sub 2/ receptor. Quantitation is necessary to determine possible washout of activity from the M/sub 2/ receptors in cortex. Similar time studies in man should provide distinctions between the M/sub 1/ and M/sub 2/ receptor rich structures and the preferential loss of a subtype of receptor due to disease.

  3. Effects of nucleotides on [3H]bradykinin binding in guinea pig: further evidence for multiple B2 receptor subtypes.

    PubMed

    Seguin, L; Widdowson, P S

    1993-02-01

    We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 microM) and the nonmetabolized analogue of GTP, guanyl-5'-yl-imidodiphosphate (GppNHp; 100 microM). Competition studies with bradykinin and with [Hyp3]bradykinin, which shows approximately 20-fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 microM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 microM) and GDP (100 microM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 microM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Interaction of psychoactive tryptamines with biogenic amine transporters and serotonin receptor subtypes

    PubMed Central

    Blough, Bruce E.; Landavazo, Antonio; Decker, Ann M.; Partilla, John S.; Baumann, Michael H.; Rothman, Richard B.

    2014-01-01

    Rationale Synthetic hallucinogenic tryptamines, especially those originally described by Alexander Shulgin, continue to be abused in the United States. The range of subjective experiences produced by different tryptamines suggests that multiple neurochemical mechanisms are involved in their actions, in addition to the established role of agonist activity at serotonin-2A (5-HT2A) receptors. Objectives This study evaluated the interaction of a series of synthetic tryptamines with biogenic amine neurotransmitter transporters and with serotonin (5-HT) receptor subtypes implicated in psychedelic effects. Methods Neurotransmitter transporter activity was determined in rat brain synaptosomes. Receptor activity was determined using calcium mobilization and DiscoveRx PathHunter® assays in HEK293, Gα16-CHO, and CHOk1 cells transfected with human receptors. Results Twenty-one tryptamines were analyzed in transporter uptake and release assays, and 5-HT2A, serotonin 1A (5-HT1A), and 5-HT2A β-arrestin functional assays. Eight of the compounds were found to have 5-HT-releasing activity. Thirteen compounds were found to be 5-HT uptake inhibitors or were inactive. All tryptamines were 5-HT2A agonists with a range of potencies and efficacies, but only a few compounds were 5-HT1A agonists. Most tryptamines recruited β-arrestin through 5-HT2A activation. Conclusions All psychoactive tryptamines are 5-HT2A agonists, but 5-HT transporter (SERT) activity may contribute significantly to the pharmacology of certain compounds. The in vitro transporter data confirm structure-activity trends for releasers and uptake inhibitors whereby releasers tend to be structurally smaller compounds. Interestingly, two tertiary amines were found to be selective substrates at SERT, which dispels the notion that 5-HT-releasing activity is limited only to primary or secondary amines. PMID:24800892

  5. Cloning and expression of a human kidney cDNA for an /alpha//sub 2/-adrenergic receptor subtype

    SciTech Connect

    Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1988-09-01

    An /alpha//sub 2/-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet /alpha//sub 2/-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet /alpha//sub 2/-adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the /alpha//sub 2/-adrenergic ligand (/sup 3/H)rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the /alpha//sub 2/B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet /alpha//sub 2/-adrenergic receptor (/alpha//sub 2/A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective /alpha/-adrenergic ligands.

  6. Cloning, expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper, Epinephelus coioides.

    PubMed

    Wang, Fei; Chen, Weimin; Lin, Haoran; Li, Wensheng

    2014-12-01

    As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp(6.59) might be a common NPY-binding site, while Asp(2.68) of the Y8b receptor and Glu(5.24) of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.

  7. Involvement of 5-HT receptor subtypes in the discriminative stimulus properties of mescaline.

    PubMed

    Appel, J B; Callahan, P M

    1989-01-01

    In order to further evaluate the extent to which particular 5-HT receptor subtypes (5-HT1, 5-HT2) might be involved in the behavioral effects of hallucinogenic drugs, rats were trained to discriminate mescaline (10 mg/kg i.p.) from saline and were given substitution (generalization) and combination (antagonism) tests with putatively selective serotonergic and related neuroactive compounds. The mescaline cue generalized to relatively high doses of the 5-HT2 agonists, 2,5-dimethoxy-4-methylamphetamine (DOM), LSD and psilocybin; the extent of generalization to 5-HT1 agonists (8-hydroxy-2-[diethylamino]tetralin (8-OHDPAT), RU-24969 and 8-hydroxy-2-[di-n-propylamino]tetralin (TFMPP] was unclear. Combinations of the training drug and sufficiently high doses of 5-HT2 antagonists (ketanserin, LY-53857, pirenperone) were followed by saline-lever responding; less selective central 5-HT (metergoline), and DA (SCH-23390, haloperidol) antagonists, did not block the mescaline cue. These data suggest that 5-HT2 receptors are involved in the stimulus properties of mescaline.

  8. M3-subtype muscarinic receptor that controls intracellular calcium release and inositol phosphate accumulation in gastric parietal cells.

    PubMed

    Leonard, A; Cuq, P; Magous, R; Bali, J P

    1991-07-25

    The muscarinic receptor subtype which triggers acid secretion was investigated in isolated rabbit gastric parietal cells. Cytosolic free Ca2+ concentration ([Ca2+]i), measured with the fluorescent indicator FURA-2, increased rapidly after full agonist (carbachol) stimulation (6-8 sec), then returned to an intermediate sustained value. Other M2-agonists, oxotremorine and arecoline, produced a partial [Ca2+]i increase, whereas M1-agonists, pilocarpine and [4-m-chlorophenylcarbamoyloxyl]-2-butynyl-trimethylammonium, were without any significant effect. [Ca2+]i rise was inhibited by selective muscarinic antagonists: atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than quinuclidinylbenzilate (QNB) greater than pirenzepine greater than 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one, this sequence being characteristic of the involvement of an M3-subtype. This inhibition was shown to be stereoselective; dexetimide and (-)QNB were more potent than levetimide and (+)QNB. The IC50 values for inhibition of [Ca2+]i increase by muscarinic antagonists were in good agreement with those obtained for inhibition of phospholipase C activation. In conclusion, the muscarinic receptor that controls acid secretion appears to be of the M3-subtype and the biochemical events coupled to the activation of this receptor system are also controlled through the same subtype. PMID:1651079

  9. Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility.

    PubMed

    Tinoco, A B; Valenciano, A I; Gómez-Boronat, M; Blanco, A M; Nisembaum, L G; De Pedro, N; Delgado, M J

    2015-09-01

    Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both goldfish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S. PMID:26051613

  10. Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility.

    PubMed

    Tinoco, A B; Valenciano, A I; Gómez-Boronat, M; Blanco, A M; Nisembaum, L G; De Pedro, N; Delgado, M J

    2015-09-01

    Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both goldfish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S.

  11. Prostaglandin E2 receptor subtype 2 regulation of scavenger receptor CD36 modulates microglial Aβ42 phagocytosis.

    PubMed

    Li, Xianwu; Melief, Erica; Postupna, Nadia; Montine, Kathleen S; Keene, C Dirk; Montine, Thomas J

    2015-01-01

    Recent studies underline the potential relevance of microglial innate immune activation in Alzheimer disease. Primary mouse microglia that lack prostaglandin E2 receptor subtype 2 (EP2) show decreased innate immune-mediated neurotoxicity and increased amyloid β (Aβ) peptide phagocytosis, features that were replicated in vivo. Here, we tested the hypothesis that scavenger receptor CD36 is an effector of EP2-regulated Aβ phagocytosis. CD36 expression was 143-fold greater in mouse primary microglia than in primary astrocytes. Three different means of suppressing EP2 signaling increased and an agonist of EP2 decreased CD36 expression in primary wild-type microglia. Activation of Toll-like receptor (TLR) 3, TLR4, and TLR7, but not TLR2 or TLR9, reduced primary microglial CD36 transcription and cell surface CD36 protein and reduced Aβ42 phagocytosis as well. At each step, the effects of innate immune activation on CD36 were reversed by at least 50% by an EP2 antagonist, and this partial rescue of microglia Aβ42 phagocytosis was largely mediated by CD36 activity. Finally, we showed in hippocampus of wild-type mice that innate immune activation suppressed CD36 expression by an EP2-dependent mechanism. Taken together with results of others that found brain clearance of Aβ peptides and behavioral improvements mediated by CD36 in mice, regulation of CD36-mediated Aβ phagocytosis by suppression of EP2 signaling may provide a new approach to suppressing some aspects of Alzheimer disease pathogenesis.

  12. Attribution to Heterogeneous Risk Factors for Breast Cancer Subtypes Based on Hormone Receptor and Human Epidermal Growth Factor 2 Receptor Expression in Korea.

    PubMed

    Park, Boyoung; Choi, Ji-Yeob; Sung, Ho Kyung; Ahn, Choonghyun; Hwang, Yunji; Jang, Jieun; Lee, Juyeon; Kim, Heewon; Shin, Hai-Rim; Park, Sohee; Han, Wonshik; Noh, Dong-Young; Yoo, Keun-Young; Kang, Daehee; Park, Sue K

    2016-04-01

    We conducted a heterogeneous risk assessment of breast cancer based on the hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) calculating the risks and population-based attributable fractions (PAFs) for modifiable and nonmodifiable factors.Using matched case-control study design from the Seoul Breast Cancer Study and the national prevalence of exposure, the risks and PAFs for modifiable and nonmodifiable factors were estimated for total breast cancers and subtypes.The attribution to modifiable factors was different for each subtype (luminal A, PAF = 61.4% [95% confidence interval, CI = 54.3%-69.8%]; luminal B, 21.4% [95% CI = 18.6-24.9%]; HER2-overexpression, 59.4% [95% CI = 47.8%-74.3%], and triple negative tumors [TNs], 27.1% [95% CI = 22.9%-32.4%)], and the attribution to the modifiable factors for the luminal A and HER2-overexpression subtypes was higher than that of the luminal B and TN subtypes (P heterogeneity  ≤  0.001). The contribution of modifiable reproductive factors to luminal A type in premenopausal women was higher than that of the other subtypes (18.2% for luminal A; 3.1%, 8.1%, and -3.1% for luminal B, HER2-overexpression, and TN subtypes, respectively; P heterogeneity  ≤  0.001). Physical activity had the highest impact preventing 32.6% of luminal A, 14.5% of luminal B, 38.0% of HER2-overexpression, and 26.9% of TN subtypes (P heterogeneity = 0.014). Total reproductive factors were also heterogeneously attributed to each breast cancer subtype (luminal A, 65.4%; luminal B, 24.1%; HER2-overexpression, 57.9%, and TN subtypes, -3.1%; P heterogeneity  ≤  0.001).Each pathological subtype of breast cancer by HRs and HER2 status may be associated with heterogeneous risk factors and their attributable risk, suggesting a different etiology. The luminal B and TN subtypes seemed to be less preventable despite intervention for alleged risk factors, even though physical activity had a high

  13. Attribution to Heterogeneous Risk Factors for Breast Cancer Subtypes Based on Hormone Receptor and Human Epidermal Growth Factor 2 Receptor Expression in Korea

    PubMed Central

    Park, Boyoung; Choi, Ji-Yeob; Sung, Ho Kyung; Ahn, Choonghyun; Hwang, Yunji; Jang, Jieun; Lee, Juyeon; Kim, Heewon; Shin, Hai-Rim; Park, Sohee; Han, Wonshik; Noh, Dong-Young; Yoo, Keun-Young; Kang, Daehee; Park, Sue K.

    2016-01-01

    Abstract We conducted a heterogeneous risk assessment of breast cancer based on the hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) calculating the risks and population-based attributable fractions (PAFs) for modifiable and nonmodifiable factors. Using matched case–control study design from the Seoul Breast Cancer Study and the national prevalence of exposure, the risks and PAFs for modifiable and nonmodifiable factors were estimated for total breast cancers and subtypes. The attribution to modifiable factors was different for each subtype (luminal A, PAF = 61.4% [95% confidence interval, CI = 54.3%–69.8%]; luminal B, 21.4% [95% CI = 18.6–24.9%]; HER2-overexpression, 59.4% [95% CI = 47.8%–74.3%], and triple negative tumors [TNs], 27.1% [95% CI = 22.9%–32.4%)], and the attribution to the modifiable factors for the luminal A and HER2-overexpression subtypes was higher than that of the luminal B and TN subtypes (P heterogeneity ≤ 0.001). The contribution of modifiable reproductive factors to luminal A type in premenopausal women was higher than that of the other subtypes (18.2% for luminal A; 3.1%, 8.1%, and −3.1% for luminal B, HER2-overexpression, and TN subtypes, respectively; P heterogeneity ≤ 0.001). Physical activity had the highest impact preventing 32.6% of luminal A, 14.5% of luminal B, 38.0% of HER2-overexpression, and 26.9% of TN subtypes (P heterogeneity = 0.014). Total reproductive factors were also heterogeneously attributed to each breast cancer subtype (luminal A, 65.4%; luminal B, 24.1%; HER2-overexpression, 57.9%, and TN subtypes, −3.1%; P heterogeneity ≤ 0.001). Each pathological subtype of breast cancer by HRs and HER2 status may be associated with heterogeneous risk factors and their attributable risk, suggesting a different etiology. The luminal B and TN subtypes seemed to be less preventable despite intervention for alleged risk factors, even though physical

  14. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Breyer, Richard M.; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2012-01-01

    A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor. PMID:22499581

  15. Somatostatin-14 mainly binds the somatostatin receptor subtype 2 in human neuroblastoma tumors.

    PubMed

    Prevost, G; Veber, N; Viollet, C; Roubert, V; Roubert, P; Benard, J; Eden, P

    1996-02-01

    Neuroblastoma is a pediatric cancer for which a cure is elusive for most children with disseminated disease. Neuroblastomas possess receptors for somatostatin (SS). Some SS analogues can inhibit their proliferation. In addition, when SS analogues were used as agents for scintigraphy, neuroblastoma tumor sites can be localized with high efficiency. In this study, to better characterize the SS receptor subtype(s) (sst1-5) present in primary tumors and metastases of neuroblastoma, we show that: (1) The ligand 125I-Tyr11-SS-14 binding on membrane proteins from primary tumors and metastases of neuroblastoma cell line IGR-N-91 developed in nude mice shows similar values of Kd (in order of 0.1 nM) and Bmax (in order of fmol/mg) by filter-retention assay. These data are close to those measured on two other neuroblastoma cell lines: SK-N-SH and IGR-N-835 or to that measured on the rat cerebral cortex. (2) The IGR-N-91 sublines derived from primary tumor and metastases show one major complex of 57 kD by the chemical cross-linking assay using the ligands: 125I-SS-14 and 125I-BIM23014. One similar major complex of 57 kD was also detected in SK-N-SH and IGR-N-835 or in the cerebral cortex. (3) Addition of excess nonlabeled peptides selective for sst2 (BIM23014, BIM23060, BIM23068) suppressed the formation of the complex 57 kD whereas addition of BIM23052 or BIM23056 (sst5 and sst3 selective respectively) does not. This pharmacological profile corresponds to sst2. (4) Only RNA message of sst2 gene is detected in IGR-N-91 cells and its metastases derived sublines by reverse-transcription-polymerase chain reaction and Northern hybridization in keeping with the presence of sst2. (5) In human biopsies, the complex of 57 kD corresponding to sst2 is consistently detected in three samples of the histological subset of the disease: benign ganglioneuroma, ganglioneuroblastoma and immature neuroblastoma. Therefore, the sst2 should be considered as the primary target to develop more potent

  16. Somatostatin-14 mainly binds the somatostatin receptor subtype 2 in human neuroblastoma tumors.

    PubMed

    Prevost, G; Veber, N; Viollet, C; Roubert, V; Roubert, P; Benard, J; Eden, P

    1996-02-01

    Neuroblastoma is a pediatric cancer for which a cure is elusive for most children with disseminated disease. Neuroblastomas possess receptors for somatostatin (SS). Some SS analogues can inhibit their proliferation. In addition, when SS analogues were used as agents for scintigraphy, neuroblastoma tumor sites can be localized with high efficiency. In this study, to better characterize the SS receptor subtype(s) (sst1-5) present in primary tumors and metastases of neuroblastoma, we show that: (1) The ligand 125I-Tyr11-SS-14 binding on membrane proteins from primary tumors and metastases of neuroblastoma cell line IGR-N-91 developed in nude mice shows similar values of Kd (in order of 0.1 nM) and Bmax (in order of fmol/mg) by filter-retention assay. These data are close to those measured on two other neuroblastoma cell lines: SK-N-SH and IGR-N-835 or to that measured on the rat cerebral cortex. (2) The IGR-N-91 sublines derived from primary tumor and metastases show one major complex of 57 kD by the chemical cross-linking assay using the ligands: 125I-SS-14 and 125I-BIM23014. One similar major complex of 57 kD was also detected in SK-N-SH and IGR-N-835 or in the cerebral cortex. (3) Addition of excess nonlabeled peptides selective for sst2 (BIM23014, BIM23060, BIM23068) suppressed the formation of the complex 57 kD whereas addition of BIM23052 or BIM23056 (sst5 and sst3 selective respectively) does not. This pharmacological profile corresponds to sst2. (4) Only RNA message of sst2 gene is detected in IGR-N-91 cells and its metastases derived sublines by reverse-transcription-polymerase chain reaction and Northern hybridization in keeping with the presence of sst2. (5) In human biopsies, the complex of 57 kD corresponding to sst2 is consistently detected in three samples of the histological subset of the disease: benign ganglioneuroma, ganglioneuroblastoma and immature neuroblastoma. Therefore, the sst2 should be considered as the primary target to develop more potent

  17. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis. PMID:22100731

  18. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis.

  19. Early cerebral activities of the environmental estrogen bisphenol A appear to act via the somatostatin receptor subtype sst(2).

    PubMed Central

    Facciolo, Rosa Maria; Alò, Raffaella; Madeo, Maria; Canonaco, Marcello; Dessì-Fulgheri, Francesco

    2002-01-01

    Recently, considerable interest has been aroused by the specific actions of bisphenol A (BPA). The present investigation represents a first study dealing with the interaction of BPA with the biologically more active somatostatin receptor subtype (sst(2)) in the rat limbic circuit. After treating pregnant female Sprague-Dawley rats with two doses (400 microg/kg/day; 40 microg/kg/day) of BPA, the binding activity of the above receptor subtype was evaluated in some limbic regions of the offspring. The higher dose proved to be the more effective one, as demonstrated by the elevated affinity of sst(2) with its specific radioligand, [(125)I]-Tyr(0)somatostatin-14. The most dramatic effects of BPA on sst(2) levels occurred at the low-affinity states of such a subtype in some telencephalic limbic areas of postnatal rats (10 days of age; postnatal day [PND] 10). These included lower (p < 0.05) sst(2) levels in the gyrus dentate of the hippocampus and basomedial nucleus of the amygdala; significantly higher (p < 0.01) levels were observed only for the high-affinity states of the periventricular nucleus of the hypothalamus. A similar trend was maintained in PND 23 rats with the exception of much lower levels of the high-affinity sst(2) receptor subtype in the amygdala nucleus and ventromedial hypothalamic nucleus. However, greater changes produced by this environmental estrogen were reported when the binding activity of sst(2) was checked in the presence of the two more important selective agonists (zolpidem and Ro 15-4513) specific for the alpha-containing Gamma-aminobutyric acid (GABA) type A receptor complex. In this case, an even greater potentiating effect (p < 0.001) was mainly obtained for the low-affinity sst(2) receptor subtype in PND 10 animals, with the exception of the high-affinity type in the ventromedial hypothalamic nucleus and gyrus dentate. These results support the contention that an sst(2) subtype alpha-containing GABA type A receptor system might

  20. P2Y4 Nucleotide Receptor in Neuronal Precursors Induces Glutamatergic Subtype Markers in Their Descendant Neurons

    PubMed Central

    Uda, Youichi; Xu, Shuai; Matsumura, Takafumi; Takei, Yoshinori

    2016-01-01

    Summary Neural stem cells (NSCs) produce all neuronal subtypes involved in the nervous system. The mechanism regulating their subtype selection is not fully understood. We found that the expression of the nucleotide receptor P2Y4 was transiently augmented in the course of neuronal differentiation of mouse embryonic stem cells (ESCs), which was after loss of pluripotency but prior to terminal differentiation of neurons. The activation of P2Y4 in the differentiating ESCs resulted in an increased proportion of neurons expressing vesicular glutamate transporter (vGluT), a marker of glutamatergic subtype. A subpopulation of type 2 NSCs of the adult mouse hippocampus expressed P2Y4. Its activation induced the expression of glutamatergic subtype markers, vGluT and TBR1, in their descendant neurons. Reciprocally, inhibition of the P2Y4 signaling abolished the effects of nucleotides on those expressions. Our results provide evidence that differentiating NSCs pass through a stage in which nucleotides can affect subtype marker expression of their descendant neurons. PMID:26972684

  1. Analgesia and unwanted benzodiazepine effects in point-mutated mice expressing only one benzodiazepine-sensitive GABAA receptor subtype

    PubMed Central

    Ralvenius, William T.; Benke, Dietmar; Acuña, Mario A.; Rudolph, Uwe; Zeilhofer, Hanns Ulrich

    2015-01-01

    Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain. PMID:25865415

  2. Identification of Metabotropic Glutamate Receptor Subtype 5 Potentiators Using Virtual High-Throughput Screening

    PubMed Central

    2010-01-01

    Selective potentiators of glutamate response at metabotropic glutamate receptor subtype 5 (mGluR5) have exciting potential for the development of novel treatment strategies for schizophrenia. A total of 1,382 compounds with positive allosteric modulation (PAM) of the mGluR5 glutamate response were identified through high-throughput screening (HTS) of a diverse library of 144,475 substances utilizing a functional assay measuring receptor-induced intracellular release of calcium. Primary hits were tested for concentration-dependent activity, and potency data (EC50 values) were used for training artificial neural network (ANN) quantitative structure−activity relationship (QSAR) models that predict biological potency from the chemical structure. While all models were trained to predict EC50, the quality of the models was assessed by using both continuous measures and binary classification. Numerical descriptors of chemical structure were used as input for the machine learning procedure and optimized in an iterative protocol. The ANN models achieved theoretical enrichment ratios of up to 38 for an independent data set not used in training the model. A database of ∼450,000 commercially available drug-like compounds was targeted in a virtual screen. A set of 824 compounds was obtained for testing based on the highest predicted potency values. Biological testing found 28.2% (232/824) of these compounds with various activities at mGluR5 including 177 pure potentiators and 55 partial agonists. These results represent an enrichment factor of 23 for pure potentiation of the mGluR5 glutamate response and 30 for overall mGluR5 modulation activity when compared with those of the original mGluR5 experimental screening data (0.94% hit rate). The active compounds identified contained 72% close derivatives of previously identified PAMs as well as 28% nontrivial derivatives of known active compounds. PMID:20414370

  3. Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

    PubMed Central

    Green, Jill A.; Schmid, Cullen L.; Bley, Elizabeth; Monsma, Paula C.; Brown, Anthony; Bohn, Laura M.

    2015-01-01

    Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling. PMID:26503786

  4. Regulation of body temperature and brown adipose tissue thermogenesis by bombesin receptor subtype-3.

    PubMed

    Lateef, Dalya M; Abreu-Vieira, Gustavo; Xiao, Cuiying; Reitman, Marc L

    2014-03-01

    Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, with Brs3 knockout (Brs3(-/y)) mice being hypometabolic, hypothermic, and hyperphagic and developing obesity. We now report that the reduced body temperature is more readily detected if body temperature is analyzed as a function of physical activity level and light/dark phase. Physical activity level correlated best with body temperature 4 min later. The Brs3(-/y) metabolic phenotype is not due to intrinsically impaired brown adipose tissue function or in the communication of sympathetic signals from the brain to brown adipose tissue, since Brs3(-/y) mice have intact thermogenic responses to stress, acute cold exposure, and β3-adrenergic activation, and Brs3(-/y) mice prefer a cooler environment. Treatment with the BRS-3 agonist MK-5046 increased brown adipose tissue temperature and body temperature in wild-type but not Brs3(-/y) mice. Intrahypothalamic infusion of MK-5046 increased body temperature. These data indicate that the BRS-3 regulation of body temperature is via a central mechanism, upstream of sympathetic efferents. The reduced body temperature in Brs3(-/y) mice is due to altered regulation of energy homeostasis affecting higher center regulation of body temperature, rather than an intrinsic defect in brown adipose tissue.

  5. Nerve Demyelination Increases Metabotropic Glutamate Receptor Subtype 5 Expression in Peripheral Painful Mononeuropathy

    PubMed Central

    Ko, Miau-Hwa; Hsieh, Yu-Lin; Hsieh, Sung-Tsang; Tseng, To-Jung

    2015-01-01

    Wallerian degeneration or nerve demyelination, arising from spinal nerve compression, is thought to bring on chronic neuropathic pain. The widely distributed metabotropic glutamate receptor subtype 5 (mGluR5) is involved in modulating nociceptive transmission. The purpose of this study was to investigate the potential effects of mGluR5 on peripheral hypersensitivities after chronic constriction injury (CCI). Sprague-Dawley rats were operated on with four loose ligatures around the sciatic nerve to induce thermal hyperalgesia and mechanical allodynia. Primary afferents in dermis after CCI exhibited progressive decreases, defined as partial cutaneous denervation; importantly, mGluR5 expressions in primary afferents were statistically increased. CCI-induced neuropathic pain behaviors through the intraplantar injections of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, were dose-dependently attenuated. Furthermore, the most increased mGluR5 expressions in primary afferents surrounded by reactive Schwann cells were observed at the distal CCI stumps of sciatic nerves. In conclusion, these results suggest that nerve demyelination results in the increases of mGluR5 expression in injured primary afferents after CCI; and further suggest that mGluR5 represents a main therapeutic target in developing pharmacological strategies to prevent peripheral hypersensitivities. PMID:25739080

  6. Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs*

    PubMed Central

    Pediani, John D.; Ward, Richard J.; Godin, Antoine G.; Marsango, Sara

    2016-01-01

    Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm−2 human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior. PMID:27080256

  7. Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs.

    PubMed

    Pediani, John D; Ward, Richard J; Godin, Antoine G; Marsango, Sara; Milligan, Graeme

    2016-06-17

    Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior. PMID:27080256

  8. Isolation of estrogen receptor subtypes and vitellogenin genes: expression in female Chalcalburnus tarichi.

    PubMed

    Unal, Guler; Marquez, Emily C; Feld, Mara; Stavropoulos, Pericles; Callard, Ian P

    2014-01-01

    Reproductively arrested gonadal development has been previously described in the teleost pearl mullet (Chalcalburnus tarichi, Cyprinidae) from Van Edremit Gulf of Lake Van, Turkey. Oocyte development in some females was arrested at the previtellogenic stage, while gonadosomatic index (GSI) and plasma 17β-estradiol (E2) level were low. A subset of the females was found to have normal ovaries and relatively higher plasma E2 and GSI. These two groups were termed reproductively arrested (RA) and reproductively non-arrested (RN) females. In this study, we cloned estrogen receptor (ER) isoforms (ERα, ERβ1 and ERβ2) and vitellogenin (Vtg), and their mRNA levels were measured in RA and RN fish tissues. C. tarichi ERs fell in the same clade with other fish ERs and ERα and ERβ1 had 97% and 98% identity with the roach (Rutilus rutilus) ERs, respectively. Both Vtg and ER isoforms' mRNA abundance were higher in the liver than in the ovary and hypothalamus (liver>ovary>hypothalamus). The level of ERα mRNA was significantly lower in the liver, ovary and brain of RA fish than in the RN fish tissues. ERβ1 mRNA levels were not different in the liver and ovary from RA and RN fish while ERβ2 expression significantly increased in the liver and ovary from RA fish. All ER subtype expression was found to be lower in the brain from RA fish than RN fish. The level of Vtg mRNA was significantly lower in the liver and ovary from RA fish than RN fish tissue. These results suggest that ER subtypes are differentially regulated by E2, and their functions are also different in vitellogenesis. Analysis of organic contaminants in sediments revealed that C. tarichi living in Van Edremit Gulf of Lake Van are exposed to the contaminants bis(2-ethylhexyl) phthalate and 4,4(') DDT. We suggest that the RA fish represent a segment of the population that is more sensitive to exposure to endocrine disrupting compounds. PMID:24747933

  9. Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

    PubMed

    Báez-Pagán, Carlos A; Del Hoyo-Rivera, Natalie; Quesada, Orestes; Otero-Cruz, José David; Lasalde-Dominicci, José A

    2016-08-01

    The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane. PMID:27116687

  10. Isolation of estrogen receptor subtypes and vitellogenin genes: expression in female Chalcalburnus tarichi.

    PubMed

    Unal, Guler; Marquez, Emily C; Feld, Mara; Stavropoulos, Pericles; Callard, Ian P

    2014-01-01

    Reproductively arrested gonadal development has been previously described in the teleost pearl mullet (Chalcalburnus tarichi, Cyprinidae) from Van Edremit Gulf of Lake Van, Turkey. Oocyte development in some females was arrested at the previtellogenic stage, while gonadosomatic index (GSI) and plasma 17β-estradiol (E2) level were low. A subset of the females was found to have normal ovaries and relatively higher plasma E2 and GSI. These two groups were termed reproductively arrested (RA) and reproductively non-arrested (RN) females. In this study, we cloned estrogen receptor (ER) isoforms (ERα, ERβ1 and ERβ2) and vitellogenin (Vtg), and their mRNA levels were measured in RA and RN fish tissues. C. tarichi ERs fell in the same clade with other fish ERs and ERα and ERβ1 had 97% and 98% identity with the roach (Rutilus rutilus) ERs, respectively. Both Vtg and ER isoforms' mRNA abundance were higher in the liver than in the ovary and hypothalamus (liver>ovary>hypothalamus). The level of ERα mRNA was significantly lower in the liver, ovary and brain of RA fish than in the RN fish tissues. ERβ1 mRNA levels were not different in the liver and ovary from RA and RN fish while ERβ2 expression significantly increased in the liver and ovary from RA fish. All ER subtype expression was found to be lower in the brain from RA fish than RN fish. The level of Vtg mRNA was significantly lower in the liver and ovary from RA fish than RN fish tissue. These results suggest that ER subtypes are differentially regulated by E2, and their functions are also different in vitellogenesis. Analysis of organic contaminants in sediments revealed that C. tarichi living in Van Edremit Gulf of Lake Van are exposed to the contaminants bis(2-ethylhexyl) phthalate and 4,4(') DDT. We suggest that the RA fish represent a segment of the population that is more sensitive to exposure to endocrine disrupting compounds.

  11. Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus

    PubMed Central

    Lee, Peter S.; Ohshima, Nobuko; Stanfield, Robyn L.; Yu, Wenli; Iba, Yoshitaka; Okuno, Yoshinobu; Kurosawa, Yoshikazu; Wilson, Ian A.

    2015-01-01

    Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here, we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2, and H13, using the enhanced avidity of its IgG to overcome lower affinity Fab binding, as observed with other receptor-binding site antibodies. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small molecule therapeutics. PMID:24717798

  12. Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus

    SciTech Connect

    Lee, Peter S.; Ohshima, Nobuko; Stanfield, Robyn L.; Yu, Wenli; Iba, Yoshitaka; Okuno, Yoshinobu; Kurosawa, Yoshikazu; Wilson, Ian A.

    2014-04-10

    Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012–2013. Here we describe an antibody, F045–092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045–092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045–092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhanced avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics.

  13. The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies

    PubMed Central

    Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

    2014-01-01

    BACKGROUND AND PURPOSE Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. PMID:24750073

  14. Receptor-Defined Subtypes of Breast Cancer in Indigenous Populations in Africa: A Systematic Review and Meta-Analysis

    PubMed Central

    Eng, Amanda; McCormack, Valerie; dos-Santos-Silva, Isabel

    2014-01-01

    Background Breast cancer is the most common female cancer in Africa. Receptor-defined subtypes are a major determinant of treatment options and disease outcomes but there is considerable uncertainty regarding the frequency of poor prognosis estrogen receptor (ER) negative subtypes in Africa. We systematically reviewed publications reporting on the frequency of breast cancer receptor-defined subtypes in indigenous populations in Africa. Methods and Findings Medline, Embase, and Global Health were searched for studies published between 1st January 1980 and 15th April 2014. Reported proportions of ER positive (ER+), progesterone receptor positive (PR+), and human epidermal growth factor receptor-2 positive (HER2+) disease were extracted and 95% CI calculated. Random effects meta-analyses were used to pool estimates. Fifty-four studies from North Africa (n = 12,284 women with breast cancer) and 26 from sub-Saharan Africa (n = 4,737) were eligible. There was marked between-study heterogeneity in the ER+ estimates in both regions (I2>90%), with the majority reporting proportions between 0.40 and 0.80 in North Africa and between 0.20 and 0.70 in sub-Saharan Africa. Similarly, large between-study heterogeneity was observed for PR+ and HER2+ estimates (I2>80%, in all instances). Meta-regression analyses showed that the proportion of ER+ disease was 10% (4%–17%) lower for studies based on archived tumor blocks rather than prospectively collected specimens, and 9% (2%–17%) lower for those with ≥40% versus those with <40% grade 3 tumors. For prospectively collected samples, the pooled proportions for ER+ and triple negative tumors were 0.59 (0.56–0.62) and 0.21 (0.17–0.25), respectively, regardless of region. Limitations of the study include the lack of standardized procedures across the various studies; the low methodological quality of many studies in terms of the representativeness of their case series and the quality of the procedures for collection

  15. Exploring amino acids derivatives as potent, selective, and direct agonists of sphingosine-1-phosphate receptor subtype-1.

    PubMed

    Evindar, Ghotas; Deng, Hongfeng; Bernier, Sylvie G; Doyle, Elisabeth; Lorusso, Jeanine; Morgan, Barry A; Westlin, William F

    2013-01-15

    In the quest to discover a potent and selective class of direct agonists to the sphingosine-1-phosphate receptor, we explored the carboxylate functional group as a replacement to previously reported lead phosphates. This has led to the discovery of potent and selective direct agonists with moderate to substantial in vivo lymphopenia. The previously reported selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) in the phenylamide and phenylimidazole scaffolds were crucial to obtaining selectivity for S1P receptor subtype 1 over 3. PMID:23245510

  16. Galanin-neuropeptide Y (NPY) interactions in central cardiovascular control: involvement of the NPY Y receptor subtype.

    PubMed

    Díaz-Cabiale, Zaida; Parrado, Concepción; Rivera, Alicia; de la Calle, Adelaida; Agnati, Luigi; Fuxe, Kjell; Narváez, José A

    2006-07-01

    The interactions between neuropeptide Y (NPY), specifically through NPY Y(1) and Y(2) receptor subtypes, and galanin [GAL(1-29)] have been analysed at the cardiovascular level. The cardiovascular effects of intracisternal coinjections of GAL(1-29) with NPY or NPY Y(1) or Y(2) agonists, as well as quantitative receptor autoradiography of the binding characteristics of NPY Y(1) and Y(2) receptor subtypes in the nucleus of the solitary tract (NTS), in the presence or absence of GAL(1-29), have been investigated. The effects of coinjections of GAL(1-29) and the NPY Y(1) agonist on the expression of c-FOS immunoreactivity in the NTS were also studied. The coinjection of NPY with GAL(1-29) induced a significant vasopressor and tachycardic action with a maximum 40% increase (P < 0.001). The coinjection of the NPY Y(1) agonist and GAL(1-29) induced a similar increase in mean arterial pressure and heart rate as did NPY plus GAL(1-29), actions that were not observed with the NPY Y(2) agonist plus GAL(1-29). GAL(1-29), 3 nm, significantly and substantially (by approximately 40%) decreased NPY Y(1) agonist binding in the NTS. This effect was significantly blocked (P < 0.01) in the presence of the specific galanin antagonist M35. The NPY Y(2) agonist binding was not modified in the presence of GAL(1-29). At the c-FOS level, the coinjection of NPY Y(1) and GAL(1-29) significantly reduced the c-FOS-immunoreactive response induced by either of the two peptides. The present findings suggest the existence of a modulatory antagonistic effect of GAL(1-29) mediated via galanin receptors on the NPY Y(1) receptor subtype and its signalling within the NTS.

  17. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    SciTech Connect

    Buck, M.A.; Fraser, C.M. )

    1990-12-14

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of (3H)-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of (3H)-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. (3H)-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

  18. Negative Allosteric Modulators of Metabotropic Glutamate Receptors Subtype 5 in Addiction: a Therapeutic Window

    PubMed Central

    2016-01-01

    Background: Abundant evidence at the anatomical, electrophysiological, and molecular levels implicates metabotropic glutamate receptor subtype 5 (mGluR5) in addiction. Consistently, the effects of a wide range of doses of different mGluR5 negative allosteric modulators (NAMs) have been tested in various animal models of addiction. Here, these studies were subjected to a systematic review to find out if mGluR5 NAMs have a therapeutic potential that can be translated to the clinic. Methods: Literature on consumption/self-administration and reinstatement of drug seeking as outcomes of interest published up to April 2015 was retrieved via PubMed. The review focused on the effects of systemic (i.p., i.v., s.c.) administration of the mGluR5 NAMs 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) and 2-Methyl-6-(phenylethynyl)pyridine (MPEP) on paradigms with cocaine, ethanol, nicotine, and food in rats. Results: MTEP and MPEP were found to reduce self-administration of cocaine, ethanol, and nicotine at doses ≥1mg/kg and 2.5mg/kg, respectively. Dose-response relationship resembled a sigmoidal curve, with low doses not reaching statistical significance and high doses reliably inhibiting self-administration of drugs of abuse. Importantly, self-administration of cocaine, ethanol, and nicotine, but not food, was reduced by MTEP and MPEP in the dose range of 1 to 2mg/kg and 2.5 to 3.2mg/kg, respectively. This dose range corresponds to approximately 50% to 80% mGluR5 occupancy. Interestingly, the limited data found in mice and monkeys showed a similar therapeutic window. Conclusion: Altogether, this review suggests a therapeutic window for mGluR5 NAMs that can be translated to the treatment of substance-related and addictive disorders. PMID:26802568

  19. GABA transporter subtype 1 and GABA transporter subtype 3 modulate glutamatergic transmission via activation of presynaptic GABA(B) receptors in the rat globus pallidus.

    PubMed

    Jin, Xiao-Tao; Paré, Jean-Francois; Smith, Yoland

    2012-08-01

    The intra-pallidal application of γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) or GABA transporter subtype 3 (GAT-3) transporter blockers [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA(B) heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole-cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA(A) receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA(B) receptor blockade by the intra-cellular application of N-(2,6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (OX314), bath application of SKF 89976A (10 μM) or SNAP 5114 (10 μM) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid, a GABA(B) receptor antagonist. The paired-pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA(B) heteroreceptors following GAT-1 or GAT-3 blockade. In conclusion, our findings demonstrate that presynaptic GABA(B) heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced

  20. Structure-based prediction of subtype-selectivity of Histamine H3 receptor selective antagonists in clinical trials

    PubMed Central

    Kim, Soo-Kyung; Fristrup, Peter; Abrol, Ravinder; Goddard, William A.

    2011-01-01

    Histamine receptors (HRs) are excellent drug targets for the treatment of diseases such as schizophrenia, psychosis, depression, migraine, allergies, asthma ulcers, and hypertension. Among them, the human H3 Histamine receptor (hH3HR) antagonists have been proposed for specific therapeutic applications, including treatment of Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity.1 However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity and activity for such treatments it would be useful to have the three dimensional structures for all four HRs. We report here the predicted structures of four HR subtypes (H1, H2, H3, and H4) using the GEnSeMBLE (GPCR Ensemble of Structures in Membrane BiLayer Environment) Monte Carlo protocol.2 sampling ~ 35 million combinations of helix packings to predict the 10 most stable packings for each of the four subtypes. Then we used these best 10 protein structures with the DarwinDock Monte Carlo protocol to sample ~ 50,000*20 poses to predict the optimum ligand-protein structures for various agonists and antagonists. We find that E2065.46 contributes most in binding H3 selective agonists (5, 6, 7) in agreement with experimental mutation studies. We also find that conserved E5.46/ S5.43 in both of hH3HR and hH4HR are involved in H3/ H4 subtype selectivity. In addition, we find that M3786.55 in hH3HR provides additional hydrophobic interactions different from hH4HR (the corresponding amino acid of T3236.55 in hH4HR) to provide additional subtype bias. From these studies we developed a pharmacophore model based on our predictions for known hH3HR selective antagonists in clinical study [ABT-239 1, GSK-189,254 2, PF-3654746 3, and BF2.649 (Tiprolisant) 4] that suggests critical selectivity directing elements are: the basic proton interacting with D1143.32, the spacer, the aromatic

  1. Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Analogues of IP3

    PubMed Central

    Saleem, Huma; Tovey, Stephen C.; Rahman, Taufiq; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

    2013-01-01

    Most animal cells express mixtures of the three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R) encoded by vertebrate genomes. Activation of each subtype by different agonists has not hitherto been examined in cells expressing defined homogenous populations of IP3R. Here we measure Ca2+ release evoked by synthetic analogues of IP3 using a Ca2+ indicator within the lumen of the endoplasmic reticulum of permeabilized DT40 cells stably expressing single subtypes of mammalian IP3R. Phosphorylation of (1,4,5)IP3 to (1,3,4,5)IP4 reduced potency by ∼100-fold. Relative to (1,4,5)IP3, the potencies of IP3 analogues modified at the 1-position (malachite green (1,4,5)IP3), 2-position (2-deoxy(1,4,5)IP3) or 3-position (3-deoxy(1,4,5)IP3, (1,3,4,5)IP4) were similar for each IP3R subtype. The potency of an analogue, (1,4,6)IP3, in which the orientations of the 2- and 3-hydroxyl groups were inverted, was also reduced similarly for all three IP3R subtypes. Most analogues of IP3 interact similarly with the three IP3R subtypes, but the decrease in potency accompanying removal of the 1-phosphate from (1,4,5)IP3 was least for IP3R3. Addition of a large chromophore (malachite green) to the 1-phosphate of (1,4,5)IP3 only modestly reduced potency suggesting that similar analogues could be used to measure (1,4,5)IP3 binding optically. These data provide the first structure-activity analyses of key IP3 analogues using homogenous populations of each mammalian IP3R subtype. They demonstrate broadly similar structure-activity relationships for all mammalian IP3R subtypes and establish the potential utility of (1,4,5)IP3 analogues with chromophores attached to the 1-position. PMID:23372785

  2. Metabotropic glutamate receptor modulation of voltage-gated Ca2+ channels involves multiple receptor subtypes in cortical neurons.

    PubMed

    Choi, S; Lovinger, D M

    1996-01-01

    Metabotropic glutamate receptor (mGluR) modulation of voltage-gated Ca2+ channels was examined in isolated deep layer frontoparietal cortical neurons under conditions designed to isolate calcium-independent modulatory pathways. Trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD), a nonspecific mGluR agonist, produced rapid and reversible inhibition of Ca2+ channels. This effect was mimicked by agonists for group I and group II, but not group III, mGluRs. Effects of group I and II agonists often were observed in the same neurons, but separate subgroups of neurons were unresponsive to the group I agonist quisqualate or the group II agonist 2-(2,3-dicarboxycyclopropyl) glycine (DCG-IV). Inhibition by quisqualate and DCG-IV was nonocclusive in neurons responding to both agonists. These agonists thus appear to act on different mGluRs. The mGluR antagonist alpha-methyl-4-carboxylphenylglycine attenuated inhibition by t-ACPD, quisqualate, and DCG-IV. Inhibition by quisqualate and DCG-IV was voltage-dependent. Although the effects of both agonists were greatly reduced by N-ethylmaleimide (NEM), inhibition by DCG-IV was more sensitive to NEM than inhibition by quisqualate. t-ACPD-induced inhibition was reduced by omega-conotoxin GVIA (omega-CgTx) and omega-agatoxin IVA (omega-AgTx) but was affected little by nifedipine. Inhibition by DCG-IV and quisqualate also was reduced by omega-CgTx. We conclude that multiple mGluR subtypes inhibit Ca2+ channels in cortical neurons and that N- and possibly P-type channels are inhibited. Modulation is via a rapid-onset, voltage-dependent mechanism that likely involves a pertussis toxin (PTX)-sensitive G-protein. Type I mGluRs may work via additional PTX-insensitive pathways.

  3. Prostaglandin E2-prostaglandin E receptor subtype 4 (EP4) signaling mediates UV irradiation-induced systemic immunosuppression.

    PubMed

    Soontrapa, Kitipong; Honda, Tetsuya; Sakata, Daiji; Yao, Chengcan; Hirata, Takako; Hori, Shohei; Matsuoka, Toshiyuki; Kita, Yoshihiro; Shimizu, Takao; Kabashima, Kenji; Narumiya, Shuh

    2011-04-19

    UV radiation induces systemic immunosuppression. Because nonsteroidal anti-inflammatory drugs suppress UV-induced immunosuppression, prostanoids have been suspected as a crucial mediator of this UV effect. However, the identity of the prostanoid involved and its mechanism of action remain unclear. Here, we addressed this issue by subjecting mice deficient in each prostanoid receptor individually or mice treated with a subtype-specific antagonist to UV irradiation. Mice treated with an antagonist for prostaglandin E receptor subtype 4 (EP4), but not those deficient in other prostanoid receptors, show impaired UV-induced immunosuppression, whereas administration of an EP4 agonist rescues the impairment of the UV-induced immunosuppression in indomethacin-treated mice. The EP4 antagonist treatment suppresses an increase in the number of CD4(+)/forkhead box P3-positive (Foxp3(+)) regulatory T cells (Treg cells) in the peripheral lymph nodes (LNs) and dendritic cells expressing DEC205 in the LNs and the skin after UV irradiation. Furthermore, the EP4 antagonist treatment down-regulates UV-induced expression of receptor activator of NF-κB ligand (RANKL) in skin keratinocytes. Finally, administration of anti-RANKL antibody abolishes the restoration of UV-induced immunosuppression by EP4 agonism in indomethacin-treated mice. Thus, prostaglandin E(2) (PGE(2))-EP4 signaling mediates UV-induced immunosuppression by elevating the number of Treg cells through regulation of RANKL expression in the epidermis.

  4. Cartography of 5-HT1A and 5-HT2A Receptor Subtypes in Prefrontal Cortex and Its Projections.

    PubMed

    Mengod, Guadalupe; Palacios, José M; Cortés, Roser

    2015-07-15

    Since the development of chemical neuroanatomical tools in the 1960s, a tremendous wealth of information has been generated on the anatomical components of the serotonergic system, at the microscopic level in the brain including the prefrontal cortex (PFC). The PFC receives a widespread distribution of serotonin (5-hydroxytryptamine, 5-HT) terminals from the median and dorsal raphe nuclei. 5-HT receptors were first visualized using radioligand autoradiography in the late 1980s and early 1990s and showed, in contrast to 5-HT innervation, a differential distribution of binding sites associated with different 5-HT receptor subtypes. Due to the cloning of the different 5-HT receptor subtype genes in the late 1980s and early 1990s, it was possible, using in situ hybridization histochemistry, to localize cells expressing mRNA for these receptors. Double in situ hybridization histochemistry and immunohistochemistry allowed for the chemical characterization of the phenotype of cells expressing 5-HT receptors. Tract tracing technology allowed a detailed cartography of the neuronal connections of PFC and other brain areas. Based on these data, maps have been constructed that reflect our current understanding of the different circuits where 5-HT receptors can modulate the electrophysiological, pharmacological, and behavioral functions of the PFC. We will review current knowledge regarding the cellular localization of 5-HT1A and 5-HT2A receptors in mammalian PFC and their possible functions in the neuronal circuits of the PFC. We will discuss data generated in our laboratory as well as in others, focusing on localization in the pyramidal and GABAergic neuronal cell populations in different mammalian species using molecular neuroanatomy and on the connections with other brain regions. PMID:25739427

  5. Age-dependent effects on social interaction of NMDA GluN2A receptor subtype-selective antagonism.

    PubMed

    Green, Torrian L; Burket, Jessica A; Deutsch, Stephen I

    2016-07-01

    NMDA receptor-mediated neurotransmission is implicated in the regulation of normal sociability in mice. The heterotetrameric NMDA receptor is composed of two obligatory GluN1 and either two "modulatory" GluN2A or GluN2B receptor subunits. GluN2A and GluN2B-containing receptors differ in terms of their developmental expression, distribution between synaptic and extrasynaptic locations, and channel kinetic properties, among other differences. Because age-dependent differences in disruptive effects of GluN2A and GluN2B subtype-selective antagonists on sociability and locomotor activity have been reported in rats, the current investigation explored age-dependent effects of PEAQX, a GluN2A subtype-selective antagonist, on sociability, stereotypic behaviors emerging during social interaction, and spatial working memory in 4- and 8-week old male Swiss Webster mice. The data implicate an age-dependent contribution of GluN2A-containing NMDA receptors to the regulation of normal social interaction in mice. Specifically, at a dose of PEAQX devoid of any effect on locomotor activity and mouse rotarod performance, the social interaction of 8-week old mice was disrupted without any effect on the social salience of a stimulus mouse. Moreover, PEAQX attenuated stereotypic behavior emerging during social interaction in 4- and 8-week old mice. However, PEAQX had no effect on spontaneous alternations, a measure of spatial working memory, suggesting that neural circuits mediating sociability and spatial working memory may be discrete and dissociable from each other. Also, the data suggest that the regulation of stereotypic behaviors and sociability may occur independently of each other. Because expression of GluN2A-containing NMDA receptors occurs at a later developmental stage, they may be more involved in mediating the pathogenesis of ASDs in patients with histories of "regression" after a period of normal development than GluN2B receptors.

  6. Cartography of 5-HT1A and 5-HT2A Receptor Subtypes in Prefrontal Cortex and Its Projections.

    PubMed

    Mengod, Guadalupe; Palacios, José M; Cortés, Roser

    2015-07-15

    Since the development of chemical neuroanatomical tools in the 1960s, a tremendous wealth of information has been generated on the anatomical components of the serotonergic system, at the microscopic level in the brain including the prefrontal cortex (PFC). The PFC receives a widespread distribution of serotonin (5-hydroxytryptamine, 5-HT) terminals from the median and dorsal raphe nuclei. 5-HT receptors were first visualized using radioligand autoradiography in the late 1980s and early 1990s and showed, in contrast to 5-HT innervation, a differential distribution of binding sites associated with different 5-HT receptor subtypes. Due to the cloning of the different 5-HT receptor subtype genes in the late 1980s and early 1990s, it was possible, using in situ hybridization histochemistry, to localize cells expressing mRNA for these receptors. Double in situ hybridization histochemistry and immunohistochemistry allowed for the chemical characterization of the phenotype of cells expressing 5-HT receptors. Tract tracing technology allowed a detailed cartography of the neuronal connections of PFC and other brain areas. Based on these data, maps have been constructed that reflect our current understanding of the different circuits where 5-HT receptors can modulate the electrophysiological, pharmacological, and behavioral functions of the PFC. We will review current knowledge regarding the cellular localization of 5-HT1A and 5-HT2A receptors in mammalian PFC and their possible functions in the neuronal circuits of the PFC. We will discuss data generated in our laboratory as well as in others, focusing on localization in the pyramidal and GABAergic neuronal cell populations in different mammalian species using molecular neuroanatomy and on the connections with other brain regions.

  7. Modulation of agonist binding to human dopamine receptor subtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog.

    PubMed

    Verma, Vaneeta; Mann, Amandeep; Costain, Willard; Pontoriero, Giuseppe; Castellano, Jessica M; Skoblenick, Kevin; Gupta, Suresh K; Pristupa, Zdenek; Niznik, Hyman B; Johnson, Rodney L; Nair, Venugopalan D; Mishra, Ram K

    2005-12-01

    The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur. PMID:16126839

  8. Dissociation of β1- and β2-adrenergic receptor subtypes in the retrieval of cocaine-associated memory.

    PubMed

    Fitzgerald, Michael K; Otis, James M; Mueller, Devin

    2016-01-01

    Drug seeking is maintained by encounters with drug-associated cues, and disrupting retrieval of these drug-cue associations would reduce the risk of relapse. Retrieval of cocaine-associated memories is dependent on β-adrenergic receptor (β-AR) activation, and blockade of these receptors induces a persistent retrieval deficit. Whether retrieval of cocaine-associated memory is mediated by a specific β-AR subtype, however, remains unclear. Using a cocaine conditioned place preference (CPP) procedure, we examined whether retrieval of a cocaine CPP memory is mediated collectively by β1- and β2-ARs, or by one of these β-AR subtypes alone. We show that co-blockade of β1- and β2-ARs abolished CPP expression on that and subsequent drug-free CPP tests, resulting in a long-lasting retrieval deficit that prevented subsequent cocaine-induced reinstatement. To dissociate the necessity of either β1- or β2-ARs alone, we administered subtype-specific antagonists prior to retrieval. Administration of a β1-AR antagonist before the initial CPP trial dose-dependently reduced expression of a CPP on that and subsequent drug-free trials as compared to vehicle administration. In contrast, administration of a β2-AR antagonist had no effect on initial CPP expression, although the highest dose reduced subsequent CPP expression. Importantly, either β1- or β2-AR blockade prior to an initial retrieval trial prevented subsequent cocaine-induced reinstatement. Our findings indicate that the β1-AR subtype mediates retrieval of a cocaine CPP, and that acutely blocking either β1- or β2-ARs can prevent subsequent cocaine-induced reinstatement. Thus, β-AR antagonists, particularly β1-ARs antagonists, could serve as adjuncts for addiction therapies to prevent retrieval of drug-associated memories and provide protection against relapse.

  9. Nicotinic Receptor Subtypes Mediating Relaxation of the Normal Human Clasp and Sling Fibers of the Upper Gastric Sphincter

    PubMed Central

    Ruggieri, Michael R.; Braverman, Alan S.; Vegesna, Anil K.; Miller, Larry S.

    2014-01-01

    Background Proper function of the gastroesophageal high pressure zone is essential for the integrity of the antireflux barrier. Mechanisms include tonic contractions as well as the decreased tone during transient lower esophageal sphincter relaxations. Methods We characterized the pharmacology of nicotinic receptors mediating relaxations of the human upper gastric sphincter (clasp and sling fibers) using currently available subtype selective nicotinic antagonists in tissue from organ transplant donors. Donors with either a history of gastroesophageal reflux disease or histologic evidence of Barrett’s esophagus were excluded. Clasp and sling muscle fiber strips were used for one of three paradigms. For paradigm 1, each strip was exposed to carbachol, washed, exposed to nicotinic antagonists then re-exposed to carbachol. In paradigm 2, strips were exposed to a near maximally effective bethanechol concentration then nicotine was added. Strips then were washed, exposed to nicotinic antagonists then re-exposed to bethanechol followed by nicotine. In paradigm 3, strips were exposed to bethanechol then choline or cytisine. Key Results 100 µM methyllycaconitine has no inhibitory effects on relaxations, eliminating homomeric α7 subtypes. Subtypes composed of α4β2 subunits are also eliminated because choline acts as an agonist and dihydro-beta-erythroidine is ineffective. Conclusions & Inferences Because mecamylamine blocks the relaxations and both choline and cytisine act as agonists in both clasp and sling fibers, the nicotinic receptor subtypes responsible for these relaxations could be composed of α3β4β2, α2β4 or α4β4 subunits. PMID:24827539

  10. Subtype-selective nicotinic acetylcholine receptor agonists enhance the responsiveness to citalopram and reboxetine in the mouse forced swim test.

    PubMed

    Andreasen, Jesper T; Nielsen, Elsebet Ø; Christensen, Jeppe K; Olsen, Gunnar M; Peters, Dan; Mirza, Naheed R; Redrobe, John P

    2011-10-01

    Nicotine increases serotonergic and noradrenergic neuronal activity and facilitates serotonin and noradrenaline release. Accordingly, nicotine enhances antidepressant-like actions of reuptake inhibitors selective for serotonin or noradrenaline in the mouse forced swim test and the mouse tail suspension test. Both high-affinity α4β2 and low-affinity α7 nicotinic acetylcholine receptor subtypes are implicated in nicotine-mediated release of serotonin and noradrenaline. The present study therefore investigated whether selective agonism of α4β2 or α7 nicotinic acetylcholine receptors would affect the mouse forced swim test activity of two antidepressants with distinct mechanisms of action, namely the selective serotonin reuptake inhibitor citalopram and the noradrenaline reuptake inhibitor reboxetine. Subthreshold and threshold doses of citalopram (3 and 10 mg/kg) or reboxetine (10 and 20 mg/kg) were tested alone and in combination with the novel α4β2-selective partial nicotinic acetylcholine receptor agonist, NS3956 (0.3 and 1.0 mg/kg) or the α7-selective nicotinic acetylcholine receptor agonist, PNU-282987 (10 and 30 mg/kg). Alone, NS3956 and PNU-282987 were devoid of activity in the mouse forced swim test, but both 1.0 mg/kg NS3956 and 30 mg/kg PNU-282987 enhanced the effect of citalopram and also reboxetine. The data suggest that the activity of citalopram and reboxetine in the mouse forced swim test can be enhanced by agonists at either α4β2 or α7 nicotinic acetylcholine receptors, suggesting that both nicotinic acetylcholine receptor subtypes may be involved in the nicotine-enhanced action of antidepressants.

  11. Stereoselective recognition of the enantiomers of phenglutarimide and of six related compounds by four muscarinic receptor subtypes.

    PubMed Central

    Waelbroeck, M.; Lazareno, S.; Pfaff, O.; Friebe, T.; Tastenoy, M.; Mutschler, E.; Lambrecht, G.

    1996-01-01

    1. We have compared the binding properties of the enantiomers of phenglutarimide (1) and of six related compounds to M1 receptors in NB-OK-1 cells, M2 receptors in rat heart, M3 receptors in rat pancreas and the M4 receptors of rat striatum, with their functional (antimuscarinic) properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2) and guinea-pig ileum (M3) receptors. The binding properties of the enantiomers of three of the compounds were also measured on cloned human m1-m4 receptors expressed by CHO cells, using [3H]-N-methylscopolamine ([3H]-NMS) as radioligand. 2. The high affinity enantiomers behaved as competitive antagonists in binding and pharmacological studies. (S)-phenglutarimide (pKi-M1 = 9.0/9.3) and (R)-thienglutarimide (pKi-M1 = 8.6/9.2) recognized selectively the native M1 > M4 > M3 > M2 receptors in tissues as well as the respective cloned receptors. 3. The pA2 values at the inhibitory heteroreceptors in the rabbit vas deferens, and at the guinea-pig atria and ileum for the seven more potent enantiomers were compatible with the previous classification of these receptors as M1/M4-like, M2 and M3, respectively. 4. Replacement of the phenyl by a thienyl ring or of the diethylamino by a piperidino group in the phenglutarimide molecule did not affect markedly the potencies of the high affinity enantiomer. In contrast, replacement of the phenyl by a cyclohexyl ring decreased 20 fold the active enantiomers potency. Methylation of the piperidine-2,6-dione nitrogen also reduced markedly the eutomers' affinities, more on the M1 than on the other subtypes. 5. The selectivity profiles (recognition of four receptor subtypes) of six of the seven less active enantiomers were different from the corresponding more active enantiomers selectivity profiles, suggesting that the preparations used in this study were pure. However, we cannot not exclude the hypothesis that the batch of (S)-thienglutarimide used in this study was contaminated by less than

  12. Cloning, mRNA expression and transcriptional regulation of five retinoid X receptor subtypes in yellow catfish Pelteobagrus fulvidraco by insulin.

    PubMed

    Pan, Ya-Xiong; Luo, Zhi; Wu, Kun; Zhang, Li-Han; Xu, Yi-Huan; Chen, Qi-Liang

    2016-01-01

    Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and mediate development, reproduction, homeostasis and cell differentiation processes in vertebrates. In this study, full-length cDNA sequences of five rxr subtypes from yellow catfish Pelteobagrus fulvidraco were cloned. Their mRNA expression patterns in different tissues and transcriptional regulation by insulin were determined. Five P. fulvidraco rxr (Pf-rxr) subtypes differed in the length of cDNA sequence and the open reading frame, but shared the similar domain structures as in typical nuclear receptors. Phylogenetic analysis revealed that the five Pf-rxr subtypes were paralogous genes, and that Pf-rxrβa and Pf-rxrβb had arisen during a teleost-specific genome duplication event. Five subtypes of Pf-rxr were detected in all the tested tissues. Overlapping and distinct expression patterns were found for different Pf-rxr subtypes, suggesting functional redundancy and divergence of these duplicates. Intraperitoneal insulin injection and incubation reduced the mRNA expression of Pf-rxrgb, but not other subtypes, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that Pf-rxrgb is the dominant rxr subtype involved in the insulin signaling pathway in P. fulvidraco. PMID:26519760

  13. Effects of the xenoestrogen bisphenol A in diencephalic regions of the teleost fish Coris julis occur preferentially via distinct somatostatin receptor subtypes.

    PubMed

    Alo', Raffaella; Facciolo, Rosa Maria; Madeo, Maria; Giusi, Giuseppina; Carelli, Antonio; Canonaco, Marcello

    2005-04-15

    The xenoestrogen bisphenol A, a contaminant used in the manufacturing of polymers for many consumer products, has been shown to mimic estrogenic actions. This xenoestrogen regulates secretion and expression of pituitary lactotrophs plus morphological and structural features of estrogen target tissues in rodents. Recently, ecological hazards produced by bisphenol A have drawn interests towards the effects of this environmental chemical on neurobiological functions of aquatic vertebrates of which little is known. In this study, the effects of bisphenol A on the distribution of the biologically more active somatostatin receptor subtypes in diencephalic regions of the teleost fish Coris julis were assessed using nonpeptide agonists (L-779, 976 and L-817, 818) that are highly selective for subtype(2) and subtype(5), respectively. Bisphenol A proved to be responsible for highly significant increased binding levels of subtype(2) in hypothalamic areas, while markedly decreased levels of subtype(5) were found in these diencephalic areas, as well as in the medial preglomerular nucleus. The extensive distribution of somatostatin receptor subtype(2) and subtype(5) in the teleost diencephalic areas suggests that, like in mammals, this receptor system may not only be involved in enhanced hypophysiotropic neurohormonal functions but might also promote neuroplasticity events.

  14. Synthesis and biological evaluation of spirocyclic antagonists of CCR2 (chemokine CC receptor subtype 2).

    PubMed

    Strunz, Ann Kathrin; Zweemer, Annelien J M; Weiss, Christina; Schepmann, Dirk; Junker, Anna; Heitman, Laura H; Koch, Michael; Wünsch, Bernhard

    2015-07-15

    Activation of chemokine CC receptors subtype 2 (CCR2) plays an important role in chronic inflammatory processes such as atherosclerosis, multiple sclerosis and rheumatoid arthritis. A diverse set of spirocyclic butanamides 4 (N-benzyl-4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)butanamides) was prepared by different combination of spirocyclic piperidines 8 (3,4-dihydrospiro[[2]benzopyran-1,4'-piperidines]) and γ-halobutanamides 11. A key step in the synthesis of spirocyclic piperidines 8 was an Oxa-Pictet-Spengler reaction of β-phenylethanols 5 with piperidone acetal 6. The substituted γ-hydroxybutanamides 11c-e were prepared by hydroxyethylation of methyl acetates 13 with ethylene sulfate giving the γ-lactones 14c and 14e. Aminolysis of the γ-lactones 14c and 14e with benzylamines provided the γ-hydroxybutanamides 15c-e, which were converted into the bromides 11c-e by an Appel reaction using polymer-bound PPh3. In radioligand binding assays the spirocyclic butanamides 4 did not displace the iodinated radioligand (125)I-CCL2 from the human CCR2. However, in the Ca(2+)-flux assay using human CCR2 strong antagonistic activity of butanamides 4 was detected. Analysis of the IC50-values led to clear relationships between the structure and the inhibition of the Ca(2+)-flux. 4g (4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)-N-[3,5-bis(trifluoromethylbenzyl)]-2-(4-fluorophenyl)butanamide) and 4o (N-[3,5-bis(trifluoromethyl)benzyl]-2-cyclopropyl-4-(3,4-dihydrospiro[[2]benzopyran-1,4'-piperidin]-1'-yl)butanamide) represent the most potent CCR2 antagonists with IC50-values of 89 and 17nM, respectively. Micromolar activities were found in the β-arrestin recruitment assay with murine CCR2, but the structure-activity-relationships detected in the Ca(2+)-flux assay were confirmed. PMID:25766632

  15. Hyperspectral multiplex single-particle tracking of different receptor subtypes labeled with quantum dots in live neurons

    NASA Astrophysics Data System (ADS)

    Labrecque, Simon; Sylvestre, Jean-Philippe; Marcet, Stephane; Mangiarini, Francesca; Bourgoin, Brice; Verhaegen, Marc; Blais-Ouellette, Sébastien; De Koninck, Paul

    2016-04-01

    The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders.

  16. Estrogenic Regulation of Histamine Receptor Subtype H1 Expression in the Ventromedial Nucleus of the Hypothalamus in Female Rats

    PubMed Central

    Mori, Hiroko; Matsuda, Ken-Ichi; Yamawaki, Masanaga; Kawata, Mitsuhiro

    2014-01-01

    Female sexual behavior is controlled by central estrogenic action in the ventromedial nucleus of the hypothalamus (VMN). This region plays a pivotal role in facilitating sex-related behavior in response to estrogen stimulation via neural activation by several neurotransmitters, including histamine, which participates in this mechanism through its strong neural potentiating action. However, the mechanism through which estrogen signaling is linked to the histamine system in the VMN is unclear. This study was undertaken to investigate the relationship between estrogen and histamine receptor subtype H1 (H1R), which is a potent subtype among histamine receptors in the brain. We show localization of H1R exclusively in the ventrolateral subregion of the female VMN (vl VMN), and not in the dorsomedial subregion. In the vl VMN, abundantly expressed H1R were mostly colocalized with estrogen receptor α. Intriguingly, H1R mRNA levels in the vl VMN were significantly elevated in ovariectomized female rats treated with estrogen benzoate. These data suggest that estrogen can amplify histamine signaling by enhancing H1R expression in the vl VMN. This enhancement of histamine signaling might be functionally important for allowing neural excitation in response to estrogen stimulation of the neural circuit and may serve as an accelerator of female sexual arousal. PMID:24805361

  17. Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hsstr 1-5).

    PubMed

    Patel, Y C; Srikant, C B

    1994-12-01

    Recent reports (Raynor et al) have claimed the identification of potent somatostatin (SST) agonists exhibiting binding affinities of 1-2 pM and up to 30,000 fold binding selectivity for several of the 5 cloned sstr subtypes. These conclusions, however, are based on binding comparisons of sstr subtypes from different species expressed in different cell lines and studied with different radioligands. To eliminate the effect of species and/or methodological variations, we have investigated agonist selectivity of 32 synthetic SST analogs for all 5 hsstrs stably expressed in CHO-K1 cells under identical binding conditions. We show that hsstr2, 3, 5 react potently with hexapeptide as well as cyclic and linear octapeptide analogs and belong to a similar sstr subclass. hsstr1 and 4 react poorly with these analogs and belong to a separate subclass. The present generation of SST analogs exhibit a modest-50 fold increase in binding potency compared to SST-14 for 2 subtypes (hsstr2, 3), and relative selectivity for only 1 subtype (hsstr2) which is at best only 35 fold. The potency and degree of selectivity of these analogs is several orders of magnitude less than that reported earlier and suggests the need for caution in using these compounds as putative superagonists or subtype selective compounds for any of the individual sstrs.

  18. Subtype-selective nicotinic acetylcholine receptor agonists can improve cognitive flexibility in an attentional set shifting task.

    PubMed

    Wood, Christopher; Kohli, Shivali; Malcolm, Emma; Allison, Claire; Shoaib, Mohammed

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are considered to be viable targets to enhance cognition in patients diagnosed with schizophrenia. Activation of nAChRs with selective nicotinic receptor agonists may provide effective means to pharmacologically treat cognitive deficits observed in schizophrenia. Cognitive flexibility is one aspect of cognition, which can be assessed in a rodent model of the attentional set-shifting task (ASST). The aim of the present study was two-fold, firstly, to evaluate the efficacy of a series of subtype selective nAChR agonists, such as those that target α7 and α4β2 nAChR subtypes in non-compromised rodents. Secondly, nicotine as a prototypic agonist was evaluated for its effects to restore attentional deficits produced by sub-chronic ketamine exposure in the ASST. Male hooded Lister rats underwent habituation, consisting of a simple odour and medium discrimination with subsequent assessment 24 h later. In experimentally naïve rats, α7 subtype selective agonists, compound-A and SSR180711 along with PNU-120596, an α7 positive allosteric modulator (PAM), were compared against the β2* selective agonist, 5IA-85380. All compounds except for PNU-120596 were observed to significantly improve extra-dimensional (ED) shift performance, nicotine, 5IA-85380 and SSR180711 further enhanced the final reversal (REV3) stage of the task. In another experiment, sub-chronic ketamine treatment produced robust deficits during the ED and the REV3 stages of the discriminations; rodents required significantly more trials to reach criterion during these discriminations. These deficits were attenuated in rodents treated acutely with nicotine (0.1 mg/kg SC) 10 min prior to the ED shift. These results highlight the potential utility of targeting nAChRs to enhance cognitive flexibility, particularly the α7 and β2* receptor subtypes. The improvement with nicotine was much greater in rodents that were impaired following the sub-chronic ketamine

  19. Diamine Derivatives as Novel Small-Molecule, Potent, and Subtype-Selective Somatostatin SST3 Receptor Agonists

    PubMed Central

    2014-01-01

    A novel class of small-molecule, highly potent, and subtype-selective somatostatin SST3 agonists was discovered through modification of a SST3 antagonist. As an example, (1R,2S)-9 demonstrated not only potent in vitro SST3 agonist activity but also in vivo SST3 agonist activity in a mouse oral glucose tolerance test (OGTT). These agonists may be useful reagents for studying the physiological roles of the SST3 receptor and may potentially be useful as therapeutic agents. PMID:24944745

  20. mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain

    PubMed Central

    Shukla, Rammohan; Watakabe, Akiya; Yamamori, Tetsuo

    2014-01-01

    To better understand serotonin function in the primate brain, we examined the mRNA expression patterns of all the 13 members of the serotonin receptor (5HTR) family, by in situ hybridization (ISH) and the distribution of serotonergic terminations by serotonin transporter (SERT) protein immunohistochemical analysis. Ten of the 13 5HTRs showed significant mRNA expressions in the marmoset brain. Our study shows several new features of the organization of serotonergic systems in the marmoset brain. (1) The thalamus expressed only a limited number of receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there are layer-selective and area-selective mRNA expressions of 5HTRs. (3) Highly localized mRNA expressions of 5HT1F and 5HT3A were observed. (4) There was a conspicuous overlap of the mRNA expressions of receptor subtypes known to have somatodendritic localization of receptor proteins with dense serotonergic terminations in the visual cortex, the central lateral (CL) nucleus of the thalamus, the presubiculum, and the medial mammillary nucleus of the hypothalamus. This suggests a high correlation between serotonin availability and receptor expression at these locations. (5) The 5HTRs show differences in mRNA expression pattern between the marmoset and mouse cortices whereas the patterns of both the species were much similar in the hippocampus. We discuss the possible roles of 5HTRs in the marmoset brain revealed by the analysis of their overall mRNA expression patterns. PMID:24904298

  1. A melanoma subtype with intrinsic resistance to BRAF inhibition identified by receptor tyrosine kinases gene-driven classification

    PubMed Central

    Dugo, Matteo; Nicolini, Gabriella; Tragni, Gabrina; Bersani, Ilaria; Tomassetti, Antonella; Colonna, Valentina; Del Vecchio, Michele; De Braud, Filippo; Canevari, Silvana

    2015-01-01

    Dysregulation of receptor tyrosine kinases (RTKs) contributes to several aspects of oncogenesis including drug resistance. In melanoma, distinct RTKs have been involved in BRAF inhibitors (BRAFi) resistance, yet the utility of RTKs expression pattern to identify intrinsically resistant tumors has not been assessed. Transcriptional profiling of RTKs and integration with a previous classification, reveals three robust subtypes in two independent datasets of melanoma cell lines and one cohort of melanoma samples. This classification was validated by Western blot in a panel of patient-derived melanoma cell lines. One of the subtypes identified here for the first time displayed the highest and lowest expression of EGFR and ERBB3, respectively, and included BRAF-mutant tumors all intrinsically resistant to BRAFi PLX4720, as assessed by analysis of the Cancer Cell Line Encyclopedia pharmacogenomic study and by in vitro growth inhibition assays. High levels of EGFR were detected, even before therapy, in tumor cells of one of three melanoma patients unresponsive to BRAFi. Use of different pharmacological inhibitors highlighted the relevance of PI3K/mTOR signaling for growth of this PLX4720-resistant subtype. Our results identify a specific molecular profile of melanomas intrinsically resistant to BRAFi and suggest the PI3K/mTOR pathway as a potential therapeutic target for these tumors. PMID:25742786

  2. Delineation of gastric cancer subtypes by co-regulated expression of receptor tyrosine kinases and chemosensitivity genes

    PubMed Central

    Li, Shu-Chun; Ma, Rong; Wu, Jian-Zhong; Xiao, Xia; Wu, Wei; Li, Gang; Chen, Bo; Sharma, Ashok; Bai, Shan; Dun, Bo-Ying; She, Jin-Xiong; Tang, Jin-Hai

    2015-01-01

    Chemotherapy plays a key role in improving disease-free survival and overall survival of gastric cancer (GC); however, response rates are variable and a non-negligible proportion of patients undergo toxic and costly chemotherapeutic regimens without a survival benefit. Several studies have shown the existence of GC subtypes which may predict survival and respond differently to chemotherapy. It is also known that the expression level of chemotherapy-related and target therapy-related genes correlates with response to specific antitumor drugs. Nevertheless, these genes have not been considered jointly to define GC subtypes. In this study, we evaluated seven genes known to influence chemotherapeutic response (ERCC1, BRCA1, RRM1, TUBB3, STMN1, TYMS and TOP2A) and five receptor tyrosine kinases (RTKs) (EGFR, ERBB2, PDGFRB, VEGFR1 and VEGFR2). We demonstrate significant heterogeneity of gene expression among GC patients and identified four GC subtypes using the expression profiles of eight genes in two co-regulation groups: chemosensitivity (BRCA1, STMN1, TYMS and TOP2A) and RTKs (EGFR, PDGFRB, VEGFR1 and VEGFR2). The results are of immediate translational value regarding GC diagnostics and therapeutics, as many of these genes are curently widely used in relevant clinical testing. PMID:26396673

  3. Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

    PubMed Central

    Saleem, Huma; Tovey, Stephen C.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

    2013-01-01

    Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca2+ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca2+ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes. PMID:23469136

  4. The effects of estrogen on the α2-adrenergic receptor subtypes in rat uterine function in late pregnancy in vitro

    PubMed Central

    Hajagos-Tóth, Judit; Bóta, Judit; Ducza, Eszter; Csányi, Adrienn; Tiszai, Zita; Borsodi, Anna; Samavati, Reza; Benyhe, Sándor; Gáspár, Róbert

    2016-01-01

    Aim To assess the effect of 17β-estradiol pretreatment on the function and expression of α2- adrenergic receptors (ARs) subtypes in late pregnancy in rats. Methods Sprague-Dawley SPD rats (n = 37) were treated with 17β-estradiol for 4 days starting from the 18th day of pregnancy. The myometrial expression of the α2-AR subtypes was determined by real time polymerase chain reaction and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C), and spiroxatrine (α2A). The cyclic adenosine monophosphate (cAMP) accumulation was also measured. The activated G-protein level was investigated by guanosine 5′-O-[gamma-thio]triphosphate (GTPγS) binding assay. Results 17β-estradiol pretreatment decreased the contractile effect of (-)-noradrenaline via the α2-ARs, and abolished the contractile effect via the α2B-ARs. All the α2-AR subtypes’ mRNA was significantly decreased. 17β-estradiol pretreatment significantly increased the myometrial cAMP level in the presence of BRL 44408 (P = 0.001), ARC 239 (P = 0.007), and spiroxatrine (P = 0.045), but did not modify it in the presence of spiroxatrine + BRL 44408 combination (P = 0.073). It also inhibited the G-protein-activating effect of (-)-noradrenaline by 25% in the presence of BRL 44408 + spiroxatrine combination. Conclusions The expression of the α2-AR subtypes is sensitive to 17β-estradiol, which decreases the contractile response of (-)-noradrenaline via the α2B-AR subtype, and might cause changes in G-protein signaling pathway. Estrogen dysregulation may be responsible for preterm labor or uterine inertia via the α2-ARs. PMID:27106352

  5. Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes

    PubMed Central

    Bolcato, Chiara; Cusan, Claudia; Pastorin, Giorgia; Cacciari, Barbara; Klotz, Karl Norbert; Morizzo, Erika

    2007-01-01

    In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach. PMID:18368532

  6. Muscarinic receptor subtypes differentially control synaptic input and excitability of cerebellum-projecting medial vestibular nucleus neurons.

    PubMed

    Zhu, Yun; Chen, Shao-Rui; Pan, Hui-Lin

    2016-04-01

    Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We

  7. Muscarinic receptor subtypes differentially control synaptic input and excitability of cerebellum-projecting medial vestibular nucleus neurons.

    PubMed

    Zhu, Yun; Chen, Shao-Rui; Pan, Hui-Lin

    2016-04-01

    Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We

  8. A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters*

    PubMed Central

    Shibasaki, Koji; Ikenaka, Kazuhiro; Tamalu, Fuminobu; Tominaga, Makoto; Ishizaki, Yasuki

    2014-01-01

    Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca2+ transients in astrocytes, and these Ca2+ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4+ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4+ astrocytes. After activation, both TRPV4+ and TRPV4− astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4+ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4+ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands. PMID:24737318

  9. Differential regulation of primary afferent input to spinal cord by muscarinic receptor subtypes delineated using knockout mice.

    PubMed

    Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

    2014-05-16

    Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732

  10. Co-Expression of Two Subtypes of Melatonin Receptor on Rat M1-Type Intrinsically Photosensitive Retinal Ganglion Cells

    PubMed Central

    Sheng, Wen-Long; Chen, Wei-Yi; Yang, Xiong-Li; Zhong, Yong-Mei; Weng, Shi-Jun

    2015-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions. PMID:25714375

  11. New 4-Functionalized Glutamate Analogues Are Selective Agonists at Metabotropic Glutamate Receptor Subtype 2 or Selective Agonists at Metabotropic Glutamate Receptor Group III.

    PubMed

    Huynh, Tri H V; Erichsen, Mette N; Tora, Amélie S; Goudet, Cyril; Sagot, Emmanuelle; Assaf, Zeinab; Thomsen, Christian; Brodbeck, Robb; Stensbøl, Tine B; Bjørn-Yoshimoto, Walden E; Nielsen, Birgitte; Pin, Jean-Philippe; Gefflaut, Thierry; Bunch, Lennart

    2016-02-11

    The metabotropic glutamate (Glu) receptors (mGluRs) play key roles in modulating excitatory neurotransmission in the brain. In all, eight subtypes have been identified and divided into three groups, group I (mGlu1,5), group II (mGlu2,3), and group III (mGlu4,6-8). In this article, we present a L-2,4-syn-substituted Glu analogue, 1d, which displays selective agonist activity at mGlu2 over the remaining mGluR subtypes. A modeling study and redesign of the core scaffold led to the stereoselective synthesis of four new conformationally restricted Glu analogues, 2a-d. Most interestingly, 2a retained a selective agonist activity profile at mGlu2 (EC50 in the micromolar range), whereas 2c/2d were both selective agonists at group III, subtypes mGlu4,6,8. In general, 2d was 20-fold more potent than 2c and potently activated mGlu4,6,8 in the low-mid nanomolar range.

  12. Heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116

    SciTech Connect

    Bloom, J.W.; Halonen, M.; Seaver, N.A.; Yamamura, H.I.

    1987-07-27

    Recent studies have demonstrated that the majority of muscarinic receptors in rabbit peripheral lung homogenates bind pirenzepine with high affinity (putative M1 subtype). In experiments of AF-DX 116 inhibiting (TH)(-)quinuclidinyl benzilate or (TH)pirenzepine, the authors found similar inhibitory constants for AF-DX 116 binding in rat heart and rabbit peripheral lung that were 4-fold smaller (i.e. of higher affinity) than the inhibitory constant for rat cerebral cortex. This results demonstrates heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex. 20 references, 1 figure, 2 tables.

  13. Rectal antinociceptive properties of alverine citrate are linked to antagonism at the 5-HT1A receptor subtype.

    PubMed

    Coelho, A M; Jacob, L; Fioramonti, J; Bueno, L

    2001-10-01

    Serotonin (5-HT) is considered as a major mediator causing hyperalgesia and is involved in inflammatory reactions and irritable bowel syndrome. Alverine citrate may possess visceral antinociceptive properties in a rat model of rectal distension-induced abdominal contractions. This study was designed to evaluate the pharmacological properties of alverine citrate in a rat model of rectal hyperalgesia induced by 5-HTP (5-HT precursor) and by a selective 5-HT1A agonist (8-OH-DPAT) and to compare this activity with a reference 5-HT1A antagonist (WAY 100635). At 4 h after their administration, 5-HTP and 8-OH-DPAT increased the number of abdominal contractions in response to rectal distension at the lowest volume of distension (0.4 mL). When injected intraperitoneally before 8-OH-DPAT and 5-HTP, WAY 100635 (1 mg kg(-1)) blocked their nociceptive effect, but also reduced the response to the highest volume of distension (1.6 mL). Similarly, when injected intraperitoneally, alverine citrate (20 mg kg(-1)) suppressed the effect of 5-HTP, but not that of 8-OH-DPAT. However, when injected intracerebroventricularly (75 microg/rat) alverine citrate reduced 8-OH-DPAT-induced enhancement of rectal distension-induced abdominal contractions. In-vitro binding studies revealed that alverine citrate had a high affinity for 5-HT1A receptors and a weak affinity for 5-HT3 and 5-HT4 subtypes. These results suggest that 5-HTP-induced rectal hypersensitivity involves 5-TH1A receptors and that alverine citrate acts as a selective antagonist at the 5-HT1A receptor subtype to block both 5-HTP and 8-OH-DPAT-induced rectal hypersensitivity. PMID:11697552

  14. Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats.

    PubMed

    Ting, K N; Blaylock, N A; Sugden, D; Delagrange, P; Scalbert, E; Wilson, V G

    1999-06-01

    1. In this study reverse transcriptase-polymerase chain reaction (RT-PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4-phenyl-2-propionamidotetraline (4-P-PDOT) and 2-benzyl-N-pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. 2. Using RT-PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. 3. At low concentrations of the MT2 selective ligands, neither 10 nM 4-P-PDOT (pEC50=8.70+/-0.31 (control) vs 8.73+/-0.16, n=6) nor 60 nM 2-benzyl-NV-pentanoyltryptamine (pEC50= 8.53+/-0.20 (control) vs 8.83+/-0.38, n = 6) significantly altered the potency of melatonin in the rat tail artery. 4. At concentrations non-selective for mt1 and MT2 receptors. 4-P-PDOT (3 microM) and 2-benzyl-N-pentanoyltryptamine (5 microM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4-P-PDOT, the estimated pKB (6.17+/-0.16, n=8) is similar to the binding affinity for mt1 receptor. 5. Pre-incubation with 1 microM melatonin did not affect the conversion of [3H]-adenine to [3H]-cyclic AMP under basal condition (0.95+/-0.19% conversion (control) vs 0.92+/-0.19%, n=4) or following exposure to 30 microM forskolin (5.20+/-1.30% conversion (control) vs 5.35+/-0.90%, n=4). 6. Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

  15. A Novel Selective Muscarinic Acetylcholine Receptor Subtype 1 Antagonist Reduces Seizures without Impairing Hippocampus-Dependent LearningS⃞

    PubMed Central

    Sheffler, Douglas J.; Williams, Richard; Bridges, Thomas M.; Xiang, Zixiu; Kane, Alexander S.; Byun, Nellie E.; Jadhav, Satyawan; Mock, Mathew M.; Zheng, Fang; Lewis, L. Michelle; Jones, Carrie K.; Niswender, Colleen M.; Weaver, Charles D.; Lindsley, Craig W.; Conn, P. Jeffrey

    2009-01-01

    Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M1 mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M1 mAChRs relative to M2-M5. Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M1 mAChRs, a surprising finding given the high level of M1 mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-d-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M1 mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders. PMID:19407080

  16. Radioligand binding analysis of receptor subtypes in two FP receptor preparations that exhibit different functional rank orders of potency in response to prostaglandins.

    PubMed

    Woodward, D F; Fairbairn, C E; Krauss, A H; Lawrence, R A; Protzman, C E

    1995-04-01

    The rat colon and Swiss 3T3 cells have been proposed as FP receptor preparations. However, the rank orders of potency for contraction of the rat colon and Ca++ signaling in Swiss 3T3 cells were found to be disparate. Although both appeared to be FP receptor preparations in that PGF2 alpha and FP receptor selective analogs were the most potent agonists, the potency ranking for other PGs and their analogs differed markedly. This presented two alternative major hypotheses for interpreting these data: (1) Swiss 3T3 cells and the rat colon possess different FP receptor subtypes and (2) the rat colon contains a heterogeneous population of prostanoid receptors. To further characterize prostanoid receptor populations in these two preparations, radioligand binding studies were performed with 3H-PGE2 and 3H-17-phenyl-PGF2 alpha. The rank order of potency for inhibition of 3H-PGE2 binding in the rat colon was consistent with EP3 receptor pharmacology. Thus, MB 28767, sulprostone and PGE2 were potent inhibitors, whereas PGF2 alpha, PGD2 and other analogs were substantially less potent. The rank order of potency for inhibition of 3H-17-phenyl-PGF2 alpha binding in the rat colon was consistent with the presence of an FP receptor. Thus, the potency rank order for the natural PGs was PGF2 alpha > PGD2 > PGE2 and among the synthetic analogs only PGF2 alpha analogs were potent competitors. In Swiss 3T3 cells an identical rank order of potency for eliciting a Ca++ transient signal and inhibition of 3H-17-phenyl-PGF2 alpha binding was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Role of astrocytic leptin receptor subtypes on leptin permeation across hCMEC/D3 human brain endothelial cells.

    PubMed

    Hsuchou, Hung; Kastin, Abba J; Tu, Hong; Joan Abbott, N; Couraud, Pierre-Olivier; Pan, Weihong

    2010-12-01

    Astrocytic leptin receptors (ObR) can be up-regulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb over-expression in C6 cells increased leptin permeation whereas ObRa over-expression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to the sodium fluorescein control was unchanged by over-expression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the over-expression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the evidence that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability.

  18. Ligand Independent and Subtype-Selective Actions of Thyroid Hormone Receptors in Human Adipose Derived Stem Cells

    PubMed Central

    Cvoro, Aleksandra; Bajic, Aleksandar; Zhang, Aijun; Simon, Marisa; Golic, Igor; Sieglaff, Douglas H.; Maletic-Savatic, Mirjana; Korac, Aleksandra; Webb, Paul

    2016-01-01

    Thyroid hormone (TH) receptors (TRs α and β) are homologous ligand-dependent transcription factors (TFs). While the TRs display distinct actions in development, metabolic regulation and other processes, comparisons of TRα and TRβ dependent gene regulation mostly reveal similar mechanisms of action and few TR subtype specific genes. Here, we show that TRα predominates in multipotent human adipose derived stem cells (hADSC) whereas TRβ is expressed at lower levels and is upregulated during hADSC differentiation. The TRs display several unusual properties in parental hADSC. First, TRs display predominantly cytoplasmic intracellular distribution and major TRα variants TRα1 and TRα2 colocalize with mitochondria. Second, knockdown experiments reveal that endogenous TRs influence hADSC cell morphology and expression of hundreds of genes in the absence of hormone, but do not respond to exogenous TH. Third, TRα and TRβ affect hADSC in completely distinct ways; TRα regulates cell cycle associated processes while TRβ may repress aspects of differentiation. TRα splice variant specific knockdown reveals that TRα1 and TRα2 both contribute to TRα-dependent gene expression in a gene specific manner. We propose that TRs work in a non-canonical and hormone independent manner in hADSC and that prominent subtype-specific activities emerge in the context of these unusual actions. PMID:27732649

  19. Differential effects of subtype-specific nicotinic acetylcholine receptor agonists on early and late hippocampal LTP.

    PubMed

    Kroker, Katja S; Rast, Georg; Rosenbrock, Holger

    2011-12-01

    Brain nicotinic acetylcholine receptors are involved in several neuropsychiatric disorders, e.g. Alzheimer's and Parkinson's diseases, Tourette's syndrome, schizophrenia, depression, autism, attention deficit hyperactivity disorder, and anxiety. Currently, approaches selectively targeting the activation of specific nicotinic acetylcholine receptors are in clinical development for treatment of memory impairment of Alzheimer's disease patients. These are α4β2 and α7 nicotinic acetylcholine receptor agonists which are believed to enhance cholinergic and glutamatergic neurotransmission, respectively. In order to gain a better insight into the mechanistic role of these two nicotinic acetylcholine receptors in learning and memory, we investigated the effects of the α4β2 nicotinic acetylcholine receptor agonist TC-1827 and the α7 nicotinic acetylcholine receptor partial agonist SSR180711 on hippocampal long-term potentiation (LTP), a widely accepted cellular experimental model of memory formation. Generally, LTP is distinguished in an early and a late form, the former being protein-synthesis independent and the latter being protein-synthesis dependent. TC-1827 was found to increase early LTP in a bell-shaped dose dependent manner, but did not affect late LTP. In contrast, the α7 nicotinic acetylcholine receptor partial agonist SSR180711 showed enhancing effects on both early and late LTP in a bell-shaped manner. Furthermore, SSR180711 not only increased early LTP, but also transformed it into late LTP, which was not observed with the α4β2 nicotinic acetylcholine receptor agonist. Therefore, based on these findings α7 nicotinic acetylcholine receptor (partial) agonists appear to exhibit stronger efficacy on memory improvement than α4β2 nicotinic acetylcholine receptor agonists. PMID:21968142

  20. Feeding condition and the relative contribution of different dopamine receptor subtypes to the discriminative stimulus effects of cocaine in rats

    PubMed Central

    Baladi, Michelle G; Newman, Amy H; France, Charles P

    2013-01-01

    Rationale The contribution of dopamine receptor subtypes in mediating the discriminative stimulus effects of cocaine is not fully established. Many drug discrimination studies use food to maintain responding, necessitating food restriction, which can alter drug effects. Objective This study established stimulus control with cocaine (10 mg/kg) in free-feeding and food-restricted rats responding under a schedule of stimulus shock termination (SST) and in food-restricted rats responding under a schedule of food presentation to examine whether feeding condition or the reinforcer used to maintain responding impacts the effects of cocaine. Method Dopamine receptor agonists and antagonists were examined for their ability to mimic or attenuate, respectively, the effects of cocaine. Result Apomorphine, quinpirole, and lisuride occasioned >90% responding on the cocaine-associated lever in free-feeding rats responding under a schedule of SST; apomorphine, but not quinpirole or lisuride, occasioned >90% responding on the cocaine lever in food-restricted rats responding under a schedule of SST. In food-restricted rats responding for food these drugs occasioned little cocaine lever responding and were comparatively more potent in decreasing responding. In free-feeding rats, the effects of cocaine were attenuated by the D2/D3 receptor antagonist raclopride and the D3 receptor-selective antagonist PG01037. In food-restricted rats, raclopride and the D2 receptor-selective antagonist L-741,626 attenuated the effects of cocaine. Raclopride antagonized quinpirole in all groups while PG01037 antagonized quinpirole only in free-feeding rats. Conclusion These results demonstrate significant differences in the discriminative stimulus of cocaine that are due to feeding conditions and not to the use of different reinforcers across procedures. PMID:24030470

  1. Determinants involved in subtype-specific functions of rat trace amine-associated receptors 1 and 4

    PubMed Central

    Stäubert, C; Bohnekamp, J; Schöneberg, T

    2013-01-01

    Aims The trace amine-associated receptor (Taar) family displays high species- and subtype-specific pharmacology. Several trace amines such as β-phenylethylamine (β-PEA), p-tyramine and tryptamine are agonists at TA1 but poorly activate rat and mouse Taar4. Principal Results Using rat TA1 and Taar4 chimera, we identified determinants in transmembrane helices 3 and 6, which, when replaced by the corresponding portion of rat TA1, can rescue cell surface expression of rat Taar4. When expressed at the cell surface, rat Taar4 pharmacology was very similar to that of TA1 and coupled to the Gαs-protein/AC pathway. Our data suggest that binding pockets of Taar for surrogate agonists overlap between paralogs. Conclusions This implicates that the repertoire of Taar ensures functional redundancy, tissue- and cell-specific expression and/or different downstream signalling rather than different agonist specificity. PMID:23072560

  2. Two Affinity Sites of the Cannabinoid Subtype 2 Receptor Identified by a Novel Homogeneous Binding Assay.

    PubMed

    Martínez-Pinilla, Eva; Rabal, Obdulia; Reyes-Resina, Irene; Zamarbide, Marta; Navarro, Gemma; Sánchez-Arias, Juan A; de Miguel, Irene; Lanciego, José L; Oyarzabal, Julen; Franco, Rafael

    2016-09-01

    Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-yl]sulfonyl-2-pyridyl]oxy]propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may

  3. Role of selective blocking of bradykinin receptor subtypes in attenuating allergic airway inflammation in guinea pigs.

    PubMed

    El-Kady, Mohamed M; Girgis, Zarif I; Abd El-Rasheed, Eman A; Shaker, Olfat; Attallah, Magdy I; Soliman, Ahmed A

    2016-10-01

    The present study was designed to evaluate the potential role of bradykinin antagonists (R-715; bradykinin B1 receptor antagonist and icatibant; bradykinin B2 receptor antagonist) in treatment of allergic airway inflammation in comparison to dexamethasone and montelukast. R-715 as dexamethasone significantly decreased peribronchial leukocyte infiltration, bronchoalveolar lavage fluid (BALF) albumin and interleukin 1β as well as serum OVA-specific IgE level. Also, R-715 like montelukast significantly decreased BALF cell count (total and eosinophils). Icatibant showed negative results. The current findings suggest that selective bradykinin B1 receptor antagonists may have the therapeutic potential for the treatment of allergic airway inflammation. PMID:27321873

  4. miR-155 functions downstream of angiotensin II receptor subtype 1 and calcineurin to regulate cardiac hypertrophy

    PubMed Central

    Yang, Yong; Zhou, Yong; Cao, Zheng; Tong, Xin Zhu; Xie, Hua Qiang; Luo, Tao; Hua, Xian Ping; Wang, Han Qin

    2016-01-01

    Cardiac hypertrophy is characterized by maladaptive tissue remodeling that may lead to heart failure or sudden death. MicroRNAs (miRs) are negative regulators of angiotensin II and the angiotensin II receptor subtype 1 (AGTR1), which are two components involved in cardiac hypertrophy. In the present study, the interaction between angiotensin II receptor subtype 1 (AGTR1) signaling and miR-155 was investigated. Rat H9C2 (2–1) cardiomyocytes were transfected with miR-155 analogues or inhibitors, then stimulated with angiotensin II to induce cardiac hypertrophy. miR-155 expression was revealed to be altered following transfection with chemically-modified miR-155 analogues and inhibitors in rat cardiomyocytes. In cell cardiac hypertrophy models, the cell surface area, AGTR1, atrial natriuretic peptide and myosin heavy chain-β mRNA expression levels were revealed to be lower in cells stimulated with miR-155 analogue-transfected cells treated with angiotensin II compared with cells stimulated with angiotensin alone (P<0.05), as determined using reverse transcription-polymerase chain reaction (PCR), quantitative PCR and western blot analyses. Furthermore, calcineurin mRNA and protein, intracellular free calcium and nuclear factor of activated T-cells-4 proteins were downregulated in miR-155 analogue-transfected cells treated with angiotensin II, as compared with cells stimulated with angiotensin II alone (P<0.05). In conclusion, the current study indicates that miR-155 may improve cardiac hypertrophy by downregulating AGTR1 and suppressing the calcium signaling pathways activated by AGTR1. PMID:27588076

  5. Impaired Bone Resorption by Lipopolysaccharide In Vivo in Mice Deficient in the Prostaglandin E Receptor EP4 Subtype

    PubMed Central

    Sakuma, Yoko; Tanaka, Kiyoshi; Suda, Michio; Komatsu, Yasato; Yasoda, Akihiro; Miura, Masako; Ozasa, Ami; Narumiya, Shuh; Sugimoto, Yukihiko; Ichikawa, Atsushi; Ushikubi, Fumitaka; Nakao, Kazuwa

    2000-01-01

    In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions. PMID:11083800

  6. Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

    PubMed Central

    Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

    2013-01-01

    Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

  7. alpha. -Adrenergic vasoconstriction and receptor subtypes in large coronary arteries of calves

    SciTech Connect

    Young, M.A.; Vatner, D.E.; Knight, D.R.; Graham, R.M.; Homcy, C.J.; Vatner, S.F. New England Regional Primate Research Center, Southborough, MA )

    1988-12-01

    The authors investigated {alpha}-adrenoceptor subtype distribution in large coronary arteries from both functional and biochemical perspectives. The effects of intracoronary administration of the selective {alpha}{sub 1}-adrenoceptor agonist phenylephrine, of the selective {alpha}{sub 2}-adrenoceptor agonist B-HT 920 and of the mixed {alpha}{sub 1+2}-adrenoceptor agonist norepinephrine were examined on measurements of left circumflex coronary artery diameter in conscious calves. After {beta}-adrenergic blockade, equivalent reductions in large coronary artery diameter were observed with phenylephrine, B-HT, and norepinephrine. Phenylephrine-induced constrictions were abolished by prazosin, an {alpha}{sub 1}-selective antagonist, but unaffected by rauwolscine, an {alpha}{sub 2}-selective antagonist. Conversely, the B-HT-induced constriction was abolished by rauwolscine but unaffected by prazosin. Coronary constriction with norepinephrine was attenuated with either prazosin or rauwolscine and abolished by the two antagonists combined. Ligand-binding studies in which ({sup 3}H)prazosin and ({sup 3}H)rauwolscine and sarcolemmal membranes were used revealed an {alpha}{sub 1}-adrenoceptor density of 15 {plus minus} 3.1 fmol/mg protein with a dissociation constant (K{sub D}) of 0.7 {plus minus} 0.2 nM and an {alpha}{sub 2}-adrenoceptor density of 68 {plus minus} 5.1 fmol/mg protein, with a K{sub D} of 7.4 {plus minus} 1.2 nM. Thus large coronary arteries of the calf contain both {alpha}{sub 1}- and {alpha}{sub 2}-adrenoceptor subtypes, each of which elicits constriction of the large coronary artery in the conscious animal.

  8. Alpha-2A adrenergic receptor subtype gene expression in the intestines of cocaine-exposed rat embryos.

    PubMed

    Ward, Laura P; Hill, Joanna M; McCune, Susan K

    2002-10-01

    Cocaine has become a popular illicit drug in our society, and pregnant women are not immune from this epidemic. Recently, there have been several references in the literature describing an association between prenatal cocaine exposure and the subsequent development of necrotizing enterocolitis in the neonate, but the mechanism underlying this relationship remains speculative. Because alpha-2 adrenergic receptors are thought to play a role in the autoregulatory mechanism in the newborn intestine that responds to hypoxia and ischemia, we examined the expression of this receptor in the intestine of embryonic rats exposed to low- and high-dose cocaine in utero. Pregnant Sprague Dawley rats were injected daily with either saline, low-dose cocaine, or high-dose cocaine beginning on embryonic d 5 (E 5) and continuing to E 20. Mothers were killed on E 16, E 17, E 18, E 19, and E 20. Embryos were frozen and stored at -80 degrees C. In situ hybridization was performed on 20- micro m sections with 35S-labeled oligonucleotide probes specific for the alpha-2A adrenergic receptor subtype. Densitometric analysis revealed a significant decrease in the alpha-2A receptor expression in the intestine of both the low-dose and high-dose cocaine-exposed animals compared with controls. This down-regulation was demonstrated by E 17, and continued through the remainder of gestation. These changes may limit the normal adaptation to vasoconstriction, thus exacerbating the already insufficient compensatory mechanisms for responding to ischemic injury, and thus may be one of the important factors predisposing cocaine-exposed infants to necrotizing enterocolitis.

  9. Receptor subtypes mediating depressor responses to microinjections of nicotine into medial NTS of the rat.

    PubMed

    Dhar, S; Nagy, F; McIntosh, J M; Sapru, H N

    2000-07-01

    Microinjections (50 nl) of nicotine (0.01-10 microM) into the nucleus of the solitary tract (NTS) of adult, urethan-anesthetized, artificially ventilated, male Wistar rats, elicited decreases in blood pressure and heart rate. Prior microinjections of alpha-bungarotoxin (alpha-BT) and alpha-conotoxin ImI (specific toxins for nicotinic receptors containing alpha7 subunits) elicited a 20-38% reduction in nicotine responses. Similarly, prior microinjections of hexamethonium, mecamylamine, and alpha-conotoxin AuIB (specific blockers or toxin for nicotinic receptors containing alpha3beta4 subunits) elicited a 47-79% reduction in nicotine responses. Nicotine responses were completely blocked by prior sequential microinjections of alpha-BT and mecamylamine into the NTS. Complete blockade of excitatory amino acid receptors (EAARs) in the NTS did not attenuate the responses to nicotine. It was concluded that 1) the predominant type of nicotinic receptor in the NTS contains alpha3beta4 subunits, 2) a smaller proportion contains alpha7 subunits, 3) the presynaptic nicotinic receptors in the NTS do not contribute to nicotine-induced responses, and 4) EAARs in the NTS are not involved in mediating responses to nicotine.

  10. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor

    SciTech Connect

    Sobolevsky, Alexander I.; Rosconi, Michael P.; Gouaux, Eric

    2010-02-02

    Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact ionotropic glutamate receptor are unknown. Here we report the crystal structure of the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 {angstrom} resolution in complex with a competitive antagonist. The receptor harbours an overall axis of two-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting four-fold symmetry. A symmetry mismatch between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis, we suggest that GluN1 and GluN2A NMDA (N-methyl-D-aspartate) receptors have a similar architecture, with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non-competitive antagonists and pore blockers.

  11. Identification of four areas each enriched in a unique muscarinic receptor subtype

    SciTech Connect

    Hoss, W.; Ellerbrock, B.R.; Goldman, P.S.; Collins, D.A.; Messer, W.S. Jr. )

    1990-01-01

    The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC{sub 50} values and Hill coefficients for the inhibition of the binding of 0.2 nM ({sup 3}H)-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol >> arecoline > bethanechol > McN A343 = oxotremorine = pilocarpine.

  12. Axospinous synaptic subtype-specific differences in structure, size, ionotropic receptor expression, and connectivity in apical dendritic regions of rat hippocampal CA1 pyramidal neurons

    PubMed Central

    Nicholson, Daniel A.; Geinisman, Yuri

    2008-01-01

    The morphology of axospinous synapses and their parent spines varies widely. Additionally, many of these synapses are contacted by multiple synapse boutons (MSBs) and show substantial variability in receptor expression. The two major axospinous synaptic subtypes are perforated and nonperforated, but there are several subcategories within these two classes. The present study used serial section electron microscopy to determine whether perforated and nonperforated synaptic subtypes differed with regard to their distribution, size, receptor expression, and connectivity to MSBs in three apical dendritic regions of rat hippocampal area CA1: the proximal and distal thirds of stratum radiatum, and stratum lacunosum-moleculare. All synaptic subtypes were present throughout the apical dendritic regions, but there were several subclass-specific differences. First, segmented, completely partitioned synapses changed in number, proportion, and AMPA receptor expression with distance from the soma beyond that found within other perforated synaptic subtypes. Second, atypically large nonperforated synapses showed NMDA receptor immunoreactivity identical to perforated synapses, levels of AMPA receptor expression intermediate to nonperforated and perforated synapses, and perforated synapse-like changes in structure with distance from the soma. Finally, MSB connectivity was highest in proximal stratum radiatum, but only for those MSBs comprised of nonperforated synapses. The immunogold data suggest that most MSBs would not generate simultaneous depolarizations in multiple neurons or spines, however, because the vast majority of MSBs are comprised of two synapses with abnormally low levels of receptor expression, or involve one synapse with a high level of receptor expression and another with only a low level. PMID:19006199

  13. Dual effects of nicotine on dopamine neurons mediated by different nicotinic receptor subtypes.

    PubMed

    Schilström, Björn; Rawal, Nina; Mameli-Engvall, Monica; Nomikos, George G; Svensson, Torgny H

    2003-03-01

    Burst firing of dopaminergic neurons has been found to represent a particularly effective means of increasing dopamine release in terminal areas as well as activating immediate early genes in dopaminoceptive cells. Spontaneous burst firing is largely controlled by the level of activation of NMDA receptors in the ventral tegmental area (VTA) as a consequence of glutamate released from afferents arising mainly in the prefrontal cortex. Nicotine has been found to effectively increase burst firing of dopaminergic cells. This effect of nicotine may be due to an alpha 7 nicotinic receptor-mediated presynaptic facilitation of glutamate release in the VTA. By the use of in-vivo single-cell recordings and immunohistochemistry we here evaluated the role of alpha 7 nicotinic receptors in nicotine-induced burst firing of dopamine cells in the VTA and the subsequent activation of immediate early genes in dopaminoceptive target areas. Nicotine (0.5 mg/kg s.c.) was found to increase firing rate and burst firing of dopaminergic neurons. In the presence of methyllycaconitine (MLA, 6.0 mg/kg i.p.) nicotine only increased firing rate. Moreover, in the presence of dihydro-beta-erythroidine (DH beta E, 1.0 mg/kg i.p.), an antagonist at non-alpha 7 nicotinic receptors, nicotine produced an increase in burst firing without increasing the firing rate. Nicotine also increased Fos-like immunoreactivity in dopamine target areas, an effect that was antagonized with MLA but not with DH beta E. Our data suggest that nicotine's augmenting effect on burst firing is, indeed, due to stimulation of alpha 7 nicotinic receptors whereas other nicotinic receptors seem to induce an increase in firing frequency.

  14. The oxytocin/vasopressin receptor family has at least five members in the gnathostome lineage, inclucing two distinct V2 subtypes.

    PubMed

    Ocampo Daza, Daniel; Lewicka, Michalina; Larhammar, Dan

    2012-01-01

    The vertebrate oxytocin and vasopressin receptors form a family of G-protein-coupled receptors (GPCRs) that mediate a large variety of functions, including social behavior and the regulation of blood pressure, water balance and reproduction. In mammals four family members have been identified, three of which respond to vasopressin (VP) named V1A, V1B and V2, and one of which is activated by oxytocin (OT), called the OT receptor. Four receptors have been identified in chicken as well, but these have received different names. Until recently only V1-type receptors have been described in several species of teleost fishes. We have identified family members in several gnathostome genomes and performed phylogenetic analyses to classify OT/VP-receptors across species and determine orthology relationships. Our phylogenetic tree identifies five distinct ancestral gnathostome receptor subtypes in the OT/VP receptor family: V1A, V1B, V2A, V2B and OT receptors. The existence of distinct V2A and V2B receptors has not been previously recognized. We have found these two subtypes in all examined teleost genomes as well as in available frog and lizard genomes and conclude that the V2A-type is orthologous to mammalian V2 receptors whereas the V2B-type is orthologous to avian V2 receptors. Some teleost fishes have acquired additional and more recent gene duplicates with up to eight receptor family members. Thus, this analysis reveals an unprecedented complexity in the gnathostome repertoire of OT/VP receptors, opening interesting research avenues regarding functions such as regulation of water balance, reproduction and behavior, particularly in reptiles, amphibians, teleost fishes and cartilaginous fishes. PMID:22057000

  15. Human alpha 2-adrenergic receptor subtype distribution: widespread and subtype-selective expression of alpha 2C10, alpha 2C4, and alpha 2C2 mRNA in multiple tissues.

    PubMed

    Eason, M G; Liggett, S B

    1993-07-01

    At present, molecular cloning and pharmacological studies have delineated three human alpha 2-adrenergic receptor (alpha 2AR) subtypes, alpha 2C10, alpha 2C4, and alpha 2C2. Assignment of the alpha 2AR subtypes to specific functions has been limited by an unclear definition of tissue alpha 2AR expression outside of the central nervous system. It has been suggested that alpha 2C4 expression is confined to the brain, that alpha 2C2 expression is only in the liver and kidney, and that there is nearly ubiquitous expression of alpha 2C10. However, this is based on studies of a limited number of rat tissues or on studies using non-species-specific approaches. Therefore, to define alpha 2C10, alpha 2C4, and alpha 2C2 tissue expression, we used reverse transcription of total RNA isolated from 20 human tissues, followed by amplification of alpha 2AR cDNA using the polymerase chain reaction. This technique provided two advantages: high sensitivity and, with the use of subtype-specific oligonucleotide primers and probes, differentiation between the alpha 2AR subtypes. The tissues studied were aorta, vena cava, heart (epicardium and endocardium), lung, skeletal muscle, liver, pancreas (head and tail), fat (perinephric and subcutaneous), kidney (cortex and medulla), prostate, stomach, ileum, jejunum, colon, adrenal gland, and spleen. We found that the majority of these tissues expressed alpha 2C10, with the exceptions being the head of the pancreas, subcutaneous fat, colon, and spleen. In marked distinction to other studies, however, we found a prolific expression of the alpha 2C4 and alpha 2C2 subtypes. Expression of alpha 2C4 was found in all tissues with the exception of liver, fat, stomach, and colon, and a virtually ubiquitous expression of alpha 2C2 was found, with the exception of epicardium. Of all tissues studied, only colon and subcutaneous fat expressed a single alpha 2AR subtype, which was alpha 2C2. Thus, the alpha 2AR subtypes do not have a confined expression but

  16. Human alpha 2-adrenergic receptor subtype distribution: widespread and subtype-selective expression of alpha 2C10, alpha 2C4, and alpha 2C2 mRNA in multiple tissues.

    PubMed

    Eason, M G; Liggett, S B

    1993-07-01

    At present, molecular cloning and pharmacological studies have delineated three human alpha 2-adrenergic receptor (alpha 2AR) subtypes, alpha 2C10, alpha 2C4, and alpha 2C2. Assignment of the alpha 2AR subtypes to specific functions has been limited by an unclear definition of tissue alpha 2AR expression outside of the central nervous system. It has been suggested that alpha 2C4 expression is confined to the brain, that alpha 2C2 expression is only in the liver and kidney, and that there is nearly ubiquitous expression of alpha 2C10. However, this is based on studies of a limited number of rat tissues or on studies using non-species-specific approaches. Therefore, to define alpha 2C10, alpha 2C4, and alpha 2C2 tissue expression, we used reverse transcription of total RNA isolated from 20 human tissues, followed by amplification of alpha 2AR cDNA using the polymerase chain reaction. This technique provided two advantages: high sensitivity and, with the use of subtype-specific oligonucleotide primers and probes, differentiation between the alpha 2AR subtypes. The tissues studied were aorta, vena cava, heart (epicardium and endocardium), lung, skeletal muscle, liver, pancreas (head and tail), fat (perinephric and subcutaneous), kidney (cortex and medulla), prostate, stomach, ileum, jejunum, colon, adrenal gland, and spleen. We found that the majority of these tissues expressed alpha 2C10, with the exceptions being the head of the pancreas, subcutaneous fat, colon, and spleen. In marked distinction to other studies, however, we found a prolific expression of the alpha 2C4 and alpha 2C2 subtypes. Expression of alpha 2C4 was found in all tissues with the exception of liver, fat, stomach, and colon, and a virtually ubiquitous expression of alpha 2C2 was found, with the exception of epicardium. Of all tissues studied, only colon and subcutaneous fat expressed a single alpha 2AR subtype, which was alpha 2C2. Thus, the alpha 2AR subtypes do not have a confined expression but

  17. Estrogen Receptors Alpha (ERα) and Beta (ERβ): Subtype-Selective Ligands and Clinical Potential

    PubMed Central

    Paterni, Ilaria; Granchi, Carlotta; Katzenellenbogen, John A.; Minutolo, Filippo

    2014-01-01

    Estrogen receptors alpha (ERα) and beta (ERβ) are nuclear transcription factors that are involved in the regulation of many complex physiological processes in humans. Modulation of these receptors by prospective therapeutic agents is currently being considered for prevention and treatment of a wide variety of pathological conditions, such as, cancer, metabolic and cardiovascular diseases, neurodegeneration, inflammation, and osteoporosis. This review provides an overview and update of compounds that have been recently reported as modulators of ERs, with a particular focus on their potential clinical applications. PMID:24971815

  18. Characterization of multiple membrane progestin receptor (mPR) subtypes from the goldfish ovary and their roles in the induction of oocyte maturation.

    PubMed

    Tokumoto, Toshinobu; Tokumoto, Mika; Oshima, Takayuki; Shimizuguchi, Kumi; Fukuda, Tatsuya; Sugita, Etsuko; Suzuki, Manami; Sakae, Yu-ta; Akiyama, Yu-ichi; Nakayama, Ryo; Roy, Shimi Rani; Saydur Rahman, Md; Pang, Yefei; Dong, Jing; Thomas, Peter

    2012-05-15

    Oocyte maturation (OM) in goldfish is induced by the maturation inducing hormone (MIH) via its membrane receptor. Previously, we described the cloning of the membrane progesterone receptor alpha (mPRα or paqr7b) cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIH induction of OM in goldfish. Three mPR subtypes have been identified in fish by cDNA cloning or by in silico analysis of genome sequence databases. In order to investigate the potential roles of the mPR subtypes in oocyte maturation, we cloned additional mPRs from a goldfish ovarian cDNA library. RACE amplification, and screening of the cDNA library identified one β (paqr8) and two γ subtypes (paqr5) (hereafter referred to as γ-1 and γ-2), respectively. Tissue distribution of mPR subtypes showed differential expression pattern. However, in addition to mPRα, the β, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Cell lines expressing the β, γ-1 and γ-2 genes were established and their steroid binding properties compared. The β subtype exhibited higher binding affinity than the γ subtypes for 17,20β-DHP, the MIH in goldfish. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRβ blocked the induction of oocyte maturational competence, whereas injection of antisense oliogonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that the goldfish mPRβ protein acts as an intermediary during MIH induction of OM in goldfish, in a manner similar to that described previously for mPRα.

  19. Cytoplasmic domain of δ subunit is important for the extra-synaptic targeting of GABAA receptor subtypes.

    PubMed

    Arslan, Ayla; von Engelhardt, Jakob; Wisden, William

    2014-12-01

    GABA(A) receptors (GABA(A)Rs) are hetero-pentameric chloride channels and the primary sites for fast synaptic inhibition. We have expressed recombinant γ2 and δ subunits of GABA(A)Rs in cultured hippocampal neurons to analyze the membrane targeting of synaptic and extra-synaptic GABA(A)Rs, a phenomenon not well understood. Our data demonstrate that the synaptic targeting of γ2-containing GABA(A)Rs (γ2-GABA(A)Rs) does not depend on the cytoplasmic loop of γ2 subunit, in parallel with previous findings, showing that the synaptic localization of γ2-GABA(A)Rs requires the TM4 domain of γ2 rather than the large cytoplasmic loop. On the other hand, we showed here that the extrasynaptic targeting of the δ-containing GABA(A)Rs (δ-GABA(A)Rs) depends on the cytoplasmic loop of δ subunit via an active or a passive mechanism. We also show that the amino acid sequences of δ loop is highly conserved across the whole span of vertebrate evolution suggesting an active role of δ loop in extra-synaptic targeting of corresponding receptor subtypes. PMID:25233879

  20. Anatomical characterization of bombesin receptor subtype-3 mRNA expression in the rodent central nervous system.

    PubMed

    Zhang, Li; Parks, Gregory S; Wang, Zhiwei; Wang, Lien; Lew, Michelle; Civelli, Olivier

    2013-04-01

    Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) involved in the regulation of energy homeostasis. Mice deficient in BRS-3 develop late-onset mild obesity with metabolic defects, while synthetic agonists activating BRS-3 show antiobesity profiles by inhibiting food intake and increasing metabolic rate in rodent models. The molecular mechanisms and the neural circuits responsible for these effects, however, remain elusive and demand better characterization. We report here a comprehensive mapping of BRS-3 mRNA in the rat and mouse brain through in situ hybridization. Furthermore, to investigate the neurochemical characteristics of the BRS-3-expressing neurons, double in situ hybridization was performed to determine whether BRS-3 colocalizes with other neurotransmitters or neuropeptides. Many, but not all, of the BRS-3-expressing neurons were found to be glutamatergic, while few were found to be cholinergic or GABAergic. BRS-3-containing neurons do not express some of the well-characterized neuropeptides, such as neuropeptide Y (NPY), proopiomelanocortin (POMC), orexin/hypocretin, melanin-concentrating hormone (MCH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and kisspeptin. Interestingly, BRS-3 mRNA was found to partially colocalize with corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), suggesting novel interactions of BRS-3 with stress- and growth-related endocrine systems. Our study provides important information for evaluating BRS-3 as a potential therapeutic target for the treatment of obesity. PMID:22911445

  1. Cytoplasmic domain of δ subunit is important for the extra-synaptic targeting of GABAA receptor subtypes.

    PubMed

    Arslan, Ayla; von Engelhardt, Jakob; Wisden, William

    2014-12-01

    GABA(A) receptors (GABA(A)Rs) are hetero-pentameric chloride channels and the primary sites for fast synaptic inhibition. We have expressed recombinant γ2 and δ subunits of GABA(A)Rs in cultured hippocampal neurons to analyze the membrane targeting of synaptic and extra-synaptic GABA(A)Rs, a phenomenon not well understood. Our data demonstrate that the synaptic targeting of γ2-containing GABA(A)Rs (γ2-GABA(A)Rs) does not depend on the cytoplasmic loop of γ2 subunit, in parallel with previous findings, showing that the synaptic localization of γ2-GABA(A)Rs requires the TM4 domain of γ2 rather than the large cytoplasmic loop. On the other hand, we showed here that the extrasynaptic targeting of the δ-containing GABA(A)Rs (δ-GABA(A)Rs) depends on the cytoplasmic loop of δ subunit via an active or a passive mechanism. We also show that the amino acid sequences of δ loop is highly conserved across the whole span of vertebrate evolution suggesting an active role of δ loop in extra-synaptic targeting of corresponding receptor subtypes.

  2. Early emergence of three dopamine D1 receptor subtypes in vertebrates. Molecular phylogenetic, pharmacological, and functional criteria defining D1A, D1B, and D1C receptors in European eel Anguilla anguilla.

    PubMed

    Cardinaud, B; Sugamori, K S; Coudouel, S; Vincent, J D; Niznik, H B; Vernier, P

    1997-01-31

    The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum. PMID:9006917

  3. In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment

    PubMed Central

    Yang, Li; Islam, Mohammad R; Karamyan, Vardan T.; Abbruscato, Thomas J.

    2015-01-01

    To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10 nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10 nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype. PMID:25801116

  4. Lack of the Metabotropic Glutamate Receptor Subtype 7 Selectively Modulates Theta Rhythm and Working Memory

    ERIC Educational Resources Information Center

    Holscher, Christian; Schmid, Susanne; Pilz, Peter K. D.; Sansig, Gilles; van der Putten, Herman; Plappert, Claudia F.

    2005-01-01

    Metabotropic glutamate receptors (mGluRs) are known to play a role in synaptic plasticity and learning. We have previously shown that mGluR7 deletion in mice produces a selective working memory (WM) impairment, while other types of memory such as reference memory remain unaffected. Since WM has been associated with Theta activity (6-12 Hz) in…

  5. Data on alteration of hormone and growth factor receptor profiles over progressive passages of breast cancer cell lines representing different clinical subtypes.

    PubMed

    Nair, Madhumathy G; Desai, Krisha; Prabhu, Jyothi S; Hari, P S; Remacle, Jose; Sridhar, T S

    2016-09-01

    Human breast cancers are a highly heterogeneous group of tumours consisting of several molecular subtypes with a variable profile of hormone, growth factor receptors and cytokeratins [1]. Here, the data shows immunofluorescence profiling of four different cell lines belonging to distinct clinical subtypes of breast cancer. Post revival, the cell lines were passaged in culture and immunophenotyping was done for ER, HER-2, AR and EGFR. Data for the markers from early passage (5th) through passages as late as 25 for the different cell lines is presented. PMID:27508248

  6. Improvements in the methodology for analyzing receptor subtypes and neuronal populations affected by anticholinesterase exposure. Annual summary report, 15 November 1983-14 November 1984

    SciTech Connect

    Wamsley, J.K.

    1984-11-14

    Conditions were defined that provide a means of selectively labeling subtypes of muscarinic receptors. The so-called M1 receptor population can be labeled with tritiated pirenzepine, while the receptor population labeled with tritiated quinuclidinyl benzilate (QNB) but not labeled with pirenzepine represents M2 receptor population. High- and low-affinity states of the receptors were also defined on the basis of agonist displacement of antagonist binding. Both the M1 and M2 receptor populations undergo axonal transport and the affinity states of these receptors are altered by neurochemical and neurosurgical lesions. Radioactive standards were developed that provide a means of quantitating the femtomoles of receptor bound with each ligand in microscopic regions of the brain. The technology was also devised to directly localize nicotinic cholinergic receptors using tritiated nicotine. It is now possible to localize several peptide receptors associated with cholinergic function including receptors for thyrotropin-releasing hormone (TRH) and somatostatin. The receptor autoradiographic technique was also carried beyond the receptor level of localization by using compounds to label adenylate cyclase and the GTP binding protein. This methodology should provide an elegant means of determining how anticholinesterase exposure has affected these many parameters of cholinergic nerve function.

  7. An immune response gene expression module identifies a good prognosis subtype in estrogen receptor negative breast cancer

    PubMed Central

    Teschendorff, Andrew E; Miremadi, Ahmad; Pinder, Sarah E; Ellis, Ian O; Caldas, Carlos

    2007-01-01

    Background Estrogen receptor (ER)-negative breast cancer specimens are predominantly of high grade, have frequent p53 mutations, and are broadly divided into HER2-positive and basal subtypes. Although ER-negative disease has overall worse prognosis than does ER-positive breast cancer, not all ER-negative breast cancer patients have poor clinical outcome. Reliable identification of ER-negative tumors that have a good prognosis is not yet possible. Results We apply a recently proposed feature selection method in an integrative analysis of three major microarray expression datasets to identify molecular subclasses and prognostic markers in ER-negative breast cancer. We find a subclass of basal tumors, characterized by over-expression of immune response genes, which has a better prognosis than the rest of ER-negative breast cancers. Moreover, we show that, in contrast to ER-positive tumours, the majority of prognostic markers in ER-negative breast cancer are over-expressed in the good prognosis group and are associated with activation of complement and immune response pathways. Specifically, we identify an immune response related seven-gene module and show that downregulation of this module confers greater risk for distant metastasis (hazard ratio 2.02, 95% confidence interval 1.2-3.4; P = 0.009), independent of lymph node status and lymphocytic infiltration. Furthermore, we validate the immune response module using two additional independent datasets. Conclusion We show that ER-negative basal breast cancer is a heterogeneous disease with at least four main subtypes. Furthermore, we show that the heterogeneity in clinical outcome of ER-negative breast cancer is related to the variability in expression levels of complement and immune response pathway genes, independent of lymphocytic infiltration. PMID:17683518

  8. Electroacupuncture Inhibition of Hyperalgesia in Rats with Adjuvant Arthritis: Involvement of Cannabinoid Receptor 1 and Dopamine Receptor Subtypes in Striatum

    PubMed Central

    Shou, Yin; Yang, Yang; Xu, Ming-Shu; Zhao, Ying-Qian; Ge, Lin-Bao; Zhang, Bi-Meng

    2013-01-01

    Electroacupuncture (EA) has been regarded as an alternative treatment for inflammatory pain for several decades. However, the molecular mechanisms underlying the antinociceptive effect of EA have not been thoroughly clarified. Previous studies have shown that cannabinoid CB1 receptors are related to pain relief. Accumulating evidence has shown that the CB1 and dopamine systems sometimes interact and may operate synergistically in rat striatum. To our knowledge, dopamine D1/D2 receptors are involved in EA analgesia. In this study, we found that repeated EA at Zusanli (ST36) and Kunlun (BL60) acupoints resulted in marked improvements in thermal hyperalgesia. Both western blot assays and FQ-PCR analysis results showed that the levels of CB1 expression in the repeated-EA group were much higher than those in any other group (P = 0.001). The CB1-selective antagonist AM251 inhibited the effects of repeated EA by attenuating the increases in CB1 expression. The two kinds of dopamine receptors imparted different actions on the EA-induced CB1 upregulation in AA rat model. These results suggested that the strong activation of the CB1 receptor after repeated EA resulted in the concomitant phenomenon of the upregulation of D1 and D2 levels of gene expression. PMID:23762129

  9. Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

    PubMed Central

    Zholos, Alexander V; Bolton, Thomas B

    1997-01-01

    The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4±1.4 nS and a mean −log EC50 of 5.12±0.03 (mean±s.e.mean) (n=114). Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of

  10. Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells.

    PubMed

    Murillo, Laurence; Hamon, Alain; Es-Salah-Lamoureux, Zeineb; Itier, Valérie; Quinchard, Sophie; Lapied, Bruno

    2011-12-01

    Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil. PMID:21684305

  11. The role of histological subtype in hormone receptor positive metastatic breast cancer: similar survival but different therapeutic approaches

    PubMed Central

    Lobbezoo, Dorien; Truin, Wilfred; Voogd, Adri; Roumen, Rudi; Vreugdenhil, Gerard; Dercksen, Marcus Wouter; van den Berkmortel, Franchette; Smilde, Tineke; van de Wouw, Agnes; van Kampen, Roel; van Riel, Johanna; Peters, Natascha; Peer, Petronella; Tjan-Heijnen, Vivianne C.G.

    2016-01-01

    Introduction This study describes the differences between the two largest histological breast cancer subtypes (invasive ductal carcinoma (IDC) and invasive (mixed) lobular carcinoma (ILC) with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. Results Patients with ILC were older at diagnosis of primary breast cancer and had more often initial bone metastasis (46.5% versus 34.8%, P = 0.01) and less often multiple metastatic sites compared to IDC (23.7% versus 30.9%, P = 0.11). Six months after diagnosis of metastatic breast cancer, 28.1% of patients with ILC and 39.8% of patients with IDC had received chemotherapy with a longer median time to first chemotherapy for those with ILC (P = 0.001). After six months 84.8% of patients with ILC had received endocrine therapy versus 72.5% of patients with IDC (P = 0.0001). Median overall survival was 29 months for ILC and 25 months for IDC (P = 0.53). Materials and Methods We included 437 patients with hormone receptor-positive IDC and 131 patients with hormone receptor-positive ILC, all diagnosed with metastatic breast cancer between 2007–2009, irrespective of date of the primary diagnosis. Patient and tumor characteristics and data on treatment and outcome were collected. Survival curves were obtained using the Kaplan-Meier method. Conclusions Treatment strategies of hormone receptor-positive metastatic breast cancer were remarkably different for patients with ILC and IDC. Further research is required to understand tumor behavior and treatment-choices in real-life. PMID:27121067

  12. Conformational Constraint of the Glycerol Moiety of Lysophosphatidylserine Affords Compounds with Receptor Subtype Selectivity.

    PubMed

    Jung, Sejin; Inoue, Asuka; Nakamura, Sho; Kishi, Takayuki; Uwamizu, Akiharu; Sayama, Misa; Ikubo, Masaya; Otani, Yuko; Kano, Kuniyuki; Makide, Kumiko; Aoki, Junken; Ohwada, Tomohiko

    2016-04-28

    Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator that specifically activates membrane proteins of the P2Y and its related families of G protein-coupled receptors (GPCR), GPR34 (LPS1), P2Y10 (LPS2), and GPR174 (LPS3). Here, in order to increase potency and receptor selectivity, we designed and synthesized LysoPS analogues containing the conformational constraints of the glycerol moiety. These reduced structural flexibility by fixation of the glycerol framework of LysoPS using a 2-hydroxymethyl-3-hydroxytetrahydropyran skeleton, and related structures identified compounds which exhibited high potency and selectivity for activation of GPR34 or P2Y10. Morphing of the structural shape of the 2-hydroxymethyl-3-hydroxytetrahydropyran skeleton into a planar benzene ring enhanced the P2Y10 activation potentcy rather than the GPR34 activation. PMID:27077565

  13. Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena)

    PubMed Central

    2013-01-01

    Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72 hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163+Sialoadhesin (Sn)+, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163+Sn+, CD163+Sn- and to a lesser extent CD163-Sn- monocytic subtypes were involved in infection. CD163+Sn- cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163+Sn+ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163+Sn- cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena. PMID:24007551

  14. Muscarinic receptor subtypes mediating the mucosal response to neural stimulation of guinea pig ileum

    SciTech Connect

    Carey, H.V.; Tien, X.Y.; Wallace, L.J.; Cooke, H.J.

    1987-09-01

    Muscarinic receptors involved in the secretory response evoked by electrical stimulation of submucosal neutrons were investigated in muscle-stripped flat sheets of guinea pig ileum set up in flux chambers. Neural stimulation produced a biphasic increase in short-circuit current due to active chloride secretion. Atropine and 4-diphenylacetoxy-N-methylpiperadine methiodide (4-DAMP) (10/sup -7/ M) were more potent inhibitors of the cholinergic phase of the response than was pirenzepine. Dose-dependent increases in base-line short-circuit current were evoked by carbachol and bethanechol; 4-hydroxy-2-butynyl trimethylammonium chloride (McN A343) produced a much smaller effect. Tetrodotoxin abolished the effects of McN A343 but did not alter the responses of carbachol and bethanechol. McN A343 significantly reduced the cholinergic phase of the neurally evoked response and caused a rightward shift of the carbachol dose-response curve. All muscarinic compounds inhibited (/sup 3/H)quinuclidinyl benzilate binding to membranes from muscosal scrapings, with a rank order of potency of 4-DAMP > pirenzepine > McN A343 > carbachol > bethanechol. These results suggest that acetylcholine released from submucosal neurons mediates chloride secretion by interacting with muscarinic cholinergic receptors that display a high binding affinity for 4-DAMP. Activation of neural muscarinic receptors makes a relatively small contribution to the overall secretory response.

  15. The Role of NMDA Receptor Subtypes in Short-Term Plasticity in the Rat Entorhinal Cortex

    PubMed Central

    Chamberlain, Sophie E. L.; Yang, Jian; Jones, Roland S. G.

    2008-01-01

    We have previously shown that spontaneous release of glutamate in the entorhinal cortex (EC) is tonically facilitated via activation of presynaptic NMDA receptors (NMDAr) containing the NR2B subunit. Here we show that the same receptors mediate short-term plasticity manifested by frequency-dependent facilitation of evoked glutamate release at these synapses. Whole-cell patch-clamp recordings were made from layer V pyramidal neurones in rat EC slices. Evoked excitatory postsynaptic currents showed strong facilitation at relatively low frequencies (3 Hz) of activation. Facilitation was abolished by an NR2B-selective blocker (Ro 25-6981), but unaffected by NR2A-selective antagonists (Zn2+, NVP-AAM077). In contrast, postsynaptic NMDAr-mediated responses could be reduced by subunit-selective concentrations of all three antagonists. The data suggest that NMDAr involved in presynaptic plasticity in layer V are exclusively NR1/NR2B diheteromers, whilst postsynaptically they are probably a mixture of NR1/NR2A, NR1/NR2B diheteromers and NR1/NR2A/NR2B triheteromeric receptors. PMID:18989370

  16. Differential gene expression of the three natriuretic peptides and natriuretic peptide receptor subtypes in human liver.

    PubMed Central

    Vollmar, A M; Paumgartner, G; Gerbes, A L

    1997-01-01

    BACKGROUND: Various effects of atrial natriuretic peptide (ANP) on the liver have been observed. However, there is limited information about the types of receptors for natriuretic peptides expressed by the human liver. AIM: To investigate gene expression of the three NP receptor types (NPR) as well as of the NP in human liver. METHODS: Presence of mRNA coding for all three NPR and for ANP, brain and C-type natriuretic peptide (BNP, CNP) was investigated by reverse transcription-polymerase chain reaction (RT-PCR). Human liver tissues and hepatocellular carcinoma tissues were examined. RESULTS: Specific PCR products for all three NPR, namely NPR-A, B, and C, could be detected. Moreover, ANP and CNP, but not BNP mRNA was detectable. The concentration of ANP transcripts was up to fivefold higher in hepatocellular carcinoma compared with non-tumorous liver tissue of the same subjects. No difference in the expression of NP receptors relative to GAPDH mRNA of tumorous and non-tumorous tissue was observed except of slightly increased NPR-A transcripts. CONCLUSION: These data show that NPR transcripts are coexpressed with ANP and CNP mRNA in the human liver. This provides evidence for a local NP system in the human liver. Images PMID:9155593

  17. Competitive molecular docking approach for predicting estrogen receptor subtype α agonists and antagonists

    PubMed Central

    2014-01-01

    Background Endocrine disrupting chemicals (EDCs) are exogenous compounds that interfere with the endocrine system of vertebrates, often through direct or indirect interactions with nuclear receptor proteins. Estrogen receptors (ERs) are particularly important protein targets and many EDCs are ER binders, capable of altering normal homeostatic transcription and signaling pathways. An estrogenic xenobiotic can bind ER as either an agonist or antagonist to increase or inhibit transcription, respectively. The receptor conformations in the complexes of ER bound with agonists and antagonists are different and dependent on interactions with co-regulator proteins that vary across tissue type. Assessment of chemical endocrine disruption potential depends not only on binding affinity to ERs, but also on changes that may alter the receptor conformation and its ability to subsequently bind DNA response elements and initiate transcription. Using both agonist and antagonist conformations of the ERα, we developed an in silico approach that can be used to differentiate agonist versus antagonist status of potential binders. Methods The approach combined separate molecular docking models for ER agonist and antagonist conformations. The ability of this approach to differentiate agonists and antagonists was first evaluated using true agonists and antagonists extracted from the crystal structures available in the protein data bank (PDB), and then further validated using a larger set of ligands from the literature. The usefulness of the approach was demonstrated with enrichment analysis in data sets with a large number of decoy ligands. Results The performance of individual agonist and antagonist docking models was found comparable to similar models in the literature. When combined in a competitive docking approach, they provided the ability to discriminate agonists from antagonists with good accuracy, as well as the ability to efficiently select true agonists and antagonists from

  18. Impact of Breast Cancer Subtype Defined by Immunohistochemistry Hormone Receptor and HER2 Status on the Incidence of Immediate Postmastectomy Reconstruction

    PubMed Central

    Wu, Wei; Cheng, Shi; Deng, Heran; Wu, Jiannan; Mao, Kai; Cao, Minghui

    2016-01-01

    Abstract Immediate postmastectomy reconstruction has become an increasingly popular choice for breast cancer patients recently. However, whether molecular subtype of cancer impacts the incidence of breast reconstruction is unclear. We aimed to investigate the association between breast cancer subtype defined by immunohistochemistry hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status and recent rates of immediate postmastectomy reconstruction in the United States. The National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate stage I–III breast cancer patients with different subtypes who underwent either mastectomy alone or mastectomy plus reconstruction between 2010 and 2012. Univariate and multivariate analyses were conducted to identify factors influencing the incidence of immediate reconstruction. Of 47,123 women included, 33.1% (10,712/32,376) of HR+/HER2−, 33.1% (1912/5768) of HR+/HER2+, 29.6% (850/2875) of HR−/HER2+, and 27.7% (1689/6104) of triple negative breast cancer patients received immediate breast reconstruction (chi-square test, P < 0.001), respectively. Thus, HER2-overexpressing and triple negative breast cancer patients received significantly less breast reconstruction. After adjusting for demographic, socioeconomic, geographic, or clinicopathologic factors, HER2-overexpressing (OR 0.896, 95% CI 0.817–0.984) and triple negative (OR 0.806, 95% CI 0.751–0.866) breast cancer patients remained less likely to undergo immediate postmastectomy reconstruction compared with HR+/HER2− or HR+/HER2+ patients. No significant difference was found in the type of reconstruction among different subtypes. Subgroup analysis showed that the difference of breast reconstruction rates among distinct subtypes varied with different grade and stage groups, and the association between breast cancer subtype and the reconstruction rate was not significant in low grade and early stage

  19. Impact of Breast Cancer Subtype Defined by Immunohistochemistry Hormone Receptor and HER2 Status on the Incidence of Immediate Postmastectomy Reconstruction.

    PubMed

    Wu, Wei; Cheng, Shi; Deng, Heran; Wu, Jiannan; Mao, Kai; Cao, Minghui

    2016-01-01

    Immediate postmastectomy reconstruction has become an increasingly popular choice for breast cancer patients recently. However, whether molecular subtype of cancer impacts the incidence of breast reconstruction is unclear. We aimed to investigate the association between breast cancer subtype defined by immunohistochemistry hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status and recent rates of immediate postmastectomy reconstruction in the United States.The National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate stage I-III breast cancer patients with different subtypes who underwent either mastectomy alone or mastectomy plus reconstruction between 2010 and 2012. Univariate and multivariate analyses were conducted to identify factors influencing the incidence of immediate reconstruction.Of 47,123 women included, 33.1% (10,712/32,376) of HR+/HER2-, 33.1% (1912/5768) of HR+/HER2+, 29.6% (850/2875) of HR-/HER2+, and 27.7% (1689/6104) of triple negative breast cancer patients received immediate breast reconstruction (chi-square test, P < 0.001), respectively. Thus, HER2-overexpressing and triple negative breast cancer patients received significantly less breast reconstruction. After adjusting for demographic, socioeconomic, geographic, or clinicopathologic factors, HER2-overexpressing (OR 0.896, 95% CI 0.817-0.984) and triple negative (OR 0.806, 95% CI 0.751-0.866) breast cancer patients remained less likely to undergo immediate postmastectomy reconstruction compared with HR+/HER2- or HR+/HER2+ patients. No significant difference was found in the type of reconstruction among different subtypes. Subgroup analysis showed that the difference of breast reconstruction rates among distinct subtypes varied with different grade and stage groups, and the association between breast cancer subtype and the reconstruction rate was not significant in low grade and early stage patients

  20. Contribution of regional brain melanocortin receptor subtypes to elevated activity energy expenditure in lean, active rats.

    PubMed

    Shukla, C; Koch, L G; Britton, S L; Cai, M; Hruby, V J; Bednarek, M; Novak, C M

    2015-12-01

    Physical activity and non-exercise activity thermogenesis (NEAT) are crucial factors accounting for individual differences in body weight, interacting with genetic predisposition. In the brain, a number of neuroendocrine intermediates regulate food intake and energy expenditure (EE); this includes the brain melanocortin (MC) system, consisting of MC peptides as well as their receptors (MCR). MC3R and MC4R have emerged as critical modulators of EE and food intake. To determine how variance in MC signaling may underlie individual differences in physical activity levels, we examined behavioral response to MC receptor agonists and antagonists in rats that show high and low levels of physical activity and NEAT, that is, high- and low-capacity runners (HCR, LCR), developed by artificial selection for differential intrinsic aerobic running capacity. Focusing on the hypothalamus, we identified brain region-specific elevations in expression of MCR 3, 4, and also MC5R, in the highly active, lean HCR relative to the less active and obesity-prone LCR. Further, the differences in activity and associated EE as a result of MCR activation or suppression using specific agonists and antagonists were similarly region-specific and directly corresponded to the differential MCR expression patterns. The agonists and antagonists investigated here did not significantly impact food intake at the doses used, suggesting that the differential pattern of receptor expression may by more meaningful to physical activity than to other aspects of energy balance regulation. Thus, MCR-mediated physical activity may be a key neural mechanism in distinguishing the lean phenotype and a target for enhancing physical activity and NEAT.

  1. Benzoxazole piperidines as selective and potent somatostatin receptor subtype 5 antagonists.

    PubMed

    Martin, Rainer E; Mohr, Peter; Maerki, Hans Peter; Guba, Wolfgang; Kuratli, Christoph; Gavelle, Olivier; Binggeli, Alfred; Bendels, Stefanie; Alvarez-Sánchez, Rubén; Alker, André; Polonchuk, Liudmila; Christ, Andreas D

    2009-11-01

    SAR studies of a recently described SST5R selective benzoxazole piperidine lead series are described with particular focus on the substitution pattern on the benzyl and benzoxazole side-chains. Introduction of a second meta substituent at the benzyl unit significantly lowers residual hH1 activity and insertion of substituents onto the benzoxazole periphery entirely removes remaining h5-HT2B activity. Compounds with single digit nM activity, functional antagonism and favorable physicochemical properties endowed with a good pharmacokinetic profile in rats are described which should become valuable tools for exploring the pharmacological role of the SST5 receptor in vivo.

  2. Nipecotic acid ethyl ester: a cholinergic agonist that may differentiate muscarinic receptor subtypes

    SciTech Connect

    Zorn, S.H.; Duman, R.S.; Enna, S.J.; Krogsgaard-Larsen, P.; Micheletti, R.; Giraldo, E.; Giachetti, A.

    1986-03-05

    Reports indicate that nipecotic acid ethyl ester (NAEE) displays cholinomimetic properties in vivo. In the present study a series of physiological and biochemical tests were conducted to characterize this action. NAEE had a negative inotropic effect on the guinea pig atrium, and stimulated contraction of the guinea pig ileum and isolated mouse stomach strip at concentrations similar to bethanechol (BCH). The atrial and ilial effects were reversed by atropine. Unlike BCH, NAEE had no effect on basal acid secretion in the isolated mouse stomach at concentrations < 100 ..mu..M. NAEE was more potent than carbachol (CCH) in displacing /sup 3/H-ONB binding from rat brain membranes. The potency of NAEE to inhibit antagonist binding in rat heart membranes was enhanced by Mg/sup + +/ (Hill coefficient < 1.0) and reduced by Gpp(NH)p. Like CCH, NAEE inhibited GTP-stimulated adenylate cyclase in rat brain striatal membranes. As compared to CCH, NAEE had little effect (< 5%) as a stimulator of inositol phosphate (IP) production in rat brain slices. The results indicate that NAEE is a direct-acting muscarinic receptor agonist. Moreover, its differential effects on acid secretion, IP accumulation, and adenylate cyclase suggest that it may be useful for defining cholinergic receptor subclasses.

  3. Metabotropic Glutamate Subtype 5 Receptors Are Quantified in the Human Brain with a Novel Radioligand for PET

    PubMed Central

    Brown, Amira K.; Kimura, Yasuyuki; Zoghbi, Sami S.; Siméon, Fabrice G.; Liow, Jeih-San; Kreisl, William C.; Taku, Andrew; Fujita, Masahiro; Pike, Victor W.; Innis, Robert B.

    2009-01-01

    We developed a radioligand, 3-fluoro-5-(2-(2-18F-(fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile (18F-SP203), for metabotropic glutamate subtype 5 (mGluR5) receptors that showed both promising (high specific binding) and problematic (defluorination) imaging characteristics in animals. The purposes of this initial evaluation in human subjects were to determine whether 18F-SP203 is defluorinated in vivo (as measured by uptake of radioactivity in the skull) and to determine whether the uptake in the brain can be quantified as distribution volume relative to concentrations of 18F-SP203 in plasma. Methods Seven healthy subjects were injected with18F-SP203 (323 ± 87 MBq) and scanned over5 h, with rest periods outside the camera. The concentrations of 18F-SP203, separated from radiometabolites, were measured in arterial plasma. Results The skull was difficult to visualize on PET images in the initial 2 h, because of high radioactivity in the brain. Although radioactivity in the skull and adjacent cortex showed some cross-contamination, the concentration of radioactivity in the skull was less than half of that in the adjacent cortex during the initial 2 h. Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model, consistent with measurable amounts of both receptor-specific and nonspecific binding. The concentrations of activity in the brain measured with PET were consistently greater than the modeled values at late but not early time points and may well have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume, we selected a scan duration (i.e., 2 h) associated with maximal or near-maximal identifiability. Distribution volume was well identified (~2%) by only 2 h (and even just 1) of image acquisition. Conclusion This initial evaluation of 18F-SP203 in healthy human subjects showed that defluorination

  4. The restructuring of muscarinic receptor subtype gene transcripts in c-fos knock-out mice.

    PubMed

    Benes, Jan; Mravec, Boris; Kvetnansky, Richard; Myslivecek, Jaromir

    2013-05-01

    Although c-Fos plays a key role in intracellular signalling, the disruption of the c-fos gene has only minor consequences on the central nervous system (CNS) function. As muscarinic receptors (MR) play important roles in many CNS functions (attention, arousal, and cognition), the c-fos knock-out might be compensated through MR changes. The aim of this study was to evaluate changes in the M1-M5 MR mRNA in selected CNS areas: frontal, parietal, temporal and occipital cortex, striatum, hippocampus, hypothalamus and cerebellum (FC, PC, TC, OC, stria, hip, hypo, and crbl, respectively). Knocking out the c-fos gene changed the expression of MR in FC (reduced M1R, M4R and M5R expression), TC (increased M4R expression), OC (decreased M2R and M3R expression) and hippocampus (reduced M3R expression). Moreover, gender differences were observed in WT mice: increased expression of all M1-M5R in the FC in males and M1-M4R in the striatum in females. A detailed analysis of MR transcripts showed pre-existing correlations in the amount of MR-mRNA between specific regions. WT mice showed three major types of cortico-cortical correlations: fronto-occipital, temporo-parietal and parieto-occipital. The cortico-subcortical correlations involved associations between the FC, PC, TC and striatum. In KO mice, a substantial rearrangement of the correlation pattern was observed: only a temporo-parietal correlation and correlations between the FC and striatum remained, and a new correlation between the hypothalamus and cerebellum appeared. Thus, in addition to the previously described dopamine receptor restructuring, the restructuring of MR mRNA correlations reveals an additional mechanism for adaptation to the c-fos gene knockout.

  5. Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation.

    PubMed

    Kuriyama, Shuhko; Kashiwagi, Hitoshi; Yuhki, Koh-ichi; Kojima, Fumiaki; Yamada, Takehiro; Fujino, Takayuki; Hara, Akiyoshi; Takayama, Koji; Maruyama, Takayuki; Yoshida, Akitoshi; Narumiya, Shuh; Ushikubi, Fumitaka

    2010-10-01

    The effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2-/- platelets) and EP4 (EP4-/- platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1-259 and AE1-329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2-/- and EP4-/- platelets. In human platelets, AE1-259 and AE1-329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1-329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1-259. AE1-329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3-208, an EP4 antagonist. In addition, AE1-329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.

  6. Sphingosine 1-phosphate counteracts insulin signaling in pancreatic β-cells via the sphingosine 1-phosphate receptor subtype 2.

    PubMed

    Japtok, Lukasz; Schmitz, Elisabeth I; Fayyaz, Susann; Krämer, Stephanie; Hsu, Leigh J; Kleuser, Burkhard

    2015-08-01

    Glucolipotoxic stress has been identified as a key player in the progression of pancreatic β-cell dysfunction contributing to insulin resistance and the development of type 2 diabetes mellitus (T2D). It has been suggested that bioactive lipid intermediates, formed under lipotoxic conditions, are involved in these processes. Here, we show that sphingosine 1-phosphate (S1P) levels are not only increased in palmitate-stimulated pancreatic β-cells but also regulate β-cell homeostasis in a divergent manner. Although S1P possesses a prosurvival effect in β-cells, an enhanced level of the sphingolipid antagonizes insulin-mediated cell growth and survival via the sphingosine 1-phosphate receptor subtype 2 (S1P2) followed by an inhibition of Akt-signaling. In an attempt to investigate the role of the S1P/S1P2 axis in vivo, the New Zealand obese (NZO) diabetic mouse model, characterized by β-cell loss under high-fat diet (HFD) conditions, was used. The occurrence of T2D was accompanied by an increase of plasma S1P levels. To examine whether S1P contributes to the morphologic changes of islets via S1P2, the receptor antagonist JTE-013 was administered. Most interestingly, JTE-013 rescued β-cell damage clearly indicating an important role of the S1P2 in β-cell homeostasis. Therefore, the present study provides a new therapeutic strategy to diminish β-cell dysfunction and the development of T2D. PMID:25911610

  7. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains.

    PubMed

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains.

  8. The Metabotropic Glutamate Receptor Subtype 1 Mediates Experience-Dependent Maintenance of Mature Synaptic Connectivity in the Visual Thalamus.

    PubMed

    Narushima, Madoka; Uchigashima, Motokazu; Yagasaki, Yuki; Harada, Takeshi; Nagumo, Yasuyuki; Uesaka, Naofumi; Hashimoto, Kouichi; Aiba, Atsu; Watanabe, Masahiko; Miyata, Mariko; Kano, Masanobu

    2016-09-01

    Neural circuits formed during postnatal development have to be maintained stably thereafter, but their mechanisms remain largely unknown. Here we report that the metabotropic glutamate receptor subtype 1 (mGluR1) is essential for the maintenance of mature synaptic connectivity in the dorsal lateral geniculate nucleus (dLGN). In mGluR1 knockout (mGluR1-KO) mice, strengthening and elimination at retinogeniculate synapses occurred normally until around postnatal day 20 (P20). However, during the subsequent visual-experience-dependent maintenance phase, weak retinogeniculate synapses were newly recruited. These changes were similar to those of wild-type (WT) mice that underwent visual deprivation or inactivation of mGluR1 in the dLGN from P21. Importantly, visual deprivation was ineffective in mGluR1-KO mice, and the changes induced by visual deprivation in WT mice were rescued by pharmacological activation of mGluR1 in the dLGN. These results demonstrate that mGluR1 is crucial for the visual-experience-dependent maintenance of mature synaptic connectivity in the dLGN. PMID:27545713

  9. Microglia Lacking E Prostanoid Receptor Subtype 2 Have Enhanced Aβ Phagocytosis yet Lack Aβ-Activated Neurotoxicity

    PubMed Central

    Shie, Feng-Shiun; Breyer, Richard M.; Montine, Thomas J.

    2005-01-01

    Experimental therapies for Alzheimer’s disease (AD) are focused on enhanced clearance of neurotoxic Aβ peptides from brain. Microglia can be neuroprotective by phagocytosing Aβ; however, this comes at the cost of activated innate immunity that causes paracrine damage to neurons. Here, we show that ablation of E prostanoid receptor subtype 2 (EP2) significantly increased microglial-mediated clearance of Aβ peptides from AD brain sections and enhanced microglial Aβ phagocytosis in cell culture. The enhanced phagocytosis was PKC-dependent and was associated with elevated microglial secretion of the chemoattractant chemokines, macrophage inflammatory protein-1α and macrophage chemoattractant protein-1. This suggested that microglial activation is negatively regulated by EP2 signaling through suppression of prophagocytic cytokine secretion. However, despite this enhancement of Aβ phagocytosis, lack of EP2 completely suppressed Aβ-activated microglia-mediated paracrine neurotoxicity. These data demonstrate that blockade of microglial EP2 is a highly desirable mechanism for AD therapy that can maximize neuroprotective actions while minimizing bystander damage to neurons. PMID:15793296

  10. Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity; pharmacological and conformational studies

    PubMed Central

    Doedens, Lucas; Opperer, Florian; Cai, Minying; Beck, Johannes G.; Dedek, Matt; Palmer, Erin; Hruby, Victor J.; Kessler, Horst

    2010-01-01

    Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but non-selective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4 and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation which resembles other a-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains. PMID:20496895

  11. Protection of Hippocampal Neurogenesis from Toll-Like Receptor 4-Dependent Innate Immune Activation by Ablation of Prostaglandin E2 Receptor Subtype EP1 or EP2

    PubMed Central

    Keene, C. Dirk; Chang, Rubens; Stephen, Christina; Nivison, Mary; Nutt, Samuel E.; Look, Amy; Breyer, Richard M.; Horner, Phillip J.; Hevner, Robert; Montine, Thomas J.

    2009-01-01

    Prostaglandin E2 is one of several eicosanoid products of the cyclooxygenase isozymes and is a key regulator of innate immune responses; it also possesses paracrine effects on mature neurons. The prostaglandin E2 receptor family consists of four subtypes of which EP1 and EP2 are known to be expressed by microglia. Lipopolysaccharide (LPS)-induced innate immune activation leads to the degeneration of intermediate progenitor cells (IPCs) that are destined for neuronal maturation in the hippocampal subgranular zone (SGZ); these cells can be identified by the expression of the transcription factor T-box brain gene 2 (Tbr2). Importantly, depletion of LPS-induced IPCs from the SGZ is suppressed by cyclooxygenase inhibitors. We therefore tested the hypothesis that either EP1 or EP2 is critical to LPS-induced depletion of Tbr2+ IPCs from the SGZ. Expression of either EP1 or EP2 was necessary for Toll-like receptor 4-dependent innate immune-mediated depletion of these Tbr2+ IPCs in mice. Moreover, EP1 activation was directly toxic to murine adult hippocampal progenitor cells; EP2 was not expressed by these cells. Finally, EP1 modulated the response of murine primary microglia cultures to LPS but in a manner distinct from EP2. These results indicate that prostaglandin E2 signaling via either EP1 or EP2 is largely to completely necessary for Toll-like receptor 4-dependent depletion of IPCs from the SGZ and suggest further pharmacological strategies to protect this important neurogenic niche. PMID:19389932

  12. Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease

    PubMed Central

    Cortés-Ramírez, Dionisio A.; Rodríguez-Tojo, María J.; Coca-Meneses, Juan C.; Marichalar-Mendia, Xabier

    2014-01-01

    The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. Objectives: To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Material and Methods: Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Results: Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk. Key words:Oral lichenoid disease, oral lichen planus, oral lichenoid lesion, oral carcinogenesis, EGFR. PMID:24880441

  13. Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats

    PubMed Central

    Martín-Fernández, Beatriz; Rubio-Navarro, Alfonso; Cortegano, Isabel; Ballesteros, Sandra; Alía, Mario; Cannata-Ortiz, Pablo; Olivares-Álvaro, Elena; Egido, Jesús; de Andrés, Belén; Gaspar, María Luisa; de las Heras, Natalia; Lahera, Vicente; Moreno, Juan Antonio

    2016-01-01

    We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt–treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt–treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation. PMID:26730742

  14. Inositol 1,4,5-trisphosphate receptor subtypes differentially recognize regioisomers of D-myo-inositol 1,4,5-trisphosphate.

    PubMed Central

    Hirata, M; Takeuchi, H; Riley, A M; Mills, S J; Watanabe, Y; Potter, B V

    1997-01-01

    The Ins(1,4,5)P3 regioisomers, Ins(1,4,6)P3 and Ins(1,3,6)P3, which can mimic the 1,4,5-arrangement on the inositol ring of Ins(1,4,5)P3, were examined for Ca2+ release by using four types of saponin-permeabilized cell possessing various abundances of receptor subtypes, with special reference to the relation of potency to receptor subtype. Ins(1,4,6)P3 and Ins(1,3,6)P3 were weak agonists in rat basophilic leukaemic cells (RBL cells), which possess predominantly subtype II receptors, with respective potencies of 1/200 and less than 1/500 that of Ins(1,4,5)P3 [the EC50 values were 0.2, 45 and more than 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively]. Similar rank order potencies were also evaluated for the displacement of [3H]Ins(1,4,5)P3 bound to RBL cell membranes by these regioisomers. However, they caused Ca2+ release from GH3 rat pituitary cells possessing predominantly subtype I receptors more potently; Ins(1,4,6)P3 and Ins(1,3,6)P3 evoked release at respective concentrations of only one-third and one-twentieth that of Ins(1,4,5)P3 (the EC50 values were 0.4, 1.2 and 8 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In COS-1 African green-monkey kidney cells, with the relative abundances of 37% of the subtype II and of 62% of the subtype III receptor, potencies of 1/40 and approx. 1/200 for Ins(1, 4,6)P3 and Ins(1,3,6)P3 respectively were exhibited relative to Ins(1,4,5)P3 (the EC50 values were 0.4, 15 and approx. 80 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In HL-60 human leukaemic cells, in spite of the dominant presence of subtype I receptors (71%), similar respective potencies to those seen with COS-1 cells were exhibited (the EC50 values were 0.3, 15 and approx. 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). These results indicate that these regioisomers are the first ligands that distinguish between receptor subtypes; the present observations are of

  15. Toxicological profiles of selected synthetic cannabinoids showing high binding affinities to the cannabinoid receptor subtype CB₁.

    PubMed

    Koller, Verena J; Zlabinger, Gerhard J; Auwärter, Volker; Fuchs, Sabine; Knasmueller, Siegfried

    2013-07-01

    Products containing synthetic cannabinoids are consumed as a surrogate for marihuana due to their non-detectability with commonly used drug tests and their strong cannabimimetic effects. Because data concerning their toxicological properties are scarce, the cytotoxic, genotoxic, immunomodulatory, and hormonal activities of four naphthoylindole compounds (JWH-018, JWH-073, JWH-122 and JWH-210) and of one benzoylindole (AM-694) were studied in human cell lines and primary cells; tetrahydrocannabinol was included as the classical non-endogenous cannabinoid receptor ligand. All compounds induced damage to the cell membranes of buccal (TR146) and breast (MCF-7) derived cells at concentrations of ≥75-100 μM. No cytotoxic responses were seen in other assays which reflect mitochondrial damage, protein synthesis, and lysosomal activities. JWH-073 and JWH-122 induced DNA migration in buccal and liver cells (HepG2) in single cell gel electrophoresis assays, while JWH-210 was only in the latter cell line active. No estrogenic activities were detected in bone marrow cells (U2-OS), but all compounds caused anti-estrogenic effects at levels between 2.1 and 23.0 μM. Furthermore, no impact on cytokine release (i.e., on IL-10, IL-6, IL-12/23p40 and TNFα levels) was seen in LPS-stimulated human PBMCs, except with JWH-210 and JWH-122 which caused a decrease of TNFα and IL-12/23p40. All toxic effects were observed with concentrations higher than those expected in body fluids of users. Since genotoxic effects are in general linear over a wide concentration range and the exposure levels may be higher in epithelial cells than [corrected] in serum, further experimental work is required to find out if DNA damage takes place in drug users.

  16. Expression and distribution patterns of Mas-related gene receptor subtypes A-H in the mouse intestine: inflammation-induced changes.

    PubMed

    Avula, Leela Rani; Buckinx, Roeland; Favoreel, Herman; Cox, Eric; Adriaensen, Dirk; Van Nassauw, Luc; Timmermans, Jean-Pierre

    2013-05-01

    Mas-related gene (Mrg) receptors constitute a subfamily of G protein-coupled receptors that are implicated in nociception, and are as such considered potential targets for pain therapies. Furthermore, some Mrgs have been suggested to play roles in the regulation of inflammatory responses to non-immunological activation of mast cells and in mast cell-neuron communication. Except for MrgD, E and F, whose changed expression has been revealed during inflammation in the mouse intestine in our earlier studies, information concerning the remaining cloned mouse Mrg subtypes in the gastrointestinal tract during (patho) physiological conditions is lacking. Therefore, the present study aimed at identifying the presence and putative function of these remaining cloned Mrg subtypes (n = 19) in the (inflamed) mouse intestine. Using reverse transcriptase-PCR, quantitative-PCR and multiple immunofluorescence staining with commercial and newly custom-developed antibodies, we compared the ileum and the related dorsal root ganglia (DRG) of non-inflamed mice with those of two models of intestinal inflammation, i.e., intestinal schistosomiasis and 2,4,6-trinitrobenzene sulfonic acid-induced ileitis. In the non-inflamed ileum and DRG, the majority of the Mrg subtypes examined were sparsely expressed, showing a neuron-specific expression pattern. However, significant changes in the expression patterns of multiple Mrg subtypes were observed in the inflamed ileum; for instance, MrgA4, MrgB2and MrgB8 were expressed in a clearly increased number of enteric sensory neurons and in nerve fibers in the lamina propria, while de novo expression of MrgB10 was observed in enteric sensory neurons and in newly recruited mucosal mast cells (MMCs). The MrgB10 expressing MMCs were found to be in close contact with nerve fibers in the lamina propria. This is the first report on the expression of all cloned Mrg receptor subtypes in the (inflamed) mouse intestine. The observed changes in the expression and

  17. The α3β4* nicotinic acetylcholine receptor subtype mediates nicotine reward and physical nicotine withdrawal signs independently of the α5 subunit in the mouse

    PubMed Central

    Jackson, Kia J.; Sanjakdar, Sarah S.; Muldoon, Pretal P.; McIntosh, J. Michael; Damaj, M. Imad

    2013-01-01

    The 15q25 gene cluster contains genes that code for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the α5 and β4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the α3β4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the α3β4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective α3β4* nAChR antagonist, α-conotoxin AuIB, we assessed the role of α3β4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because α5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with α3β4*, we also evaluated the role of the α3β4α5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the α3β4* nAChR subtype in α5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in α5 nAChR knockout mice. This study shows that α3β4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The α5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the α3β4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome. PMID:23416040

  18. The α3β4* nicotinic acetylcholine receptor subtype mediates nicotine reward and physical nicotine withdrawal signs independently of the α5 subunit in the mouse.

    PubMed

    Jackson, Kia J; Sanjakdar, Sarah S; Muldoon, Pretal P; McIntosh, J Michael; Damaj, M Imad

    2013-07-01

    The 15q25 gene cluster contains genes that code for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the α5 and β4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the α3β4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the α3β4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective α3β4* nAChR antagonist, α-conotoxin AuIB, we assessed the role of α3β4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because α5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with α3β4*, we also evaluated the role of the α3β4α5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the α3β4* nAChR subtype in α5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in α5 nAChR knockout mice. This study shows that α3β4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The α5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the α3β4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome.

  19. Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition.

    PubMed

    Mitsui, Takaaki; Watanabe-Takahashi, Miho; Shimizu, Eiko; Zhang, Baihao; Funamoto, Satoru; Yamasaki, Shinji; Nishikawa, Kiyotaka

    2016-09-01

    Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC. PMID:27382021

  20. Adaptive increases in expression and vasodilator activity of estrogen receptor subtypes in a blood vessel-specific pattern during pregnancy.

    PubMed

    Mata, Karina M; Li, Wei; Reslan, Ossama M; Siddiqui, Waleed T; Opsasnick, Lauren A; Khalil, Raouf A

    2015-11-15

    Normal pregnancy is associated with adaptive hemodynamic, hormonal, and vascular changes, and estrogen (E2) may promote vasodilation during pregnancy; however, the specific E2 receptor (ER) subtype, post-ER signaling mechanism, and vascular bed involved are unclear. We tested whether pregnancy-associated vascular adaptations involve changes in the expression/distribution/activity of distinct ER subtypes in a blood vessel-specific manner. Blood pressure (BP) and plasma E2 were measured in virgin and pregnant (day 19) rats, and the thoracic aorta, carotid artery, mesenteric artery, and renal artery were isolated for measurements of ERα, ERβ, and G protein-coupled receptor 30 [G protein-coupled ER (GPER)] expression and tissue distribution in parallel with relaxation responses to E2 (all ERs) and the specific ER agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERα), diarylpropionitrile (DPN; ERβ), and G1 (GPER). BP was slightly lower and plasma E2 was higher in pregnant versus virgin rats. Western blots revealed increased ERα and ERβ in the aorta and mesenteric artery and GPER in the aorta of pregnant versus virgin rats. Immunohistochemistry revealed that the increases in ERs were mainly in the intima and media. In phenylephrine-precontracted vessels, E2 and PPT caused relaxation that was greater in the aorta and mesenteric artery but similar in the carotid and renal artery of pregnant versus virgin rats. DPN- and G1-induced relaxation was greater in the mesenteric and renal artery than in the aorta and carotid artery, and aortic relaxation to G1 was greater in pregnant versus virgin rats. The nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester with or without the cyclooxygenase inhibitor indomethacin with or without the EDHF blocker tetraethylammonium or endothelium removal reduced E2, PPT, and G1-induced relaxation in the aorta of pregnant rats, suggesting an endothelium-dependent mechanism, but did not affect E2-, PPT

  1. Adaptive increases in expression and vasodilator activity of estrogen receptor subtypes in a blood vessel-specific pattern during pregnancy.

    PubMed

    Mata, Karina M; Li, Wei; Reslan, Ossama M; Siddiqui, Waleed T; Opsasnick, Lauren A; Khalil, Raouf A

    2015-11-15

    Normal pregnancy is associated with adaptive hemodynamic, hormonal, and vascular changes, and estrogen (E2) may promote vasodilation during pregnancy; however, the specific E2 receptor (ER) subtype, post-ER signaling mechanism, and vascular bed involved are unclear. We tested whether pregnancy-associated vascular adaptations involve changes in the expression/distribution/activity of distinct ER subtypes in a blood vessel-specific manner. Blood pressure (BP) and plasma E2 were measured in virgin and pregnant (day 19) rats, and the thoracic aorta, carotid artery, mesenteric artery, and renal artery were isolated for measurements of ERα, ERβ, and G protein-coupled receptor 30 [G protein-coupled ER (GPER)] expression and tissue distribution in parallel with relaxation responses to E2 (all ERs) and the specific ER agonist 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERα), diarylpropionitrile (DPN; ERβ), and G1 (GPER). BP was slightly lower and plasma E2 was higher in pregnant versus virgin rats. Western blots revealed increased ERα and ERβ in the aorta and mesenteric artery and GPER in the aorta of pregnant versus virgin rats. Immunohistochemistry revealed that the increases in ERs were mainly in the intima and media. In phenylephrine-precontracted vessels, E2 and PPT caused relaxation that was greater in the aorta and mesenteric artery but similar in the carotid and renal artery of pregnant versus virgin rats. DPN- and G1-induced relaxation was greater in the mesenteric and renal artery than in the aorta and carotid artery, and aortic relaxation to G1 was greater in pregnant versus virgin rats. The nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester with or without the cyclooxygenase inhibitor indomethacin with or without the EDHF blocker tetraethylammonium or endothelium removal reduced E2, PPT, and G1-induced relaxation in the aorta of pregnant rats, suggesting an endothelium-dependent mechanism, but did not affect E2-, PPT

  2. Investigation of the role of 5-HT2 receptor subtypes in the control of the bladder and the urethra in the anaesthetized female rat

    PubMed Central

    Mbaki, Y; Ramage, A G

    2008-01-01

    Background and purpose: Micturition is controlled by central 5-HT-containing pathways. 5-HT2 receptors have been implicated in this system especially in control of the urethra, which is a drug target for treating urinary incontinence. This study investigates the role of each of the three subtypes of this receptor with emphasis on sphincter regulation. Experimental approach: Recordings of urethral and bladder pressure, external urethral sphincter (EUS) EMG, as well as the micturition reflex induced by bladder distension along with blood pressure and heart rate were made in anaesthetized rats. The effects of agonists and antagonists for 5-HT2 receptor subtypes were studied on these variables. Key results: The 5-HT2C agonists Ro 60-0175, WAY 161503 and mCPP, i.v., activated the EUS, increased urethral pressure and inhibited the micturition reflex. The effects of Ro 60-0175 on the EUS were blocked by the 5-HT2C antagonist SB 242084 and the 5-HT2A antagonists, ketanserin and MDL 100907. SB 242084 also blocked the inhibitory action on the reflex, while the 5-HT2B antagonist RS 127445 only blocked the increase in urethral pressure. The 5-HT2A receptor agonist DOI given i.v. or i.t. but not i.c.v. activated the EUS. Conclusions and implications: 5-HT2A/2C receptors located in the sacral spinal cord activate the EUS, while central 5-HT2C receptors inhibit the micturition reflex and 5-HT2B receptors, probably at the level of the urethra, increase urethral smooth muscle tone. Furthermore, 5-HT2B and 5-HT2C receptors do not seem to play an important role in the physiological regulation of micturition. PMID:18604238

  3. Metabotropic Glutamate Receptor Subtype 7 in the Bed Nucleus of the Stria Terminalis is Essential for Intermale Aggression.

    PubMed

    Masugi-Tokita, Miwako; Flor, Peter J; Kawata, Mitsuhiro

    2016-02-01

    Metabotropic glutamate receptor subtype 7 (mGluR7) is a member of group III mGluRs, which localize to the presynaptic active zones of the mammalian central nervous system. Although histological, genetic, and electrophysiological studies ensure the importance of mGluR7, its roles in behavior and physiology remain largely unknown. Using a resident-intruder paradigm, we found a severe reduction in intermale aggressive behavior in mGluR7 knockout (KO) mice. We also found alterations in other social behaviors in male mGluR7 KO mice, including sexual behavior toward male intruders. Because olfaction is critical for rodent social behavior, including aggression, we performed an olfaction test, finding that mGluR7 KO mice failed to show interest in the smell of male urine. To clarify the olfactory deficit, we then exposed mice to urine and analyzed c-Fos-immunoreactivity, discovering a remarkable reduction in neural activity in the bed nucleus of the stria terminalis (BNST) of mGluR7 KO mice. Finally, intra-BNST administration of the mGluR7-selective antagonist 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP) also reproduced the phenotype of mGluR7 KO mice, including reduced aggression and altered social interaction. Thus mGluR7 may work as an 'enhancer of neural activity' in the BNST and is important for intermale aggression. Our findings demonstrate that mGluR7 is essential for social behavior and innate behavior. Our study on mGluR7 in the BNST will shed light on future therapies for emotional disorders in humans. PMID:26149357

  4. Histamine induces the production of matrix metalloproteinase-9 in human astrocytic cultures via H1-receptor subtype.

    PubMed

    Patel, Aarti; Vasanthan, Vishnu; Fu, Wen; Fahlman, Richard P; MacTavish, David; Jhamandas, Jack H

    2016-05-01

    Accumulation of β-amyloid (Aβ) protein within the brain is a neuropathological hallmark of Alzheimer's disease (AD). One strategy to facilitate Aβ clearance from the brain is to promote Aβ catabolism. Matrix metalloproteinase-9 (MMP-9), a member of the family of Zn(+2)-containing endoproteases, known to be expressed and secreted by astrocytes, is capable of degrading Aβ. Histamine, a major aminergic brain neurotransmitter, stimulates the production of MMP-9 in keratinocytes through the histamine H1 receptor (H1R). In the present study, we show that histamine evokes a concentration- and calcium-dependent release of MMP-9 from human astrocytic U373 cells and primary cultures of human and rat astrocytes through the H1R subtype. Activation of H1R on astrocytes elevated intracellular levels of Ca(2+) that was accompanied by time-dependent increases in MAP kinase p44/p42 and PKC. In-cell western blots revealed dose-dependent increases in both enzymes, confirming involvement of these signal transduction pathways. We next investigated the extent of recombinant human MMP-9 (rhMMP-9) proteolytic activity on soluble oligomeric Aβ (soAβ). Mass spectrometry demonstrated time-dependent cleavage of soAβ (20 μM), but not another amyloidogenic protein amylin, upon incubation with rhMMP-9 (100 nM) at 1, 4 and 17 h. Furthermore, Western blots showed a shift in soAβ equilibrium toward lower order, less toxic monomeric species. In conclusion, both MAPK p44/p42 and PKC pathways appear to be involved in histamine-upregulated MMP-9 release via H1Rs in astrocytes. Furthermore, MMP-9 appears to cleave soAβ into less toxic monomeric species. Given the key role of histamine in MMP-9 release, this neurotransmitter may serve as a potential therapeutic target for AD.

  5. 'Distinct cellular localization' of the messenger ribonucleic acid for prostaglandin E receptor subtypes in the mouse uterus during pseudopregnancy.

    PubMed

    Katsuyama, M; Sugimoto, Y; Morimoto, K; Hasumoto, K; Fukumoto, M; Negishi, M; Ichikawa, A

    1997-01-01

    As an initial step to clarify the mechanisms of various uterine actions of PGE2, expression patterns of the messenger RNAs (mRNAs) for four subtypes of PGE receptors, EP1, EP2, EP3, and EP4, were investigated in the mouse uterus during pseudopregnancy. Relative expression levels were investigated by Northern blot analysis of mRNA levels in uteri obtained on days 0, 1, 3, 5, 7, and 9 of pseudopregnancy (day 0 = 48 h after PMSG injection), and cellular localization was determined by in situ hybridization in uteri obtained on days 0 and 5. EP2 mRNA was specifically expressed on day 5, and its expression was confined to the luminal epithelium. On the other hand, the level of the EP3 mRNA expression progressively increased until day 5. Cell populations expressing the EP3 mRNA were confined to the longitudinal smooth muscle on day 0, but they changed to the circular smooth muscle on day 5. The expression level of EP4 mRNA was low on days 0 and 1, but it became high on days 3 and 5. On day 0, EP4 mRNA was localized to the luminal epithelium. On day 5, diffuse, but significant, EP4 expression was observed over the endometrial stroma and epithelium. No EP1 mRNA signals were observed. Transient expression of EP2 on day 5 of pseudopregnancy in the luminal epithelium suggests its involvement in blastocyst implantation signaling. EP4 in the endometrial stroma is suggested to be involved in decidual transformation of the stromal cells, whereas EP3 in the myometrium is believed to be involved in regulation of myometrial activity.

  6. Metabotropic Glutamate Receptor Subtype 7 in the Bed Nucleus of the Stria Terminalis is Essential for Intermale Aggression.

    PubMed

    Masugi-Tokita, Miwako; Flor, Peter J; Kawata, Mitsuhiro

    2016-02-01

    Metabotropic glutamate receptor subtype 7 (mGluR7) is a member of group III mGluRs, which localize to the presynaptic active zones of the mammalian central nervous system. Although histological, genetic, and electrophysiological studies ensure the importance of mGluR7, its roles in behavior and physiology remain largely unknown. Using a resident-intruder paradigm, we found a severe reduction in intermale aggressive behavior in mGluR7 knockout (KO) mice. We also found alterations in other social behaviors in male mGluR7 KO mice, including sexual behavior toward male intruders. Because olfaction is critical for rodent social behavior, including aggression, we performed an olfaction test, finding that mGluR7 KO mice failed to show interest in the smell of male urine. To clarify the olfactory deficit, we then exposed mice to urine and analyzed c-Fos-immunoreactivity, discovering a remarkable reduction in neural activity in the bed nucleus of the stria terminalis (BNST) of mGluR7 KO mice. Finally, intra-BNST administration of the mGluR7-selective antagonist 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP) also reproduced the phenotype of mGluR7 KO mice, including reduced aggression and altered social interaction. Thus mGluR7 may work as an 'enhancer of neural activity' in the BNST and is important for intermale aggression. Our findings demonstrate that mGluR7 is essential for social behavior and innate behavior. Our study on mGluR7 in the BNST will shed light on future therapies for emotional disorders in humans.

  7. Prevention by blockade of angiotensin subtype1-receptors of the development of genetic hypertension but not its heritability.

    PubMed Central

    Madeddu, P.; Anania, V.; Varoni, M. V.; Parpaglia, P. P.; Demontis, M. P.; Fattaccio, M. C.; Palomba, D.; Pollock, D.; Glorioso, N.

    1995-01-01

    1. We determined whether early inhibition of angiotensin II subtype1 (AT1) receptors by the newly synthesized nonpeptidic antagonist, A-81988, can attenuate the development of hypertension in spontaneously hypertensive rats (SHR) and if the altered blood pressure phenotype can be passed on to the subsequent generation, not exposed to the antagonist. 2. Pairs of SHR were mated while drinking tap water or A-81988 in tap water, and the progeny was maintained on the parental regimen until 14 weeks of age. At this stage, A-81988-treated rats showed lower systolic blood pressure and body weight values (136 +/- 5 versus 185 +/- 4 mmHg and 247 +/- 4 versus 283 +/- 4 g in controls, P < 0.01); while heart rate was similar. In addition, mean blood pressure was reduced (101 +/- 7 versus 170 +/- 7 mmHg in controls, P < 0.01), and the pressor responses to intravenous or intracerebroventricular angiotensin II were inhibited by 27 and 59%, respectively. Heart/body weight ratio was smaller in A-81988-treated rats (3.2 +/- 0.1 versus 3.8 +/- 0.1 in controls, P < 0.01). 3. The antihypertensive and antihypertrophic effect of A-81988 persisted in rats removed from therapy for 7 weeks (systolic blood pressure: 173 +/- 4 versus 220 +/- 4 mmHg, heart/body weight ratio: 3.4 +/- 0.1 versus 4.1 +/- 0.1 in controls at 21 weeks of age, P < 0.01 for both comparisons), whereas the cardiovascular hypertensive phenotype was fully expressed in the subsequent generation that was maintained without treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582472

  8. Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment

    PubMed Central

    Lecoutey, Cédric; Hedou, Damien; Freret, Thomas; Giannoni, Patrizia; Gaven, Florence; Since, Marc; Bouet, Valentine; Ballandonne, Céline; Corvaisier, Sophie; Malzert Fréon, Aurélie; Mignani, Serge; Cresteil, Thierry; Boulouard, Michel; Claeysen, Sylvie; Rochais, Christophe; Dallemagne, Patrick

    2014-01-01

    RS67333 is a partial serotonin subtype 4 receptor (5-HT4R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-β peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer’s disease (AD). We show herein that RS67333 is also a submicromolar acetylcholinesterase (AChE) inhibitor and therefore, could contribute, through this effect, to the restoration of the cholinergic neurotransmission that becomes altered in AD. We planned to pharmacomodulate RS67333 to enhance its AChE inhibitory activity to take advantage of this pleiotropic pharmacological profile in the design of a novel multitarget-directed ligand that is able to exert not only a symptomatic but also, a disease-modifying effect against AD. These efforts allowed us to select donecopride as a valuable dual (h)5-HT4R partial agonist (Ki = 10.4 nM; 48.3% of control agonist response)/(h)AChEI (IC50 = 16 nM) that further promotes sAPPα release (EC50 = 11.3 nM). Donecopride, as a druggable lead, was assessed for its in vivo procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improvement of memory performances observed at 0.3 and 1 mg/kg on the object recognition test. On the basis of these in vitro and in vivo activities, donecopride seems to be a promising drug candidate for AD treatment. PMID:25157130

  9. ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

    PubMed Central

    Matsuo, Katsuichi; Katsuragi, Takeshi; Fujiki, Sono; Sato, Chiemi; Furukawa, Tatsuo

    1997-01-01

    The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED50 values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.Although the contraction caused by α,β-mATP was strongly diminished by Ca2+-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release. PMID:9283712

  10. Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries.

    PubMed

    Tangsucharit, Panot; Takatori, Shingo; Zamami, Yoshito; Goda, Mitsuhiro; Pakdeechote, Poungrat; Kawasaki, Hiromu; Takayama, Fusako

    2016-01-01

    The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation.

  11. The role of dopamine D2, but not D3 or D4, receptor subtypes, in quinpirole-induced inhibition of the cardioaccelerator sympathetic outflow in pithed rats

    PubMed Central

    Altamirano-Espinoza, A H; González-Hernández, A; Manrique-Maldonado, G; Marichal-Cancino, B A; Ruiz-Salinas, I; Villalón, C M

    2013-01-01

    Background and Purpose Quinpirole (a dopamine D2-like receptor agonist) inhibits the cardioaccelerator sympathetic outflow in pithed rats by sympathoinhibitory D2-like receptors. The present study was designed to identify pharmacologically the specific D2-like receptor subtypes (i.e. D2, D3 and D4) involved in this sympathoinhibition by quinpirole. Experimental Approach One hundred fourteen male Wistar rats were pithed, artificially ventilated with room air and prepared for either preganglionic spinal (C7-T1) stimulation of the cardioaccelerator sympathetic outflow (n = 102) or i.v. bolus injections of exogenous noradrenaline (n = 12). This approach resulted in frequency-dependent and dose-dependent tachycardic responses, respectively, as previously reported by our group. Key Results I.v. continuous infusions of quinpirole (0.1–10 μg kg−1 min−1), but not of saline (0.02 mL min−1), dose-dependently inhibited the sympathetically induced tachycardic responses. Moreover, the cardiac sympathoinhibition induced by 3 μg kg−1 min−1 quinpirole (which failed to affect the tachycardic responses to i.v. noradrenaline) was: (i) unchanged after i.v. injections of the antagonists SB-277011-A (D3; 100–300 μg kg−1) or L-745,870 (D4; 30–100 μg kg−1); and (ii) markedly blocked and abolished by, respectively, 100 and 300 μg kg−1 of the D2 preferring receptor subtype antagonist L-741,626. These doses of antagonists, which did not affect per se the sympathetically induced tachycardic responses, were high enough to completely block their respective receptors. Conclusions and Implications The cardiac sympathoinhibition induced by 3 μg kg−1 min−1 quinpirole involves the dopamine D2 receptor subtype, with no evidence for the involvement of the D3 or D4 subtypes. This provides new evidence for understanding the modulation of the cardioaccelerator sympathetic outflow. PMID:24032529

  12. Binding and functional properties of antimuscarinics of the hexocyclium/sila-hexocyclium and hexahydro-diphenidol/hexahydro-sila-diphenidol type to muscarinic receptor subtypes.

    PubMed Central

    Waelbroeck, M.; Tastenoy, M.; Camus, J.; Christophe, J.; Strohmann, C.; Linoh, H.; Zilch, H.; Tacke, R.; Mutschler, E.; Lambrecht, G.

    1989-01-01

    1. In an attempt to assess the structural requirements for the muscarinic receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a series of structurally related C/Si pairs were investigated, along with atropine, pirenzepine and methoctramine, for their binding affinities in NB-OK 1 cells as well as in rat heart and pancreas. 2. The action of these antagonists at muscarinic receptors mediating negative inotropic responses in guinea-pig atria and ileal contractions has also been assessed. 3. Antagonist binding data indicated that NB-OK 1 cells (M1 type) as well as rat heart (cardiac type) and pancreas (glandular/smooth muscle type) possess different muscarinic receptor subtypes. 4. A highly significant correlation was found between the binding affinities of the antagonists to muscarinic receptors in rat heart and pancreas, respectively, and the affinities to muscarinic receptors in guinea-pig atria and ileum. This implies that the muscarinic binding sites in rat heart and the receptors in guinea-pig atria are essentially similar, but different from those in pancreas and ileum. 5. The antimuscarinic potency of hexahydro-diphenidol and hexahydro-sila-diphenidol at the three subtypes was influenced differently by structural modifications (e.g. quaternization). Different selectivity profiles for the antagonists were obtained, which makes these compounds useful tools to investigate further muscarinic receptor heterogeneity. Indeed, the tertiary analogues hexahydro-diphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) had an M1 = glandular/smooth muscle greater than cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M1 preferring (M1 greater than glandular/smooth muscle, cardiac). PMID:2804545

  13. [3H]-LY341495 as a novel antagonist radioligand for group II metabotropic glutamate (mGlu) receptors: characterization of binding to membranes of mGlu receptor subtype expressing cells.

    PubMed

    Johnson, B G; Wright, R A; Arnold, M B; Wheeler, W J; Ornstein, P L; Schoepp, D D

    1999-10-01

    Metabotropic glutamate (mGlu) receptors are a family of eight known subtypes termed mGlu1-8. Currently, few ligands are available to study the pharmacology of mGlu receptor subtypes. In functional assays, we previously described LY341495 as a highly potent and selective mGlu2 and mGlu3 receptor antagonist. In this study, radiolabeled [3H]-LY341495 was used to investigate the characteristics of receptor binding to membranes from cells expressing human mGlu receptor subtypes. Using membranes from cells expressing human mGlu2 and mGlu3 receptors, [3H]-LY341495 (1 nM) specific binding was > 90% of total binding. At an approximate K(D) concentration for [3H]-LY341495 binding to human mGlu2 and mGlu3 receptors (1 nM), no appreciable specific binding of [3H-]LY341495 was found in membranes of cells expressing human mGlu1a, mGlu5a, mGlu4a, mGlu6, or mGlu7a receptors. However, modest (approximately 20% of mGlu2/3) specific [3H]-LY341495 (1 nM) binding was observed in human mGlu8 expressing cells. [3H]-LY341495 bound to membranes expressing human mGlu2 and mGlu3 receptors in a reversible and saturable manner with relatively high affinities (Bmax 20.5 +/- 5.4 and 32.0 +/- 7.0 pmol/mg protein; and K(D) = 1.67 +/- 0.20 and 0.75 +/- 0.43 nM, respectively). The pharmacology of [3H]-LY341495 binding in mGlu2 and mGlu3 expressing cells was consistent with that previously described for LY341495 in functional assays. [3H]-LY341495 binding provides a useful way to further investigate regulation of receptor expression and pharmacological properties of mGlu2 and mGlu3 receptor subtypes in recombinant systems. PMID:10530814

  14. Single cell laser dissection with molecular beacon polymerase chain reaction identifies 2A as the predominant serotonin receptor subtype in hypoglossal motoneurons.

    PubMed

    Zhan, G; Shaheen, F; Mackiewicz, M; Fenik, P; Veasey, S C

    2002-01-01

    We hypothesize that sleep state-dependent withdrawal of serotonin (5-hydroxytryptamine, 5-HT) at upper airway (UAW) dilator motoneurons contributes significantly to sleep-related suppression of dilator muscle activity in obstructive sleep apnea. Identification of 5-HT receptor subtypes involved in postsynaptic facilitation of UAW motoneuron activity may provide pharmacotherapies for this prevalent disorder. We have adapted two assays to provide semi-quantitative measurements of mRNA copy numbers for 5-HT receptor subtypes in single UAW motoneurons. Specifically, soma of 111 hypoglossal (XII) motoneurons in 10 adult male rats were captured using a laser dissection microscope, and then used individually in single round molecular beacon polymerase chain reaction (PCR) for real-time quantitation of 5-HT(2A), 5-HT(2C), 5-HT(3), 5-HT(4), 5-HT(5A), 5-HT(5B), 5-HT(6) or 5-HT(7) receptor. Receptor mRNA copy numbers from single XII motoneurons were compared to control samples from within the XII nucleus and lateral medulla. All 20 motoneuronal soma assayed for the 5-HT(2A) receptor had measurable copy numbers (7028+/-2656 copies/cell). In contrast, copy numbers for the 5-HT(2A) receptor in XII non-motoneuronal (n=17) and lateral medulla (n=15) samples were 81+/-51 copies and 83+/-35 copies, respectively, P<0.05. Seven of 13 XII motoneurons assayed had measurable 5-HT(2C) receptor copy numbers of mRNA (287+/-112 copies/cell). XII soma had minimal 5-HT(3), 5-HT(4), 5-HT(5A), 5-HT(5B), 5-HT(6) or 5-HT(7) receptor mRNA. 5-HT(2A) receptor mRNA presence within XII motoneurons was confirmed with digoxigenin-labeled in situ hybridization. In summary, combined use of laser dissection and molecular beacon PCR revealed 5-HT(2A) receptor as the predominant 5-HT receptor mRNA in XII motoneurons, and identified small quantities of 5-HT(2C) receptor. This information will allow a more complete understanding of serotonergic control of respiratory activity.

  15. Synthesis and in vitro autoradiographic evaluation of a novel high-affinity radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors.

    PubMed

    Donohue, Sean R; Varnäs, Katarina; Jia, Zhisheng; Gulyás, Balázs; Pike, Victor W; Halldin, Christer

    2009-11-01

    There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB1) receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB1 receptor ligand ([125I]8, [125I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxylate, [125I]SD7015). By autoradiography in vitro, [125I]8 showed selective binding to CB1 receptors on human brain postmortem cryosections and now merits labeling with iodine-123 for further evaluation as a SPECT radioligand in non-human primate. PMID:19767206

  16. Quantification of metabotropic glutamate subtype 5 receptors in brain by an equilibrium method using 18F-SP203

    PubMed Central

    Kimura, Yasuyuki; Siméon, Fabrice G.; Zoghbi, Sami S.; Zhang, Yi; Hatazawa, Jun; Pike, Victor W.; Innis, Robert B.; Fujita, Masahiro

    2011-01-01

    A new PET ligand, 3-fluoro-5-(2-(2-18F-(fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile (18F-SP203) can quantify metabotropic glutamate subtype 5 receptors (mGluR5) in human brain by a bolus injection and kinetic modeling. As an alternative approach to a bolus injection, binding can simply be measured as a ratio of tissue to metabolite-corrected plasma at a single time point under equilibrium conditions achieved by administering the radioligand with a bolus injection followed by a constant infusion. The purpose of this study was to validate the equilibrium method as an alternative to the standard kinetic method for measuring 18F-SP203 binding in brain. Nine healthy subjects were injected with 18F-SP203 using a bolus plus constant infusion for 300 minutes. A single ratio of bolus-to-constant infusion (the activity of bolus equaled to that of infusion over 219 minutes) was applied to all subjects to achieve equilibrium in approximately 120 minutes. As a measure of ligand binding, we compared total distribution volume (VT) calculated by the equilibrium and kinetic methods in each scan. The equilibrium method calculated VT by the ratio of radioactivity in brain to the concentration of 18 F-SP203 in arterial plasma at 120 minutes, and the kinetic method calculated VT by a two-tissue compartment model using brain and plasma dynamic data from 0 to 120 minutes. VT obtained via the equilibrium method was highly correlated with VT obtained via kinetic modeling. Inter-subject variability of VT obtained via the equilibrium method was slightly smaller than VT obtained via the kinetic method. VT obtained via the equilibrium method was ~10% higher than VT obtained via the kinetic method, indicating a small difference between the measurements. Taken together, the results of this study show that using the equilibrium method is an acceptable alternative to the standard kinetic method when using 18F-SP203 to measure mGluR5. Although small differences in the measurements obtained via

  17. Quantification of metabotropic glutamate subtype 5 receptors in the brain by an equilibrium method using 18F-SP203.

    PubMed

    Kimura, Yasuyuki; Siméon, Fabrice G; Zoghbi, Sami S; Zhang, Yi; Hatazawa, Jun; Pike, Victor W; Innis, Robert B; Fujita, Masahiro

    2012-02-01

    A new PET ligand, 3-fluoro-5-(2-(2-(18)F-(fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile (18F-SP203) can quantify metabotropic glutamate subtype 5 receptors (mGluR5) in human brain by a bolus injection and kinetic modeling. As an alternative approach to a bolus injection, binding can simply be measured as a ratio of tissue to metabolite-corrected plasma at a single time point under equilibrium conditions achieved by administering the radioligand with a bolus injection followed by a constant infusion. The purpose of this study was to validate the equilibrium method as an alternative to the standard kinetic method for measuring 18F-SP203 binding in the brain. Nine healthy subjects were injected with 18F-SP203 using a bolus plus constant infusion for 300 min. A single ratio of bolus-to-constant infusion (the activity of bolus equaled to that of infusion over 219 min) was applied to all subjects to achieve equilibrium in approximately 120 min. As a measure of ligand binding, we compared total distribution volume (VT) calculated by the equilibrium and kinetic methods in each scan. The equilibrium method calculated VT by the ratio of radioactivity in the brain to the concentration of 18F-SP203 in arterial plasma at 120 min, and the kinetic method calculated VT by a two-tissue compartment model using brain and plasma dynamic data from 0 to 120 min. VT obtained via the equilibrium method was highly correlated with VT obtained via kinetic modeling. Inter-subject variability of VT obtained via the equilibrium method was slightly smaller than VT obtained via the kinetic method. VT obtained via the equilibrium method was ~10% higher than VT obtained via the kinetic method, indicating a small difference between the measurements. Taken together, the results of this study show that using the equilibrium method is an acceptable alternative to the standard kinetic method when using 18F-SP203 to measure mGluR5. Although small differences in the measurements obtained via the

  18. The alpha7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca(2+) dependent mechanism.

    PubMed

    Dajas-Bailador, F A; Lima, P A; Wonnacott, S

    2000-10-01

    Neuronal nicotinic acetylcholine receptors (nAChR) have been suggested to play a role in a variety of modulatory and regulatory processes, including neuroprotection. Here we have characterized the neuroprotective effects of nicotine against an excitotoxic insult in primary hippocampal cultures. Exposure of hippocampal neurons to 200 microM NMDA for 1 h decreased cell viability by 25+/-5%, an effect blocked by NMDA receptor antagonists. Nicotine (10 microM) counteracted the NMDA-induced cell death when co-incubated with NMDA or when present subsequent to the NMDA treatment. Nicotine protection was prevented by 1 microM MLA, confirming that it was mediated by nAChR, and by 1 microM alpha-bungarotoxin, demonstrating that the alpha7 nAChR subtype was responsible. Both the NMDA evoked neurotoxicity and nicotine neuroprotection were Ca(2+)-dependent. In Fura-2-loaded hippocampal neurons, nicotine (10 microM) and NMDA (200 microM) acutely increased intracellular resting Ca(2+) from 70 nM to 200 and 500 nM, respectively. Responses to NMDA were unaffected by the presence of nicotine. (45)Ca(2+) uptake after a 1 h exposure to nicotine or NMDA also demonstrated quantitative differences between the two drugs. This study demonstrates that the alpha7 subtype of nAChR can support neuronal survival after an excitotoxic stimulus, through a Ca(2+) dependent mechanism that operates downstream of NMDA receptor activation.

  19. Involvement of subtype 1 metabotropic glutamate receptors in apoptosis and caspase-7 over-expression in spinal cord of neuropathic rats

    PubMed Central

    Siniscalco, Dario; Giordano, Catia; Fuccio, Carlo; Luongo, Livio; Ferraraccio, Franca; Rossi, Francesca; de Novellis, Vito; Roth, Kevin A.; Maione, Sabatino

    2008-01-01

    The effect of the non-selective, 1-aminoindan-1,5-dicarboxylic acid (AIDA), and selective (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4- methoxycyclohexyl) methanone (JNJ16259685), metabotropic glutamate subtype 1 (mGlu1) receptor antagonists, on rat sciatic nerve chronic constrictive injury (CCI)- induced hyperalgesia, allodynia, spinal dorsal horn apoptosis, and gliosis was examined at 3 and 7 days post-injury. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), nestin, GFAP, and caspase-7 mRNA in the dorsal horn spinal cord by 3 days post-CCI. At 7 days post-CCI, only over-expression of bcl-2, nestin and GFAP mRNA was observed. Administration of AIDA reduced thermal hyperalgesia and mechanical allodynia at 3 and 7 days post-CCI; administration of JNJ16259685 reduced thermal hyperalgesia at 3 and 7 days post-CCI, but not mechanical allodynia. AIDA decreased the mRNA levels of bax, apaf-1, GFAP and caspase-7 genes. JNJ16259685 increased the mRNA levels of bcl- 2 and GFAP gene, and decreased APAF-1 and caspases-7 genes. Inhibiting mGlu1 receptors also reduced TUNEL-positive profiles and immunohistochemical reactivity for caspase-7. We report here that despite inhibiting CCI-induced over-expression of pro-apoptotic genes in the spinal cord dorsal horn, the selective mGlu1 receptor antagonist JNJ16259685 exerted only a slight and transient allodynic effect. Moreover, JNJ16259685, but not the non-selective AIDA, increased astrogliosis which may account for its decreased analgesic efficacy. This study provides evidence that the contemporary and partial blockade of group I and likely ionotropic glutamate receptors may be a more suitable therapy than selective blockade of mGlu1 subtype receptors condition to decrease neuropathic pain symptoms. PMID:18325779

  20. Implication of 5-HT2A subtype receptors in DOI activity in the four-plates test-retest paradigm in mice.

    PubMed

    Ripoll, Nadège; Hascoët, Martine; Bourin, Michel

    2006-01-01

    The four-plates test (FPT) is an animal model of anxiety which allows the detection of anxiolytic effect not only of benzodiazepines (BZDs) but also of other non-BZDs anxiolytic compounds such as antidepressants (ADs). Furthermore, DOI, a 5-HT(2A/2C) agonist, has been shown to exert an anxiolytic-like effect in this model. Retesting mice in animal models of anxiety (test-retest paradigm) induces an anxiogenic-like and a loss of anxiolytic-like effects in response to BZDs and ADs. On the contrary, DOI has been reported to oppose the fear potentiation induced by trial 1 in the FPT. Despite DOI is considered as one of the most selective 5-HT(2A) available, it acts as agonist at all three 5-HT(2) receptor subtypes (5-HT(2A), 5-HT(2B) and 5-HT(2C)). The aim of this study was thus to investigate in the FPT test-retest paradigm, which 5-HT(2) receptor subtype(s) was involved in the DOI-induced effect in experienced mice. The effect of DOI (0.25-4 mg/kg) and the agonists, 5-HT(2B), BW 723C86 (1-16 mg/kg) and 5-HT(2C), RO 60-0175 (0.25-4 mg/kg) have also been studied. Then, antagonism studies were conducted combinating the 5-HT(2A) receptor antagonist SR 46349B, the 5-HT(2B/2C) receptor antagonist SB 206553 or the selective 5-HT(2C) receptor antagonist RS 10-2221 (at the doses of 0.1 and 1 mg/kg) with the DOI (1 mg/kg). Our study shows that the BW 723C86 had no effect on retesting mice, whereas it exerted an anxiolytic-like effect in naive mice. By contrast to DOI, the RO 60-0175 had no effect neither in naive nor experienced mice. Furthermore, only the SR 46349B antagonized the DOI-induced anti-punishment effect. Diazepam included as a positive control also increased in each case the number of punished passages in naive mice. Our findings altogether also suggest that DOI exerts its anxiolytic-like effect in the FPT test-retest paradigm through 5-HT(2A) receptors.

  1. Curcumin pretreatment mediates antidiabetogenesis via functional regulation of adrenergic receptor subtypes in the pancreas of multiple low-dose streptozotocin-induced diabetic rats.

    PubMed

    Naijil, George; Anju, T R; Jayanarayanan, S; Paulose, C S

    2015-09-01

    Lifestyle modification pivoting on nutritional management holds tremendous potential to meet the challenge of management of diabetes. The current study hypothesizes that regular uptake of curcumin lowers the incidence of diabetes by functional regulation of pancreatic adrenergic receptor subtypes. The specific objective of the study was to identify the regulatory pathways implicated in the antidiabetogenesis effect of curcumin in multiple low-dose streptozotocin (MLD-STZ)-induced diabetic Wistar rats. Administration of MLD-STZ to curcumin-pretreated rats induced a prediabetic condition. Scatchard analysis, real-time polymerase chain reaction, and confocal microscopic studies confirmed a significant increase in α2-adrenergic receptor expression in the pancreas of diabetic rats. Pretreatment with curcumin significantly decreased α2-adrenergic receptor expression. The diabetic group showed a significant decrease in the expression of β-adrenergic receptors when compared with control. Pretreatment significantly increased β-adrenergic receptor expression to near control. When compared with the diabetic rats, a significant up-regulation of CREB, phospholipase C, insulin receptor, and glucose transporter 2 were observed in the pretreated group. Curcumin pretreatment was also able to maintain near control levels of cyclic adenosine monophosphate, cyclic guanosine monophosphate, and inositol triphosphate. These results indicate that a marked decline in α2-adrenergic receptor function relents sympathetic inhibition of insulin release. It also follows that escalated signaling through β-adrenergic receptors mediates neuronal stimulation of hyperglycemia-induced β-cell compensatory response. Curcumin-mediated functional regulation of adrenergic receptors and modulation of key cell signaling molecules improve pancreatic glucose sensing, insulin gene expression, and insulin secretion. PMID:26255758

  2. Curcumin pretreatment mediates antidiabetogenesis via functional regulation of adrenergic receptor subtypes in the pancreas of multiple low-dose streptozotocin-induced diabetic rats.

    PubMed

    Naijil, George; Anju, T R; Jayanarayanan, S; Paulose, C S

    2015-09-01

    Lifestyle modification pivoting on nutritional management holds tremendous potential to meet the challenge of management of diabetes. The current study hypothesizes that regular uptake of curcumin lowers the incidence of diabetes by functional regulation of pancreatic adrenergic receptor subtypes. The specific objective of the study was to identify the regulatory pathways implicated in the antidiabetogenesis effect of curcumin in multiple low-dose streptozotocin (MLD-STZ)-induced diabetic Wistar rats. Administration of MLD-STZ to curcumin-pretreated rats induced a prediabetic condition. Scatchard analysis, real-time polymerase chain reaction, and confocal microscopic studies confirmed a significant increase in α2-adrenergic receptor expression in the pancreas of diabetic rats. Pretreatment with curcumin significantly decreased α2-adrenergic receptor expression. The diabetic group showed a significant decrease in the expression of β-adrenergic receptors when compared with control. Pretreatment significantly increased β-adrenergic receptor expression to near control. When compared with the diabetic rats, a significant up-regulation of CREB, phospholipase C, insulin receptor, and glucose transporter 2 were observed in the pretreated group. Curcumin pretreatment was also able to maintain near control levels of cyclic adenosine monophosphate, cyclic guanosine monophosphate, and inositol triphosphate. These results indicate that a marked decline in α2-adrenergic receptor function relents sympathetic inhibition of insulin release. It also follows that escalated signaling through β-adrenergic receptors mediates neuronal stimulation of hyperglycemia-induced β-cell compensatory response. Curcumin-mediated functional regulation of adrenergic receptors and modulation of key cell signaling molecules improve pancreatic glucose sensing, insulin gene expression, and insulin secretion.

  3. Synthesis and structure-affinity relationships of novel small molecule natural product derivatives capable of discriminating between serotonin 5-HT1A, 5-HT2A, 5-HT2C receptor subtypes

    PubMed Central

    Cummings, David F.; Canseco, Diana C.; Sheth, Pratikkumar; Johnson, James E.; Schetz, John A.

    2010-01-01

    Efforts to develop ligands that distinguish between clinically relevant 5-HT2A and 5-HT2C serotonin receptor subtypes have been challenging, because their sequences have high homology. Previous studies reported that a novel aplysinopsin belonging to a chemical class of natural products isolated from a marine sponge was selective for the 5-HT2C over the 5-HT2A receptor subtype. Our goal was to explore the 5-HT2A/2C receptor structure-affinity relationships of derivatives based on the aplysinopsin natural product pharmacophore. Twenty aplysinopsin derivatives were synthesized, purified and tested for their affinities for cloned human serotonin 5-HT1A, 5-HT2A and 5-HT2C receptor subtypes. Four compounds in this series had >30-fold selectivity for 5-HT2A or 5-HT2C receptors. The compound (E)-5-((5,6-dichloro-1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one (UNT-TWU-22, 16) had approximately 2100-fold selectivity for the serotonin 5-HT2C receptor subtype: an affinity for 5-HT2C equal to 46 nM and no detectable affinity for the 5-HT1A or 5-HT2A receptor subtypes. The two most important factors controlling 5-HT2A or 5-HT2C receptor subtype selectivity were the combined R1, R3-alkylation of the imidazolidinone ring and the type and number of halogens on the indole ring of the aplysinopsin pharmacophore. PMID:20570529

  4. Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences.

    PubMed Central

    Ramsay, Douglas; Kellett, Elaine; McVey, Mary; Rees, Stephen; Milligan, Graeme

    2002-01-01

    Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET(2) (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human delta-opioid receptor was confirmed using BRET(2). Homo-oligomerization of the kappa-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both delta- and kappa-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50,000-100,000 copies of the receptor energy acceptor construct per cell. The effectiveness of delta-kappa-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the beta(2)-adrenoceptor was also assessed. Although such interactions were detected, at least 250,000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the kappa-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100,000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression. PMID:11971762

  5. Expansion of the. alpha. sub 2 -adrenergic receptor family: Cloning and characterization of a human. alpha. sub 2 -adrenergic receptor subtype, the gene for which is located on chromosome 2

    SciTech Connect

    Lomasney, J.W.; Lorenz, W.; Allen, L.F.; King, K.; Caron, M.G.; Lefkowitz, R.J. ); Regan, J.W. ); Yang-Feng, T.L. )

    1990-07-01

    Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple {alpha}{sub 2}-adrenergic receptor ({alpha}{sub 2}AR) subtypes. The authors have cloned a human {alpha}{sub 2}AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned {alpha}{sub 2}ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the {alpha}{sub 2}ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique {alpha}{sub 2}AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an {alpha}{sub 2}AR subtype not previously identified by classical pharmacological or ligand binding approaches.

  6. Murine embryonic stem cell line CGR8 expresses all subtypes of muscarinic receptors and multiple nicotinic receptor subunits: Down-regulation of α4- and β4-subunits during early differentiation.

    PubMed

    Kaltwasser, Susanne; Schmitz, Luise; Michel-Schmidt, Rosmarie; Anspach, Laura; Kirkpatrick, Charles James; Wessler, Ignaz

    2015-11-01

    Non-neuronal acetylcholine mediates its cellular effects via stimulation of the G-protein-coupled muscarinic receptors and the ligand-gated ion channel nicotinic receptors. The murine embryonic stem cell line CGR8 synthesizes and releases non-neuronal acetylcholine. In the present study a systematic investigation of the expression of nicotinic receptor subunits and muscarinic receptors was performed, when the stem cells were grown in the presence or absence of LIF, as the latter condition induces early differentiation. CGR8 cells expressed multiple nicotinic receptor subtypes (α3, α4, α7, α9, α10, β1, β2, β3, β4, γ, δ, ε) and muscarinic receptors (M1, M3, M4, M5); M2 was detected only in 2 out of 8 cultures. LIF removal caused a down-regulation only of the α4- and β4-subunit. In conclusion, more or less the whole repertoire of cholinergic receptors is expressed on the murine embryonic stem cell line CGR8 for mediating cellular signaling of non-neuronal acetylcholine which acts via auto- and paracrine pathways. During early differentiation of the murine CGR8 stem cell signaling via nicotinic receptors containing α4- or β4 subunits is reduced. Thus, the so-called neuronal α4 nicotine receptor composed of these subunits may be involved in the regulation of pluripotency in this murine stem cell line.

  7. Discovery and Labeling of High Affinity 3,4-Diarylpyrazolines as Candidate Radioligands for In Vivo Imaging of Cannabinoid Subtype-1 (CB1) Receptors

    PubMed Central

    Donohue, Sean R.; Pike, Victor W.; Finnema, Sjoerd J.; Truong, Phong; Andersson, Jan; Gulyás, Balázs; Halldin, Christer

    2008-01-01

    Imaging of cannabinoid subtype-1 (CB1) receptors in vivo with positron emission tomography (PET) is likely to be important for understanding their role in neuropsychiatric disorders and for drug development. Radioligands for imaging with PET are required for this purpose. We synthesized new ligands from a 3,4-diarylpyrazoline platform of which (-)-12a ((-)-3-(4-chlorophenyl)-N’-[(4-cyanophenyl)sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine) was found to have high-affinity and selectivity for binding to CB1 receptors. (-)-12a and its lower affinity enantiomer ((+)-12a) were labeled with carbon-11 (t1/2 = 20.4 min) using [11C]cyanide ion as labeling agent and evaluated as PET radioligands in cynomolgus monkey. After injection of [11C](-)-12a there was high uptake and retention of radioactivity across brain according to the rank order of CB1 receptor densities. The distomer, [11C](+)-12a, failed to give a sustained CB1 receptor-specific distribution. Polar radiometabolites of [11C](-)-12a appeared moderately slowly in plasma. Radioligand [11C](-)-12a is promising for the study of brain CB1 receptors and merits further investigation in human subjects. PMID:18754613

  8. Rescue of amyloid-Beta-induced inhibition of nicotinic acetylcholine receptors by a peptide homologous to the nicotine binding domain of the alpha 7 subtype.

    PubMed

    Nery, Arthur A; Magdesian, Margaret H; Trujillo, Cleber A; Sathler, Luciana B; Juliano, Maria A; Juliano, Luiz; Ulrich, Henning; Ferreira, Sergio T

    2013-01-01

    Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

  9. Subtype-selective induction of wild-type p53 and apoptosis, but not cell cycle arrest, by human somatostatin receptor 3.

    PubMed

    Sharma, K; Patel, Y C; Srikant, C B

    1996-12-01

    Somatostatin (SST) exerts direct antiproliferative effects in tumor cells, triggering either growth arrest or apoptosis. The cellular actions of SST are transduced through a family of five distinct somatostatin receptor subtypes (SSTR1-5). Whereas growth inhibition has been reported to follow stimulation of protein tyrosine phosphatase via SSTR2 or inhibition of Ca2+ channels via SSTR5 in heterologous expression systems, the subtype selectivity for signaling apoptosis has not been investigated. The tumor suppressor protein p53 and the protooncogene product c-Myc regulate cell cycle progression (growth factors present) or apoptosis (growth factors absent). The p53-induced G1 arrest requires induction of p21, an inhibitor of cyclin-dependent kinases, whereas apoptosis requires induction of Bax. c-Myc is capable of abrogating p53-induced G1 arrest by interfering with the inhibitory action of p21 on cyclin-dependent kinases. We have, therefore, investigated the regulation of p53, p21, c-Myc, and Bax and cellular apoptosis in relation to cell cycle progression in CHO-K1 cells stably expressing individual human SSTR1-5. We demonstrate that apoptosis is signaled uniquely through human SSTR3 and is associated with dephosphorylation-dependent conformational change in wild-type (wt) p53 as well as induction of Bax. The induction of wt p53 occurs rapidly and precedes the onset of apoptosis. We show that the increase in wt p53 is not associated with the induction of p21 or c-Myc when octreotide-induced apoptosis becomes evident, suggesting that such apoptosis does not require G1 arrest and is not c-Myc dependent. These findings provide the first evidence for hormonal induction of wt p53-associated apoptosis via G protein-coupled receptor in a subtype-selective manner.

  10. Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness.

    PubMed

    Ikegami, Taro; Maruyama, Yusuke; Doi, Hiroyuki; Hattori, Atsuhiko; Ando, Hironori

    2015-01-01

    Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel 1a 1.4, mel 1a 1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0-15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer.

  11. (/sup 3/H)U-69593 labels a subtype of kappa opiate receptor with characteristics different from that labeled by (/sup 3/H)ethylketocyclazocine

    SciTech Connect

    Nock, B.; Rajpara, A.; O'Connor, L.H.; Cicero, T.J.

    1988-01-01

    (/sup 3/H)U-69593 is an opiate agonist that has been reported to bind in vitro with high affinity and selectivity to the kappa receptor subtype. The studies reported here were designed to determine the optimal conditions for labeling kappa receptors with (/sup 3/H)U-69593 and to further characterize the binding site. The effects of temperature and NaCl on (/sup 3/H)U-69593 binding were of particular interest because previous studies reported that (/sup 3/H)ethylketocyclazocine ((/sup 3/H)EKC) and (/sup 3/H)bremazocine binding to kappa receptors was optimal at 4/sup 0/C in the presence of NaCl. Those conditions were not found to be optimal for (/sup 3/H)U-69593 binding. Although the pharmacological specificity and Bmax of (/sup 3/H)U-69593 binding was similar at room temperature and at 4/sup 0/C, the binding affinity was approximately three times lower at 4/sup 0/C than at room temperature. In addition, NaCl had an effect on (/sup 3/H)U-69593 binding that was opposite that on (/sup 3/H)EKC binding at 4/sup 0/C. These differences between (/sup 3/H)U-69593 and (/sup 3/H)EKC binding at 4/sup 0/C were accentuated by a vast difference in the density of the binding sites and suggested that (/sup 3/H)U-69593 might bind selectively to a kappa receptor subtype.

  12. Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice

    PubMed Central

    Mulak, Agata; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Rivier, Jean; Taché, Yvette

    2014-01-01

    Somatostatin interacts with 5 G-protein-coupled receptor (sst1–5). Octreotide, a stable sst2≫3≥5 agonist, octreotide, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1–5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10 μg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (4 sets of 3 CRD, each at 55 mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between 2 sets of graded CRD (15, 30, 45, and 60 mmHg, 3 times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60 mmHg CRD, respectively. ODT8-SST (10 μg) and the sst2 agonist, S-346-011 (3 and 10 μg) prevented mechanically-induced visceral hypersensitivity in the 3 sets of CRD, the sst1 agonist (10 μg) blocked only the 2nd set and showed a trend at 3 μg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10 μg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10 μg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization. PMID:25451334

  13. Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness

    PubMed Central

    Ikegami, Taro; Maruyama, Yusuke; Doi, Hiroyuki; Hattori, Atsuhiko; Ando, Hironori

    2015-01-01

    Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel1a1.4, mel1a1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0–15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer. PMID:25688184

  14. Classification of distinct subtypes of peripheral T-cell lymphoma unspecified, identified by chemokine and chemokine receptor expression: Analysis of prognosis.

    PubMed

    Ohshima, Koichi; Karube, Kennosuke; Kawano, Riko; Tsuchiya, Takeshi; Suefuji, Hiroaki; Yamaguchi, Takahiro; Suzumiya, Junji; Kikuchii, Masahiro

    2004-09-01

    WHO classification for malignant lymphoma was recently proposed. However, PTCL is heterogeneous. Chemokines and its receptors are closely associated with the T-cell subtypes. To clarify the T-cell subtype in PTCL, we conducted DNA chips of chemokine, its receptor (R) and cytokines. Angioimmunoblastic T-cell lymphoma (AILD, n=4), anaplastic large cell lymphoma (ALCL, n=4), adult T-cell leukemia lymphoma (ATLL, n=7), NK-cell lymphoma (NKL, n=2) and PTCL, unspecified (PTCL-U, n=6) were analyzed using DNA chips. In addition, immunological stainings were performed in 280 cases. In DNA chip, AILD, ALCL, NKL and ATLL showed a tendency for respective clusters, otherwise, PTCL-U clustered with AILD, ALCL and ATLL. From the gene expression profiling, CCR4, CCR3, MIG, CXCR3 and BLC were selected for immunohistochemistry. ATLL (n=48) expressed CCR4. ALCL (n=26) expressed CCR3, NKL (n=20) expressed MIG, and AILD (n=29) expressed CXCR3 and/or BLC. From the expression patterns, PTCL-U (n=134) were classified into three groups; CCR4 type (CCR4(+), n=42), CCR3 type (CCR3(+), n=31) and CXCR3 type (CXCR3(+) BLC(+/-), n=54). The prognosis was poor for ATLL, intermediate for AILD and favorable for ALCL (P=0.0014). Among PTCL-U, CCR4 type, CXCR3 type and CCR3 type had prognoses equivalent to ATLL, AILD and ALCL, respectively (P<0.0001).

  15. Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4β2 and α3β4 subtypes.

    PubMed

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna; Kaczor, Agnieszka A; Poso, Antti; Jozwiak, Krzysztof

    2015-02-01

    To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca2+ influx results established that: (a) varenicline activates (μM range) nAChR subtypes with the following rank sequence: hα7>hα4β4>hα4β2>hα3β4>hα1β1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4β2~hα4β4>hα3β4>hα7>Torpedo α1β1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs. PMID:25475645

  16. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  17. Activation of Muscarinic Acetylcholine Receptor Subtype 4 Is Essential for Cholinergic Stimulation of Gastric Acid Secretion: Relation to D Cell/Somatostatin

    PubMed Central

    Takeuchi, Koji; Endoh, Takuya; Hayashi, Shusaku; Aihara, Takeshi

    2016-01-01

    Background/Aim: Muscarinic acetylcholine receptors exist in five subtypes (M1∼M5), and they are widely expressed in various tissues to mediate diverse autonomic functions, including gastric secretion. In the present study, we demonstrated, using M1∼M5 KO mice, the importance of M4 receptors in carbachol (CCh) stimulation of acid secretion and investigated how the secretion is modulated by the activation of M4 receptors. Methods: C57BL/6J mice of wild-type (WT) and M1–M5 KO were used. Under urethane anesthesia, acid secretion was measured in the stomach equipped with an acute fistula. CCh (30 μg/kg) was given subcutaneously (s.c.) to stimulate acid secretion. Atropine or octreotide (a somatostatin analog) was given s.c. 20 min before the administration of CCh. CYN154806 (a somatostatin SST2 receptor antagonist) was given i.p. 20 min before the administration of octreotide or CCh. Results: CCh caused an increase of acid secretion in WT mice, and the effect was totally inhibited by prior administration of atropine. The effect of CCh was similarly observed in the animals lacking M1, M2 or M5 receptors but significantly decreased in M3 or M4 KO mice. CYN154806, the SST2 receptor antagonist, dose-dependently and significantly reversed the decreased acid response to CCh in M4 but not M3 KO mice. Octreotide, the somatostatin analog, inhibited the secretion of acid under CCh-stimulated conditions in WT mice. The immunohistochemical study showed the localization of M4 receptors on D cells in the stomach. Serum somatostatin levels in M4 KO mice were higher than WT mice under basal conditions, while those in WT mice were significantly decreased in response to CCh. Conclusions: These results suggest that under cholinergic stimulation the acid secretion is directly mediated by M3 receptors and indirectly modified by M4 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and minimizes the acid inhibitory effect of

  18. (3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

    SciTech Connect

    Norman, A.B.; Battaglia, G.; Creese, I.

    1985-12-01

    In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.

  19. Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

    PubMed Central

    Dillon, G. H.; Im, W. B.; Carter, D. B.; McKinley, D. D.

    1995-01-01

    1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582470

  20. Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

    PubMed

    Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

    2015-06-01

    We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.

  1. Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

    USGS Publications Warehouse

    Jett, D.A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.

    1993-01-01

    The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

  2. Effects of chronic nicotine treatment on expression of diverse nicotinic acetylcholine receptor subtypes. I. Dose- and time-dependent effects of nicotine treatment.

    PubMed

    Ke, L; Eisenhour, C M; Bencherif, M; Lukas, R J

    1998-08-01

    Nicotinic acetylcholine receptors (nAChRs) exist as a diverse family of physiologically important ligand-gated ion channels active in classic, excitatory neurotransmission and perhaps in more novel forms of neurochemical signaling. Because of their critical functional roles centrally and peripherally, nAChRs are ideal targets for the regulation of nervous system function. nAChRs also are targets of nicotine, which acts acutely like acetylcholine to stimulate nAChR function. Here, we report studies using model cell culture systems testing the general hypothesis that more chronic nicotine exposure has unique effects on nAChRs. Chronic nicotine treatment induces increases in numbers of human muscle-type nAChRs containing alpha-1, beta-1, gamma and delta subunits, a human ganglionic nAChR subtype containing alpha-3 and beta-4 subunits and a human ganglionic nAChR containing alpha-7 subunits in intracellular and (except for alpha-7 nAChRs) in cell surface pools. However, the half-maximal potency with which nicotine has these effects differs across these nAChR subtypes, as do rates and magnitudes of the "nicotine-induced nAChR up-regulation." These changes in nAChR numbers are not attributable to either transient or sustained changes in nAChR subunit mRNA levels. Nicotine exposure more potently, more rapidly, and with nAChR-subtype specificity, induces two phases of losses in functional responsiveness of muscle-type nAChRs and alpha-3 beta-4 nAChRs, including a "persistent inactivation" that is distinct from classicly defined "desensitization." Based on these results, we hypothesize that chronic nicotine treatment induces persistent functional inactivation and numerical up-regulation of all nAChR subtypes via distinct post-transcriptional mechanisms and with potencies, at rates and with magnitudes that are nAChR-subtype specific. We also hypothesize that chronic nicotine exposure produces long-lasting changes in nervous system function, at least in part, by disabling

  3. Racial disparities in individual breast cancer outcomes by hormone-receptor subtype, area-level socio-economic status and healthcare resources

    PubMed Central

    Akinyemiju, Tomi; Moore, Justin Xavier; Ojesina, Akinyemi I.; Waterbor, John W.; Altekruse, Sean F

    2016-01-01

    The aim of the study is to determine the influence of area-level socio-economic status and healthcare access in addition to tumor hormone-receptor subtype on individual breast cancer stage, treatment, and mortality among Non-Hispanic (NH)-Black, NH-White, and Hispanic US adults. Analysis was based on 456,217 breast cancer patients in the SEER database from 2000 to 2010. Multilevel and multivariable-adjusted logistic and Cox proportional hazards regression analysis was conducted to account for clustering by SEER registry of diagnosis. NH-Black women had greater area-level access to healthcare resources compared with women of other races. For instance, the average numbers of oncology hospitals per million population in counties with NH-Black, NH-White, and Hispanic women were 8.1, 7.7, and 5.0 respectively; average numbers of medical doctors per million in counties with NH-Black, NH-White, and Hispanic women were 100.7, 854.0, and 866.3 respectively; and average number of Ob/Gyn in counties with NH-Black, NH-White, and Hispanic women was 155.6, 127.4, and 127.3, respectively (all p values <0.001). Regardless, NH-Black women (HR 1.39, 95 % CI 1.36–1.43) and Hispanic women (HR 1.05, 95 % CI 1.03–1.08) had significantly higher breast cancer mortality compared with NH-White women even after adjusting for hormone-receptor subtype, area-level socioeconomic status, and area-level healthcare access. In addition, lower county-level socio-economic status and healthcare access measures were significantly and independently associated with stage at presentation, surgery, and radiation treatment as well as mortality after adjusting for age, race/ethnicity, and HR subtype. Although breast cancer HR subtype is a strong, important, and consistent predictor of breast cancer outcomes, we still observed significant and independent influences of area-level SES and HCA on breast cancer outcomes that deserve further study and may be critical to eliminating breast cancer outcome

  4. Synthesis and structure-activity relationships of a novel series of non-peptide angiotensin II receptor binding inhibitors specific for the AT2 subtype.

    PubMed

    Blankley, C J; Hodges, J C; Klutchko, S R; Himmelsbach, R J; Chucholowski, A; Connolly, C J; Neergaard, S J; Van Nieuwenhze, M S; Sebastian, A; Quin, J

    1991-11-01

    Structure-activity relationships are reported for a novel class of 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid derivatives that displace 125I-labeled angiotensin II from a specific subset of angiotensin II (Ang II) binding sites in rat adrenal preparations. This binding site is not the Ang II receptor mediating vascular contraction or aldosterone release, but, rather, is one whose function has not yet been fully elucidated. It has been identified in a number of tissues and has a similar affinity for Ang II and its peptide analogues as does the vascular receptor. The non-peptide compounds reported here are uniquely specific in displacing Ang II at this binding site and are inactive in antagonizing Ang II at the vascular receptor or in pharmacological assays measuring vascular effects. PD 123,319 (79), one of the most potent compounds, has an IC50 of 34 nM. Certain of these compounds may have utility in the definition and study of Ang II receptor subtypes.

  5. Radiosynthesis and evaluation of an 18F-labeled positron emission tomography (PET) radioligand for brain histamine subtype-3 receptors based on a nonimidazole 2-aminoethylbenzofuran chemotype

    PubMed Central

    Bao, Xiaofeng; Lu, Shuiyu; Liow, Jeih-San; Zoghbi, Sami S.; Jenko, Kimberly J.; Clark, David T.; Gladding, Robert L.; Innis, Robert B.; Pike, Victor W.

    2012-01-01

    A known chemotype of H3 receptor ligand was explored for development of a radioligand for imaging brain histamine subtype 3 (H3) receptors in vivo with positron emission tomography (PET), namely non-imidazole 2-aminoethylbenzofurans, represented by the compound (R)-(2-(2-(2-methylpyrrolidin-1-yl)ethyl)benzofuran-5-yl)(4-fluorophenyl)methanone (9). Compound 9 was labeled with fluorine-18 (t1/2= 109.7 min) in high specific activity by treating the prepared nitro analog (12) with cyclotron-produced [18F]fluoride ion. [18F]9 was studied with PET in mouse and in monkey after intravenous injection. [18F]9 showed favorable properties as a candidate PET radioligand, including moderately high brain uptake with a high proportion of H3 receptor-specific signal in the absence of radiodefluorination. The nitro compound 12 was found to have even higher H3 receptor affinity, indicating the potential of this chemotype for the development of further promising PET radioligands. PMID:22313227

  6. Influence of moonlight on mRNA expression patterns of melatonin receptor subtypes in the pineal organ of a tropical fish.

    PubMed

    Park, Yong-Ju; Park, Ji-Gweon; Takeuchi, Yuki; Hur, Sung-Pyo; Lee, Young-Don; Kim, Se-Jae; Takemura, Akihiro

    2014-04-01

    The goldlined spinefoot, Siganus guttatus, is a lunar-synchronized spawner, which repeatedly releases gametes around the first quarter moon during the reproductive season. A previous study reported that manipulating moonlight brightness at night disrupted synchronized spawning, suggesting involvement of this natural light source in lunar synchronization. The present study examined whether the mRNA expression pattern of melatonin receptor subtypes MT1 and Mel1c in the pineal organ of the goldlined spinefoot is related to moonlight. Real-time quantitative polymerase chain reaction analysis revealed that the abundance of MT1 and Mel1c mRNA at midnight increased during the new moon phase and decreased during the full moon phase. Exposing fish to moonlight intensity during the full moon period resulted in a decrease in Mel1c mRNA abundance within 1h. Fluctuations in the melatonin receptor genes according to changes in the moon phase agreed with those of melatonin levels in the blood. These results indicate that periodic changes in cues from the moon influence melatonin receptor mRNA expression levels. The melatonin-melatonin receptor system may play a role in predicting the moon phase through changes in night brightness. PMID:24269345

  7. Influence of moonlight on mRNA expression patterns of melatonin receptor subtypes in the pineal organ of a tropical fish.

    PubMed

    Park, Yong-Ju; Park, Ji-Gweon; Takeuchi, Yuki; Hur, Sung-Pyo; Lee, Young-Don; Kim, Se-Jae; Takemura, Akihiro

    2014-04-01

    The goldlined spinefoot, Siganus guttatus, is a lunar-synchronized spawner, which repeatedly releases gametes around the first quarter moon during the reproductive season. A previous study reported that manipulating moonlight brightness at night disrupted synchronized spawning, suggesting involvement of this natural light source in lunar synchronization. The present study examined whether the mRNA expression pattern of melatonin receptor subtypes MT1 and Mel1c in the pineal organ of the goldlined spinefoot is related to moonlight. Real-time quantitative polymerase chain reaction analysis revealed that the abundance of MT1 and Mel1c mRNA at midnight increased during the new moon phase and decreased during the full moon phase. Exposing fish to moonlight intensity during the full moon period resulted in a decrease in Mel1c mRNA abundance within 1h. Fluctuations in the melatonin receptor genes according to changes in the moon phase agreed with those of melatonin levels in the blood. These results indicate that periodic changes in cues from the moon influence melatonin receptor mRNA expression levels. The melatonin-melatonin receptor system may play a role in predicting the moon phase through changes in night brightness.

  8. Both alpha2 and alpha3 GABAA receptor subtypes mediate the anxiolytic properties of benzodiazepine site ligands in the conditioned emotional response paradigm.

    PubMed

    Morris, H V; Dawson, G R; Reynolds, D S; Atack, J R; Stephens, D N

    2006-05-01

    Mice with point-mutated alpha2 GABAA receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in unconditioned models of anxiety. We investigated the role of the alpha2 GABAA subtype in a model of conditioned anxiety. alpha2(H101R) and wildtype mice were trained in a conditioned emotional response (CER) task, in which lever-pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a conditioned stimulus (CS+) that predicted footshock. The ability of diazepam, ethanol and pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. Diazepam (0, 0.5, 1, 2, 4 and 8 mg/kg) induced a dose-dependent anxiolytic-like effect in wildtype mice. At high doses, diazepam (2, 4 and 8 mg/kg) was sedative in alpha2(H101R) mice. Analysis of the anxiolytic properties of nonsedative diazepam doses (0.5 and 1 mg/kg), showed that alpha2(H101R) mice were resistant to the anxiolytic effects of diazepam. Equivalent anxiolytic properties of pentobarbital (20 mg/kg) and ethanol (1 and 2 g/kg) were seen in both genotypes. These findings confirm the critical importance of the alpha2 GABAA subtype in mediating BZ anxiolysis. However, as a compound, L-838417, with agonist properties at alpha2, alpha3 and alpha5-containing receptors, gave rise to anxiolytic-like activity in alpha2(H101R) mice in the CER test, alpha3-containing GABA receptors are also likely to contribute to anxiolysis. Observations that alpha2(H101R) mice were more active, and displayed a greater suppression of lever pressing in response to fear-conditioned stimuli than wildtype mice, suggests that the alpha2(H101R) mutation may not be behaviourally silent.

  9. A divergent role for estrogen receptor-beta in node-positive and node-negative breast cancer classified according to molecular subtypes: an observational prospective study

    PubMed Central

    Novelli, Flavia; Milella, Michele; Melucci, Elisa; Di Benedetto, Anna; Sperduti, Isabella; Perrone-Donnorso, Raffaele; Perracchio, Letizia; Venturo, Irene; Nisticò, Cecilia; Fabi, Alessandra; Buglioni, Simonetta; Natali, Pier Giorgio; Mottolese, Marcella

    2008-01-01

    Introduction Estrogen receptor-alpha (ER-α) and progesterone receptor (PgR) are consolidated predictors of response to hormonal therapy (HT). In contrast, little information regarding the role of estrogen receptor-beta (ER-β) in various breast cancer risk groups treated with different therapeutic regimens is available. In particular, there are no data concerning ER-β distribution within the novel molecular breast cancer subtypes luminal A (LA) and luminal B (LB), HER2 (HS), and triple-negative (TN). Methods We conducted an observational prospective study using immunohistochemistry to evaluate ER-β expression in 936 breast carcinomas. Associations with conventional biopathological factors and with molecular subtypes were analyzed by multiple correspondence analysis (MCA), while univariate and multivariate Cox regression analysis and classification and regression tree analysis were applied to determine the impact of ER-β on disease-free survival in the 728 patients with complete follow-up data. Results ER-β evenly distributes (55.5%) across the four molecular breast cancer subtypes, confirming the lack of correlation between ER-β and classical prognosticators. However, the relationships among the biopathological factors, analyzed by MCA, showed that ER-β positivity is located in the quadrant containing more aggressive phenotypes such as HER2 and TN or ER-α/PgR/Bcl2- tumors. Kaplan-Meier curves and Cox regression analysis identified ER-β as a significant discriminating factor for disease-free survival both in the node-negative LA (P = 0.02) subgroup, where it is predictive of response to HT, and in the node-positive LB (P = 0.04) group, where, in association with PgR negativity, it conveys a higher risk of relapse. Conclusion Our data indicated that, in contrast to node-negative patients, in node-positive breast cancer patients, ER-β positivity appears to be a biomarker related to a more aggressive clinical course. In this context, further investigations

  10. Putative therapeutic targets for symptom subtypes of adult ADHD: D4 receptor agonism and COMT inhibition improve attention and response inhibition in a novel translational animal model.

    PubMed

    Tomlinson, Anneka; Grayson, Ben; Marsh, Samuel; Hayward, Andrew; Marshall, Kay M; Neill, Joanna C

    2015-04-01

    Prefrontal cortical dopamine plays an important role in cognitive control, specifically in attention and response inhibition; the core deficits in ADHD. We have previously shown that methylphenidate and atomoxetine differentially improve these deficits dependent on baseline performance. The present study extends this work to investigate the effects of putative therapeutic targets in our model. A selective dopamine D4 receptor agonist (A-412997) and the catechol-O-methyl-transferase (COMT) inhibitor; tolcapone, were investigated in the combined subtype of adult ADHD (ADHD-C). Adult female rats were trained to criterion in the 5C-CPT (5-Choice Continuous Performance Task) and then separated into subgroups according to baseline levels of sustained attention, vigilance, and response disinhibition. The subgroups included: high-attentive (HA) and low-attentive with high response disinhibition (ADHD-C). The ADHD-C subgroup was selected to represent the combined subtype of adult ADHD. Effects of tolcapone (3.0, 10.0, 15.0mg/kg) and A-412997 (0.1, 0.3, 1.0µmol/kg) were tested by increasing the variable inter-trial-interval (ITI) duration in the 5C-CPT. Tolcapone (15mg/kg) significantly increased sustained attention, vigilance and response inhibition in ADHD-C animals, and impaired attention in HA animals. A-412997 (1.0µmol/kg) significantly increased vigilance and response inhibition in ADHD-C animals only, with no effect in HA animals. This is the first study to use the translational 5C-CPT to model the adult ADHD-C subtype in rats and to study new targets in this model. Both tolcapone and A-412997 increased vigilance and response inhibition in the ADHD-C subgroup. D4 and COMT are emerging as important potential therapeutic targets in adult ADHD that warrant further investigation.

  11. Structure and Receptor Binding Preferences of Recombinant Hemagglutinins from Avian and Human H6 and H10 Influenza A Virus Subtypes

    PubMed Central

    Yang, Hua; Carney, Paul J.; Chang, Jessie C.; Villanueva, Julie M.

    2015-01-01

    ABSTRACT During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. IMPORTANCE Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses. PMID:25673707

  12. Orally Active Metabotropic Glutamate Subtype 2 Receptor Positive Allosteric Modulators: Structure-Activity Relationships and Assessment in a Rat Model of Nicotine Dependence

    PubMed Central

    Sidique, Shyama; Dhanya, Raveendra-Panickar; Sheffler, Douglas J.; Nickols, Hilary Highfield; Yang, Li; Dahl, Russell; Mangravita-Novo, Arianna; Smith, Layton H.; D’Souza, Manoranjan S.; Semenova, Svetlana; Conn, P. Jeffrey; Markou, Athina; Cosford, Nicholas D. P.

    2012-01-01

    Compounds that modulate metabotropic glutamate subtype 2 (mGlu2) receptors have the potential to treat several disorders of the central nervous system (CNS) including drug dependence. Herein we describe the synthesis and structure-activity relationship (SAR) studies around a series of mGlu2 receptor positive allosteric modulators (PAMs). The effects of N-substitution (R1) and substitutions on the aryl ring (R2) were identified as key areas for SAR exploration (Figure 3). Investigation of the effects of varying substituents in both the isoindolinone (2) and benzisothiazolone (3) series led to compounds with improved in vitro potency and/or efficacy. In addition, several analogues exhibited promising pharmacokinetic (PK) properties. Furthermore, compound 2 was shown to dose-dependently decrease nicotine self-administration in rats following oral administration. Our data, showing for the first time efficacy of an mGlu2 receptor PAM in this in vivo model, suggest potential utility for the treatment of nicotine dependence in humans. PMID:23009245

  13. Immunohistochemical distribution of somatostatin and somatostatin receptor subtypes (SSTR1-5) in hypothalamus of ApoD knockout mice brain.

    PubMed

    Kumar, Ujendra

    2012-11-01

    In the present study, the expression of somatostatin (SST) and somatostatin receptor subtypes (SSTR1-5) was determined in the hypothalamus of wild-type (wt) and apolipoprotein D knockout (ApoD(-/-)) mice brain. SST-like immunoreactivity, while comparable in most regions of hypothalamus, diminished significantly in arcuate nucleus of ApoD(-/-) mice. SSTR1 strongly localized in all major hypothalamic nuclei as well as in the median eminence and ependyma of the third ventricle of wt mice brain. SSTR1-like immunoreactivity increases in hypothalamus except in paraventricular nucleus of ApoD(-/-) mice. SSTR2 was well expressed in most of the hypothalamic regions whereas it decreases significantly in ventromedial and arcuate nucleus of ApoD(-/-) mice. SSTR3 and SSTR4-like immunoreactivity increases in ApoD(-/-) mice in all major nuclei of hypothalamus, median eminence, and ependymal cells of third ventricle. SSTR5 is well expressed in ventromedial and arcuate nucleus whereas weakly expressed in paraventricular nucleus. In comparison to wt, ApoD(-/-) mice exhibit increased SSTR5-like immunoreactivity in paraventricular nuclei and decreased receptor expression in ventromedial hypothalamus and arcuate nucleus. In conclusion, the changes in hypothalamus of ApoD(-/-) mice may indicate potential role of ApoD in regulation of endocrine functions of somatostatin in a receptor-dependent manner.

  14. The potency and efficacy of anticholinergics to inhibit haloperidol-induced catalepsy in rats correlates with their rank order of affinities for the muscarinic receptor subtypes.

    PubMed

    Erosa-Rivero, Helena B; Bata-García, José L; Alvarez-Cervera, Fernando J; Heredia-López, Francisco J; Góngora-Alfaro, José L

    2014-06-01

    Extrapyramidal syndromes (EPS) caused by antipsychotic therapy are currently treated with anticholinergics that lack selectivity for the five muscarinic receptor subtypes. Since these receptors are heterogeneously expressed among the different classes of striatal neurons and their afferents, it can be expected that their simultaneous blockade will cause distinct, sometimes opposed, effects within the striatal circuitry. In order to test the hypothesis that the differential blockade of the muscarinic receptor subtypes would influence their potency and efficacy to prevent EPS, here we tested four anticholinergics with varying order of affinities for the muscarinic receptor subtypes, and compared their dose-response curves to inhibit haloperidol-induced catalepsy in male rats. Drugs were applied into the lateral ventricle 15 min before haloperidol (2 mg/kg, s.c.). Catalepsy was measured in the bar test at 15 min intervals during 5 h. The preferential M1/M4 antagonist pirenzepine (3, 10, 30, 100, and 300 nmol) caused a dose-dependent inhibition of catalepsy intensity: ED50 = 5.6 nmol [95% CI, 3.9-8.1], and latency: ED50 = 5.6 nmol [95% CI, 3.7-8.6]. Pirenzepine had the steepest dose-response curve, producing maximal inhibition (84 ± 5%) at the dose of 10 nmol, while its effect tended to reverse at higher doses (62 ± 11%). The purported M1/M3 antagonist 4-DAMP (30, 100, and 300 nmol) also caused a dose-dependent inhibition of catalepsy intensity: ED50 = 29.5 nmol [95% CI, 7.0 to 123.0], and latency: ED50 = 28.5 nmol [95% CI, 2.2 to 362.0]. However, the curve for 4-DAMP had a less pronounced slope, reaching its maximal effect (63 ± 14%) at the dose of 300 nmol. The M2/M4 antagonist AF-DX 116 (10, 30, and 300 nmol) only caused a partial inhibition of catalepsy (30 ± 11%) at the dose of 30 nmol, but this changed to a non-significant increment (15 ± 10%) at the dose of 100 nmol. The alleged M4 antagonist tropicamide (30, 100, 300, and

  15. (11) C-labeled and (18) F-labeled PET ligands for subtype-specific imaging of histamine receptors in the brain.

    PubMed

    Funke, Uta; Vugts, Danielle J; Janssen, Bieneke; Spaans, Arnold; Kruijer, Perry S; Lammertsma, Adriaan A; Perk, Lars R; Windhorst, Albert D

    2013-01-01

    The signaling molecule histamine plays a key role in the mediation of immune reactions, in gastric secretion, and in the sensory system. In addition, it has an important function as a neurotransmitter in the central nervous system, acting in pituitary hormone secretion, wakefulness, motor and cognitive functions, as well as in itch and nociception. This has raised interest in the role of the histaminergic system for the treatment and diagnosis of various pathologies such as allergy, sleeping and eating disorders, neurodegeneration, neuroinflammation, mood disorders, and pruritus. In the past 20 years, several ligands targeting the four different histamine receptor subtypes have been explored as potential radiotracers for positron emission tomography (PET). This contribution provides an overview of the developments of subtype-selective carbon-11-labeled and fluorine-18-labeled compounds for imaging in the brain. Using specific radioligands, the H1 R expression in human brain could be examined in diseases such as schizophrenia, depression, and anorexia nervosa. In addition, the sedative effects of antihistamines could be investigated in terms of H1 R occupancy. The H3 R is of special interest because of its regulatory role in the release of various other neurotransmitters, and initial H3 R PET imaging studies in humans have been reported. The H4 R is the youngest member of the histamine receptor family and is involved in neuroinflammation and various sensory pathways. To date, two H4 R-specific (11) C-labeled ligands have been synthesized, and the imaging of the H4 R in vivo is in the early stage.

  16. A selective positive allosteric modulator of metabotropic glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis.

    PubMed

    Benneyworth, Michael A; Xiang, Zixiu; Smith, Randy L; Garcia, Efrain E; Conn, P Jeffrey; Sanders-Bush, Elaine

    2007-08-01

    Recent clinical studies reveal that selective agonists of group II metabotropic glutamate (mGlu) receptors have robust efficacy in treating positive and negative symptoms in patients with schizophrenia. Group II mGlu receptor agonists also modulate the in vivo activity of psychotomimetic drugs and reduce the ability of psychotomimetic hallucinogens to increase glutamatergic transmission. Because increased excitation of the medial prefrontal cortex (mPFC) has been implicated in pathophysiology of schizophrenia, the ability of group II mGlu receptor agonists to reduce hallucinogenic drug action in this region is believed to be directly related to their antipsychotic efficacy. A novel class of ligands, termed positive allosteric modulators, has recently been identified, displaying exceptional mGlu2 receptor selectivity. These compounds do not activate mGlu2 receptors directly but potentiate the ability of glutamate and other agonists to activate this receptor. We now report that the mGlu2 receptor-selective positive allosteric modulator biphenyl-indanone A (BINA) modulates excitatory neurotransmission in the mPFC and attenuates the in vivo actions of the hallucinogenic 5-HT(2A/2C) receptor agonist (-)2,5-dimethoxy-4-bromoamphetamine [(-)DOB]. BINA attenuates serotonin-induced increases in spontaneous excitatory postsynaptic currents in the mPFC, mimicking the effect of the mGlu2/3 receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV). In addition, BINA reduced (-)DOB-induced head twitch behavior and Fos expression in mPFC, effects reversed by pretreatment with the mGlu2/3 receptor antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl) -3 - (xanth-9-yl-)propionic acid (LY341495). These data confirm the relevance of excitatory signaling in the mPFC to the behavioral actions of hallucinogens and further support the targeting of mGlu2 receptors as a novel strategy for treating glutamatergic dysfunction in schizophrenia.

  17. Nicotine effects on human endothelial intercellular communication via α4β2 and α3β2 nicotinic acetylcholine receptor subtypes.

    PubMed

    Duerrschmidt, Nicole; Hagen, Anja; Gaertner, Christiane; Wermke, Alice; Nowicki, Marcin; Spanel-Borowski, Katharina; Stepan, Holger; Mohr, Friedrich-Wilhelm; Dhein, Stefan

    2012-06-01

    Since previous in vitro experiments revealed that nicotine can impair endothelial intercellular communication via the downregulation of connexin43 (Cx43), we wanted to find out which nicotinic acetylcholine receptors are involved in the molecular mechanism of communication failure. Cultured human endothelial cells were exposed to 1 μM nicotine for 5 days. Intercellular communication was measured using dye transfer study with/without subtype-specific nicotinic acetylcholine receptor (nAChR) inhibitors. Reverse transcriptase (RT)-PCR was used to further investigate the regulation of nAChR subtypes. Electron microscopy together with MAP LC3-II western blot was used to investigate possible autophagy processes. In cultured human endothelial cells, nicotine decreased the Cx43 protein amount as shown by western blot and immunohistochemistry; however, together with an unaltered mRNA expression as shown by RT-PCR. The nicotine-induced Cx43 downregulation functionally impaired intercellular dye transfer, which could be prevented by mecamylamine, κ-bungarotoxin, lobeline, and dihydro-β-erythroidine but not α-bungarotoxin, indicating that the nAChR subtypes α4β2 and α3β2 but not α7 are involved in signal cascade. RT-PCR analysis revealed that nicotine exposure resulted in the upregulation of α3 and β4 and the downregulation of α4-nAChR, while α7- and β2-nAChR-mRNA expressions remained unaltered. Furthermore, nicotine increased total protein ubiquinylation and proteasome activity as was shown by immunohistochemistry and peptide degradation analysis. Evidence of enhanced autophagic processes was assured by the occurrence of autophagic vacuoles in transmission electron microscopy and enhanced formation of MAP LC3-II in western blot. Reduced intercellular endothelial communication together with programmed cell death helps to explain the toxic effect of nicotine leading to endothelial dysfunction. The nAChR involved in the impairment of intercellular communication

  18. Opposite influence of the metabotropic glutamate receptor subtypes mGlu3 and -5 on astrocyte proliferation in culture.

    PubMed

    Ciccarelli, R; Sureda, F X; Casabona, G; Di Iorio, P; Caruso, A; Spinella, F; Condorelli, D F; Nicoletti, F; Caciagli, F

    1997-12-01

    In non-synchronized, subconfluent secondary cultures of rat cortical astrocytes, the selective group-I metabotropic glutamate (mGlu) receptor agonist 3,5-dihydroxyphenylglycine (DHPG) increased [methyl-3H]-thymidine incorporation. This effect was mediated by the activation of the mGlu5 receptor, which was shown to be present by either RT-PCR or Western blot analysis. The mixed mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine reduced the increase in both intracellular Ca2+ and [methyl-3H]-thymidine incorporation produced by DHPG. In contrast, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), a potent and selective agonist of group-II mGlu receptors, reduced [methyl-3H]-thymidine incorporation in non-synchronized astrocyte cultures. The antiproliferative effect of DCG-IV was prevented by the selective group-II mGlu receptor antagonist (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV). The opposite effect of DHPG and DCG-IV on astrocyte proliferation was confirmed in cultures deprived of serum for 48 hours and then stimulated to proliferate with either epidermal growth factor (EGF) or the metabolically stable ATP analogue adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP). We conclude that activation of mGlu5 receptors enhances proliferation in cultured astrocytes, whereas activation of a receptor with pharmacological characteristics similar to those of mGlu2/3 receptors reduces proliferation. PMID:9419014

  19. Role of Dopamine Receptors Subtypes, D1-Like and D2-Like, within the Nucleus Accumbens Subregions, Core and Shell, on Memory Consolidation in the One-Trial Inhibitory Avoidance Task

    ERIC Educational Resources Information Center

    Manago, Francesca; Castellano, Claudio; Oliverio, Alberto; Mele, Andrea; De Leonibus, Elvira

    2009-01-01

    Recent evidence demonstrated that dopamine within the nucleus accumbens mediates consolidation of both associative and nonassociative memories. However, the specific contribution of the nucleus accumbens subregions, core and shell, and of D1 and D2 receptors subtypes has not been yet clarified. The aim of this study was, therefore, to directly…

  20. Methyl substitution of 2-aminobicyclo[3.1.0]hexane 2,6-dicarboxylate (LY354740) determines functional activity at metabotropic glutamate receptors: identification of a subtype selective mGlu2 receptor agonist.

    PubMed

    Dominguez, Carmen; Prieto, Lourdes; Valli, Matthew J; Massey, Steven M; Bures, Mark; Wright, Rebecca A; Johnson, Bryan G; Andis, Sherri L; Kingston, Ann; Schoepp, Darryle D; Monn, James A

    2005-05-19

    LY354740 (1) is a highly potent and selective agonist of metabotropic glutamate (mGlu) receptors 2 and 3. In the present study, we have prepared C3- and C4-methyl-substituted variants of rac-1, compounds 5, 9, and 13. Each of these racemic methyl-substituted analogues displaced specific binding of the mGlu2/3 receptor antagonist (3)H-2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid ((3)H-LY341495) from membranes expressing mGlu2 or mGlu3 receptor subtypes. Evaluation of the functional effects of this series on second messenger responses in cells expressing human mGlu2 or mGlu3 receptors revealed C3beta-methyl analogue 5 to possess antagonist properties at both mGlu2 and mGlu3 receptors while C4beta-methyl analogue 9 acts as a full agonist at each of these targets. Unexpectedly, we found that incorporation of a methyl substituent at the C4alpha-position as in analogue 13 results in a mixed mGlu2 agonist/mGlu3 antagonist pharmacological profile. All of the mGlu2 agonist and mGlu3 antagonist activity of rac-13 was found to reside in its resolved (+)-isomer. PMID:15887967

  1. Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes.

    PubMed

    Bell, L Andrew; Bell, Karen A; McQuiston, A Rory

    2013-10-01

    Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K(+) channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function

  2. Corticotropin-releasing factor receptor subtype 2 in human colonic mucosa: Down-regulation in ulcerative colitis

    PubMed Central

    Chatzaki, Ekaterini; Anton, Peter A; Million, Mulugeta; Lambropoulou, Maria; Constantinidis, Theodoros; Kolios, George; Taché, Yvette; Grigoriadis, Dimitri E

    2013-01-01

    AIM: To assess corticotropin-releasing factor receptor 2 (CRF2) expression in the colon of healthy subjects and patients with ulcerative colitis (UC). METHODS: We examined CRF2 gene and protein expression in the distal/sigmoid colonic mucosal biopsies from healthy subjects and patients with UC (active or disease in remission), human immunodeficiency virus (HIV) and functional bowel disease (FBD) by reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: Gene expression of CRF2 was demonstrated in the normal human colonic biopsies, but not in the human colorectal adenocarcinoma cell line Caco2. Receptor protein localization showed immunoreactive CRF2 receptors in the lamina propria and in the epithelial cells of the distal/sigmoid biopsy samples. Interestingly, CRF2 immunoreactivity was no longer observed in epithelial cells of patients with mild-moderately active UC and disease in remission, while receptor protein expression did not change in the lamina propria. No differences in CRF2 expression profile were observed in distal/sigmoid intestinal biopsies from HIV infection and FBD patients, showing no signs of inflammation. CONCLUSION: The down-regulation of the CRF2 receptor in the distal/sigmoid biopsies of UC patients is indicative of change in CRF2 signalling associated with the process of inflammation. PMID:23539366

  3. The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcohol-seeking behaviors.

    PubMed

    Harvey, Scott C; Foster, Katrina L; McKay, Pete F; Carroll, Michelle R; Seyoum, Regat; Woods, James E; Grey, Collette; Jones, Cecily M; McCane, Shannan; Cummings, Rancia; Mason, Dynesha; Ma, Chunrong; Cook, James M; June, Harry L

    2002-05-01

    We investigated the potential role of the alpha1-containing GABA(A) receptor in regulating the reinforcing properties of alcohol. To accomplish this, we developed 3-propoxy-beta-carboline hydrochloride (3-PBC), a mixed agonist-antagonist benzodiazepine site ligand with binding selectivity at the alpha1 receptor. We then tested the capacity of 3-PBC to block alcohol-maintained responding in the ventral pallidum (VP), a novel alcohol reward substrate, which primarily expresses the alpha1-receptor isoform. Our results demonstrated that bilateral microinfusion of 3-PBC (0.5-40 microg) in the anterior and medial VP produced marked reductions in alcohol-maintained responding in a genetically selected rodent model of alcohol drinking. The VP infusions showed both neuroanatomical and reinforcer specificity because no effects were seen in sites dorsal to the VP (e.g., nucleus accumbens, caudate putamen). The saccharin-maintained responding was reduced only with the highest dose (40 microg). Parenteral injections of 3-PBC (1-20 mg/kg) also showed a similar selectivity on alcohol-maintained responding. Complementary in vitro studies revealed that 3-PBC exhibited a low partial agonist efficacy profile at recombinant diazepam-sensitive receptors (e.g., alpha1beta3gamma2, alpha2beta3gamma, and alpha3beta3gamma2). The selective suppression of 3-PBC on alcohol-maintained responding after central and parenteral administrations, together with its low-efficacy agonist profile, suggest that the reduction in alcohol-maintained behaviors was not attributable to a general suppression on consummatory behaviors. These results demonstrate that the alpha1-containing GABA(A) receptors in both the anterior and medial VP are important in regulating the reinforcing properties of alcohol. These receptors represent novel targets in the design and development of pharmacotherapies for alcohol-dependent subjects. PMID:11978852

  4. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.

    PubMed

    Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G

    2012-11-21

    Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

  5. A case of lithium intoxication induced by an antihypertensive angiotensin 1 subtype-specific angiotensin II receptor blocker in an elderly patient with bipolar disorder and hypertension.

    PubMed

    Hayashi, Yuichi; Nishida, Shohei; Takekoshi, Akira; Murakami, Muneharu; Yamada, Megumi; Kimura, Akio; Suzuki, Akio; Inuzuka, Takashi

    2016-01-01

    Lithium carbonate is considered to be a first-line treatment for bipolar disorder; however, this drug has a narrow therapeutic window, and lithium intoxication is commonly induced by various drugs interaction and situations. We herein report a case of lithium intoxication induced by the administration of an antihypertensive agent targeting the angiotensin 1 (AT1) subtype of the angiotensin II receptor in a 65-year-old woman with a 40-year history of bipolar disorder type 1, and 1-year history of essential hypertension. Her bipolar disorder had been well-controlled with 600 mg/day of lithium carbonate for more than 10 years. She was later diagnosed with hypertension and the AT1 receptor blocker, azilsartan was thereafter administrated on a daily basis. After 3 weeks of azilsartan administration, she presented with progressive action tremor and showed a gradual deterioration of her physical state. Four months after the start of azilsartan administration, she presented with alternating episodes of diarrhea and constipation. Two weeks before admission to our hospital, she presented with mild consciousness disturbances, myoclonus, truncal ataxia, and appetite loss. She was diagnosed to have lithium intoxication based on an elevated serum lithium concentration of 3.28 mEq/l.It is therefore important to evaluate the serum lithium concentration after the administration of antihypertensive agents, and consider lithium-antihypertensive agent interactions when selecting antihypertensive agents in elderly patients receiving long-term lithium carbonate treatment. PMID:27535187

  6. Syntheses of 2-amino and 2-halothiazole derivatives as high-affinity metabotropic glutamate receptor subtype 5 ligands and potential radioligands for in vivo imaging.

    PubMed

    Siméon, Fabrice G; Wendahl, Matthew T; Pike, Victor W

    2011-02-10

    The structure of the potent selective mGlu(5) ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu(5) ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed subnanomolar mGlu(5) affinity. 10b also displayed >10000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [(18)F]fluoride ion (t(1/2) = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu(5) with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu(5) and may also serve as a lead to a potential (123)I-labeled ligand for imaging brain mGlu(5) with single photon emission computed tomography. PMID:21207959

  7. Syntheses of 2-Amino and 2-Halothiazole Derivatives as High-Affinity Metabotropic Glutamate Receptor Subtype 5 Ligands and Potential Radioligands for In Vivo Imaging

    PubMed Central

    Siméon, Fabrice G; Wendahl, Matthew T.; Pike, Victor W.

    2011-01-01

    The structure of the potent selective mGlu5 ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu5 ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed sub-nanomolar mGlu5 affinity. 10b also displayed >10,000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [18F]fluoride ion (t1/2 = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu5 with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu5 and may also serve as a lead to a potential 123I-labeled ligand for imaging brain mGlu5 with single photon emission computed tomography. PMID:21207959

  8. 68Ga-DOTATOC PET and gene expression profile in patients with neuroendocrine carcinomas: strong correlation between PET tracer uptake and gene expression of somatostatin receptor subtype 2

    PubMed Central

    Olsen, Ingrid H; Langer, Seppo W; Federspiel, Birgitte H; Oxbøl, Jytte; Loft, Annika; Berthelsen, Anne Kiil; Mortensen, Jann; Oturai, Peter; Knigge, Ulrich; Kjær, Andreas

    2016-01-01

    Somatostatin receptor expression on both protein and gene expression level was compared with in vivo 68Ga-DOTATOC PET/CT in patients with neuroendocrine carcinomas (NEC). Twenty-one patients with verified NEC who underwent a 68Ga-DOTATOC PET/CT between November 2012 and May 2014, were retrospectively included. By real-time polymerase chain reaction, we quantitatively determined the gene expression of several genes and compared with 68Ga-DOTATOC PET uptake. By immunohistochemistry we qualitatively studied the expression of assorted proteins in NEC. The median age at diagnosis was 68 years (range 41-84) years. All patients had WHO performance status 0-1. Median Ki67 index was 50% (range 20-100%). Gene expression of somatostatin receptor subtype (SSTR) 2 and Ki67 were both positively correlated to the 68Ga-DOTATOC uptake (r=0.89; p<0.0001 and r=0.5; p=0.021, respectively). Furthermore, SSTR2 and SSTR5 gene expression were strongly and positively correlated (r=0.57; p=0.006). This study as the first verifies a positive and close correlation of 68Ga-DOTATOC uptake and gene expression of SSTR2 in NEC. SSTR2 gene expression has a stronger correlation to 68Ga-DOTATOC uptake than SSTR5. In addition, the results indicate that the gene expression levels of SSTR2 and SSTR5 at large follow one another. PMID:27069766

  9. MRI Kinetics With Volumetric Analysis in Correlation With Hormonal Receptor Subtypes and Histologic Grade of Invasive Breast Cancers

    PubMed Central

    Leong, Lester Chee Hao; Gombos, Eva C.; Jagadeesan, Jayender; Fook-Chong, Stephanie Man Chung

    2016-01-01

    OBJECTIVE The aim of this study was to assess whether computer-assisted detection–processed MRI kinetics data can provide further information on the biologic aggressiveness of breast tumors. MATERIALS AND METHODS We identified 194 newly diagnosed invasive breast cancers presenting as masses on contrast-enhanced MRI by a HIPAA-compliant pathology database search. Computer-assisted detection–derived data for the mean and median peak signal intensity percentage increase, most suspicious kinetic curve patterns, and volumetric analysis of the different kinetic patterns by mean percentage tumor volume were compared against the different hormonal receptor (estrogen-receptor [ER], progesterone-receptor [PR], ERRB2 (HER2/neu), and triple-receptor expressivity) and histologic grade subgroups, which were used as indicators of tumor aggressiveness. RESULTS The means and medians of the peak signal intensity percentage increase were higher in ER-negative, PR-negative, and triple-negative (all p ≤ 0.001), and grade 3 tumors (p = 0.011). Volumetric analysis showed higher mean percentage volume of rapid initial enhancement in biologically more aggressive ER-negative, PR-negative, and triple-negative tumors compared with ER-positive (64% vs 53.6%, p = 0.013), PR-positive (65.4% vs 52.5%, p = 0.001), and nontriple-negative tumors (65.3% vs 54.6%, p = 0.028), respectively. A higher mean percentage volume of rapid washout component was seen in ERRB2-positive tumors compared with ERRB2-negative tumors (27.5% vs 17.9%, p = 0.020). CONCLUSION Peak signal intensity percentage increase and volume analysis of the different kinetic patterns of breast tumors showed correlation with hormonal receptor and histologic grade indicators of cancer aggressiveness. Computer-assisted detection–derived MRI kinetics data have the potential to further characterize the aggressiveness of an invasive cancer. PMID:25714321

  10. Role of NPY and its receptor subtypes in foraging, food hoarding, and food intake by Siberian hamsters.

    PubMed

    Day, Diane E; Keen-Rhinehart, Erin; Bartness, Timothy J

    2005-07-01

    Fasting has widespread physiological and behavioral effects such as increases in arcuate nucleus neuropeptide Y (NPY) gene expression in rodents, including Siberian hamsters. Fasting also stimulates foraging and food hoarding (appetitive ingestive behaviors) by Siberian hamsters but does relatively little to change food intake (consummatory ingestive behavior). Therefore, we tested the effects of third ventricular NPY Y1 ([Pro(34)]NPY) or Y5 ([D-Trp(34)]NPY) receptor agonists on these ingestive behaviors using a wheel running-based food delivery system coupled with simulated burrow housing. Siberian hamsters had 1) no running wheel access and free food, 2) running wheel access and free food, or 3) foraging requirements (10 or 50 revolutions/pellet). NPY (1.76 nmol) stimulated food intake only during the first 4 h postinjection ( approximately 200-1,000%) and mostly in hamsters with a foraging requirement. The Y1 receptor agonist markedly increased food hoarding (250-1,000%), increased foraging as well as wheel running per se, and had relatively little effect on food intake (<250%). Unlike NPY, the Y5 agonist significantly increased food intake, especially in foraging animals ( approximately 225-800%), marginally increased food hoarding (250-500%), and stimulated foraging and wheel running 4-24 h postinjection, with the distribution of earned pellets favoring eating versus hoarding across time. Across treatments, food hoarding predominated early postinjection, whereas food intake tended to do so later. Collectively, NPY stimulated both appetitive and consummatory ingestive behaviors in Siberian hamsters involving Y1/Y5 receptors, with food hoarding and foraging/wheel running (appetitive) more involved with Y1 receptors and food intake (consummatory) with Y5 receptors.

  11. Differences in Estrogen Receptor Subtype According to Family History of Breast Cancer among Hispanic, but not Non-Hispanic White Women

    PubMed Central

    Hines, Lisa M.; Risendal, Betsy; Slattery, Martha L.; Baumgartner, Kathy B.; Giuliano, Anna R.; Byers, Tim

    2008-01-01

    Background Pathologic differences have been reported among breast tumors when comparing ethnic populations. Limited research has been done to evaluate the ethnic-specific relationships between breast cancer risk factors and the pathologic features of breast tumors. Methods Given that genetic variation may contribute to ethnic-related etiologic differences in breast cancer, we hypothesized that tumor characteristics differ according to family history of breast cancer among Hispanic and non-Hispanic White (NHW) women. Logistic regression models were used to compute odds ratios (OR) and 95% confidence intervals (95% CI) to assess this relationship in the population-based, case-control 4-Corners Breast Cancer Study (1,537 cases and 2,452 controls). Results Among Hispanic women, having a family history was associated with a 2.7-fold increased risk of estrogen receptor (ER) negative (95% CI, 1.59-4.44), but not ER positive tumors (OR, 1.04; 95% CI, 0.71-1.54) when compared with women without breast cancer. In contrast, there was an increased risk for ER positive (OR, 1.89; 95% CI, 1.50-2.38) and a marginally significant increased risk for ER negative tumors (OR, 1.41; 95% CI, 0.92-2.17) among NHW women. When comparing tumor characteristics among invasive cases, those with a family history also had a significantly higher proportion of ER negative tumors among Hispanics (39.2% versus 25.8%; P = 0.02), but not among NHWs (16.3% versus 21.1%; P = 0.13). Conclusions These results may reflect ethnic-specific predisposing genetic factors that promote the development of specific breast tumor subtypes, and emphasize the importance of evaluating the relationship between breast cancer risk factors and breast tumor subtypes among different ethnic populations. PMID:18843012

  12. The role of the serotonin receptor subtypes 5-HT1A and 5-HT7 and its interaction in emotional learning and memory

    PubMed Central

    Stiedl, Oliver; Pappa, Elpiniki; Konradsson-Geuken, Åsa; Ögren, Sven Ove

    2015-01-01

    Serotonin [5-hydroxytryptamine (5-HT)] is a multifunctional neurotransmitter innervating cortical and limbic areas involved in cognition and emotional regulation. Dysregulation of serotonergic transmission is associated with emotional and cognitive deficits in psychiatric patients and animal models. Drugs targeting the 5-HT system are widely used to treat mood disorders and anxiety-like behaviors. Among the fourteen 5-HT receptor (5-HTR) subtypes, the 5-HT1AR and 5-HT7R are associated with the development of anxiety, depression and cognitive function linked to mechanisms of emotional learning and memory. In rodents fear conditioning and passive avoidance (PA) are associative learning paradigms to study emotional memory. This review assesses the role of 5-HT1AR and 5-HT7R as well as their interplay at the molecular, neurochemical and behavioral level. Activation of postsynaptic 5-HT1ARs impairs emotional memory through attenuation of neuronal activity, whereas presynaptic 5-HT1AR activation reduces 5-HT release and exerts pro-cognitive effects on PA retention. Antagonism of the 5-HT1AR facilitates memory retention possibly via 5-HT7R activation and evidence is provided that 5HT7R can facilitate emotional memory upon reduced 5-HT1AR transmission. These findings highlight the differential role of these 5-HTRs in cognitive/emotional domains of behavior. Moreover, the results indicate that tonic and phasic 5-HT release can exert different and potentially opposing effects on emotional memory, depending on the states of 5-HT1ARs and 5-HT7Rs and their interaction. Consequently, individual differences due to genetic and/or epigenetic mechanisms play an essential role for the responsiveness to drug treatment, e.g., by SSRIs which increase intrasynaptic 5-HT levels thereby activating multiple pre- and postsynaptic 5-HTR subtypes. PMID:26300776

  13. Functional characterization of estrogen receptor subtypes, ER{alpha} and ER{beta}, mediating vitellogenin production in the liver of rainbow trout

    SciTech Connect

    Leanos-Castaneda, Olga Kraak, Glen van der

    2007-10-15

    The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ER{alpha} and ER{beta}, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ER{alpha} selective agonist, methyl-piperidino-pyrazole (MPP) an ER{alpha} selective antagonist, and diarylpropionitrile (DPN) an ER{beta} selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [{sup 3}H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ER{alpha} could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ER{beta}. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ER{alpha}. On the other hand, once blocked ER{alpha} with MPP, the only manifestation of agonist activity of estradiol would be achieved via ER{beta}. In conclusion, the present results indicate that vitellogenin production is

  14. The stereotypy-inducing and OCD-like effects of chronic ‘binge’ cocaine are modulated by distinct subtypes of nicotinic acetylcholine receptors

    PubMed Central

    Metaxas, A; Keyworth, HL; Yoo, JH; Chen, Y; Kitchen, I; Bailey, A

    2012-01-01

    BACKGROUND AND PURPOSE High rates of cigarette smoking occur in cocaine-dependent individuals, reflecting an involvement of nicotinic acetylcholine receptors (nAChRs) in cocaine-elicited behaviour. This study was designed to assess the contribution of different nAChR subtypes to the behavioural and neurochemical effects of chronic cocaine treatment. EXPERIMENTAL APPROACH Cocaine (15 mg·kg−1, i.p.) was administered to male C57BL/6J mice in a chronic ‘binge’ paradigm, with and without the coadministration of the α7 preferring nAChR antagonist methyllycaconitine (MLA; 5 mg·kg−1, i.p.) or the β2* nAChR antagonist dihydro-β-erythroidine (DHβE; 2 mg·kg−1, i.p.). Quantitative autoradiography was used to examine the effect of cocaine exposure on α7 and α4β2* nAChRs, and on the high-affinity choline transporter. KEY RESULTS MLA+cocaine administration induced an intense self-grooming behaviour, indicating a likely role for α7 nAChRs in modulating this anxiogenic, compulsive-like effect of cocaine. In the major island of Calleja, a key area of action for neuroleptics, MLA+cocaine reduced choline transporter binding compared with cocaine (with or without DHβE) administration. DHβE treatment prevented the induction of stereotypy sensitisation to cocaine but prolonged locomotor sensitisation, implicating heteromeric β2* nAChRs in the neuroadaptations mediating cocaine-induced behavioural sensitisation. ‘Binge’ cocaine treatment region-specifically increased α4β2* nAChR binding in the midbrain dopaminergic regions: ventral tegmental area and substantia nigra pars compacta. CONCLUSIONS AND IMPLICATIONS We have shown a differential, subtype-selective, contribution of nAChRs to the behavioural and neurochemical sequelae of chronic cocaine administration. These data support the clinical utility of targeting specific nAChR subtypes for the alleviation of cocaine-abuse symptomatology. PMID:22568685

  15. ETS-1-mediated transcriptional up-regulation of CD44 is required for sphingosine-1-phosphate receptor subtype 3-stimulated chemotaxis.

    PubMed

    Zhang, Wenliang; Zhao, Jiawei; Lee, Jen-Fu; Gartung, Allison; Jawadi, Hiba; Lambiv, Wanyu Louis; Honn, Kenneth V; Lee, Menq-Jer

    2013-11-01

    Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response.

  16. Selective stimulation of excitatory amino acid receptor subtypes and the survival of cerebellar granule cells in culture: effect of kainic acid.

    PubMed

    Balázs, R; Hack, N; Jørgensen, O S

    1990-01-01

    Our previous studies showed that the survival of cerebellar granule cells in culture is promoted by treatment with N-methyl-D-aspartate. Here we report on the influence of another glutamate analogue, kainic acid, which, in contrast to N-methyl-D-aspartate, is believed to stimulate transmitter receptors mediating fast excitatory postsynaptic potentials. The kainate effect was complex: increased survival at low concentrations (the maximum, at 25-50 microM, was about 50% promotion), whereas concentrations exceeding 50 microM resulted first in a loss of the effect, and then at concentrations of 2-5 x 10(-4) M cells became vulnerable to kainate. The trophic influence of kainate is mediated through receptors other than the N-methyl-D-aspartate preferring subtype. In contrast to the effect of N-methyl-D-aspartate, that of kainate did not depend on the medium K+ level and was potently blocked by dinitroquinoxalinedione, which--at the concentration used here--did not counteract the promotion of cell survival evoked by N-methyl-D-aspartate. Quisqualate was a potent inhibitor of the rescue by kainate. Furthermore, blockade of N-methyl-D-aspartate receptors with the selective antagonists MK-801 or aminophosphonovalerate did not inhibit, but rather potentiated the trophic effect of kainate. Possible mechanisms underlying the trophic effect of chronic depolarization or treatment with excitatory amino acids are discussed, and it is proposed that they involve elevated free cytoplasmic calcium activity following increased influx through voltage-sensitive Ca2+ channels (high K+ and kainate) or receptorgated channels (N-methyl-D-aspartate).

  17. An examination of the pharmacology of two substance P antagonists and the evidence for tachykinin receptor subtypes.

    PubMed Central

    Bailey, S. J.; Featherstone, R. L.; Jordan, C. C.; Morton, I. K.

    1986-01-01

    The potencies of two tachykinin antagonists [D-Pro4,D-Trp7,9,10]-SP(4-11) and [D-Arg1,D-Pro2, D-Trp7,9,Leu11]-SP(1-11) against four tachykinins were examined in a range of smooth muscle preparations, including guinea-pig ileum and bladder and rat colon muscularis mucosae and duodenum. Parallel shifts in the log dose-response curves of all the tachykinins tested were observed in all tissues, except in the case of the guinea-pig bladder where [D-Pro4, D-Trp7,9,10]-SP(4-11) was without effect at concentrations up to 32 microM. The slopes of the Schild plots for the two antagonists did not differ significantly from unity, with the exception of [D-Pro4, D-Trp7,9,10]-SP(4-11) in the rat duodenum, which may indicate a heterogeneous receptor population in this tissue. The antagonists displayed agonist selectivity in the case of the guinea-pig ileum where log dose-response curves to substance P and physalaemin were shifted less than those to eledoisin and kassinin. Rank orders of potency for eledoisin, kassinin, physalaemin and substance P in the five preparations studied allowed classification of the tissues by the predominant receptor type according to the 'SP-P' and 'SP-E' scheme. It is concluded that [D-Pro4, D-Trp7,9,10]-SP(4-11), in particular, displays tissue selectivity that may indicate different receptor populations, but classification of receptor and tissue types on this basis does not fully correspond with classifications based on agonist potencies. Such schemes should therefore be treated with caution at this stage. PMID:2420403

  18. Prostaglandin E Receptor Subtype 4 Signaling in the Heart: Role in Ischemia/Reperfusion Injury and Cardiac Hypertrophy

    PubMed Central

    Cai, Yin; Tang, Eva Hoi Ching; Ma, Haichun

    2016-01-01

    Prostaglandin E2 (PGE2) is an endogenous lipid mediator, produced from the metabolism of arachidonic acids, upon the sequential actions of phospholipase A2, cyclooxygenases, and prostaglandin E synthases. The various biological functions governed by PGE2 are mediated through its four distinct prostaglandin E receptors (EPs), designated as EP1, EP2, EP3, and EP4, among which the EP4 receptor is the one most widely distributed in the heart. The availability of global or cardiac-specific EP4 knockout mice and the development of selective EP4 agonists/antagonists have provided substantial evidence to support the role of EP4 receptor in the heart. However, like any good drama, activation of PGE2-EP4 signaling exerts both protective and detrimental effects in the ischemic heart disease. Thus, the primary object of this review is to provide a comprehensive overview of the current progress of the PGE2-EP4 signaling in ischemic heart diseases, including cardiac hypertrophy and myocardial ischemia/reperfusion injury. A better understanding of PGE2-EP4 signaling should promote the development of more effective therapeutic approaches to treat the ischemic heart diseases without triggering unwanted side effects. PMID:27190998

  19. Schaffer collateral and perforant path inputs activate different subtypes of NMDA receptors on the same CA1 pyramidal cell

    PubMed Central

    Arrigoni, Elda; Greene, Robert W

    2004-01-01

    The two major inputs to CA1 pyramidal neurons, the perforant pathway (PP) that terminates on distal dendrites and the Schaffer collaterals (SCH) that terminate on proximal dendrites, activate both AMPA and N-methyl-D-aspartate (NMDA) receptors. In an in vitro slice preparation, the pharmacologically isolated NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) (NMDA-EPSCs) of either pathway can be selectively activated onto a single CA1 pyramidal neuron. Analysis of the decay phase of PP and SCH NMDA-EPSCs revealed no significant difference in their time constants, suggesting no apparent different distribution in NR2-subunit composition in the NMDA receptors (NMDAR) activated by the two synaptic inputs. However, application of the NR2B-selective antagonist, ifenprodil, differently affected the NMDA-EPSCs activated by the PP and SCH inputs. The reduction of the PP responses was only 30% compared to 75% for the SCH responses. In addition, for both pathways, the ifenprodil-insensitive component of the NMDA-EPSCs had significantly more rapid decay kinetics than those prior to application of ifenprodil. Our results show a greater NR2B subunit contribution to the NMDA component of the SCH EPSC, compared to the NMDA component of the PP EPSC and that in single CA1 pyramidal neurons NMDA composition is anatomically specific to the afferent input. PMID:15155538

  20. Autocrine interferon-γ may affect malignant behavior and sensitivity to tamoxifen of MCF-7 via estrogen receptor β subtype.

    PubMed

    Niu, Xiu Long; Wang, Yue; Yao, Zhi; Duan, Hongjie; Li, Zhijun; Liu, Wenxing; Zhang, Hongjian; Deng, Wei Min

    2015-12-01

    Mitogenic actions of estrogens are mediated by two distinct estrogen receptors (ERs), which are critical in the progression and therapeutic response of breast cancer. ER expression is a dynamic phenomenon that is regulated by numerous factors, including cytokines, in the tumor microenvironment. Recently, studies have shown that autocrine production of IL-4 promotes cancer cell growth and there is negative correlation between tumor IL-4 and hormone receptor levels, suggesting that there is crosstalk between cytokine receptors and ER. Thus, we evaluated for interaction between the two ERs and the cytokines IL-4 and IFN-γ, and if this interaction modulates malignant behavior. We identified that ERβ exerts protective activity in the progression of breast cancer cell line MCF-7, which co-expresses ERα and ERβ. IFN-γ and IL-4 have the opposite effects on malignant biological behavior. Furthermore, we found positive correlation between IFN-γ and ERβ expression in MCF-7. We also determined that autocrine IFN-γ in MCF-7 increases mRNA expression of ERβ resulting in enhanced sensitivity to tamoxifen (TAM). These results indicate that ERβ and autocrine IFN-γ represent two putative targets for breast cancer therapy.

  1. Somatostatin receptor subtype 4 activation is involved in anxiety and depression-like behavior in mouse models.

    PubMed

    Scheich, Bálint; Gaszner, Balázs; Kormos, Viktória; László, Kristóf; Ádori, Csaba; Borbély, Éva; Hajna, Zsófia; Tékus, Valéria; Bölcskei, Kata; Ábrahám, István; Pintér, Erika; Szolcsányi, János; Helyes, Zsuzsanna

    2016-02-01

    Somatostatin regulates stress-related behavior and its expression is altered in mood disorders. However, little is known about the underlying mechanisms, especially about the importance of its receptors (sst1-sst5) in anxiety and depression-like behavior. Here we analyzed the potential role of sst4 receptor in these processes, since sst4 is present in stress-related brain regions, but there are no data about its functional relevance. Genetic deletion of sst4 (Sstr4(-/-)) and its pharmacological activation with the newly developed selective non-peptide agonist J-2156 were used. Anxiety was examined in the elevated plus maze (EPM) and depression-like behavior in the forced swim (FST) and tail suspension tests (TST). Neuronal activation during the TST was monitored by Fos immunohistochemistry, receptor expression was identified by sst4(LacZ) immunostaining in several brain regions. Sstr4(-/-) mice showed increased anxiety in the EPM and enhanced depression-like behavior in the FST. J-2156 (100 μg/kg i.p.) exhibited anxiolytic effect in the EPM and decreased immobility in the TST. J-2156 alone did not influence Fos immunoreactivity in intact mice, but significantly increased the stress-induced Fos response in the dorsal raphe nucleus, central projecting Edinger-Westphal nucleus, periaqueductal gray matter, the magnocellular, but not the parvocellular part of the hypothalamic paraventricular nucleus, lateral septum, bed nucleus of the stria terminalis and the amygdala. Notably, sst4(LacZ) immunoreactivity occurred in the central and basolateral amygdala. Together, these studies reveal that sst4 mediates anxiolytic and antidepressant-like effects by enhancing the stress-responsiveness of several brain regions with special emphasis on the amygdala.

  2. Somatostatin receptor subtype 4 activation is involved in anxiety and depression-like behavior in mouse models.

    PubMed

    Scheich, Bálint; Gaszner, Balázs; Kormos, Viktória; László, Kristóf; Ádori, Csaba; Borbély, Éva; Hajna, Zsófia; Tékus, Valéria; Bölcskei, Kata; Ábrahám, István; Pintér, Erika; Szolcsányi, János; Helyes, Zsuzsanna

    2016-02-01

    Somatostatin regulates stress-related behavior and its expression is altered in mood disorders. However, little is known about the underlying mechanisms, especially about the importance of its receptors (sst1-sst5) in anxiety and depression-like behavior. Here we analyzed the potential role of sst4 receptor in these processes, since sst4 is present in stress-related brain regions, but there are no data about its functional relevance. Genetic deletion of sst4 (Sstr4(-/-)) and its pharmacological activation with the newly developed selective non-peptide agonist J-2156 were used. Anxiety was examined in the elevated plus maze (EPM) and depression-like behavior in the forced swim (FST) and tail suspension tests (TST). Neuronal activation during the TST was monitored by Fos immunohistochemistry, receptor expression was identified by sst4(LacZ) immunostaining in several brain regions. Sstr4(-/-) mice showed increased anxiety in the EPM and enhanced depression-like behavior in the FST. J-2156 (100 μg/kg i.p.) exhibited anxiolytic effect in the EPM and decreased immobility in the TST. J-2156 alone did not influence Fos immunoreactivity in intact mice, but significantly increased the stress-induced Fos response in the dorsal raphe nucleus, central projecting Edinger-Westphal nucleus, periaqueductal gray matter, the magnocellular, but not the parvocellular part of the hypothalamic paraventricular nucleus, lateral septum, bed nucleus of the stria terminalis and the amygdala. Notably, sst4(LacZ) immunoreactivity occurred in the central and basolateral amygdala. Together, these studies reveal that sst4 mediates anxiolytic and antidepressant-like effects by enhancing the stress-responsiveness of several brain regions with special emphasis on the amygdala. PMID:26387439

  3. Discrimination by benextramine between the NPY-Y1 receptor subtypes present in rabbit isolated vas deferens and saphenous vein.

    PubMed Central

    Palea, S.; Corsi, M.; Rimland, J. M.; Trist, D. G.

    1995-01-01

    1. In order to characterize the neuropeptide Y (NPY) Y1 receptors known to be present in rabbit isolated vas deferens and saphenous vein, the pharmacological activity of the selective NPY Y1 receptor agonists, [Leu31,Pro34] NPY and various other peptide agonists, together with the putative NPY antagonist, benextramine, were compared in the two tissues. 2. In rabbit isolated saphenous vein, cumulative dose-response curves to various NPY agonists were obtained. All the peptides tested caused contractions which developed quite slowly. The rank order of potency obtained was: PYY > NPY > [Leu31,Pro34] NPY = NPY2-36 > hPP >> NPY13-36 = NPY18-36. Incubation with benextramine (BXT) at 100 microM for 30 min irreversibly abolished the contractile response to [Leu31,Pro34] NPY but was ineffective against NPY18-36-induced contractions. 3. Cumulative dose-response curves to [Leu31,Pro34] NPY were performed in the same preparation before and after incubation with 100 microM BXT for 20 min in order to inactivate NPY Y1 receptors. The pKA (-logKA) estimation for [Leu31,Pro34] NPY was 7.60 +/- 0.30 using the operational model and 7.20 +/- 0.33 using the null method; the difference between the two methods was not statistically significant (P = 0.36). 4. Prostatic segments of rabbit vas deferens were electrically stimulated with single pulses. Immediately after stabilization of the contractile response, a cumulative dose-response curve to various NPY agonists was obtained in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7647980

  4. Contrasting signaling pathways of alpha1A- and alpha1B-adrenergic receptor subtype activation of phosphatidylinositol 3-kinase and Ras in transfected NIH3T3 cells.

    PubMed

    Hu, Z W; Shi, X Y; Lin, R Z; Hoffman, B B

    1999-01-01

    Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including alpha1-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human alpha1-receptors, namely, alpha1A, alpha1B and alpha1D to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human alpha1A and alpha1B via pertussis toxin-insensitive G proteins; alpha1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with the alpha-subunit of the G protein transducin (alpha(t)) a scavenger of betagamma-subunits released from activated G proteins, inhibited alpha1B-receptor but not alpha1A-receptor-stimulated PI 3-kinase activity. Stimulation of both alpha1A- and alpha1B-receptors activated p21ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21ras attenuated alpha1B-receptor but not alpha1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuated alpha1A- but not alpha1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of the alpha1-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

  5. Effect of bombesin receptor subtype-3 and its synthetic agonist on signaling, glucose transport and metabolism in myocytes from patients with obesity and type 2 diabetes

    PubMed Central

    GONZÁLEZ, NIEVES; MARTÍN-DUCE, ANTONIO; MARTÍNEZ-ARRIETA, FÉLIX; MORENO-VILLEGAS, ZAIDA; PORTAL-NÚÑEZ, SERGIO; SANZ, RAÚL; EGIDO, JESÚS

    2015-01-01

    Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) member of the bombesin receptor family. Several studies have suggested an association between obesity, alterations in glucose metabolism, diabetes and the BRS-3 receptor. In this study, we focused on patients simultaneously diagnosed with obesity and type 2 diabetes (OB/T2D). The analysis of BRS-3 expression in the skeletal muscle of these patients revealed a marked decrease in the expression of BRS-3 at the mRNA (23.6±1.3-fold downregulation, p<0.0001) and protein level (49±7% decrease, p<0.05) compared to the normal patients (no obesity and diabetes). Moreover, in cultured primary myocytes from patients with OB/T2D, the synthetic BRS-3 agonist, [D-Try6,β-Ala11,Phe13,Nle14]bombesin6–14, significantly increased the phosphorylation levels of mitogen-activated protein kinase (MAPK), p90RSK1, protein kinase B (PKB) and p70s6K. Specifically, the ligand at 10−11 M induced the maximal phosphorylation of MAPKs (p42, 159±15% of the control; p44, 166±11% of the control; p<0.0001) and p90RSK1 (148±2% of the control, p<0.0001). The basal phosphorylation levels of all kinases were reduced (p<0.05) in the patients with OB/T2D compared to the normal patients. Furthermore, the BRS-3 agonist stimulated glucose transport, which was already detected at 10−12 M (133±9% of the control), reached maximal levels at 10−11 M (160±9%, p<0.0001) and was maintained at up to 10−8 M (overall mean, 153±7%; p<0.007). This effect was less promiment than that attained with 10−8 M insulin (202±9%, p=0.009). The effect of the agonist on glycogen synthase a activity achieved the maximum effect at 10−11 M (165±16% of the control; p<0.0001), which did not differ from that observed with higher concentrations of the agonist. These results suggest that muscle cells isolated from patients with OB/T2D have extremely high sensitivity to the synthetic ligand, and the effects are particularly observed on

  6. Facilitation of fear extinction by novelty depends on dopamine acting on D1-subtype dopamine receptors in hippocampus.

    PubMed

    Menezes, Jefferson; Alves, Niége; Borges, Sidnei; Roehrs, Rafael; de Carvalho Myskiw, Jociane; Furini, Cristiane Regina Guerino; Izquierdo, Ivan; Mello-Carpes, Pâmela B

    2015-03-31

    Extinction is the learned inhibition of retrieval. Recently it was shown that a brief exposure to a novel environment enhances the extinction of contextual fear in rats, an effect explainable by a synaptic tagging-and-capture process. Here we examine whether this also happens with the extinction of another fear-motivated task, inhibitory avoidance (IA), and whether it depends on dopamine acting on D1 or D5 receptors. Rats were trained first in IA and then in extinction of this task. The retention of extinction was measured 24 h later. A 5-min exposure to a novel environment 30 min before extinction training enhanced its retention. Right after exposure to the novelty, animals were given bilateral intrahippocampal infusions of vehicle (VEH), of the protein synthesis inhibitor anisomycin, of the D1/D5 dopaminergic antagonist SCH23390, of the PKA inhibitor Rp-cAMP or of the PKC inhibitor Gö6976, and of the PKA stimulator Sp-cAMP or of the PKC stimulator PMA. The novelty increased hippocampal dopamine levels and facilitated the extinction, which was inhibited by intrahippocampal protein synthesis inhibitor anisomysin, D1/D5 dopaminerdic antagonist SCH23390, or PKA inhibitor Rp-cAMP and unaffected by PKC inhibitor Gö6976; additionally, the hippocampal infusion of PKA stimulator Sp-cAMP reverts the effect of D1/D5 dopaminergic antagonist SCH 23390, but the infusion of PKC stimulator PMA does not. The results attest to the generality of the novelty effect on fear extinction, suggest that it relies on synaptic tagging and capture, and show that it depends on hippocampal dopamine D1 but not D5 receptors.

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

    PubMed

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

    2008-04-15

    Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth. PMID:17891781

  8. Lack of the metabotropic glutamate receptor subtype 7 selectively impairs short-term working memory but not long-term memory.

    PubMed

    Hölscher, Christian; Schmid, Susanne; Pilz, Peter K D; Sansig, Gilles; van der Putten, Herman; Plappert, Claudia F

    2004-10-01

    Metabotropic glutamate receptors (mGluRs), and in particular the mGluR group III receptors (subtypes 4, 6, 7, 8) are known to play a role in synaptic plasticity and learning. Here, we report the effect of mGluR7 gene ablation in different learning paradigms. In the acoustic startle response (ASR), no differences were seen between knockout (KO) mice and wildtype (WT) littermates in parameters including prepulse inhibition and habituation. In an open field test, no differences were seen between genotypes in motor activity, exploratory behaviour, and fearful behaviour. In a T-maze reinforced alternation working memory (WM) task, again no difference was seen between groups. However, when increasing the demands on working-memory in a 4-arm and 8-arm maze task, KO mice committed more WM errors than WT littermates thereby uncovering a highly significant difference between the two groups that persisted every day for the whole 9 days of the experiment. In a 4-arm maze with 2 arms baited, KO and wildtype mice committed the same number of LTM errors, whereas KOs committed more WM errors. Altogether, these findings suggest that a lack of mGluR7 mainly impairs short-term working but not long-term memory performance while having no effect on sensorimotor processing, non-associative learning, motor activity and spatial orientation. The effects on WM are task-dependent and become apparent in more complex but not simple learning tasks. We discuss how mGluR7 could influence WM.

  9. Synaptically driven endocannabinoid release requires Ca2+-assisted metabotropic glutamate receptor subtype 1 to phospholipase Cbeta4 signaling cascade in the cerebellum.

    PubMed

    Maejima, Takashi; Oka, Saori; Hashimotodani, Yuki; Ohno-Shosaku, Takako; Aiba, Atsu; Wu, Dianqing; Waku, Keizo; Sugiura, Takayuki; Kano, Masanobu

    2005-07-20

    Endocannabinoids mediate retrograde signaling and modulate synaptic transmission in various regions of the CNS. Depolarization-induced elevation of intracellular Ca2+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G(q/11)-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also causes endocannabinoid-mediated suppression of synaptic transmission. However, precise mechanisms of endocannabinoid production initiated by physiologically relevant synaptic activity remain to be determined. To address this problem, we made whole-cell recordings from Purkinje cells (PCs) in mouse cerebellar slices and examined their excitatory synapses arising from climbing fibers (CFs) and parallel fibers (PFs). We first characterized three distinct modes to induce endocannabinoid release by analyzing CF to PC synapses. The first mode is strong activation of mGluR subtype 1 (mGluR1)-phospholipase C (PLC) beta4 cascade without detectable Ca2+ elevation. The second mode is Ca2+ elevation to a micromolar range without activation of the mGluR1-PLCbeta4 cascade. The third mode is the Ca2+-assisted mGluR1-PLCbeta4 cascade that requires weak mGluR1 activation and Ca2+ elevation to a submicromolar range. By analyzing PF to PC synapses, we show that the third mode is essential for effective endocannabinoid release from PCs by excitatory synaptic activity. Furthermore, our biochemical analysis demonstrates that combined weak mGluR1 activation and mild depolarization in PCs effectively produces 2-arachidonoylglycerol (2-AG), a candidate of endocannabinoid, whereas either stimulus alone did not produce detectable 2-AG. Our results strongly suggest that under physiological conditions, excitatory synaptic inputs to PCs activate the Ca2+-assisted mGluR1-PLCbeta4 cascade, and thereby produce 2-AG, which retrogradely modulates synaptic transmission to PCs.

  10. Blocking metabotropic glutamate receptor subtype 7 (mGlu7) via the Venus flytrap domain (VFTD) inhibits amygdala plasticity, stress, and anxiety-related behavior.

    PubMed

    Gee, Christine E; Peterlik, Daniel; Neuhäuser, Christoph; Bouhelal, Rochdi; Kaupmann, Klemens; Laue, Grit; Uschold-Schmidt, Nicole; Feuerbach, Dominik; Zimmermann, Kaspar; Ofner, Silvio; Cryan, John F; van der Putten, Herman; Fendt, Markus; Vranesic, Ivo; Glatthar, Ralf; Flor, Peter J

    2014-04-18

    The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

  11. Sensitivity of bronchopulmonary receptors to cold and heat mediated by transient receptor potential cation channel subtypes in an ex vivo rat lung preparation.

    PubMed

    Zhou, Yun; Sun, Biying; Li, Qian; Luo, Pin; Dong, Li; Rong, Weifang

    2011-08-15

    Changes in airway temperature can result in respiratory responses such as cough, bronchoconstriction and mucosal secretion after cold exposure and hyperventilation after heat exposure. In the present investigation, we examined the activity of bronchopulmonary receptors in response to activators of thermo-sensitive transient receptor potential (TS-TRP) cation channels using an ex vivo rat lung preparation. Receptive fields in small bronchioles were probed with von Frey hair monofilaments, warm (50°C) or cold (8°C) saline or saline containing TS-TRP agonists. Among 233 fibers tested, 159 (68.2%) responded to heat (50°C). A large proportion of heat-responsive receptors (107/145) were also activated by capsaicin. Heat and capsaicin-evoked responses were both blocked by TRPV1 antagonist, capsazepine. Only 15.3% of airway receptors responded to cold, which was associated with sensitivity to TRPM8 agonist menthol but not to TRPA1 agonist cinnamaldehyde (CA). Moreover, cold-evoked responses was unaffected by TRPA1 antagonist HC-03001. Our observations suggest that TRPV1 and TRPM8 are involved in transducing heat and cold in the lower respiratory tract, respectively.

  12. Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca(2+) oscillations in mouse pancreatic acinar cells.

    PubMed

    Huang, Zebing; Wang, Haiyan; Wang, Jingke; Zhao, Mengqin; Sun, Nana; Sun, Fangfang; Shen, Jianxin; Zhang, Haiying; Xia, Kunkun; Chen, Dejie; Gao, Ming; Hammer, Ronald P; Liu, Qingrong; Xi, Zhengxiong; Fan, Xuegong; Wu, Jie

    2016-01-01

    Emerging evidence demonstrates that the blockade of intracellular Ca(2+) signals may protect pancreatic acinar cells against Ca(2+) overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca(2+) signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca(2+) oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca(2+) oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca(2+) oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca(2+) oscillations and L-arginine-induced enhancement of Ca(2+) signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis. PMID:27432473

  13. Internalization of RGD peptide conjugates of near-infrared fluorescent probes in different cell lines occurs via different integrin receptor subtypes

    NASA Astrophysics Data System (ADS)

    Bloch, S.; Xu, B.; Ye, Y.; Liang, K.; Achilefu, S.

    2006-02-01

    Expression of integrin α vβ 3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Previous studies demonstrated that near infrared (NIR) fluorescent probes designed to target α vβ 3 accumulated both in vitro and in vivo in α vβ 3-positive tumor cells. To evaluate the selectivity of some NIR-labeled RGD peptides for α vβ 3, the molecular probes were incubated in different cells, including the α vβ 3-positive U87 and A549 cells, and α vβ 3-negative HT29 cells. Whereas the RGD compounds tested internalized in the A549 cells, their uptake by the HT29 cell line, which is positive for α vβ 5 and α vβ 6, was low. The uptake of these probes in U87 depended on the structural features of the compounds. Further studies with functional blocking antibodies showed that the internalization in the α vβ 3-positive cells may be mediated by different integrin receptor subtypes. The preliminary results suggest that the internalization of linear RGD peptides is mediated by the α vβ 3 heterodimer but rearrangement of the peptide sequence could alter the selectivity of the molecular probes for different integrin subunits in the dimeric α and β proteins. Thus, a careful choice of RGD peptides can be used to monitor the functional status of different integrins in cells and tissues.

  14. Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca2+ oscillations in mouse pancreatic acinar cells

    PubMed Central

    Huang, Zebing; Wang, Haiyan; Wang, Jingke; Zhao, Mengqin; Sun, Nana; Sun, Fangfang; Shen, Jianxin; Zhang, Haiying; Xia, Kunkun; Chen, Dejie; Gao, Ming; Hammer, Ronald P.; Liu, Qingrong; Xi, Zhengxiong; Fan, Xuegong; Wu, Jie

    2016-01-01

    Emerging evidence demonstrates that the blockade of intracellular Ca2+ signals may protect pancreatic acinar cells against Ca2+ overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca2+ signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca2+ oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca2+ oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca2+ oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca2+ oscillations and L-arginine-induced enhancement of Ca2+ signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis. PMID:27432473

  15. Virtual screening workflow development guided by the "receiver operating characteristic" curve approach. Application to high-throughput docking on metabotropic glutamate receptor subtype 4.

    PubMed

    Triballeau, Nicolas; Acher, Francine; Brabet, Isabelle; Pin, Jean-Philippe; Bertrand, Hugues-Olivier

    2005-04-01

    The "receiver operating characteristic" (ROC) curve method is a well-recognized metric used as an objective way to evaluate the ability of a given test to discriminate between two populations. This facilitates decision-making in a plethora of fields in which a wrong judgment may have serious consequences including clinical diagnosis, public safety, travel security, and economic strategies. When virtual screening is used to speed-up the drug discovery process in pharmaceutical research, taking the right decision upon selecting or discarding a molecule prior to in vitro evaluation is of paramount importance. Characterizing both the ability of a virtual screening workflow to select active molecules and the ability to discard inactive ones, the ROC curve approach is well suited for this critical decision gate. As a case study, the first virtual screening workflow focused on metabotropic glutamate receptor subtype 4 (mGlu4R) agonists is reported here. Six compounds out of 38 selected and tested in vitro were shown to have agonist activity on this target of therapeutic interest.

  16. Biodistribution and radiation dosimetry of a positron emission tomographic ligand, 18F-SP203, to image metabotropic glutamate subtype 5 receptors in humans

    PubMed Central

    Kimura, Yasuyuki; Siméon, Fabrice G.; Hatazawa, Jun; Mozley, P. David; Pike, Victor W.; Innis, Robert B.; Fujita, Masahiro

    2011-01-01

    Purpose A new PET ligand, 3-fluoro-5-(2-(2-18F-(fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile (18F-SP203), is a positron emission tomographic radioligand selective for metabotropic glutamate subtype 5 receptors. The purposes of this study were to estimate the radiation-absorbed doses of 18F-SP203 in humans and to determine from the distribution of radioactivity in bone structures with various proportions of bone and red marrow whether 18F-SP203 undergoes defluorination. Methods Whole-body images were acquired for 5 h after injecting 18F-SP203 in seven healthy humans. Urine was collected at various time points. Radiation-absorbed doses were estimated by the Medical Internal Radiation Dose scheme. Results After injecting 18F-SP203, the two organs with highest radiation exposure were urinary bladder wall and gallbladder wall, consistent with both urinary and fecal excretion. In the skeleton, most of the radioactivity was in bone structures that contain red marrow and not in those without red marrow. Although the dose to red marrow (30.9 μSv/MBq) was unusually high, the effective dose (17.8 μSv/MBq) of 18F-SP203 was typical of that of other 18F radiotracers. Conclusion 18F-SP203 causes an effective dose in humans typical of several other 18F radioligands and undergoes little defluorination. PMID:20585776

  17. Acute alertness-promoting effects of a novel histamine subtype-3 receptor inverse agonist in healthy sleep-deprived male volunteers.

    PubMed

    Iannone, R; Palcza, J; Renger, J J; Calder, N; Cerchio, K; Gottesdiener, K; Hargreaves, R; Dijk, D J; Boyle, J; Murphy, M G

    2010-12-01

    The alertness-promoting effect of MK-0249 (10 or 50 mg), a histamine subtype-3 receptor (HRH3) inverse agonist (IA), was evaluated in the stimulant reference sleep deprivation model (SRSDM) using a double-blind, double-dummy, placebo- and modafinil- (200 mg) controlled, four-period crossover design in 24 healthy young men. The two primary hypotheses were related to sleep latency (first appearance of one epoch of stage 2, 3, or 4 or REM sleep, as detected using polysomnography (PSG)) at 8:00 AM on day 2. Statistically significant increases in sleep latency were observed in association with the use of modafinil 200 mg (9.07 min; P < 0.0001), MK-0249 50 mg (5.17 min; P = 0.008), and MK-0249 10 mg (5.45 min; P = 0.005) at the maintenance of wakefulness test (MWT) at 8:00 AM. Sleep latency was higher when averaged over all MWT time points (P < 0.0001 for modafinil and for both doses of MK-0249). The alertness-promoting effect with the use of MK-0249 in the SRSDM suggests that HRH3 IAs may be effective in disorders involving excessive somnolence. PMID:20981000

  18. Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

    PubMed

    Hu, Jianping; Feng, Zhiwei; Ma, Shifan; Zhang, Yu; Tong, Qin; Alqarni, Mohammed Hamed; Gou, Xiaojun; Xie, Xiang-Qun

    2016-06-27

    Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral

  19. Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

    PubMed

    Hu, Jianping; Feng, Zhiwei; Ma, Shifan; Zhang, Yu; Tong, Qin; Alqarni, Mohammed Hamed; Gou, Xiaojun; Xie, Xiang-Qun

    2016-06-27

    Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems-the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist-to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the "ionic lock" between R131(3.50) and D240(6.30) as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W258(6.48) in TM6 and residues in TM4 (V164(4.56)-L169(4.61)) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180-F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral

  20. Actions of the prototypical 5-HT1A receptor agonist 8-OH-DPAT at human alpha2-adrenoceptors: (+)8-OH-DPAT, but not (-)8-OH-DPAT is an alpha2B subtype preferential agonist.

    PubMed

    Heusler, Peter; Rauly-Lestienne, Isabelle; Tourette, Amélie; Tardif, Stéphanie; Ailhaud, Marie-Christine; Croville, Guillaume; Cussac, Didier

    2010-08-25

    8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] is the prototypical agonist at serotonin 5-HT1A receptors; however, activity at other targets contributes to the functional effects of the compound as well. We examined the properties of 8-OH-DPAT and its enantiomers at recombinant human (h)alpha2-adrenoceptor subtypes, using a panel of radioligand binding and functional tests. In competition binding experiments using [3H]-RX821002, about 10-fold selectivity of (+)8-OH-DPAT for the halpha2B subtype (pKi about 7) over halpha2A- and halpha2C-adrenoceptors was observed. In contrast, the S(-) enantiomer of 8-OH-DPAT showed similar weak affinities for the three receptor subtypes (pKis<6). The binding affinity of (+)8-OH-DPAT at the halpha2B- and the halpha2A-adrenoceptor was found sensitive to GTPgammaS, a receptor/G protein-uncoupling agent, indicating agonist properties of the drug. Furthermore, using [35S]GTPgammaS binding determination at CHO-halpha2B or CHO-halpha2A cell membranes and G protein coupled inwardly rectifying potassium (GIRK) current recordings in Xenopus oocytes expressing halpha2B, partial agonist activity of (+)8-OH-DPAT at the respective receptors was confirmed in these two different functional assays. Potency of (+)8-OH-DPAT for stimulation of [35S]GTPgammaS incorporation was lower at the halpha2A- than at the halpha2B-adrenoceptor, consistent with binding affinities. Thus, (+)8-OH-DPAT and, as a consequence, racemic (+/-)8-OH-DPAT are partial agonists at halpha2-adrenoceptors with selectivity for the halpha2B subtype, a property that might contribute to the effects of the compound described in native systems.

  1. Stress-induced switch in Numb isoforms enhances Notch-dependent expression of subtype-specific transient receptor potential channel.

    PubMed

    Kyriazis, George A; Belal, Cherine; Madan, Meenu; Taylor, David G; Wang, Jang; Wei, Zelan; Pattisapu, Jogi V; Chan, Sic L

    2010-02-26

    The Notch signaling pathway plays an essential role in the regulation of cell specification by controlling differentiation, proliferation, and apoptosis. Numb is an intrinsic regulator of the Notch pathway and exists in four alternative splice variants that differ in the length of their phosphotyrosine-binding domain (PTB) and proline-rich region domains. The physiological relevance of the existence of the Numb splice variants and their exact regulation are still poorly understood. We previously reported that Numb switches from isoforms containing the insertion in PTB to isoforms lacking this insertion in neuronal cells subjected to trophic factor withdrawal (TFW). The functional relevance of the TFW-induced switch in Numb isoforms is not known. Here we provide evidence that the TFW-induced switch in Numb isoforms regulates Notch signaling strength and Notch target gene expression. PC12 cells stably overexpressing Numb isoforms lacking the PTB insertion exhibited higher basal Notch activity and Notch-dependent transcription of the transient receptor potential channel 6 (TRPC6) when compared with those overexpressing Numb isoforms with the PTB insertion. The differential regulation of TRPC6 expression is correlated with perturbed calcium signaling and increased neuronal vulnerability to TFW-induced death. Pharmacological inhibition of the Notch pathway or knockdown of TRPC6 function ameliorates the adverse effects caused by the TFW-induced switch in Numb isoforms. Taken together, our results indicate that Notch and Numb interaction may influence the sensitivity of neuronal cells to injurious stimuli by modulating calcium-dependent apoptotic signaling cascades.

  2. Evaluation of the Biological Activity of Opuntia ficus indica as a Tissue- and Estrogen Receptor Subtype-Selective Modulator.

    PubMed

    An, Byoung Ha; Jeong, Hyesoo; Zhou, Wenmei; Liu, Xiyuan; Kim, Soolin; Jang, Chang Young; Kim, Hyun-Sook; Sohn, Johann; Park, Hye-Jin; Sung, Na-Hye; Hong, Cheol Yi; Chang, Minsun

    2016-06-01

    Phytoestrogens are selective estrogen receptor modulators (SERMs) with potential for use in hormone replacement therapy (HRT) to relieve peri/postmenopausal symptoms. This study was aimed at elucidating the molecular mechanisms underlying the SERM properties of the extract of Korean-grown Opuntia ficus-indica (KOFI). The KOFI extract induced estrogen response element (ERE)-driven transcription in breast and endometrial cancer cell lines and the expression of endogenous estrogen-responsive genes in breast cancer cells. The flavonoid content of different KOFI preparations affected ERE-luciferase activities, implying that the flavonoid composition likely mediated the estrogenic activities in cells. Oral administration of KOFI decreased the weight gain and levels of both serum glucose and triglyceride in ovariectomized (OVX) rats. Finally, KOFI had an inhibitory effect on the 17β-estradiol-induced proliferation of the endometrial epithelium in OVX rats. Our data demonstrate that KOFI exhibited SERM activity with no uterotrophic side effects. Therefore, KOFI alone or in combination with other botanical supplements, vitamins, or minerals may be an effective and safe alternative active ingredient to HRTs, for the management of postmenopausal symptoms. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Estradiol receptors in combination with neu or myc oncogene amplifications might define new subtypes of breast cancer.

    PubMed

    Bolufer, P; Molina, R; Ruiz, A; Hernandez, M; Vazquez, C; Lluch, A

    1994-09-01

    Amplifications of neu and c-myc were evaluated in 218 and 145 breast cancers (BC), respectively. Oncogene amplifications were determined for the most part by Southern blot. An association between the proportion of nodes affected and the intensity of neu amplification in estadiol receptor negative (ER-) BC was found (P = 0.028), which was confirmed by the multi-factor analysis of variance (P = 0.05). A significantly greater incidence in neu amplifications among BC with metastases was also found (P = 0.031). A strong association (P = 0.01) between the neu and myc amplification was observed. There is a strong association between myc amplification and ER- BC (P < 0.01). It is concluded that (1) the combination ER- with neu amplification might define a new group of more aggressive BC, as is suggested by their associated nodal involvement; (2) the linkage of myc amplifications with ER- BC and high grade of neu amplification might reflect a trait of tumor aggressivity.

  4. Evaluation of the Biological Activity of Opuntia ficus indica as a Tissue- and Estrogen Receptor Subtype-Selective Modulator.

    PubMed

    An, Byoung Ha; Jeong, Hyesoo; Zhou, Wenmei; Liu, Xiyuan; Kim, Soolin; Jang, Chang Young; Kim, Hyun-Sook; Sohn, Johann; Park, Hye-Jin; Sung, Na-Hye; Hong, Cheol Yi; Chang, Minsun

    2016-06-01

    Phytoestrogens are selective estrogen receptor modulators (SERMs) with potential for use in hormone replacement therapy (HRT) to relieve peri/postmenopausal symptoms. This study was aimed at elucidating the molecular mechanisms underlying the SERM properties of the extract of Korean-grown Opuntia ficus-indica (KOFI). The KOFI extract induced estrogen response element (ERE)-driven transcription in breast and endometrial cancer cell lines and the expression of endogenous estrogen-responsive genes in breast cancer cells. The flavonoid content of different KOFI preparations affected ERE-luciferase activities, implying that the flavonoid composition likely mediated the estrogenic activities in cells. Oral administration of KOFI decreased the weight gain and levels of both serum glucose and triglyceride in ovariectomized (OVX) rats. Finally, KOFI had an inhibitory effect on the 17β-estradiol-induced proliferation of the endometrial epithelium in OVX rats. Our data demonstrate that KOFI exhibited SERM activity with no uterotrophic side effects. Therefore, KOFI alone or in combination with other botanical supplements, vitamins, or minerals may be an effective and safe alternative active ingredient to HRTs, for the management of postmenopausal symptoms. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26989859

  5. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-11-27

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.

  6. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-11-27

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

  7. Molecular modeling and mutagenesis of the ligand-binding pocket of the mGlu3 subtype of metabotropic glutamate receptor.

    PubMed

    Yao, Yi; Pattabiraman, N; Michne, William F; Huang, Xi-Ping; Hampson, David R

    2003-08-01

    A homology model of the extracellular domain of the mGlu3 subtype of metabotropic glutamate (mGlu) receptor was generated and tested using site-directed mutagenesis, a radioligand-binding assay using the Group II selective agonist (2S,2'R,3'R)-2-(2',3'-[3H]dicarboxycyclopropyl) glycine ([3H]DCG-IV), and in a fluorescence-based functional assay in live transiently transfected human embryonic kidney cells. Ten of the 12 mGlu3 mutants (R64A, R68A, Y150A, S151A, T174A, D194A, Y222A, R277A, D301A and K389) showed either no binding or a 90% or greater loss of specific [3H]DCG-IV binding. Several analogous mutations in mGlu2 supported the results obtained with mGlu3. These results demonstrate that the binding of [3H]DCG-IV to mGlu3 is exceptionally sensitive to mutagenesis-induced perturbations. In silico docking of DCG-IV into the agonist binding pocket of mGlu3 facilitated the interpretation the mutagenesis results. Tyrosines 150 and 222, and arginine 277 show close contacts with the third carboxylic acid group in DCG-IV, which is not present in glutamate or (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Mutation of these three amino acids to alanine resulted in a near complete loss of receptor activation by DCG-IV and retention of near wild-type affinity for L-CCG-I. It is proposed that hydrogen bonding between this carboxylate and tyrosines 150 and 222 and arginine 277 provide a partial explanation for the high affinity and Group II selectivity of DCG-IV. These findings define the essential features of the ligand-binding pocket of mGlu3 and, together with other recent studies on mGlu receptors, provide new opportunities for structure-based drug design. PMID:12887692

  8. Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice. [Ethanol effects

    SciTech Connect

    Watson, M.; Ming, X.; McArdle, J.J. )

    1989-02-09

    Differences in voluntary ethanol consumption have been noted in various inbred strains of mice and pharmacogenetic approaches have been used to study the mechanisms of action of many drugs such as ethanol. Long-sleep (LS) and short-sleep (SS) mice, selectively bred for differences in ethanol induced narcosis, provide a method by which a relationship between the differential responsiveness of these geno-types and muscarinic acetylcholine receptors (mAChR) may be evaluated. Sleep times after injection of 3ml ethanol/kg (i.p.) verified the higher sensitivity of LS vs. SS. Mean body weights of LS (26.5g) vs. SS (22g) were also significantly (p<.01) greater. Binding assays for ({sup 3}H)(-) quinuclidinylbenzilate (({sup 3}H)(-)QNB), a specific but nonsubtype selective mAChR antagonist, ({sup 3}H)pirenzepine (({sup 3}H)PZ), a specific M1 mAChR antagonist and ({sup 3}H)11-2-((2-((diethylamino) methyl)-1-piperidinyl) acetyl)-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, (({sup 3}H)AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (K{sub d}) and density (B{sub max}) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) ({sup 3}H)(-)QNB binding suggests that SS have fewer mAChR's than LS in many areas. These differences may relate to their differential ethanol sensitivity.

  9. The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells.

    PubMed

    Molè, Daniela; Gentilin, Erica; Ibañez-Costa, Alejandro; Gagliano, Teresa; Gahete, Manuel D; Tagliati, Federico; Rossi, Roberta; Pelizzo, Maria Rosa; Pansini, Giancarlo; Luque, Raúl M; Castaño, Justo P; degli Uberti, Ettore; Zatelli, Maria Chiara

    2015-11-01

    The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

  10. Oral Mineralocorticoid-Receptor Antagonists: Real-Life Experience in Clinical Subtypes of Nonresolving Central Serous Chorioretinopathy With Chronic Epitheliopathy

    PubMed Central

    Daruich, Alejandra; Matet, Alexandre; Dirani, Ali; Gallice, Mathilde; Nicholson, Luke; Sivaprasad, Sobha; Behar-Cohen, Francine

    2016-01-01

    Purpose To evaluate the efficacy and safety of oral mineralocorticoid-receptor antagonist (MRa) therapy in three clinical presentations of nonresolving central serous chorioretinopathy (CSCR) with chronic epitheliopathy. Methods Retrospective case series of consecutive patients with nonresolving CSCR treated with oral eplerenone or spironolactone. Treatment criteria were: persistent CSCR with subretinal fluid (SRF) lasting longer than 4 months; recurrent CSCR with SRF lasting longer than 2 months; persistent CSCR (SRF ≥ 4 months) with fundus autofluorescence gravitational tracks. Outcomes at 1, 3, and 6 months were: foveal SRF height, central macular thickness (CMT), subfoveal choroidal thickness (SFCT), best-corrected visual acuity (BCVA), and occurrence of side effects. Results Among 54 eyes from 42 patients (mean age: 53 years), mean foveal SRF, CMT, and SFCT decreased significantly at 1, 3, and 6 months after treatment initiation. Mean BCVA improved significantly at 6 months. In the subgroup analysis, mean foveal SRF, CMT, and SFCT decreased significantly at 3 and 6 months in the persistent and recurrent groups. In persistent cases with tracks, a significant diminution of mean CMT and SFCT was achieved at 6 months. Treatment-related side effects were observed in 6 patients, prompting treatment discontinuation in one case. Conclusion Response to treatment was observed in the three subgroups. In persistent CSCR with tracks the response was delayed compared with persistent and recurrent cases, suggesting that longer treatment durations would be beneficial in patients with gravitational tracks of RPE alteration. Translational Relevance The clinical response to oral MRa is consistent with the involvement of the mineralocorticoid pathway in CSCR pathogenesis. PMID:26966638

  11. Afferent Input Selects NMDA Receptor Subtype to Determine the Persistency of Hippocampal LTP in Freely Behaving Mice

    PubMed Central

    Ballesteros, Jesús J.; Buschler, Arne; Köhr, Georg; Manahan-Vaughan, Denise

    2016-01-01

    The glutamatergic N-methyl-D-aspartate receptor (NMDAR) is critically involved in many forms of hippocampus-dependent memory that may be enabled by synaptic plasticity. Behavioral studies with NMDAR antagonists and NMDAR subunit (GluN2) mutants revealed distinct contributions from GluN2A- and GluN2B-containing NMDARs to rapidly and slowly acquired memory performance. Furthermore, studies of synaptic plasticity, in genetically modified mice in vitro, suggest that GluN2A and GluN2B may contribute in different ways to the induction and longevity of synaptic plasticity. In contrast to the hippocampal slice preparation, in behaving mice, the afferent frequencies that induce synaptic plasticity are very restricted and specific. In fact, it is the stimulus pattern and not variations in afferent frequency that determine the longevity of long-term potentiation (LTP) in vivo. Here, we explored the contribution of GluN2A and GluN2B to LTP of differing magnitudes and persistence in freely behaving mice. We applied differing high-frequency stimulation (HFS) patterns at 100 Hz to the hippocampal CA1 region, to induce NMDAR-dependent LTP in wild-type (WT) mice, that endured for <1 h (early (E)-LTP), (LTP, 2–4 h) or >24 h (late (L)-LTP). In GluN2A-knockout (KO) mice, E-LTP (HFS, 50 pulses) was significantly reduced in magnitude and duration, whereas LTP (HFS, 2 × 50 pulses) and L-LTP (HFS, 4 × 50 pulses) were unaffected compared to responses in WT animals. By contrast, pharmacological antagonism of GluN2B in WT had no effect on E-LTP but significantly prevented LTP. E-LTP and LTP were significantly impaired by GluN2B antagonism in GluN2A-KO mice. These data indicate that the pattern of afferent stimulation is decisive for the recruitment of distinct GluN2A and GluN2B signaling pathways that in turn determine the persistency of hippocampal LTP. Whereas brief bursts of patterned stimulation preferentially recruit GluN2A and lead to weak and short-lived forms of LTP, prolonged

  12. Protective effect of transient receptor potential vanilloid subtype 1 (TRPV1) modulator, against behavioral, biochemical and structural damage in experimental models of Alzheimer's disease.

    PubMed

    Jayant, Shalini; Sharma, B M; Sharma, Bhupesh

    2016-07-01

    Alzheime's disease (AD) is an overwhelming neurodegenerative disorder, characterized by synaptic dysfunction, memory loss, neuro-inflammation and neural cell death. Very few treatments are in hand for the management of AD and they are only concentrating on peculiar aspects. Hence, an immense thrust is required to find utmost therapeutic targets to conquer this condition. This study investigates a potential role of vanillin, a selective agonist of transient receptor potential vanilloid subtype 1 (TRPV1) in the experimental models of AD viz. intracerebroventricular (i.c.v.) streptozotocin (STZ) and aluminum trichloride (AlCl3)+d-galactose induced AD in mice. The i.c.v. administration of STZ and intraperitoneally administration of AlCl3+d-galactose have significantly impaired learning-memory (Morris water maze and attentional set-shifting test), brain structure (hematoxylin, eosin and Congo red staining), enhanced brain oxidative stress (thiobarbituric acid reactive substance - TBARS and glutathione - GSH), nitrosative stress (nitrite/nitrate), acetylcholinesterase activity (AChE), inflammation (MPO), and calcium levels (Ca(++)). Treatment with vanillin in different doses and donepezil have significantly ameliorated i.c.v. STZ and AlCl3+d-galactose induced reduction in executive function, impaired reversal learning, cognition, memory and brain damage. Treatment with these drugs has also reduced the brain oxidative stress (TBARS and GSH), nitrosative stress (nitrite/nitrate), and AChE, MPO, and Ca(++) levels. These results indicate that vanillin, a selective agonist of TRPV1 and donepezil, a potent acetylcholine esterase inhibitor have attenuated i.c.v. STZ and AlCl3+d-galactose induced experimental AD. Hence, pharmacological positive modulation of TRPV1 channels may be a potential research target for mitigation of AD. PMID:27084583

  13. Admixture Mapping of African–American Women in the AMBER Consortium Identifies New Loci for Breast Cancer and Estrogen-Receptor Subtypes

    PubMed Central

    Ruiz-Narváez, Edward A.; Sucheston-Campbell, Lara; Bensen, Jeannette T.; Yao, Song; Haddad, Stephen; Haiman, Christopher A.; Bandera, Elisa V.; John, Esther M.; Bernstein, Leslie; Hu, Jennifer J.; Ziegler, Regina G.; Deming, Sandra L.; Olshan, Andrew F.; Ambrosone, Christine B.; Palmer, Julie R.; Lunetta, Kathryn L.

    2016-01-01

    Recent genetic admixture coupled with striking differences in incidence of estrogen receptor (ER) breast cancer subtypes, as well as severity, between women of African and European ancestry, provides an excellent rationale for performing admixture mapping in African American women with breast cancer risk. We performed the largest breast cancer admixture mapping study with in African American women to identify novel genomic regions associated with the disease. We conducted a genome-wide admixture scan using 2,624 autosomal ancestry informative markers (AIMs) in 3,629 breast cancer cases (including 1,968 ER-positive, 1093 ER-negative, and 601 triple-negative) and 4,658 controls from the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium, a collaborative study of four large geographically different epidemiological studies of breast cancer in African American women. We used an independent case-control study to test for SNP association in regions with genome-wide significant admixture signals. We found two novel genome-wide significant regions of excess African ancestry, 4p16.1 and 17q25.1, associated with ER-positive breast cancer. Two regions known to harbor breast cancer variants, 10q26 and 11q13, were also identified with excess of African ancestry. Fine-mapping of the identified genome-wide significant regions suggests the presence of significant genetic associations with ER-positive breast cancer in 4p16.1 and 11q13. In summary, we identified three novel genomic regions associated with breast cancer risk by ER status, suggesting that additional previously unidentified variants may contribute to the racial differences in breast cancer risk in the African American population. PMID:27708667

  14. The metabotropic glutamate receptor subtype 5 antagonist fenobam is analgesic and has improved in vivo selectivity compared with the prototypical antagonist 2-methyl-6-(phenylethynyl)-pyridine.

    PubMed

    Montana, Michael C; Cavallone, Laura F; Stubbert, Kristi K; Stefanescu, Andrei D; Kharasch, Evan D; Gereau, Robert W

    2009-09-01

    Metabotropic glutamate receptor subtype 5 (mGlu5) has been demonstrated to play a role in the modulation of numerous nociceptive modalities. When administered via peripheral, intrathecal, or systemic routes, mGlu5 antagonists have analgesic properties in a variety of preclinical pain models. Despite a wealth of data supporting the use of mGlu5 antagonists to treat pain, studies have been limited to preclinical animal models due to a lack of mGlu5 antagonists that are approved for use in humans. It has been demonstrated previously that fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], an anxiolytic shown to be safe and effective in human trials, is a selective and potent noncompetitive antagonist of mGlu5 (J Pharmacol Exp Ther 315:711-721, 2005). Here, we report a series of studies aimed at testing whether fenobam, similar to the prototypical mGlu5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has analgesic properties in mice. We show that fenobam reduces formalin-induced pain behaviors and relieves established inflammation-induced thermal hypersensitivity in mice. Similar results were seen with MPEP. Administration of fenobam resulted in an increase in locomotor activity in the open-field task but did not impair performance on the accelerating Rotarod. Analysis of brain and plasma fenobam levels indicated that fenobam is rapidly concentrated in brain after intraperitoneal administration in mice but is essentially cleared from circulation within 1 h after injection. Fenobam had no analgesic effect in mGlu5 knockout mice, whereas the prototypical antagonist MPEP retained significant analgesic efficacy in mGlu5 knockouts. These results demonstrate that fenobam is analgesic in mice and has an improved in vivo selectivity for mGlu5 over MPEP. PMID:19515968

  15. Dose-dependent, Saturable Occupancy of the Metabotropic Glutamate Subtype 5 Receptor by Fenobam as Measured with [11C]ABP688 PET Imaging

    PubMed Central

    KUWABARA, HIROTO; STANSFIELD, KIRSTIE; VALENTINE, HEATHER; ALEXANDER, MOHAB; KUMAR, ANIL; HILTON, JOHN; DANNALS, ROBERT F.; WONG, DEAN F.; GASPARINI, FABRIZIO

    2014-01-01

    Fenobam is a negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5) with inverse agonist activity and is expected to contribute to the treatment of neuropsychiatric disorders involving dysfunction of mGluR5 including Fragile × syndrome. This study examined whether [11C]ABP688, an antagonist PET radioligand, competes with fenobam for the same binding site in the non-human primate brain and would allow examination of occupancy-plasma concentration relationships in the evaluation of the drug for target disorders in the human brain. Four paired PET studies with [11C]ABP688 were performed in baboons at a baseline condition and after intravenous treatment with fenobam at different dose levels (0.3 - 1.33 mg/kg). Total distribution volume (VT) and binding potential (BPND) using the cerebellum as a reference region were obtained by the plasma reference graphical method. Then it was examined whether occupancy follows a dose-dependent, saturating pattern that was predicted by a modified first-order Hill equation in individual regions. Baseline regional VT and BPND values agreed with previously published data. Occupancy showed dose-dependent and saturating patterns in individual regions, reaching >90% occupancy at 1.33 mg/kg dose of fenobam in the majority of regions. To our knowledge, this is the first use of PET to characterize the mGluR5 therapeutic drug fenobam. This study demonstrates a proof of principle for determining the in vivo occupancy of fenobam in primates. The results indicate that [11C]ABP688 and PET may be useful for examination of occupancy of mGluR5 by fenobam, which should prove to be useful for designing future studies and treatment of human disease states. PMID:25098663

  16. Synthesis, radiolabelling and in vitro and in vivo evaluation of a novel fluorinated ABP688 derivative for the PET imaging of metabotropic glutamate receptor subtype 5

    PubMed Central

    Sephton, Selena Milicevic; Dennler, Patrick; Leutwiler, Dominique S; Mu, Linjing; Wanger-Baumann, Cindy A; Schibli, Roger; Krämer, Stefanie D; Ametamey, Simon M

    2012-01-01

    (E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone O-(2-(3-18F-fluoropropoxy)ethyl) oxime ([18F]-PSS223) was evaluated in vitro and in vivo to establish its potential as a PET tracer for imaging metabotropic glutamate receptor subtype 5 (mGluR5). [18F]-PSS223 was obtained in 20% decay corrected radiochemical yield whereas the non-radioactive PSS223 was accomplished in 70% chemical yield in a SN2 reaction of common intermediate mesylate 8 with potassium fluoride. The in vitro binding affinity of [18F]-PSS223 was measured directly in a Scatchard assay to give Kd = 3.34 ± 2.05 nM. [18F]-PSS223 was stable in PBS and rat plasma but was significantly metabolized by rat liver microsomal enzymes, but to a lesser extent by human liver microsomes. Within 60 min, 90% and 20% of [18F]-PSS223 was metabolized by rat and human microsome enzymes, respectively. In vitro autoradiography on horizontal rat brain slices showed heterogeneous distribution of [18F]-PSS223 with the highest accumulation in brain regions where mGluR5 is highly expressed (hippocampus, striatum and cortex). Autoradiography in vitro under blockade conditions with ABP688 confirmed the high specificity of [18F]-PSS223 for mGluR5. Under the same blocking conditions but using the mGluR1 antagonist, JNJ16259685, no blockade was observed demonstrating the selectivity of [18F]-PSS223 for mGluR5 over mGluR1. Despite favourable in vitro properties of [18F]-PSS223, a clear-cut visualization of mGluR5-rich brain regions in vivo in rats was not possible mainly due to a fast clearance from the brain and low metabolic stability of [18F]-PSS223. PMID:23133799

  17. Expression of multiple somatostatin receptor genes in AtT-20 cells. Evidence for a novel somatostatin-28 selective receptor subtype.

    PubMed

    Patel, Y C; Panetta, R; Escher, E; Greenwood, M; Srikant, C B

    1994-01-14

    The pattern of expression of somatostatin receptor (SSTR) genes and gene products in AtT-20 cells was characterized in an attempt to explain the SST-28 binding selectivity that typifies these cells. AtT-20 cells expressed multiple SSTR mRNAs. Paradoxically, this included mRNA for three of the four SST-14 selective receptors: SSTR2 ( +), SSTR1 (+), SSTR4 (+). The SST-28 selective SSTR5 was expressed as a 3.8-kilobase (kb) transcript of relatively low abundance (+) in contrast to normal mouse pituitary which displayed high levels ( ) of a 2.4-kb SSTR5 mRNA. Immunoblot analysis of solubilized membranes with an antipeptide SSTR2 antibody revealed a single SSTR2 protein of 72 +/- 2 kDa. Preincubation of AtT-20 cell membranes with SSTR2 antibody reduced 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding sites by 38%. Residual binding sites exhibited a 4.9-fold increase in affinity for SST-28, a 2.6-fold decrease in affinity for SST-14, and an SST-28:SST-14 potency ratio of 40:1 compared with a potency ratio of 3.5:1 in control membranes. These results demonstrate the expression of four SSTR genes in AtT-20 cells of which SSTR2 predominates. Blockade of SSTR2 with antibody exposes high affinity SST-28 selective sites with comparable binding characteristics to those reported for cloned SSTR5. These SST-28 binding sites may arise from a SSTR5 variant encoded by a high molecular weight 3.8-kb transcript or more likely from another as yet undiscovered member of the SST-28 selective SSTR subfamily.

  18. Cardiac physiologic regulation of sub-type specific adrenergic receptors in transgenic mice overexpressing β1- and β2-adrenergic receptors

    PubMed Central

    Kim, Ka Eul; Tae, Hyun-Jin; Natalia, Petrashevskaya; Lee, Jae-Chul; Ahn, Ji Hyeon; Park, Joon Ha; Kim, In Hye; Ohk, Taek Geun; Park, Chan Woo; Cho, Jun Hwi; Won, Moo-Ho

    2016-01-01

    Objective Combination of β1-adrenergic receptor (AR) blockade and β2-AR activation might be a potential novel therapy for treating heart failure. However, use of β-AR agonists and/or antagonists in the clinical setting is controversial because of the lack of information on cardiac inotropic or chronotropic regulation by AR signaling. Methods In this study, we performed hemodynamic evaluation by examining force frequency response (FFR), Frank-Starling relationship, and response to a non-selective β-AR agonist (isoproterenol) in hearts isolated from 6-month-old transgenic (TG) mice overexpressing β1- and β2-ARs (β1- and β2-AR TG mice, respectively). Results Cardiac physiologic consequences of β1- and β2-AR overexpression resulted in similar maximal response to isoproterenol and faster temporary decline of positive inotropic response in β2-AR TG mice. β1-AR TG mice showed a pronounced negative limb of FFR, whereas β2-AR TG mice showed high stimulation frequencies with low contractile depression during FFR. In contrast, Frank-Starling relationship was equally enhanced in both β1- and β2-AR TG mice. Conclusion Hemodynamic evaluation performed in the present showed a difference in β1- and β2-AR signaling, which may be due to the difference in the desensitization of β1- and β2-ARs. PMID:27752636

  19. First-in-class thyrotropin-releasing hormone (TRH)-based compound binds to a pharmacologically distinct TRH receptor subtype in human brain and is effective in neurodegenerative models.

    PubMed

    Kelly, Julie A; Boyle, Noreen T; Cole, Natalie; Slator, Gillian R; Colivicchi, M Alessandra; Stefanini, Chiara; Gobbo, Oliviero L; Scalabrino, Gaia A; Ryan, Sinead M; Elamin, Marwa; Walsh, Cathal; Vajda, Alice; Goggin, Margaret M; Campbell, Matthew; Mash, Deborah C; O'Mara, Shane M; Brayden, David J; Callanan, John J; Tipton, Keith F; Della Corte, Laura; Hunter, Jackie; O'Boyle, Kathy M; Williams, Carvell H; Hardiman, Orla

    2015-02-01

    JAK4D, a first-in-class thyrotropin-releasing hormone (TRH)-based compound, is a prospective therapeutic candidate offering a multifaceted approach to treating neurodegeneration and other CNS conditions. The purpose of these studies was to determine the ability of JAK4D to bind to TRH receptors in human brain and to evaluate its neuropharmacological effects in neurodegenerative animal models. Additionally, JAK4D brain permeation was examined in mouse, and initial toxicology was assessed in vivo and in vitro. We report that JAK4D bound selectively with nanomolar affinity to native TRH receptors in human hippocampal tissue and showed for the first time that these receptors are pharmacologically distinct from TRH receptors in human pituitary, thus revealing a new TRH receptor subtype which represents a promising neurotherapeutic target in human brain. Systemic administration of JAK4D elicited statistically significant and clinically-relevant neuroprotective effects in three established neurodegenerative animal models: JAK4D reduced cognitive deficits when administered post-insult in a kainate (KA)-induced rat model of neurodegeneration; it protected against free radical release and neuronal damage evoked by intrastriatal microdialysis of KA in rat; and it reduced motor decline, weight loss, and lumbar spinal cord neuronal loss in G93A-SOD1 transgenic Amyotrophic Lateral Sclerosis mice. Ability to cross the blood-brain barrier and a clean initial toxicology profile were also shown. In light of these findings, JAK4D is an important tool for investigating the hitherto-unidentified central TRH receptor subtype reported herein and an attractive therapeutic candidate for neurodegenerative disorders.

  20. First-in-class thyrotropin-releasing hormone (TRH)-based compound binds to a pharmacologically distinct TRH receptor subtype in human brain and is effective in neurodegenerative models.

    PubMed

    Kelly, Julie A; Boyle, Noreen T; Cole, Natalie; Slator, Gillian R; Colivicchi, M Alessandra; Stefanini, Chiara; Gobbo, Oliviero L; Scalabrino, Gaia A; Ryan, Sinead M; Elamin, Marwa; Walsh, Cathal; Vajda, Alice; Goggin, Margaret M; Campbell, Matthew; Mash, Deborah C; O'Mara, Shane M; Brayden, David J; Callanan, John J; Tipton, Keith F; Della Corte, Laura; Hunter, Jackie; O'Boyle, Kathy M; Williams, Carvell H; Hardiman, Orla

    2015-02-01

    JAK4D, a first-in-class thyrotropin-releasing hormone (TRH)-based compound, is a prospective therapeutic candidate offering a multifaceted approach to treating neurodegeneration and other CNS conditions. The purpose of these studies was to determine the ability of JAK4D to bind to TRH receptors in human brain and to evaluate its neuropharmacological effects in neurodegenerative animal models. Additionally, JAK4D brain permeation was examined in mouse, and initial toxicology was assessed in vivo and in vitro. We report that JAK4D bound selectively with nanomolar affinity to native TRH receptors in human hippocampal tissue and showed for the first time that these receptors are pharmacologically distinct from TRH receptors in human pituitary, thus revealing a new TRH receptor subtype which represents a promising neurotherapeutic target in human brain. Systemic administration of JAK4D elicited statistically significant and clinically-relevant neuroprotective effects in three established neurodegenerative animal models: JAK4D reduced cognitive deficits when administered post-insult in a kainate (KA)-induced rat model of neurodegeneration; it protected against free radical release and neuronal damage evoked by intrastriatal microdialysis of KA in rat; and it reduced motor decline, weight loss, and lumbar spinal cord neuronal loss in G93A-SOD1 transgenic Amyotrophic Lateral Sclerosis mice. Ability to cross the blood-brain barrier and a clean initial toxicology profile were also shown. In light of these findings, JAK4D is an important tool for investigating the hitherto-unidentified central TRH receptor subtype reported herein and an attractive therapeutic candidate for neurodegenerative disorders. PMID:25281210

  1. Non-obligatory role of prostaglandin D2 receptor subtype 1 in rosacea: laropiprant in comparison to a placebo did not alleviate the symptoms of erythematoelangiectaic rosacea.

    PubMed

    Krishna, Rajesh; Guo, Ying; Schulz, Valerie; Cord-Cruz, Evyan; Smith, Shanna; Hair, Suzanne; Nahm, Walter K; Draelos, Zoe D

    2015-02-01

    Erythematotelangiectatic rosacea shares facial flushing features with those seen after niacin. This study was performed to test the hypothesis whether prostaglandin D2 (PGD2) receptor subtype 1 antagonist (laropiprant) will improve the symptoms of rosacea. The purpose of this study was to evaluate the effect of laropiprant 100 mg administered once daily for 4 weeks on the signs and symptoms of erythematotelangiectatic rosacea. Subjects received laropiprant 100 mg once-daily (n = 30) or placebo (n = 30) for 4 weeks. The primary pharmacodynamics endpoint was change in Clinician's Erythema Assessment (CEA) score from baseline to week 4. The patient self-assessment (PSA) was a secondary endpoint. Laropiprant was generally well tolerated in this study for the primary endpoint of change in CEA score from Baseline to Week 4, the least-squares mean of change from baseline to visit 4/week 4 was -3.7 and -3.4 for placebo and laropiprant (100 mg), respectively. The least-squares mean difference (placebo minus laropiprant) with 90% confidence interval of change in CEA score from baseline to visit 4/week 4 was estimated as -0.3 (-1.6, 1.0). For the secondary endpoint, the least-squares mean difference (placebo minus laropiprant) with 90% confidence interval of change from baseline to visit 4/week 4 was estimated as -0.7 (-7.7, 6.4) for PSA total score, -4.5 (-14.2, 5.3) for PSA emotion score, -1.3 (-7.8, 5.3) for PSA symptoms score, and 3.6 (-4.3, 11.4) for PSA functioning score. Laropiprant administered once daily for 4 weeks was generally well tolerated in this population of subjects with rosacea. However, there were no clinically meaningful changes in the primary endpoint of CEA given that the response to laropiprant could not be differentiated from that to placebo. There was also no clinically meaningful change in the secondary endpoint, PSA. A DP1 antagonist is not likely to be effective in rosacea.

  2. The Novel α7β2-Nicotinic Acetylcholine Receptor Subtype Is Expressed in Mouse and Human Basal Forebrain: Biochemical and Pharmacological Characterization

    PubMed Central

    Moretti, Milena; Zoli, Michele; George, Andrew A.; Lukas, Ronald J.; Pistillo, Francesco; Maskos, Uwe

    2014-01-01

    We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit–specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites. PMID:25002271

  3. Presence of sst5TMD4, a truncated splice variant of the somatostatin receptor subtype 5, is associated to features of increased aggressiveness in pancreatic neuroendocrine tumors

    PubMed Central

    Gahete, Manuel D.; Serrano-Somavilla, Ana; Villa-Osaba, Alicia; Adrados, Magdalena; Ibáñez-Costa, Alejandro; Martín-Pérez, Elena; Culler, Michael D.

    2016-01-01

    Purpose Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous tumors, and their biological behavior is not well known. We studied the presence and potential functional roles of somatostatin receptors (sst1-5), focusing particularly on the truncated variants (sst5TMD4, sst5TMD5) and on their relationships with the angiogenic system (Ang/Tie-2 and VEGF) in human GEP-NETs. Experimental Design We evaluated 42 tumor tissue samples (26 primary/16 metastatic) from 26 patients with GEP-NETs, and 30 non-tumoral tissues (26 from adjacent non-tumor regions and 4 from normal controls) from a single center. Expression of sst1-5, sst5TMD4, sst5TMD5, Ang1-2, Tie-2 and VEGF was analyzed using real-time qPCR, immunofluorescence and immunohistochemistry. Expression levels were associated with tumor characteristics and clinical outcomes. Functional role of sst5TMD4 was analyzed in GEP-NET cell lines. Results sst1 exhibited the highest expression in GEP-NET, whilst sst2 was the most frequently observed sst-subtype (90.2%). Expression levels of sst1, sst2, sst3, sst5TMD4, and sst5TMD5 were significantly higher in tumor tissues compared to their adjacent non-tumoral tissue. Lymph-node metastases expressed higher levels of sst5TMD4 than in its corresponding primary tumor tissue. sst5TMD4 was also significantly higher in intestinal tumor tissues from patients with residual disease of intestinal origin compared to those with non-residual disease. Functional assays demonstrated that the presence of sst5TMD4 was associated to enhanced malignant features in GEP-NET cells. Angiogenic markers correlated positively with sst5TMD4, which was confirmed by immunohistochemical/fluorescence studies. Conclusions sst5TMD4 is overexpressed in GEP-NETs and is associated to enhanced aggressiveness, suggesting its potential value as biomarker and target in GEP-NETs. PMID:26673010

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

    PubMed Central

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

    2011-01-01

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

  5. 5′-AMP impacts lymphocyte recirculation through activation of A2B receptors

    PubMed Central

    Bouma, Hjalmar R.; Mandl, Judith N.; Strijkstra, Arjen M.; Boerema, Ate S.; Kok, Jan-Willem; van Dam, Annie; IJzerman, Ad; Kroese, Frans G. M.; Henning, Robert H.

    2013-01-01

    Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5′-AMP. Previously, we showed that during natural torpor, the reduction in body temperature results in lymphopenia via a reduction in plasma S1P. Here, we show that during torpor induced by 5′-AMP, there is a similar reduction in the number of circulating lymphocytes that is a result of their retention in secondary lymphoid organs. This lymphopenia could be mimicked by engagement of A2BRs by a selective A2BR agonist (LUF6210) in the absence of changes in temperature and prevented by A2BR antagonists during 5′-AMP-induced torpor. In addition, forced cooling of mice led to peripheral blood lymphopenia, independent of A2BR signaling. The induction of torpor using 5′-AMP impacted the migration of lymphocytes within and between secondary lymphoid organs. During torpor, the homing into LNs was impaired, and two-photon intravital microscopy revealed that cell motility was decreased significantly and rapidly upon 5′-AMP administration. Furthermore, the S1P plasma concentration was reduced by 5′-AMP but not by LUF6210. S1P plasma levels restored upon arousal. Likely, the reduced migration in LNs combined with the reduced S1P plasma level substantially reduces lymphocyte egress after injection of 5′-AMP. In conclusion, 5′-AMP induces a state of pharmacological torpor in mice, during which, lymphopenia is governed primarily by body temperature-independent suppression of lymphocyte egress from LNs. PMID:23682128

  6. The role of adenosine receptors in regulating production of tumour necrosis factor-α and chemokines by human lung macrophages

    PubMed Central

    Buenestado, A; Delyle, S Grassin; Arnould, I; Besnard, F; Naline, E; Blouquit-Laye, S; Chapelier, A; Bellamy, JF; Devillier, P

    2010-01-01

    Background and purpose: Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A1, A2A, A2B and A3). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-α and chemokines. Experimental approach: Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. Key results: LPS increased (about 400-fold) mRNA for A2A adenosine receptors, decreased mRNA for A1 and A2B, but had no effect on A3 adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-α production concentration dependently, whereas the A1 receptor agonist, CCPA, and the A3 receptor agonist, AB-MECA, inhibited TNF-α production only at concentrations affecting A2A receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-α and chemokine production by the selective A2A adenosine receptor antagonist ZM 241385, but not the A2B receptor antagonist, MRS 1754, or the A3 receptor antagonist, MRS 1220, indicated involvement of A2A receptors. Conclusions and implications: LPS up-regulated A2A adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-α and of a subset of chemokines in human lung macrophages. PMID:20136829

  7. Image-derived and arterial blood sampled input functions for quantitative PET imaging of the angiotensin II subtype 1 receptor in the kidney

    SciTech Connect

    Feng, Tao; Tsui, Benjamin M. W.; Li, Xin; Vranesic, Melin; Lodge, Martin A.; Gulaldi, Nedim C. M.; Szabo, Zsolt

    2015-11-15

    Purpose: The radioligand {sup 11}C-KR31173 has been introduced for positron emission tomography (PET) imaging of the angiotensin II subtype 1 receptor in the kidney in vivo. To study the biokinetics of {sup 11}C-KR31173 with a compartmental model, the input function is needed. Collection and analysis of arterial blood samples are the established approach to obtain the input function but they are not feasible in patients with renal diseases. The goal of this study was to develop a quantitative technique that can provide an accurate image-derived input function (ID-IF) to replace the conventional invasive arterial sampling and test the method in pigs with the goal of translation into human studies. Methods: The experimental animals were injected with [{sup 11}C]KR31173 and scanned up to 90 min with dynamic PET. Arterial blood samples were collected for the artery derived input function (AD-IF) and used as a gold standard for ID-IF. Before PET, magnetic resonance angiography of the kidneys was obtained to provide the anatomical information required for derivation of the recovery coefficients in the abdominal aorta, a requirement for partial volume correction of the ID-IF. Different image reconstruction methods, filtered back projection (FBP) and ordered subset expectation maximization (OS-EM), were investigated for the best trade-off between bias and variance of the ID-IF. The effects of kidney uptakes on the quantitative accuracy of ID-IF were also studied. Biological variables such as red blood cell binding and radioligand metabolism were also taken into consideration. A single blood sample was used for calibration in the later phase of the input function. Results: In the first 2 min after injection, the OS-EM based ID-IF was found to be biased, and the bias was found to be induced by the kidney uptake. No such bias was found with the FBP based image reconstruction method. However, the OS-EM based image reconstruction was found to reduce variance in the subsequent

  8. [THE THYROID STATUS OF RATS IMMUNIZED WITH PEPTIDES DERIVED FROM THE EXTRACELLULAR REGIONS OF THE TYPES 3 AND 4 MELANOCORTIN RECEPTORS AND THE 1B-SUBTYPE 5-HYDROXYTRYPTAMINE RECEPTOR].

    PubMed

    Derkach, K V; Moyseuk, I V; Shpakova, E A; Sphakov, A O

    2015-01-01

    The activity of the hypothalamic-pituitary-thyroid (HPT) axis is controlled by the brain neurotransmitter systems, including the melanocortin signaling system. Pharmacological inhibition of type 4 melanocortin receptor (M4R) leads to disruption of the functioning of HPT axis and to reduction of the level of thyroid hormones. At the same time, the data on how prolonged inhibition of M4R affects this axis and on its role in regulation of M3R are absent. The relationship between the thyroid status and the activity of 1B-subtype 5-hydroxytryptamine receptor (5-HT1BR) is scarcely explored. The aim of this work to study the effects of chronic inhibition of M3R, M4R and 5-HT1BR induced by immunization of rats with BSA-conjugated peptide derived from the extracellular regions of these receptors on the thyroid status and the activity of thyroid stimulating hormone (TSH)-sensitive adenylyl cyclase signaling system (ACSS) in the thyroid glarid (TG) of the immunized animals. In rats immunized with the peptides K-[TSLHL WNRSSHGLHG11-25]-A of M4R, A[PTNPYCICTTAH269-280]-A of M3R and. [QAKAEE-EVSEC(Acm)-VVNTDH189-205]-A of 5-HT1BR levels of thyroid hormones such as fT4, tT4 and tT3 were significantly reduced. In rats immunized with M4R and M3R peptides, an increase of TSH was detected whereas in the animals immunized with 5-HT1BR peptide the level of TSH, on the contrary, was reduced. In the TG of rats immunized with M4R and M3R peptides, the stimulatory effects of hormones (TSH, PA-CAP-3 8) and GppNHp on adenylyl cyclase activity were attenuated, and the changes were most pronounced in the case M4R peptide immunization. After immunization with 5-HT1BR peptide the stimulatory effects of TSH, PACAP-38 and GppNHp were retained. Thus, the main cause of thyroid hormones deficit in rats immunized with M4R and M3R peptides was the decreased sensitivity of ACSS thyrocytes to TSH, whereas in rats iimunized with 5-HT1BR peptide the deficit of thyroid hormones was associated with decreased

  9. The Clinically-tested S1P Receptor Agonists, FTY720 and BAF312, Demonstrate Subtype-Specific Bradycardia (S1P1) and Hypertension (S1P3) in Rat

    PubMed Central

    Fryer, Ryan M.; Muthukumarana, Akalushi; Harrison, Paul C.; Nodop Mazurek, Suzanne; Chen, Rong Rhonda; Harrington, Kyle E.; Dinallo, Roger M.; Horan, Joshua C.; Patnaude, Lori; Modis, Louise K.; Reinhart, Glenn A.

    2012-01-01

    Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function in vivo. Indeed, FTY720 (non-selective S1PX receptor agonist) produces modest hypertension in patients (2–3 mmHg in 1-yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question (e.g. bradycardia, hypertension), and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound in vitro and, in vivo, the cardiovascular effects of FTY720 and the more selective S1P1,5 agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720 (0.1, 0.3, 1.0 mg/kg/20 min) or BAF312 (0.5, 1.5, 5.0 mg/kg/20 min) elicited acute bradycardia in anesthetized rats demonstrating an S1P1 mediated mechanism-of-action. However, while FTY720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations (24-hr mean blood pressure = 8.4, 12.8, 16.2 mmHg above baseline vs. 3 mmHg in vehicle controls), BAF312 (0.3, 3.0, 30.0 mg/kg/d) had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P3 receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P1 receptors mediate bradycardia while hypertension is mediated by S1P3 receptor activation. PMID:23285242

  10. The clinically-tested S1P receptor agonists, FTY720 and BAF312, demonstrate subtype-specific bradycardia (S1P₁) and hypertension (S1P₃) in rat.

    PubMed

    Fryer, Ryan M; Muthukumarana, Akalushi; Harrison, Paul C; Nodop Mazurek, Suzanne; Chen, Rong Rhonda; Harrington, Kyle E; Dinallo, Roger M; Horan, Joshua C; Patnaude, Lori; Modis, Louise K; Reinhart, Glenn A

    2012-01-01

    Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function in vivo. Indeed, FTY720 (non-selective S1P(X) receptor agonist) produces modest hypertension in patients (2-3 mmHg in 1-yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question (e.g. bradycardia, hypertension), and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound in vitro and, in vivo, the cardiovascular effects of FTY720 and the more selective S1P₁,₅ agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720 (0.1, 0.3, 1.0 mg/kg/20 min) or BAF312 (0.5, 1.5, 5.0 mg/kg/20 min) elicited acute bradycardia in anesthetized rats demonstrating an S1P₁ mediated mechanism-of-action. However, while FTY720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations (24-hr mean blood pressure = 8.4, 12.8, 16.2 mmHg above baseline vs. 3 mmHg in vehicle controls), BAF312 (0.3, 3.0, 30.0 mg/kg/d) had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P₃ receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P₁ receptors mediate bradycardia while hypertension is mediated by S1P₃ receptor activation. PMID:23285242

  11. Expression of the five somatostatin receptor (SSTR1-5) subtypes in rat pituitary somatotrophes: quantitative analysis by double-layer immunofluorescence confocal microscopy.

    PubMed

    Kumar, U; Laird, D; Srikant, C B; Escher, E; Patel, Y C

    1997-10-01

    Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

  12. Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

    PubMed Central

    Bergson, C; Mrzljak, L; Lidow, M S; Goldman-Rakic, P S; Levenson, R

    1995-01-01

    To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7536933

  13. Reinforcing effects of compounds lacking intrinsic efficacy at α1 subunit-containing GABAA receptor subtypes in midazolam- but not cocaine-experienced rhesus monkeys.

    PubMed

    Shinday, Nina M; Sawyer, Eileen K; Fischer, Bradford D; Platt, Donna M; Licata, Stephanie C; Atack, John R; Dawson, Gerard R; Reynolds, David S; Rowlett, James K

    2013-05-01

    Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors ('α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration. PMID:23303046

  14. Mast Cell Adenosine Receptors Function: A Focus on the A3 Adenosine Receptor and Inflammation

    PubMed Central

    Rudich, Noam; Ravid, Katya; Sagi-Eisenberg, Ronit

    2012-01-01

    Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells (MCs), as an attractive drug candidate. Four subtypes (A1, A2a, A2b, and A3) of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R) in mediating hyper responsiveness to adenosine in MCs, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human MCs. The relevance of mouse studies to the human is discussed. PMID:22675325

  15. Effects of estradiol, estrogen receptor subtype-selective agonists and genistein on glucose metabolism in leptin resistant female Zucker diabetic fatty (ZDF) rats.

    PubMed

    Weigt, Carmen; Hertrampf, Torsten; Flenker, Ulrich; Hülsemann, Frank; Kurnaz, Pinar; Fritzemeier, Karl Heinrich; Diel, Patrick

    2015-11-01

    The leptin resistant Zucker diabetic fatty (ZDF) rats are hyperphagic and become obese, but whereas the males develop type 2 diabetes mellitus (T2DM), the females remain euglycaemic. As estrogen deficiency is known to increase the risk of developing T2DM, we evaluated the role of ER subtypes alpha and beta in the development of glucose tolerance in leptin resistant ovariectomized (OVX) ZDF rats. At least six rats per group were treated with either vehicle (OVX), 17β-estradiol (E2), ER subtype-selective agonists (Alpha and Beta), or genistein (Gen) for 17 weeks. At the end of the treatment period a glucose tolerance assay was performed and the metabolic flux of (13)C-glucose for the E2 group was investigated. OVX ZDF rats treated with E2, Alpha, Beta, and Gen tolerated the glucose significantly better than untreated controls. E2 treatment increased absorbance/flux of (13)C-glucose to metabolic relevant tissues such liver, adipose tissue, gastrocnemius, and soleus muscle. Moreover, whereas Alpha treatment markedly increased mRNA expression of GLUT4 in gastrocnemius muscle, Beta treatment resulted in the largest fiber sizes of the soleus muscle. Treatment with Gen increased both the mRNA expression of GLUT 4 and the fiber sizes in the skeletal muscle. In addition, E2 and Alpha treatment decreased food intake and body weight gain. In summary, estrogen-improved glucose absorption is mediated via different molecular mechanisms: while activation of ER alpha seems to stimulate muscular GLUT4 functionality, activation of ER beta results in a hypertrophy of muscle fibers. In addition, selective activation of ER alpha decreased food intake and body weight gain. Our data further indicate that ER subtype-selective agonists and genistein improve systemic glucose tolerance also in the absence of a functional leptin signaling pathway.

  16. Molecular subtypes and imaging phenotypes of breast cancer

    PubMed Central

    2016-01-01

    During the last 15 years, traditional breast cancer classifications based on histopathology have been reorganized into the luminal A, luminal B, human epidermal growth factor receptor 2 (HER2), and basal-like subtypes based on gene expression profiling. Each molecular subtype has shown varying risk for progression, response to treatment, and survival outcomes. Research linking the imaging phenotype with the molecular subtype has revealed that non-calcified, relatively circumscribed masses with posterior acoustic enhancement are common in the basal-like subtype, spiculated masses with a poorly circumscribed margin and posterior acoustic shadowing in the luminal subtype, and pleomorphic calcifications in the HER2-enriched subtype. Understanding the clinical implications of the molecular subtypes and imaging phenotypes could help radiologists guide precision medicine, tailoring medical treatment to patients and their tumor characteristics. PMID:27599892

  17. Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms.

    PubMed

    Akimova, Olga A; Grygorczyk, Alexandra; Bundey, Richard A; Bourcier, Nathalie; Gekle, Michael; Insel, Paul A; Orlov, Sergei N

    2006-10-20

    In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP > 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP > UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively

  18. The 3,7-diazabicyclo[3.3.1]nonane scaffold for subtype selective nicotinic acetylcholine receptor (nAChR) ligands. Part 1: the influence of different hydrogen bond acceptor systems on alkyl and (hetero)aryl substituents.

    PubMed

    Eibl, Christoph; Tomassoli, Isabelle; Munoz, Lenka; Stokes, Clare; Papke, Roger L; Gündisch, Daniela

    2013-12-01

    3,7-Diazabicyclo[3.3.1]nonane is a naturally occurring scaffold interacting with nicotinic acetylcholine receptors (nAChRs). When one nitrogen of the 3,7-diazabicyclo[3.3.1]nonane scaffold was implemented in a carboxamide motif displa