The Experts below are selected from a list of 1425 Experts worldwide ranked by ideXlab platform
Keiichi Kuwano - One of the best experts on this subject based on the ideXlab platform.
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a comparison of vasodilation mode among selexipag ns 304 2 4 5 6 diphenylpyrazin 2 yl isopropyl amino butoxy n methylsulfonyl acetamide its active metabolite mre 269 and various Prostacyclin Receptor agonists in rat porcine and human pulmonary arteri
European Journal of Pharmacology, 2017Co-Authors: Chiaki Fuchikami, Kohji Murakami, Koyuki Tajima, Junko Homan, Keiji Kosugi, Kazuya Kuramoto, Keiichi KuwanoAbstract:Abstract Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}- N - (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid Prostacyclin Receptor (IP Receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP Receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP Receptor antagonists (CAY10441 and 2-[4-(1 H -indol-4-yloxymethyl)-benzyloxycarbonylamino]−3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP Receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E Receptor 3 antagonist enhanced the vasodilation induced by all IP Receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP Receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP Receptor agonists, works as a selective IP Receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.
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A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various Prostacyclin Receptor agonists in rat, porcine and human pul
European Journal of Pharmacology, 2016Co-Authors: Chiaki Fuchikami, Kohji Murakami, Koyuki Tajima, Junko Homan, Keiji Kosugi, Kazuya Kuramoto, Keiichi KuwanoAbstract:Abstract Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}- N - (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid Prostacyclin Receptor (IP Receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP Receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP Receptor antagonists (CAY10441 and 2-[4-(1 H -indol-4-yloxymethyl)-benzyloxycarbonylamino]−3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP Receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E Receptor 3 antagonist enhanced the vasodilation induced by all IP Receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP Receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP Receptor agonists, works as a selective IP Receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.
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selexipag an oral and selective ip Prostacyclin Receptor agonist for the treatment of pulmonary arterial hypertension
Journal of Medicinal Chemistry, 2015Co-Authors: Tetsuo Asaki, Keiichi Kuwano, Taisuke Hamamoto, Keith Morrison, John Gatfield, Martine ClozelAbstract:Prostacyclin controls cardiovascular function via activation of the Prostacyclin Receptor. Decreased Prostacyclin production occurs in several cardiovascular diseases. However, the clinical use of Prostacyclin and its analogues is complicated by their chemical and metabolic instability. A medicinal chemistry program searched for novel nonprostanoid Prostacyclin Receptor agonists not subject to these limitations. A compound with a diphenylpyrazine structural core was synthesized. Metabolic stability and agonist potency were optimized through modification of the linear side chain. Compound 12b (MRE-269, ACT-333679) was identified as a potent and highly selective Prostacyclin Receptor agonist. Replacement of the terminal carboxyl group with an N-acylsulfonamide group yielded parent compound 26a (selexipag, NS-304, ACT-293987), which is orally active and provides sustained plasma exposure of 12b. Compound 26a was developed for the treatment of pulmonary arterial hypertension and shown to reduce the risk of th...
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structure activity studies on diphenylpyrazine derivatives a novel class of Prostacyclin Receptor agonists
Bioorganic & Medicinal Chemistry, 2007Co-Authors: Tetsuo Asaki, Keiichi Kuwano, Taisuke Hamamoto, Yukiteru Sugiyama, Kenji KuwabaraAbstract:Abstract To develop nonprostanoid Prostacyclin Receptor agonists with a high degree of metabolic resistance and an extended duration of action, a novel series of diphenylpyrazine derivatives was synthesized and evaluated for their inhibition of ADP-induced human platelet aggregation. Structure–activity relationship studies on the side chain containing the carboxylic acid moiety of the lead compound 5c showed that the length of the linker and the presence of the concatenating nitrogen atom adjacent to the pyrazine ring are critical for the antiaggregatory activity. This study led to the discovery of 2-amino-5,6-diphenylpyrazine derivatives 8c , 15a , and 15b , which showed potent inhibition of platelet aggregation with IC 50 values of 0.2 μM. Among these compounds, 15b is an orally available and long-lasting Prostacyclin Receptor agonist which is promising for the treatment of various vascular diseases.
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Structure-activity relationship study on the 6-membered heteroaromatic ring system of diphenylpyrazine-type Prostacyclin Receptor agonists.
Bioorganic & Medicinal Chemistry Letters, 2007Co-Authors: Tetsuo Asaki, Keiichi Kuwano, Taisuke Hamamoto, Yukiteru Sugiyama, Kenji Kuwabara, Tomoko NiwaAbstract:Abstract A series of Prostacyclin Receptor agonists was prepared by modifying the central heteroaromatic ring of lead compound 2 , and a docking study was performed to investigate their structure–activity relationships by using a homology-modeled structure of the Prostacyclin Receptor. Compound 2 and its derivatives could be docked to the Prostacyclin Receptor in two ways depending on the position of the nitrogen atom within the heteroaromatic ring. Furthermore, hydrogen bonding between the nitrogen atom in the heteroaromatic ring and the hydroxyl group of Ser20 or Tyr75 of the Receptor appears to be important for the potent expression of biological activity.
Yasuyoshi Watanabe - One of the best experts on this subject based on the ideXlab platform.
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rapid methylation on carbon frameworks leading to the synthesis of a pet tracer capable of imaging a novel cns type Prostacyclin Receptor in living human brain
Trends in Analytical Chemistry, 2004Co-Authors: Masaaki Suzuki, Bengt Langstrom, Takamitsu Hosoya, Yasuyoshi WatanabeAbstract:Abstract To develop novel short-lived 11 C radionuclide-incorporated PET tracers with high metabolic tolerance, we performed rapid methylation using 11 CH 3 I, a frequently used precursor for 11 C-labeled tracers, on sp- and sp 2 -carbon frameworks. Methyl iodide was trapped using an excess of tributylphenylstannane for 5 min at 60 °C in the presence of Pd[P( o -tolyl) 3 ] 2 , a copper(I) salt (CuCl or CuBr), and K 2 CO 3 , resulting in a greater than 90% yield of toluene. A trapping reaction with tributyl-1-hexynylstannane for 5 min at 60 °C in the presence of Pd[P( t -C 4 H 9 ) 3 ] 2 and a fluoride ion (KF or CsF) gave a high yield (>90%) of 2-heptyne. Since these reactions are applicable to a wide range of organic molecules, we synthesized 15 R -[ 11 C]TIC methyl ester, a PET tracer targeting the CNS-specific Prostacyclin Receptor (IP 2 ), by rapid sp 2 –sp 3 coupling with some modifications. Radiolabeled 11 CH 3 I was trapped with the methyl ester of the corresponding stannyl precursor via two steps. In the first, 11 CH 3 I and Pd[P( o -tolyl) 3 ] 2 were mixed to form a methylpalladium complex, and in the second, the methylpalladium complex was mixed with the other reagents necessary for the coupling reaction. The yield of radiolabeled 15 R -[ 11 C]TIC methyl ester was 80–90%, and it had a radioactivity of 2.0–2.5 GBq, sufficient for an in vivo human PET study with high reproducibility. We used this tracer in PET studies imaging the IP 2 Receptor in living monkey and human brains. Following its penetration of the blood–brain barrier, the tracer underwent ester hydrolysis and bound to its specific Receptor in the brain. A protocol for sp–sp 3 coupling would be used to synthesize [ 11 C]iloprost methyl ester, a PET tracer for the peripheral-type Prostacyclin Receptor (IP 1 ). A methyl group is the least bulky, unpolarized organic group, suggesting that it would have little effect on the biological activity of its parent compound. Thus, [ 11 C]methylated compounds may be applicable to a wide range of biologically active molecules, converting them to PET tracers that can be utilized for in vivo molecular imaging.
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Specific ligand for a central type Prostacyclin Receptor attenuates neuronal damage in a rat model of focal cerebral ischemia.
Brain Research, 2002Co-Authors: Hiroyuki Takamatsu, Yumiko Watanabe, Masaaki Suzuki, Hideo Tsukada, Yosky Kataoka, Takamitsu Hosoya, Yasuyoshi WatanabeAbstract:The neuroprotective effect of a central type Prostacyclin Receptor ligand was examined in a rat model of focal cerebral ischemia. Under halothane anesthesia, male Sprague–Dawley rats were subjected to left middle cerebral artery occlusion. A selective central type Prostacyclin Receptor ligand, 15-deoxy-(16-m-tolyl)-17,18,19,20-tetranorisocarbacyclin methylester, or a peripheral type Prostacyclin Receptor ligand, iloprost methylester, were administered intravenously immediately after ischemia. Twenty-four hours after ischemia, brain damage was evaluated. In separate experiments, concentrations of 15-deoxy-(16-m-tolyl)-17,18,19,20-tetranorisocarbacyclin in ischemic brain tissue were measured by injection of a tritium labeled compound. Treatment with 15-deoxy-(16-m-tolyl)-17,18,19,20-tetranorisocarbacyclin methylester (0.03 mg/kg) significantly (P
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a novel subtype of Prostacyclin Receptor in the central nervous system
Journal of Neurochemistry, 2002Co-Authors: Yumiko Watanabe, Hajime Takechi, Kiyoshi Matsumura, Koichi Kato, Ryoji Noyori, Masaaki Suzuki, Hiroshi Morii, Margareta Bjorkman, Bengt Langstrom, Yasuyoshi WatanabeAbstract:Recently, in the course of our search for the Prostacyclin Receptor in the brain, we found a novel subtype, designated as IP2, which was finely discriminated by use of the specific ligand (15R)-16- ...
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a novel subtype of the Prostacyclin Receptor expressed in the central nervous system
Journal of Biological Chemistry, 1996Co-Authors: Hajime Takechi, Kiyoshi Matsumura, Yumiko Watanabe, Koichi Kato, Ryoji Noyori, Masaaki Suzuki, Yasuyoshi WatanabeAbstract:Abstract By use of several Prostacyclin analogs and an in vitro autoradiographic technique, we have found a novel subtype of the Prostacyclin Receptor, one having different binding properties compared with those of the known Prostacyclin Receptor in the rat brain. Isocarbacyclin, which is a potent agonist for the known Prostacyclin Receptor, had high affinity for the novel subtype (dissociation constant (K) of 7.8 nM). However, iloprost, which is usually used as a stable Prostacyclin analog, showed low affinity binding (K = 159 nM) for the subtype. Other prostaglandins showed no or little affinity for the subtype. [3H]Isocarbacyclin binding was high in the thalamus, lateral septal nucleus, hippocampus, cerebral cortex, striatum, and dorsal cochlear nucleus. Although the nucleus of the solitary tract and the spinal trigeminal nucleus showed a high density of [3H]isocarbacyclin binding, [3H]iloprost also had high affinity in these regions, and the binding specificity was similar to that for the known Prostacyclin Receptor. Hemilesion studies of striatal neurons lesioned by kainate or of dopaminergic afferents lesioned by 6-hydroxydopamine revealed that the binding sites of the novel subtype exist on neuronal cells in the striatum, but not on the presynaptic terminal of afferents or on glial cells. Electrophysiological studies carried out in the CA1 region of the hippocampus revealed that Prostacyclin analogs have a facilitatory effect on the excitatory transmission through the novel Prostacyclin Receptor. The widespread expression of the Prostacyclin Receptor in the central nervous system suggests that Prostacyclin has important roles in neuronal activity.
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15r 16 m tolyl 17 18 19 20 tetranorisocarbacyclin a stable ligand with high binding affinity and selectivity for a Prostacyclin Receptor in the central nervous system
Angewandte Chemie, 1996Co-Authors: Masaaki Suzuki, Hajime Takechi, Kiyoshi Matsumura, Koichi Kato, Ryoji Noyori, Yasuyoshi Watanabe, Bengt LangstromAbstract:(15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin: A stable ligand with high binding affinity and selectivity for a Prostacyclin Receptor in the central nervous system
Jeremiah Stitham - One of the best experts on this subject based on the ideXlab platform.
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Prostacyclin Receptor regulation from transcription to trafficking
Current Molecular Medicine, 2011Co-Authors: Charles Midgett, Jeremiah Stitham, Kathleen A MartinAbstract:The Prostacyclin Receptor (IP - International Union of Pharmacology nomenclature) is a member of the seven transmembrane G-protein coupled Receptor (GPCR) superfamily. Recent concerns with selective and non-selective COX-1/COX-2 inhibition have exposed an important cardioprotective role for IP in preventing atherothrombosis. Receptor dysfunction (genetic variants) or reduced signaling (COX-2 inhibition) in high cardiovascular risk patients leads to increased cardiovascular events. These clinical observations have also been confirmed genetically by mouse knockout studies. Thus, Receptor regulation is paramount in ensuring correct function in the prevention of atherothrombosis. This review summarizes recent literature on how this important Receptor is regulated, from transcription to transport (to and from the membrane surface). These regulatory processes are critical in ensuring that IP Receptors are adequately expressed and functional on the cell surface.
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comprehensive biochemical analysis of rare Prostacyclin Receptor variants study of association of signaling with coronary artery obstruction
Journal of Biological Chemistry, 2011Co-Authors: Jeremiah Stitham, Eric Arehart, Scott Gleim, Karen Douville, Larkin Elderon, Kristina M. Fetalvero, Todd A. Mackenzie, Zsolt Kasza, John F Robb, Kathleen A MartinAbstract:Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human Prostacyclin Receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface Receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional Receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human Prostacyclin Receptor genetic variants to date.
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dominant negative actions of human Prostacyclin Receptor variant through dimerization implications for cardiovascular disease
Arteriosclerosis Thrombosis and Vascular Biology, 2010Co-Authors: Salam Ibrahim, Jeremiah Stitham, Mazell Tetruashvily, Alexander J Frey, Stephen J Wilson, Emer M SmythAbstract:Objective— Prostacyclin and thromboxane mediate opposing cardiovascular effects through their Receptors, the Prostacyclin Receptor (IP) and thromboxane Receptor (TP). Individuals heterozygous for an IP variant, IP R212C , displayed exaggerated loss of platelet IP responsiveness and accelerated cardiovascular disease. We examined association of IP R212C into homo- and heterodimeric Receptor complexes and the impact on Prostacyclin and thromboxane biology. Methods and Results— Dimerization of the IP, IP R212C , and TPα was examined by bioluminesence resonance energy transfer in transfected HEK293 cells. We observed an equal propensity for formation of IPIP homodimers and IPTPα heterodimers. Compared with the IP alone, IP R212C displayed reduced cAMP generation and increased endoplasmic reticulum localization but underwent normal homo- and heterodimerization. When the IP R212C and IP were coexpressed, a dominant negative action of the variant was evident with enhanced wild-type IP localization to the endoplasmic reticulum and reduced agonist-dependent signaling. Further, the TPα activation response, which was shifted from inositol phosphate to cAMP generation following IPTPα heterodimerization, was normalized when the TPα instead dimerized with IP R212C . Conclusion— IP R212C exerts a dominant action on the wild-type IP and TPα through dimerization. This likely contributes to accelerated cardiovascular disease in individuals carrying 1 copy of the variant allele.
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Arginine (CGC) codon targeting in the human Prostacyclin Receptor gene (PTGIR) and G-protein coupled Receptors (GPCR).
Gene, 2007Co-Authors: Jeremiah Stitham, Eric Arehart, Scott Gleim, Karen Douville, Todd A. MackenzieAbstract:Abstract The human Prostacyclin Receptor (hIP) has recently been recognized as an important seven transmembrane G-protein coupled Receptor that plays critical roles in atheroprevention and cardioprotection. To date, four non-synonymous genetic variants have been identified, two of which occur at the same Arg amino acid position (R212H, R212C). This observation instigated further genetic screening for Prostacyclin Receptor variants on 1455 human genomic samples. A total of 31 distinct genetic variants were detected, with 6 (19%) involving Arg residues. Distinct differences in location and frequencies of genetic variants were noted between Caucasian, Asian, Hispanic and African Americans, with the most changes noted in the Asian cohort. From the sequencing results, three Arg-targeted changes at the same 212 position within the third cytoplasmic loop of the human Prostacyclin (hIP) Receptor were detected: 1) R212C (CGC→TGC), 2) R212H (CGC→CAC), and 3) R212R (CGC→CGT). Three additional Arg codon variants (all exhibiting the same CGC to TGC change) were also detected, R77C, R215C, and R279C. Analysis (GPCR and SNP databases) of 200 other GPCRs, with recorded non-synonymous mutations, confirmed a high frequency of Arg-targeted missense mutations, particularly within the important cytoplasmic domain. Preferential nucleotide changes (at Arg codons), were observed involving cytosine (C) to thymine (T) (pyrimidine to pyrimidine), as well as guanine (G) to adenine (A) (purine to purine) (p
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human Prostacyclin Receptor structure and function from naturally occurring and synthetic mutations
Prostaglandins & Other Lipid Mediators, 2007Co-Authors: Jeremiah Stitham, Eric Arehart, Scott Gleim, Karen DouvilleAbstract:Abstract Prostacyclin (PGI 2 ) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation (anti-thrombotic), and moderator of vascular smooth muscle cell proliferation–migration–differentiation (anti-atherosclerotic). These actions are mediated via a seven transmembrane-spanning G-protein coupled Receptor (GPCR), known as the human Prostacyclin Receptor or hIP. Animal studies using Prostacyclin Receptor knock-out (IP−/−) mice have revealed increased propensities towards thrombosis, intimal hyperplasia, atherosclerosis, restenosis, as well as reperfusion injury. Of further importance has been the world-wide withdrawal of selective COX-2 inhibitors, due to their discriminating suppression of COX-2-derived PGI 2 and its cardioprotective effects, leading to increased cardiovascular events, including myocardial infarction and thrombotic stroke. Over the last decade, mutagenesis studies of the IP Receptor, in conjunction with in vitro functional assays and molecular modeling, have provided critical insights into the molecular mechanisms of both agonist binding and Receptor activation. Most recently, the discovery of naturally-occurring and dysfunctional mutations within the hIP has provided additional insights into the proposed cardioprotective role of Prostacyclin. The aim of this review is to summarize the most recent findings regarding hIP Receptor structure–function that have developed through the study of both synthetic and naturally-occurring mutations.
Shuh Narumiya - One of the best experts on this subject based on the ideXlab platform.
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Prostacyclin Receptor suppresses cardiac fibrosis role of creb phosphorylation
Journal of Molecular and Cellular Cardiology, 2010Co-Authors: Elsa C Chan, Shuh Narumiya, Gregory J Dusting, Hitesh Peshavariya, Caroline J Taylor, Rodney J Dilley, Fan JiangAbstract:Abstract Cardiac fibrosis is a consequence of many cardiovascular diseases and contributes to impaired ventricular function. Activation of the Prostacyclin Receptor (IP) protects against cardiac fibrosis, but the molecular mechanisms are not totally understood. Using mouse cardiac fibroblasts, we found that IP activation with cicaprost suppressed expression of collagen I and other target genes of transforming growth factor-β. This effect of cicaprost was unlikely to be mediated by inhibition of the Smad2/3 or mitogen-activated protein kinase (MAPK) activities, but was associated with cAMP elevation and phosphorylation of the transcription factor cAMP response element binding protein (CREB). Expression of a non-phosphorylated CREB mutant suppressed the inhibitory effect of cicaprost. It appears that phosphorylated CREB binds to and sequestrates the transcription coactivator CBP/p300 from binding to Smad. Inhibition of the intrinsic histone acetyl-transferase activity of CBP/p300 with garcinol significantly suppressed collagen I expression in fibroblasts. Using apolipoprotein E and IP double knockout mouse, we demonstrated that endogenous Prostacyclin/IP signaling had an inhibitory effect on angiotensin II-induced cardiac fibrosis under hypercholesterolemic conditions. Taken together, our results suggest that the Prostacyclin/IP pathway suppresses cardiac fibrosis, at least partly, by inducing CREB phosphorylation.
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in situ hybridization studies of Prostacyclin Receptor mrna expression in various mouse organs
British Journal of Pharmacology, 1995Co-Authors: Hiroji Oida, Tsunehisa Namba, Yukihiko Sugimoto, Fumitaka Ushikubi, Hitoshi Ohishi, Atushi Ichikawa, Shuh NarumiyaAbstract:1 Expression of Prostacyclin Receptor (IP Receptor) mRNA was examined in various mouse organs, and the cells expressing IP Receptor mRNA were identified by in situ hybridization studies. Co-localization of mRNA for the IP Receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E Receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2 IP Receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP Receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3 IP Receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP Receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4 IP Receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small- and large-sized neurones were labelled but no labelling was found in the glia. Expression of PPT A mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP Receptor mRNA, and about half of the IP Receptor-positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 Receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP Receptor-positive neurones co-expressed the EP1, EP3 and EP4 Receptor, respectively. 5 These results not only verified IP Receptor expression in various cells and tissues known to be sensitive to Prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of Prostacyclin in these tissues. They also indicated that the actions of Prostacyclin on blood vessels and platelets are mediated by the same type of Receptor. Absence of IP Receptor mRNA in the JG cells suggests that the action of Prostacyclin on renin release may be indirect.
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GENE EXPRESSION OF Prostacyclin Receptor IN THE HYPERTROPHIED HEART OF SPONTANEOUSLY HYPERTENSIVE RATS
Clinical and Experimental Pharmacology and Physiology, 1995Co-Authors: Osamu Nakagawa, Takeshi Usui, Hiroshi Itoh, Takaaki Yoshimasa, Yutaka Sasaki, I. Tanaka, Takehiro Sando, Seiji Muro, Kiyoshi Mori, Shuh NarumiyaAbstract:Summary 1. Prostacyclin elicits potent vasodilation and inhibition of platelet aggregation through binding to its membrane Receptor. The impairment of Prostacyclin Receptor activity is implicated in various human cardiovascular diseases. We recently succeeded in molecular cloning of cDNA for the mouse, rat, and human Prostacyclin Receptors. 2. In the present study, we examined the mRNA expression of the Prostacyclin Receptor in various rat tissues, and further investigated its gene expression in the hypertrophied cardiac ventricles of stroke-prone spontaneously hypertensive rats (SHRSP). 3. In rat tissues, a single RNA band of approximately 3.7 kb was detected by northern blotting analysis using rat Prostacyclin Receptor cDNA as a probe. In adult Wistar rats, abundant mRNA expression was observed in the aorta, lung and spleen. Substantial amounts of transcript were expressed in the heart, pancreas, thymus and stomach. In contrast, no mRNA expression was detected in the brain. 4. We further examined the mRNA expression of the Prostacyclin Receptor in the ventricles of 21 week old SHRSP. The ventricles of SHRSP showed remarkable hypertrophy, compared with those of age-matched Wistar-Kyoto (WKY) rats. The expression of Prostacyclin Receptor mRNA in the hypertrophied ventricles of SHRSP was almost equivalent to that in the ventricles of WKY. 5. The present study revealed the gene expression of the Prostacyclin Receptor in various rat tissues, and further demonstrated the Receptor mRNA expression in hypertensive cardiac hypertrophy. The present study will give a clue to investigate the clinical implication of Prostacyclin and its Receptor.
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structural organization and chromosomal assignment of the human Prostacyclin Receptor gene
Genomics, 1995Co-Authors: Yoshihiro Ogawa, Issei Tanaka, Miho Inoue, Yuka Yoshitake, Naohi Isse, Osamu Nakagawa, Takeshi Usui, Hiroshi Itoh, Takaaki Yoshimasa, Shuh NarumiyaAbstract:Prostacyclin Receptor is a member of the prostanoid Receptor family in the G protein-coupled Receptor superfamily with seven transmembrane domains. The authors report here the isolation and structural organization of the human Prostacyclin Receptor gene. Southern blot analysis demonstrated a single copy of the human Prostacyclin Receptor gene in the human genome. The human Prostacyclin Receptor gene spanned approximately 7.0 kb and was composed of three exons separated by two introns. The first intron occurred in the 5`-untranslated region, 13 bp upstream to the ATG start codon. The second intron was located at the end of the sixth transmembrane domain, thereby separating it from the downstream coding region and the 3`-untranslated region. By primer extension analysis, the transcription initiation sites were mapped 870-872 bp upstream to the ATG start codon. The 1.2-kb human Prostacyclin Receptor 5`-flanking region lacked conventional TATA and CCAAT boxes, but it contained several cis-acting regulatory elements including an inverted CCAAT box (Y box) and two copies of SP-1 binding sites. Using human-rodent somatic hybrid cell DNA, the human Prostacyclin Receptor gene was assigned to human chromosome 19. The present study helps establish the genetic basis for Prostacyclin Receptor research and provides further insight into the molecularmore » mechanisms underlying the prostanoid Receptor family. 38 refs., 6 figs.« less
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cloning and expression of a cdna for rat Prostacyclin Receptor
Biochimica et Biophysica Acta, 1994Co-Authors: Yutaka Sasaki, Issei Tanaka, Osamu Nakagawa, Takeshi Usui, Shuh Narumiya, Tsunehisa Namba, Takehiro Sando, Takayuki Takahashi, Kazuwa NakaoAbstract:A cDNA clone for rat Prostacyclin Receptor was isolated. The cDNA encodes a protein of 416 amino acid residues (Mr 44662) with putative seven transmembrane domains, and belongs to the G protein-coupled Receptor superfamily. Specific binding of [3H]iloprost was found in membrane of COS-7 cells transfected with the cDNA (Kd = 1.3 nM) and was displaced with unlabeled prostaglandins in the order of iloprost = cicaprost>PGE1>STA2 = PGE2 = PGD2>PGF2α. Northern blot analysis demonstrated that rat Prostacyclin Receptor mRNA is expressed in the lung, spleen, heart, pancreas, thymus, stomach and aorta.
Therese B Kinsella - One of the best experts on this subject based on the ideXlab platform.
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identification of α helix 4 α4 of rab11a as a novel rab11 binding domain rbd interaction of rab11a with the Prostacyclin Receptor
Biochimica et Biophysica Acta, 2017Co-Authors: Eamon P. Mulvaney, Fergal Omeara, Amir R Khan, David J Oconnell, Therese B KinsellaAbstract:Abstract The cellular trafficking of numerous G protein-coupled Receptors (GPCRs) is known to be regulated by Rab proteins that involves a direct protein:protein interaction between the Receptor and the GTPase. In the case of the human Prostacyclin Receptor (hIP), it undergoes agonist-induced internalization and subsequent Rab11a-dependent recyclization involving an interaction between a Rab11-binding domain (RBD) localized within its carboxyl-tail domain with Rab11a. However, the GPCR-interacting domain on Rab11a itself is unknown. Hence, we sought to identify the region within Rab11a that mediates its interaction with the RBD of the hIP. The α4 helix region of Rab11 was identified as a novel binding domain for the hIP, a site entirely distinct from the Switch I/Switch II -regions that act as specific binding domain for most other Rab and Ras-like GTPase interactants. Specifically, Glu138 within α4 helix of Rab11a appears to contact with key residues (e.g. Lys304) within the RBD of the hIP, where such contacts differ depending on the agonist-activated versus -inactive status of the hIP. Through mutational studies, supported by in silico homology modelling of the inactive and active hIP:Rab11a complexes, a mechanism is proposed to explain both the constitutive and agonist-induced binding of Rab11a to regulate intracellular trafficking of the hIP. Collectively, these studies are not only the first to identify α4 helix of Rab11a as a protein binding domain on the GTPase but also reveal novel mechanistic insights into the intracellular trafficking of the hIP, and potentially of other members of the GPCR superfamily, involving Rab11-dependent mechanisms.
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molecular analysis of the Prostacyclin Receptor s interaction with the pdz1 domain of its adaptor protein pdzk1
PLOS ONE, 2013Co-Authors: Gabriel Birrane, Eamon P. Mulvaney, Therese B Kinsella, Olivier KocherAbstract:The prostanoid Prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of Prostacyclin are mainly mediated through its activation of the Prostacyclin Receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting Prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK411IAACSLC417) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (411IAACSLC417). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.
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interaction of the human Prostacyclin Receptor with the pdz adapter protein pdzk1 role in endothelial cell migration and angiogenesis
Molecular Biology of the Cell, 2011Co-Authors: Elizebeth C. Turner, Helen M. Reid, Eamon P. Mulvaney, Therese B KinsellaAbstract:Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high-density lipoprotein (HDL) scavenger Receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human Prostacyclin Receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. Although the interaction is constitutive, it may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser-505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, small interfering RNA (siRNA)-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF responses. Considering the essential role played by Prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.
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interaction of the human Prostacyclin Receptor with rab11 characterization of a novel rab11 binding domain within α helix 8 that is regulated by palmitoylation
Journal of Biological Chemistry, 2010Co-Authors: Helen M. Reid, Elizebeth C. Turner, Eamon P. Mulvaney, Therese B KinsellaAbstract:Abstract The human Prostacyclin Receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys308 and Cys311, in addition to Cys309, that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val299 and Leu312, and proposed to be organized into an eighth α-helical domain (α-helix 8), comprising Val299–Val307, adjacent to the palmitoylated residues at Cys308–Cys311. From mutational and [3H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys311 in addition to agonist-regulated deacylation at Cys309 > Cys308 may dynamically position α-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within α-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled Receptor superfamily, of which many members are also predicted to contain an α-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for α-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.
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agonist dependent internalization and trafficking of the human Prostacyclin Receptor a direct role for rab5a gtpase
Biochimica et Biophysica Acta, 2008Co-Authors: Martina B Okeeffe, Helen M. Reid, Therese B KinsellaAbstract:The human Prostacyclin Receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5aS34N did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5aQ79L significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, Receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating Prostacyclin signaling.
