研究者総覧

輿水 崇鏡 (コシミズ タカアキ)

  • 薬理学講座(分子薬理学部門) 教授
Last Updated :2021/12/07

研究者情報

ホームページURL

J-Global ID

研究キーワード

  • 創薬科学   薬物標的探索   受容体   Opioid   Molecular therapeutics   Receptor   

研究分野

  • ライフサイエンス / 薬理学

所属学協会

  • 日本神経内分泌学会   日本心臓病学会   日本循環器学会   日本内科学会   日本薬学会   日本薬理学会   Japanese Pharmaceutical society   

研究活動情報

論文

  • Kazune Arai, Aki Kashiwazaki, Yoko Fujiwara, Hiroyoshi Tsuchiya, Nobuya Sakai, Katsushi Shibata, Taka-aki Koshimizu
    EUROPEAN JOURNAL OF PHARMACOLOGY 749 98 - 106 2015年02月 [査読有り][通常論文]
     
    A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V-1a, vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V-1a, and V-1b receptors; Gs-coupled V-2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas al adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. (C) 2015 Elsevier B.V. All rights reserved
  • Shizue Masuki, Eri Sumiyoshi, Taka-aki Koshimizu, Jinze Qian, Keiichi Higuchi, Gozoh Tsujimoto, Hiroshi Nose
    JOURNAL OF PHYSIOLOGY-LONDON 591 14 3651 - 3665 2013年07月 [査読有り][通常論文]
     
    We previously reported that cerebral activation suppressed baroreflex control of heart rate (HR) at the onset of voluntary locomotion. In the present study, we examined whether vasopressin V1a receptors in the brain were involved in these responses by using free-moving V1a receptor knockout (KO, n= 8), wild-type mice locally infused with a V1a receptor antagonist into the nucleus tractus solitarii (BLK, n= 8) and control mice (CNT, n= 8). Baroreflex sensitivity (HR/MAP) was determined from HR response (HR) to a spontaneous change in mean arterial pressure (MAP) every 4 s during the total resting period, which was similar to 8.7 h, of the 12 h measuring period in the three groups. HR/MAP was determined during the periods when the cross-correlation function (R(t)) between HR and MAP was significant (P < 0.05). Cerebral activity was determined from the power density ratio of to wave band (/) on the electroencephalogram every 4 s. Spontaneous changes in / were significantly correlated with R(t) during 62 +/- 3% of the total resting period in CNT (P < 0.05), but only 38 +/- 4% in KO and 47 +/- 2% in BLK (vs. CNT, both P < 0.001). When R(t) and HR/MAP were divided into six bins according to the level of /, both were positively correlated with / in CNT (both P < 0.001), while neither was correlated in KO or BLK (all P > 0.05). Moreover, the probability that mice started to move after an increase in / was 24 +/- 4% in KO and 24 +/- 6% in BLK, markedly lower than 61 +/- 5% in CNT (both P < 0.001), with no suppression of the baroreflex control of HR. Thus, central V1a receptors might play an important role in suppressing baroreflex control of HR during cerebral activation at the onset of voluntary locomotion.
  • Yodo Tamaki, Yoshitaka Iwanaga, Shinichiro Niizuma, Tsuneaki Kawashima, Takao Kato, Yasutaka Inuzuka, Takahiro Horie, Hanako Morooka, Toru Takase, Yasumitsu Akahashi, Kazuhiro Kobuke, Koh Ono, Tetsuo Shioi, Soren P. Sheikh, Noona Ambartsumian, Eugene Lukanidin, Taka-aki Koshimizu, Shunichi Miyazaki, Takeshi Kimura
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 57 72 - 81 2013年04月 [査読有り][通常論文]
     
    Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relationship with p53 in cardiac fibrosis. In Dahl-rat hypertensive heart disease model, S100A4 was upregulated in the hypertrophic myocardium and further activated during transition to heart failure (HF). It was expressed in various cells including fibroblasts. In in vitro cardiac fibroblasts, the knockdown of S100A4 significantly suppressed both cell proliferation and collagen expressions. S100A4 co-localized and interacted with p53 in the nucleus. S100A4 knock-down increased the expression of p53-downstream genes, p21 and mdm2, and concomitant knockdown of p53 recovered cell proliferation and collagen expression. Transverse aortic constriction (TAC) was performed in S100A4 knockout (1(0) mice, which showed a similar baseline-phenotype to wild type (WT) mice. Although there was no difference in hypertrophic response, KO mice showed reduced interstitial fibrosis, decreased myofibroblasts, and suppressed expressions of collagens and profibrotic cytokines in the left ventricle. Also, DNA microarray analysis showed that S100A4 knockout in vivo had a significant impact on expressions of p53-associated genes. These findings suggest that S100A4 modulates p53 function in fibroblasts and thereby mediates myocardial interstitial fibrosis through two distinct mechanisms; cell proliferation and collagen expression. Blockade of S100A4 may have therapeutic potential in cardiac hypertrophy and HF by attenuating cardiac fibrosis. (C) 2013 Elsevier Ltd. All rights reserved.
  • Kumazaki M, Ando H, Kakei M, Ushijima K, Taniguchi Y, Yoshida M, Yamato S, Washino S, Koshimizu TA, Fujimura A
    European journal of pharmacology 705 1-3 1 - 10 1-3 2013年04月 [査読有り][通常論文]
     
    Clinical use of arsenic trioxide (As2O3), which can induce the remission of relapsed or refractory acute promyelocytic leukemia, is often limited because of its cardiotoxicity. Symptoms of cardiotoxicity include acute cardiac conduction disturbances, such as QT prolongation. The present study was undertaken to evaluate the effects of alpha-lipoic acid (LA) on acute As2O3-induced ECG abnormalities (QTc interval prolongation) in anesthetized guinea pigs. Intravenous injection of As2O3 in guinea pigs caused QTc interval prolongation, which was significantly attenuated by co-treatment with LA (0.35, 3.5 and 35 mg/kg) in a dose-dependent manner. In isolated guinea pig cardiomyocytes, the decrease in I-Ks current induced by As2O3 (1 mu M) was rapidly restored to the basal level by the addition of LA (10 mu M). Consistent with this finding, the As2O3-induced QTc interval prolongation was also improved rapidly by post-treatment with LA in guinea pigs. Electrospray ionization time-of-flight mass spectrometry analysis detected an expected peak of arsenic-LA complex in vitro, indicating that LA and As2O3 form a new compound in vivo. In addition, pre-treatment with a chelating agent, British anti-Lewisite (BAL, 3.5 or 35 mg/kg), also attenuated the As2O3-induced QTc interval prolongation. In this study, co-and post-treatments with LA and pre-treatment with BAL ameliorated As2O3-induced acute QT prolongation in anesthetized guinea pigs. Because LA and probably BAL may bind to As2O3, these agents may exert protective effects through their chelating activity. Further studies are needed to determine whether LA is beneficial as a prophylactic or rescue agent for acute promyelocytic leukemia patients treated with As2O3. (C) 2013 Elsevier B.V. All rights reserved.
  • Hiroyoshi Tsuchiya, Junji Sato, Hidetoshi Tsuda, Yoko Fujiwara, Toshiyuki Yamada, Akio Fujimura, Taka-aki Koshimizu
    Toxicology 305 79 - 88 2013年03月 [査読有り][通常論文]
     
    The tocolytic agent ritodrine acts on the β2-adrenoceptor and is an effective treatment option for preterm labor. However, several adverse effects of ritodrine therapy, including liver damage, have been noted. To elucidate the underlying mechanisms of ritodrine-induced adverse effects, development of sensitive biomarkers of these adverse events is necessary. Here, we report the development and analysis of an animal model of ritodrine-induced liver damage. Female mice received daily ritodrine injections for 2 weeks liver samples were then collected and subjected to DNA microarray analysis. Ritodrine significantly altered the expression of genes related to steroid and lipid metabolism, as well as the metabolism of ritodrine itself. Importantly, expression of the acute-phase reactant serum amyloid A (SAA) significantly increased after ritodrine injection, with values indicating the largest fold-change. This large increase in blood SAA levels serves as a more sensitive biomarker than conventional liver enzymes, such as aspartate aminotransferase and alanine aminotransferase. The increase in SAA expression is specific to ritodrine-induced liver damage, because SAA expression was not induced by other hepatotoxic drugs such as acetaminophen, valproic acid, or metformin. Our in vitro studies showed that cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was not a primary cause of the ritodrine-induced SAA increase. Instead, SAA expression was enhanced by indirect phosphorylation of the signal transducer and activator of transcription-3 (STAT3) mediated by interleukin-6. Therefore, our study provides a method for sensitive and early detection of hepatic injury, and may thus help preclude serious liver damage due to ritodrine use in preterm labor. © 2013 Elsevier Ireland Ltd.
  • Taka-aki Koshimizu, Kazuaki Nakamura, Nobuaki Egashira, Masami Hiroyama, Hiroshi Nonoguchi, Akito Tanoue
    PHYSIOLOGICAL REVIEWS 92 4 1813 - 1864 2012年10月 [査読有り][通常論文]
     
    Koshimizu T, Nakamura K, Egashira N, Hiroyama M, Nonoguchi H, Tanoue A. Vasopressin V1a and V1b Receptors: From Molecules to Physiological Systems. Physiol Rev 92: 1813-1864, 2012; doi:10.1152/physrev.00035.2011.-The neurohypophysial hormone arginine vasopressin (AVP) is essential for a wide range of physiological functions, including water reabsorption, cardiovascular homeostasis, hormone secretion, and social behavior. These and other actions of AVP are mediated by at least three distinct receptor subtypes: V1a, V1b, and V2. Although the antidiuretic action of AVP and V2 receptor in renal distal tubules and collecting ducts is relatively well understood, recent years have seen an increasing understanding of the physiological roles of V1a and V1b receptors. The V1a receptor is originally found in the vascular smooth muscle and the V1b receptor in the anterior pituitary. Deletion of V1a or V1b receptor genes in mice revealed that the contributions of these receptors extend far beyond cardiovascular or hormone-secreting functions. Together with extensively developed pharmacological tools, genetically altered rodent models have advanced the understanding of a variety of AVP systems. Our report reviews the findings in this important field by covering a wide range of research, from the molecular physiology of V1a and V1b receptors to studies on whole animals, including gene knockout/knockdown studies.
  • Tsuchiya H, Ushijima K, Fujiwara Y, Fujimura A, Koshimizu TA
    Regulatory toxicology and pharmacology : RTP 62 3 561 - 567 3 2012年04月 [査読有り][通常論文]
     
    Adverse events in tocolytic therapy with beta 2-adrenergic agents compromise cardiovascular and non-cardiovascular functions, including blood glucose regulation and liver function. Here, we have examined the effects of the beta 2 agonist ritodrine on glucose metabolism and liver injury in mice. Under fasting conditions, ritodrine significantly increased serum insulin levels and decreased glucose concentrations. This contrasts with the beta 2 agonist-induced hyperglycemia observed in previous studies on humans and other animals. After 14 days of ritodrine treatment, the mice showed a decrease in the total mass of epididymal fat pads, whereas their body weights increased significantly. Chronic ritodrine treatment attenuated the glucose-lowering effect observed during acute administration. Ritodrine also significantly increased serum levels of liver enzymes, which returned to control levels after 14 days of treatment. Thus, ritodrine responsiveness changes between acute and chronic treatment, indicating that close monitoring of blood glucose and serum liver enzymes is necessary in patients with reduced glucose tolerance. The findings reported here of glucose homeostasis in mice provide a unique opportunity to understand refractoriness of beta 2-adrenoceptor signaling in response to beta 2 agonists during the course of treatment. (C) 2011 Elsevier Inc. All rights reserved.
  • Atsuhiko Ichimura, Akira Hirasawa, Odile Poulain-Godefroy, Amelie Bonnefond, Takafumi Hara, Loic Yengo, Ikuo Kimura, Audrey Leloire, Ning Liu, Keiko Iida, Helene Choquet, Philippe Besnard, Cecile Lecoeur, Sidonie Vivequin, Kumiko Ayukawa, Masato Takeuchi, Kentaro Ozawa, Maithe Tauber, Claudio Maffeis, Anita Morandi, Raffaella Buzzetti, Paul Elliott, Anneli Pouta, Marjo-Riitta Jarvelin, Antje Koerner, Wieland Kiess, Marie Pigeyre, Roberto Caiazzo, Wim Van Hul, Luc Van Gaal, Fritz Horber, Beverley Balkau, Claire Levy-Marchal, Konstantinos Rouskas, Anastasia Kouvatsi, Johannes Hebebrand, Anke Hinney, Andre Scherag, Francois Pattou, David Meyre, Taka-aki Koshimizu, Isabelle Wolowczuk, Gozoh Tsujimoto, Philippe Froguel
    NATURE 483 7389 350 - U149 2012年03月 [査読有り][通常論文]
     
    Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes(1-4). Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases(3,5-13). GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference(5,6,14-16). Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents.
  • Yoko Fujiwara, Akito Tanoue, Gozoh Tsujimoto, Taka-aki Koshimizu
    CLINICAL AND EXPERIMENTAL NEPHROLOGY 16 1 30 - 34 2012年02月 [査読有り][通常論文]
     
    A prompt rise in blood pressure occurs when arginine-vasopressin is administered in quantities adequate to activate vascular V1a subtype vasopressin receptors. However, it has been controversial whether the endogenous vasopressin-V1a system contributes to the maintenance of basal blood pressure during normal development and aging. Mutant mice lacking the V1a receptor gene (V1a(-/-)) show significantly lower blood pressure compared to control mice, without a notable change in heart rate. In V1a(-/-) mice, arterial baroreceptor reflexes were attenuated due to malfunctioning baroreflex center, and the mice's circulating blood volume was significantly reduced. In line with this reduction in circulating blood volume, adrenocortical hormone release was attenuated; plasma aldosterone levels were reduced and adrenocorticotropic hormone-stimulated corticosteroid release was attenuated. In addition, V1a receptor expression was detected in macula densa cells of the kidneys, which may have facilitated renin production from the juxtaglomerular cells. Deletion of the V1a receptor appears to impact the renin-angiotensin-aldosterone system. Studies on V1a(-/-) mice revealed that non-vascular V1a receptors in the central nervous system and peripheral tissues play critical roles in the maintenance of blood pressure homeostasis.
  • Yoko Fujiwara, Hiroyoshi Tsuchiya, Nobuya Sakai, Katsushi Shibata, Akio Fujimura, Taka-aki Koshimizu
    EUROPEAN JOURNAL OF PHARMACOLOGY 670 1 208 - 215 2011年11月 [査読有り][通常論文]
     
    Effective detection of potential nephrotoxicity is crucial for pre-clinical drug development. We evaluated a sensitive animal model for drug-induced kidney injury, which includes hemi-nephrectomy of mice. Although bucillamine and D-penicillamine are used for the treatment of rheumatoid arthritis in Japan, drug-related adverse effects on the kidney can limit their therapeutic utilities. When bucillamine (1000 or 2000 mg/kg/day) or D-penicillamine (2000 mg/kg/day) were orally administered to hemi-nephrectomised BALB/c mice, the urinary protein levels of bucillamine-treated mice, but not of those treated with D-penicillamine, the vehicle, or in bucillamine treated unnephrectomized mice, were significantly increased and remained high during the 4-week drug-loading period. Membranous glomerulonephropathy occasionally seen in bucillamine/D-penicillamine-treated arthritis patients was not reproduced in mice. Instead, our mouse model showed proximal tubular injury and podocyte foot process effacement in the bucillamine-treated kidneys. These two cell types are also the primary targets of the experimental Heymann membranous glomerulonephropathy. Gene expression profiling of the bucillamine-treated mice identified lipocalin 2 as a significantly up-regulated transcript together with cytochrome P450 CYP4a14, a group-specific component, and proprotein convertase subtilisin/kexin type 9. Moreover, large amounts of lipocalin 2 were detected in the urine of the bucillamine-treated mice, but not in the hemi-nephrectomised control mice. These results indicate that hemi-nephrectomy effectively promotes acute kidney injury by bucillamine, which is accompanied by up-regulation of the urinary biomarker lipocalin 2. Our mouse model with initial stage of kidney injury should be useful to analyse the pathogenesis of drug-induced glomerular and tubular injuries. (C) 2011 Elsevier B.V. All rights reserved.
  • Kumazaki M, Ando H, Sasaki A, Koshimizu TA, Ushijima K, Hosohata K, Oshima Y, Fujimura A
    Journal of pharmacological sciences 115 2 244 - 248 2 2011年02月 [査読有り][通常論文]
     
    The clinical use of arsenic trioxide (ATO) is often limited because of its adverse effects. We examined whether alpha-lipoic acid (LA) protects against the ATO-induced cardiac toxicity. In the chronic study, two of four rats suddenly died by the repeated dosing of ATO, whereas no deaths were observed in combination with LA. In the acute study, continuous ECG recording revealed that intravenous injection of ATO caused transient ST-T change, whereas pretreatment with LA abolished the ATO-induced ECG abnormality in all animals. These results suggest that LA protects against the ATO-induced acute cardiac toxicity and subsequent sudden death in rats.
  • Taka-aki Koshimizu, Yoko Fujiwara, Nobuya Sakai, Katsushi Shibata, Hiroyoshi Tsuchiya
    LIFE SCIENCES 86 11-12 455 - 460 2010年03月 [査読有り][通常論文]
     
    Aims: A noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in several malignant tumors. Its expression in neuroblastoma, however, is not known, and the regulatory mechanisms of MALAT1 gene expression have not been elucidated. The aim of this study is to clarify how MALAT1 gene expression is altered by extracellular signals in the SK-N-SH neuroblastoma cell line and to define its proximal promoter in order to study the mechanism of MALAT1 gene expression. Methods: Transcript amounts were analyzed by real-time semiquantitative polymerase chain reaction (qPCR). Genes coregulated with MALAT1 were identified by DNA microarray analysis. The structure of the MALAT1 transcript was delineated using a tiling microarray, and the 5'-end was determined using the rapid amplification of cDNA ends (RACE) method. We investigated binding of the cyclic AMP-responsive element binding (CREB) transcription factor to the MALAT1 promoter by using chromatin immunoprecipitation (ChIP) followed by tiling array analysis, and the results were confirmed using ChIP-qPCR. Key findings: The posterior pituitary hormone oxytocin increased the levels of MALAT1 and immediate early gene transcripts as early as 15 min after stimulation. Although the expression of immediate early genes returned to basal levels after 3 h, MALAT1 transcript levels peaked 6-24 h after stimulation. We identified a shorter transcriptional initiation site and found that CREB binds to the defined proximal promoter of the MALAT1 gene. Significance: The expression of the tumor marker MALAT1 ncRNA is sensitive to cell surface receptor activation by oxytocin in a neuroblastoma cell line. (C) 2010 Elsevier Inc. All rights reserved.
  • Taka-aki Koshimizu, Hiroyoshi Tsuchiya, Hidetoshi Tsuda, Yoko Fujiwara, Katsushi Shibata, Akira Hirasawa, Gozoh Tsujimoto, Akio Fujimura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 392 4 603 - 607 2010年02月 [査読有り][通常論文]
     
    Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment. (C) 2010 Elsevier Inc. All rights reserved.
  • Stanko S. Stojilkovic, Mu-Lan He, Taka-aki Koshimizu, Ales Balik, Hana Zemkova
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 314 2 184 - 191 2010年01月 [査読有り][通常論文]
     
    Adenosine 5'-triphosphate is frequently released by cells and acts as an agonist for G protein-coupled P2Y receptors and ligand-gated P2X cationic channels in numerous tissues. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors. In the pituitary gland, adenosine 5'-triphosphate is released from the endings of magnocellular hypothalamic neurons and by anterior pituitary cells through pathway(s) that are still not well characterized. This gland also expresses several members of each family of purinergic receptors. P2X and adenosine receptors are co-expressed in the somata and nerve terminals of vasopressin-releasing neurons as well as in some secretory pituitary cells. P2X receptors stimulate electrical activity and modulate InSP3-dependent calcium release from intracellular stores, whereas adenosine receptors terminate electrical activity. Calcium-mobilizing P2Y receptors are expressed in pituicytes, folliculo-stellate cells and some secretory cells of the anterior pituitary. Published by Elsevier Ireland Ltd.
  • Toshinori Aoyagi, Taka-aki Koshimizu, Akito Tanoue
    KIDNEY INTERNATIONAL 76 10 1035 - 1039 2009年11月 [査読有り][通常論文]
     
    [Arg(8)]-vasopressin (AVP) has several functions via its three distinct receptors, V1a, V1b, and V2. The V1a vasopressin receptor (V1aR) is expressed in blood vessels and involved in vascular contraction. Recently, we generated V1a receptor-deficient (V1aR(-/-)) mice and found that they were hypotensive. In addition, V1aR(-/-) mice exhibited (1) blunted AVP-induced vasopressor response, (2) impaired arterial baroreceptor reflex, (3) decreased sympathetic nerve activity, and (4) decreased blood volume, all of which could contribute to the observed hypotension. In relation to their decreased blood volume, V1aR(-/-) mice had decreased plasma aldosterone levels, which could result not only from decreased activity of the renin-angiotensin system (RAS), but also from impaired AVP-stimulated aldosterone release in the adrenal glands. V1aR was found to specifically co-express at the macula densa cells with cyclooxygenase (COX)-2 and with neuronal nitric oxide synthase, which produces potent stimulators of renin, PGE2, and NO. The expression levels of renin, COX-2, and nNOS were significantly decreased in V1aR(-/-) mice, which led to the suppression of RAS activity and consequent decreases in aldosterone and blood volume. Furthermore, V1aR is also expressed in collecting duct cells and involved in regulating water reabsorption by affecting V2/aquaporin 2 function. Thus, AVP regulates blood pressure and volume via V1aR by exerting diverse functions in vivo. Kidney International (2009) 76, 1035-1039; doi: 10.1038/ki.2009.319; published online 19 August 2009
  • Yoshiyuki Kojima, Shoichi Sasaki, Nobuyuki Oda, Taka-Aki Koshimizu, Yutaro Hayashi, Mamoru Kiniwa, Gozoh Tsujimoto, Kenjiro Kohri
    PROSTATE 69 14 1521 - 1528 2009年10月 [査読有り][通常論文]
     
    BACKGROUND. Recently, alpha(1)-adrenoceptors (alpha(1)-ARs) have been reported to play a prominent role in the growth of a variety of cells; however, little is known about prostate growth and subtype-specific effects on cell proliferation. We examined the role of alpha(1d)-AR in prostate growth and the effect of subtype-selective alpha(1)-AR antagonist, naftopidil, which has relatively higher affinity for alpha(1d)-AR, on prostate growth in vitro and in vivo. METHODS. First, we examined the effect of naftopidil on the cell proliferation of PrEC, PrSC, and PrSMC using WST-1 assay. Second, we performed real-time RT-PCR to quantify each alpha(1)-AR subtype mRNA expression level in a benign prostate hyperplasia (BPH) model rat, which was recently established to pathologically resemble human BPH patients. In addition, naftopidil was given to this model orally for 21 days and the proliferative and apoptotic indexes measured. Third, 18 BPH patients were administered naftopidil for 12 weeks and the proliferative and apoptotic indexes were compared before and after naftopidil administration. RESULTS. Naftopidil significantly inhibited cell proliferation dose-dependently in all cell lines that expressed alpha(1d)-AR mRNA. The expression level of alpha(1d)-AR during the growth process of the prostate in the BPH model rat was significantly higher than that in the normal prostate (P < 0.001). Naftopidil administration inhibited cell proliferation without apoptosis in the BPH model rat and BPH patients. CONCLUSIONS. alpha(1d)-AR may play an important role in the regulation of cellular proliferation in the prostate, and alpha(1d)-AR blockage by naftopidil may not only improve lower urinary tract symptoms but also inhibit prostate growth in BPH patients. Prostate 69: 1521-1528, 2009. (C) 2009 Wiley-Liss, Inc.
  • Takafumi Hara, Akira Hirasawa, Qi Sun, Keiko Sadakane, Chisato Itsubo, Tomoyo Iga, Tetsuya Adachi, Taka-aki Koshimizu, Toshihiro Hashimoto, Yoshinori Asakawa, Gozoh Tsujimoto
    NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY 380 3 247 - 255 2009年09月 [査読有り][通常論文]
     
    GPR120 and GPR40 are G-protein-coupled receptors whose endogenous ligands are medium- and long-chain free fatty acids, and they are thought to play an important physiological role in insulin release. Despite recent progress in understanding their roles, much still remains unclear about their pharmacology, and few specific ligands for GPR120 and GPR40 besides medium- to long-chain fatty acids have been reported so far. To identify new selective ligands for these receptors, more than 80 natural compounds were screened, together with a reference compound MEDICA16, which is known to activate GPR40, by monitoring the extracellular regulated kinase (ERK) and [Ca2+](i) responses in inducible and stable expression cell lines for GPR40 and GPR120, respectively. MEDICA16 selectively activated [Ca2+](i) response in GPR40-expressing cells but not in GPR120-expressing cells. Among the natural compounds tested, grifolin derivatives, grifolic acid and grifolic acid methyl ether, promoted ERK and [Ca2+](i) responses in GPR120-expressing cells, but not in GPR40-expressing cells, and inhibited the alpha-linolenic acid (LA)-induced ERK and [Ca2+](i) responses in GPR120-expressing cells. Interestingly, in accordance with the pharmacological profiles of these compounds, similar profiles of glucagon-like peptide-1 secretion were seen for mouse enteroendocrine cell line, STC-1 cells, which express GPR120 endogenously. Taken together, these studies identified a selective GPR40 agonist and several GPR120 partial agonists. These compounds would be useful probes to further investigate the physiological and pharmacological functions of GPR40 and GPR120.
  • Kentarou Ushijima, Shu-ichi Tsuruoka, Hidetoshi Tsuda, Gohki Hasegawa, Yuri Obi, Tae Kaneda, Masaki Takahashi, Tomohiro Maekawa, Tomohiro Sasaki, Taka-aki Koshimizu, Akio Fujimura
    BRITISH JOURNAL OF CLINICAL PHARMACOLOGY 68 2 194 - 200 2009年08月 [査読有り][通常論文]
     
    center dot Cranberry juice has a significant inhibitory effect on CYP2C9 activity in vitro, whereas it shows a minimal effect on the pharmacokinetics and pharmacodynamics of warfarin, a CYP2C9 substrate in vivo. center dot Information regarding the interaction between cranberry juice and other medications metabolized by CYP2C9 is limited. WHAT THIS STUDY ADDS center dot Cranberry juice suppressed the metabolism of diclofenac, another CYP2C9 substrate, by human liver microsomes. center dot Pharmacokinetic parameters of diclofenac were not altered by cranberry juice consumption in human subjects. AIM To investigate a potential interaction between cranberry juice and diclofenac, a substrate of CYP2C9. METHODS The inhibitory effect of cranberry juice on diclofenac metabolism was determined using human liver microsome assay. Subsequently, we performed a clinical trial in healthy human subjects to determine whether the repeated consumption of cranberry juice changed the diclofenac pharmacokinetics. RESULTS Cranberry juice significantly suppressed diclofenac metabolism by human liver microsomes. On the other hand, repeated consumption of cranberry juice did not influence the diclofenac pharmacokinetics in human subjects. CONCLUSIONS Cranberry juice inhibited diclofenac metabolism by human liver microsomes, but not in human subjects. Based on the present and previous findings, we think that although cranberry juice inhibits CYP2C9 activity in vitro, it does not change the pharmacokinetics of medications metabolized by CYP2C9 in clinical situations.
  • Takafumi Hara, Akira Hirasawa, Qi Sun, Taka-Aki Koshimizu, Chisato Itsubo, Keiko Sadakane, Takeo Awaji, Gozoh Tsujimoto
    MOLECULAR PHARMACOLOGY 75 1 85 - 91 2009年01月 [査読有り][通常論文]
     
    GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of similar to 3 mu M. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium-to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca2+](i) responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca2+](i) response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.
  • Taka-aki Koshimizu, Gozoh Tsujimoto
    JOURNAL OF PHARMACOLOGICAL SCIENCES 109 1 33 - 37 2009年01月 [査読有り][通常論文]
     
    Arginine vasopressin (AVP) activates three vasopressin receptors and it also has an agonistic activity on the oxytocin receptor. For ail accurate description of the target receptor subtype(s) responsible for complex AVP and oxytocin actions, a careful evaluation of ligand specificity and receptor activities are required, particularly when these receptors are co-expressed in the central nervous system. Previous studies suggest that AVP plays a regulatory role in nociception through the direct activation of central vasopressin receptors and also through the receptors that reside in the peripheral tissues. Genetically altered rodent models, including the AVP-deficient Mutant Brattleboro rat and gene knockout mice lacking an endogenous opioid peptide, advanced the understanding of the interactions between the pain perception process and AVP system. This report reviews previous findings in this important field and reconciles them with the findings of recent gene knockout/knockdown Studies.
  • Toshiki Tanaka, Susumu Katsuma, Tetsuya Adachi, Taka-Aki Koshimizu, Akira Hirasawa, Gozoh Tsujimoto
    NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY 377 4-6 523 - 527 2008年06月 [査読有り][通常論文]
     
    The ingestion of fat induces secretion of the gut peptide hormone cholecystokinin (CCK); however, the mechanism responsible for lipid-induced CCK release remains unknown. Recently, a group of free fatty acid (FFA) receptors, which includes the long-chain FFA receptors GPR120 and GPR40, has been identified. In this study, we examined whether these FFA receptors mediate lipid-induced CCK release in the mouse. We first observed that intra-gastric administration of long-chain FFAs increased plasma CCK levels. Using mouse enteroendocrine STC-1 cells as a model system, we further studied the mechanism of this FFA-induced CCK secretion. Long-chain FFAs promoted CCK secretion from STC-1 cells, which was abolished either by removal of extracellular Ca(2+)or by the L-type Ca(2+)channel blocker nicardipine. Furthermore, this FFA-induced CCK secretion was specifically inhibited by transfection of GPR120-specific, but not GPR40-specific, short hairpin RNA. These results indicate that long-chain FFAs induce CCK secretion through GPR120-coupled Ca(2+)signaling.
  • Toshiki Tanaka, Takeaki Yano, Tetsuya Adachi, Taka-aki Koshimizu, Akira Hirasawa, Gozoh Tsujimoto
    NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY 377 4-6 515 - 522 2008年06月 [査読有り][通常論文]
     
    We have recently found that GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain free fatty acids (FFAs) and that GPR120 stimulation promotes the secretion of glucagons-like peptide-1 (GLP-1) in the mouse (Hirasawa et al., Nat Med 11:90-94, 2005). In this study, we cloned and characterized rat GPR120 (rGPR120), and then we examined the in vivo effects of acute and long-term administration of the natural ligand alpha-linolenic acid (alpha-LA). The cloned rat GPR120 complimentary DNA had a seven transmembrane structure, and a homology comparison of human, mouse, and rat GPR120 revealed that the rat GPR120 (rGPR120) shares 85 and 98% sequence identity with the human and mouse GPR120 proteins, respectively. The tissue distribution and ligand properties of rGPR120 were similar to those of mouse GPR120. In addition, alpha-LA provoked a transient increase in [Ca2+](i) levels in HEK293 cells expressing rGPR120. Furthermore, administration of alpha-LA to the rat increased plasma GLP-1 levels, and long-term administration of alpha-LA led to proliferation of pancreatic beta cells, probably because of the enhanced GLP-1 secretion. These results show that rat GPR120 is a G-protein-coupled receptor whose ligand is a free fatty acid, and it may play an important role in the FFA-associated physiological responses.
  • Akira Hirasawa, Chisato Itsubo, Keiko Sadakane, Takafumi Hara, Shingo Shinagawa, Hanako Koga, Hiroshi Nose, Taka-aki Koshimizu, Gozoh Tsujimoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 365 1 22 - 28 2008年01月 [査読有り][通常論文]
     
    GPR40 is G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs), and it has been implicated to play an important role in FFA-mediated enhancement of glucose-stimulated insulin release. We have developed a monoclonal antibody against the extracellular domain of GPR40. Specificity of the antibody was demonstrated by immunoprecipitation and cell surface staining using GPR40-transfected cells. GPR40 immunoreactivity was highly abundant in mouse pancreatic P-cells and splenocytes, THP-1 cells, and human peripheral blood mononuclear cells. The anti-GPR40 monoclonal antibody should prove valuable for further studying the function of this nutrient sensing receptor. (c) 2007 Elsevier Inc. All rights reserved.
  • Taka-aki Koshimizu, Akito Tanoue, Gozoh Tsujimoto
    BIOCHEMICAL PHARMACOLOGY 73 8 1107 - 1112 2007年04月 [査読有り][通常論文]
     
    alpha 1-Adrenergic receptors (alpha 1-ARs) modulate a large number of physiological functions in cardiovascular and noncardiovascular tissues. Because individual members of the alpha 1-AR family (alpha 1A-, alpha 1B-, and alpha 1D-ARs) have overlapping expression profiles in most tissues, elucidation of the precise physiological roles of individual alpha 1-AR subtypes remains a challenging task. To alleviate this constraint, a gene targeting approach has been employed to generate mutant mice lacking one or two alpha 1-AR genes. Recent studies on these mutant mouse strains are discussed in this article, with an emphasis on the role of alpha 1-AR in the central nervous system and lower urinary tracts. These are two major tissues of particular interest for the development of new therapeutic strategies targeted to the alpha 1-ARs. By combining gene targeting techniques with pharmacological tools, the specific roles of alpha 1-AR subtypes could be delineated. (c) 2006 Elsevier Inc. All rights reserved.
  • Akira Myoumoto, Kaoru Nakatani, Taka-aki Koshimizu, Hiroshi Matsubara, Souichi Adachi, Gozoh Tsujimoto
    JOURNAL OF HUMAN GENETICS 52 4 328 - 333 2007年04月 [査読有り][通常論文]
     
    The ability of glucocorticoids (GC) to efficiently kill lymphoid cells has led to their inclusion in essentially all chemotherapy procedures used to treat acute lymphoblastic leukemia (ALL). GC sensitivity is an important prognostic factor in ALL treatment, and it is used to classify patients into risk groups. Clinical assessment for GC sensitivity is very time-consuming, however. We have recently found that granzyme A (GZMA) mediates GC-induced apoptosis in ALL-derived cell line 697. In this study we examined the correlation between GC sensitivity and GC-induced GZMA expression by using seven established cell lines derived from ALL patients. The apoptosis assay showed four cell lines were GC-sensitive and three were GC-resistant. GC treatment markedly enhanced GZMA expression in GC-sensitive cell lines only, and not in GC-resistant cell lines. GC-induced GZMA expression also correlated well with the amount of GC-induced apoptosis. GC-induced GZMA expression could thus be a useful early biomarker for "personalized" ALL therapy.
  • Taka-aki Koshimizu, Gozoh Tsujimoto
    JOURNAL OF PHARMACOLOGICAL SCIENCES 101 4 261 - 266 2006年08月 [査読有り][通常論文]
     
    P2X receptors belong to a unique family of ligand-gated channels in terms of their molecular architecture, in which the channel subunit has two transmembrane alpha-helixes with a large extracellular loop keeping amino- and carboxy-termini in the cytoplasm. Posttranscriptional modifications of P2X receptors could diversify cellular responsiveness induced by extracellular ATP in anterior pituitary cells and other cell types. Recently, we found a spliced variant P2X(2) transcript, termed P2X(2e), in mouse pituitary. The P2X(2e) has a shorter cytoplasmic carboxy-terminal tail than those of full-length P2X(2a) or splice variant P2X(2b) subunits. Although ATP induced rapid responses in all homomeric P2X(2) channels, the current induced by P2X2, declined significantly faster than those by P2X(2a) or P2X(2b). In this article, we summarize functional alterations of P2X(2) receptors after splicing reactions. Combinations of different P2X(2) subunit carboxy-termini to form homomeric and heteromeric channels could be a molecular mechanism for promoting functional diversities of ATP-induced cellular signals.
  • T Koshimizu, K Kretschmannova, ML He, S Ueno, A Tanoue, N Yanagihara, SS Stojilkovic, G Tsujimoto
    MOLECULAR PHARMACOLOGY 69 5 1588 - 1598 2006年05月 [査読有り][通常論文]
     
    Purinergic P2X receptors are ion-conducting channels composed of three subunits, each having two transmembrane domains and intracellular amino (N) and carboxyl (C) termini. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post-transcriptional modifications on receptor architecture and function remains poorly understood. In this study, we focused on mouse pituitary P2X(2) receptors. In this tissue, progressive splicing of the P2X(2a) C terminus generated two functional subunit variants, P2X(2b) and P2X(2e), which exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the intersubunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C termini of P2X2a receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C termini and between C- and N termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X(2b) or P2X(2e) subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X(2b) receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in the cytoplasmic intersubunit interactions and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.
  • T Koshimizu, Y Nasa, A Tanoue, R Oikawa, Y Kawahara, Y Kiyono, T Adachi, T Tanaka, T Kuwaki, T Mori, S Takeo, H Okamura, G Tsujimoto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 20 7807 - 7812 2006年05月 [査読有り][通常論文]
     
    Arginine-vasopressin (AVP) is a hormone that is essential for both osmotic and cardiovascular homeostasis, and exerts important physiological regulation through three distinct receptors, V1a, V1b, and V2. Although AVP is used clinically as a potent vasoconstrictor (V1a receptor-mediated) in patients with circulatory shock, the physiological role of vasopressin V1a receptors in blood pressure (BP) homeostasis is ill-defined. In this study, we investigated the functional roles of the V1a receptor in cardiovascular homeostasis using gene targeting. The basal BP of conscious mutant mice lacking the V1a receptor gene (V1a(-/-)) was significantly (P < 0.001) lower compared to the wild-type mice (V1a(+/+)) without a notable change in heart rate. There was no significant alteration in cardiac functions as assessed by echocardiogram in the mutant mice. AVP-induced vasopressor responses were abolished in the mutant mice; rather, AVP caused a decrease in BP, which occurred in part through V2 receptor-mediated release of nitric oxide from the vascular endothelium. Arterial baroreceptor reflexes were markedly impaired in mutant mice, consistent with a loss of V1a receptors in the central area of baroreflex control. Notably, mutant mice showed a significant 9% reduction in circulating blood volume. Furthermore, mutant mice had normal plasma AVP levels and a normal AVP secretory response, but had significantly lower adrenocortical responsiveness to adrenocorticotropic hormone. Taken together, these results indicate that the V1a receptor plays an important role in normal resting arterial BP regulation mainly by its regulation of circulating blood volume and baroreflex sensitivity.
  • T Adachi, T Tanaka, K Takemoto, TA Koshimizu, A Hirasawa, G Tsujimoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 340 1 332 - 337 2006年02月 [査読有り][通常論文]
     
    We examined whether free fatty acids (FFAs) promote glucagon-like peptide-1 (GLP-1) secretion when administered into the intestinal tract. We found that an unsaturated long-chain FFA, alpha-linolenic acid (alpha-LA), resulted in increased plasma GLP-1 and insulin levels when administered into the colon. Such stimulatory effects were not apparent with either vehicle or a saturated middle-chain FFA, octanoic acid (OA). Concomitant with GLP-1 secretion, the administration of alpha-LA, but not vehicle or OA, also resulted in a significant increase in the population of pERK positive cells within the GLP-1 positive cells of the colonic mucosa. Moreover, colonic administration of alpha-LA into normal C3H/He mice caused a reduction in plasma glucose levels, as well as in type 2 diabetic model NSY mice. Our results indicate that the in vivo colonic administration of alpha-LA promotes secretion of incretin GLP-1 by activating the ERK pathway in L-cells and thereby enhances the secretion of insulin. (c) 2005 Elsevier Inc. All rights reserved.
  • C Hosoda, A Tanoue, M Shibano, Y Tanaka, M Hiroyama, T Koshimizu, S Cotecchia, T Kitamura, G Tsujimoto, K Koike
    BRITISH JOURNAL OF PHARMACOLOGY 146 3 456 - 466 2005年10月 [査読有り][通常論文]
     
    1 We examined the contribution of each alpha(1)-adrenoceptor (AR) subtype in noradrenaline (NAd)evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration-response curves (CRCs) for NAd in the thoracic aortas of wild-type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD(2) value in rank order is as follows: WT mice (8.21) > alpha(1B)-adrenoceptor knockout (alpha(1B)- KO) (7.77) > alpha(1D)-AR knockout (alpha(1D)-KO) (6.44) > alpha(1B)-and alpha(1D)-AR double knockout (alpha(1BD)-KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and alpha(1B)-KO mice (7.12) or alpha(1D)-KO (6.19) and alpha(1BD)-KO (6.29) mice. However, the CRC maximum responses to NAd in alpha(1D)-and alpha(1BD)-KO mice were significantly lower than those in WT and alpha(1B)-KO mice. 2 Except in the thoracic aortas of alpha(1BD)-KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the alpha(1D)-AR gene. In the thoracic aorta, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.25 and 8.46, respectively, and in alpha(1B)-KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.34 and 7.47, respectively, and in alpha(1B)-KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and alpha(1B)-KO mice, which showed low affinities to BMY7378. 3 We performed a quantitative analysis of the mRNA expression of each alpha(1)-AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each alpha(1)-AR subtype in mediating vascular contraction. 4 The rank order of each alpha(1)-AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, alpha(1D)-AR >alpha(1B)-AR >alpha(1A)-AR in the thoracic aorta and alpha(1D)-AR >alpha(1A)-AR >alpha(1B)-AR in the mesenteric artery. No dramatic compensatory change of alpha(1)-AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.
  • M Yamada, S Katsuma, T Adachi, A Hirasawa, S Shiojima, T Kadowaki, Y Okuno, TA Koshimizu, S Fujii, Y Sekiya, Y Miyamoto, M Tamura, W Yumura, H Nihei, M Kobayashi, G Tsujimoto
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 21 7736 - 7741 2005年05月 [査読有り][通常論文]
     
    Glomerulonephritis (GN) is a progressive inflammation that may be caused by a variety of underlying disorders. It is the primary cause of chronic renal failure and end-stage renal disease, which require dialysis and transplantation worldwide. Immunosuppressive therapy has been used to treat GN clinically, but this treatment has had insufficient therapeutic effects. Here, we show that protein kinase CK2 is a key molecule in the progression of GN. cDNA microarray analysis identified CK2 alpha, the catalytic subunit of CK2, as a GN-related, differentially expressed gene. Overexpression of CK2 alpha was noted in the proliferative glomerular lesions in rat GN models and in renal biopsy specimens from lupus nephritis or IgA nephropathy patients. Administration of either antisense oligodeoxynucleotide against CK2 alpha or low molecular weight CK2-specific inhibitors effectively prevented the progression of renal pathology in the rat GN models. The resolution of GN by CK2 inhibition may result from its suppression of extracellular signal-regulated kinase-mediated cell proliferation, and its suppression of inflammatory and fibrotic processes that are enhanced in GN. Our results show that CK2 plays a critical role in the progression of immunogenic renal injury, and therefore, CK2 is a potential target for GN therapy.
  • C Hosoda, T Koshimizu, A Tanoue, Y Nasa, R Oikawa, T Tomabechi, S Fukuda, H Shinoura, S Oshikawa, S Takeo, T Kitamura, S Cotecchia, G Tsujimoto
    MOLECULAR PHARMACOLOGY 67 3 912 - 922 2005年03月 [査読有り][通常論文]
     
    To study the functional role of individual alpha(1)-adrenergic (AR) subtypes in blood pressure (BP) regulation, we used mice lacking the alpha(1B)-AR and/or alpha(1D)-AR with the same genetic background and further studied their hemodynamic and vasoconstrictive responses. Both the alpha(1D)-AR knockout and alpha(1B)-/- alpha(1D)-AR double knockout mice, but not the alpha(1B)-AR knockout mice, had significantly (p < 0.05) lower levels of basal systolic and mean arterial BP than wild-type mice in nonanesthetized condition, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. All mutants showed a significantly (p < 0.05) reduced catecholamine-induced pressor and vasoconstriction responses. It is noteworthy that the infusion of norepinephrine did not elicit any pressor response at all in alpha(1B)-/alpha(1D)-AR double knockout mice. In an attempt to further examine alpha(1)-AR subtype, which is involved in the genesis or maintenance of hypertension, BP after salt loading was monitored by tail-cuff readings and confirmed at the endpoint by direct intra-arterial recording. After salt loading, alpha(1B)-AR knockout mice developed a comparable level of hypertension to wild-type mice, whereas mice lacking alpha(1D)-AR had significantly (p < 0.05) attenuated BP and lower levels of circulating catecholamines. Our data indicated that alpha(1B)- and alpha(1D)-AR subtypes participate cooperatively in BP regulation; however, the deletion of the functional alpha(1D)-AR, not alpha(1B)-AR, leads to an antihypertensive effect. The study shows differential contributions of alpha(1B)- and alpha(1D)-ARs in BP regulation.
  • SS Stojilkovic, M Tomic, HE Mu-Lan, ZH Yan, TA Koshimizu, H Zemkova
    BIOPHYSICS FROM MOLECULES TO BRAIN: IN MEMORY OF RADOSLAV K. ANDJUS 1048 116 - 130 2005年 [査読有り][通常論文]
     
    The P2X receptors (P2XRs) are a family of ATP-gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. MRS are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X(1)-P2X(7). All subunits possess intracellular Nand C-termini, two transmembrane domains, and a relatively large extracellular ligand-binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of, extracellular ATP leads to deactivation and resensitization of receptors. Activated P2XRs generate inward currents caused by Na+ and Ca2+ influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage-gated calcium entry in excitable cells. No crystal structures are available for MRS and these receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of MRS, including heteromerization, chimerization, mutagenesis, and biochemical studies.
  • T Koshimizu, G Tsujimoto, A Hirasawa, Y Kitagawa, A Tanoue
    CARDIOVASCULAR RESEARCH 63 4 662 - 672 2004年09月 [査読有り][通常論文]
     
    Background: Increasing evidence from clinical trials indicates that carvedilol, an antagonist of alpha1- and beta-adrenergic receptors (ARs), provides an effective treatment for chronic heart failure, whereas nonselective alpha1-AR blockade has an adverse outcome in this disease. It is, however, not clear whether carvedilol exhibits a subtype-dependent impact on three distinct alpha1-adrenergic receptors (alpha1-ARs). Methods and results: We determined binding properties of human ARs for carvedilol using HEK293 human embryonic kidney cells expressing a single AR subtype. Our results showed that the affinities of alpha1D-AR and alpha1B-AR for carvedilol are higher than that of the beta1-AR subtype, a major target in heart failure treatment. The affinity rank order and pKi values of ARs for carvedilol were as follows: alpha1D-AR (8.9) > alpha1B-AR (8.6) > 1-AR (8.4) > beta2-AR (8.0) > alpha1A-AR (7.9)much greater thanalpha2C-AR (5.9) > alpha2B-AR (5.5) > alpha2A-AR (5.3). Furthermore, temporal kinetics of intracellular calcium signaling mediated via alpha1D- and alpha1B-ARs, but not via alpha1A-AR (P < 0.01), showed oscillatory patterns with frequencies ranging from 0.3 to 3 per minute in human smooth muscle and HEK293 cells, which were inhibited by the therapeutic concentrations of carvedilol (10 nM) in a subtype-dependent manner. When oscillatory alpha1B-AR and non-oscillatory alpha1A-AR were co-expressed and heteromer receptors were detected with bioluminescence resonance energy transfer and co-immunoprecipitation, carvedilol suppressed only oscillatory component of global cytosolic free calcium change. Conclusions: These results indicate that in addition to beta-ARs, receptor inhibition by carvedilol is directed to alpha1-ARs, preferably to alpha1D- and alpha1B-AR-mediated signaling events, including intracellular calcium oscillations in vascular smooth muscle. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
  • H Zemkova, ML He, T Koshimizu, SS Stojilkovic
    JOURNAL OF NEUROSCIENCE 24 31 6968 - 6978 2004年08月 [査読有り][通常論文]
     
    The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X(2a)R, P2X(2b)R, and P2X(3)R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X(3)R instead of the I66-H192 or I66-Y310 sequences of P2X(2a)R and P2X(2b)R. Chimeric P2X(2a)/V60-F301X(3)R and P2X(2b)/V60-F301X(3)R inherited the P2X(3)R ligand-selective profile, whereas the potency of agonists for P2X(2a)/V60-R180X(3)R was in between those observed at parental receptors. Furthermore, P2X(2a)/V60F301X(3)R and P2X(2a)/V60-R180X(3)R desensitized in a P2X(2a)R-specific manner, and P2X(2b)/V60-F301X(3)R desensitized with rates comparable with those of P2X(2b)R. In striking contrast to parental receptors, the rates of decay in P2X(2a)/V60-F301X(3)R and P2X(2b)/V60-F301X(3)R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X(2a)/V60-R180X(3)R-expressing cells. However, the recovery from desensitization of P2X(2a)/V60-F301X(3)R and P2X(2a)/V60-R180X(3)R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.
  • S Oshikawa, A Tanoue, T Koshimizu, Y Kitagawa, G Tsujimoto
    MOLECULAR PHARMACOLOGY 65 3 623 - 629 2004年03月 [査読有り][通常論文]
     
    Vasopressin receptor subtype(s) responsible for stimulation of insulin release from pancreatic beta cells were investigated by using subtype-selective antagonists and mice that were genetically lacking either V1a or V1b receptors. Arginine vasopressin (AVP) increased insulin release from isolated mouse islet cells in a concentration-dependent manner, with a submaximal response at 100 nM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis detected V1b and oxytocin, but not V1a or V2, receptor transcripts in mouse islet cells. We characterized the recently synthesized vasopressin receptor subtype antagonists (2S)1-[(2R 3S)-(5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-difydro-1H-indole-2-carbonyl)-pyrrolidine-2-carboxamide] (SR49059), 1-{1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinolinone (OPC-21268), and (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl) sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415) using human embryonic kidney 293 cells stably expressing the three cloned mouse vasopressin receptors (V1a, V1b, and V2). A radioligand binding study showed that SR49059 and OPC-21268 potently inhibited [H-3] AVP binding to the cloned mouse V1a receptor, with K-i values of 27 and 510 nM, respectively, whereas SSR149415 potently inhibited [H-3] AVP binding to the cloned mouse V1b receptor with a K-i value of 110 nM. The inhibitory effects of vasopressin antagonists on AVP-induced insulin release correlate well with the rank order of potency to inhibit [H-3] AVP binding to the V1b receptor; pancreatic islet cells were significantly inhibited by SSR149415 but not by SR49059 or OPC-21268. Furthermore, the AVP effect on insulin release was entirely lost in mice lacking the V1b receptor but was preserved in mice lacking the V1a receptor. Our study, which combined pharmacological and knockout approaches, clearly demonstrates that vasopressin-stimulated insulin release from islet cells is mediated via V1b receptors.
  • A Tanoue, S Ito, K Honda, S Oshikawa, Y Kitagawa, T Koshimizu, T Mori, G Tsujimoto
    JOURNAL OF CLINICAL INVESTIGATION 113 2 302 - 309 2004年01月 [査読有り][通常論文]
     
    The neurohypophyseal peptide [Arg(8)]-vasopressin (AVP) exerts major physiological actions through three distinct receptor isoforms designated V1a, V1b, and V2. Among these three subtypes, the vasopressin V1b receptor is specifically expressed in pituitary corticotrophs and mediates the stimulatory effect of vasopressin on ACTH release. To investigate the functional roles of V1b receptor subtypes in vivo, gene targeting was used to create a mouse model lacking the V1b receptor gene (V1bR(-/-)). Under resting conditions, circulating concentrations of ACTH and corticosterone were lower in V1bR(-/-) mice compared with WT mice (V1bR(+/+)). The normal increase in circulating ACTH levels in response to exogenous administration of AVP was impaired in V1bR(-/-) mice, while corticotropin-releasing hormone-stimulated ACTH release in the V1bR(-/-) mice was not significantly different from that in the V1bR(+/+) mice. AVP-induced ACTH release from primary cultured pituitary cells in V1bR(-/-) mice was also blunted. Furthermore, the increase in ACTH after a forced swim stress was significantly suppressed in V1bR(-/-) mice. Our results clearly demonstrate that the V1b receptor plays a crucial role in regulating hypothalamic-pituitary-adrenal axis activity. It does this by maintaining ACTH and corticosterone levels, not only under stress but also under basal conditions.
  • ML He, H Zemkova, T Koshimizu, M Tomic, SS Stojilkovic
    AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 285 2 C467 - C479 2003年08月 [査読有り][通常論文]
     
    Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X(3)R > P2X(2b) + X4R > P2X(2b)R > P2X(2a) + X4R > P2X(4)R > P2X(2a)R > P2X(7)R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.
  • T Koshimizu, A Tanoue, A Hirasawa, J Yamauchi, G Tsujimoto
    PHARMACOLOGY & THERAPEUTICS 98 2 235 - 244 2003年05月 [査読有り][通常論文]
     
    alpha(1)-Adrenergic receptors (ARs) mediate some of the main actions of the natural catecholamines, adrenaline and noradrenaline. They participate in many essential physiological processes, such as sympathetic neurotransmission, modulation of hepatic metabolism, control of vascular tone, cardiac contraction, and the regulation of smooth muscle activity in the genitourinary system. Here, we review recent progress on subtype-specific subcellular localization, participation in signaling cascades, and the pivotal function of alpha(1)-ARs, as delineated through studies on genetically engineered animals. Together, these findings will provide new insights into the physiological and pathophysiological roles of the alpha(1)-ARs. (C) 2003 Elsevier Science Inc. All rights reserved.
  • A Tanoue, T Koshimizu, K Shibata, Y Nasa, S Takeo, G Tsujimoto
    TRENDS IN ENDOCRINOLOGY AND METABOLISM 14 3 107 - 113 2003年04月 [査読有り][通常論文]
     
    alpha(1)-Adrenoceptors (ARs) mediate some of the main actions of the natural catecholamines, epinephrine and norepinephrine, and have a crucial role in the regulation of arterial blood pressure. Since alpha(1)-AR was subdivided into three subtypes (alpha(1A)-AR, alpha(1B)-AR and (alpha(1D)-AR), the search has been on to discover subtype-specific physiological roles and to develop subtype-selective agonists and antagonists. Recently, several strains of genetically engineered mice have become available. Studies with these mice have provided several clues to help elucidate subtype-specific physiological functions; for instance, alpha(1A)-AR and alpha(1D)-AR subtypes play an important role in the regulation of blood pressure, suggesting that subtype-selective antagonists might be desirable anti hypertensive agents. The ability to study subtype-specific functions in different mouse strains by altering the same alpha(1)-AR in different ways strengthens the conclusions drawn from pharmacological studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(1)-AR subtypes.
  • T Koshimizu, S Ueno, A Tanoue, N Yanagihara, SS Stojilkovic, G Tsujimoto
    JOURNAL OF BIOLOGICAL CHEMISTRY 277 49 46891 - 46899 2002年12月 [査読有り][通常論文]
     
    P2X purinergic receptors (P2XRs) differ among themselves with respect to their ligand preferences and channel kinetics during activation, desensitization, and recovery. However, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. Here we show that homomeric receptors having the extracellular domain of the P2X(3) subunit in the P2X(2a)-based backbone (P2X(2a)/X(3)ex) mimicked two intrinsic functions of P2X(3)R, sensitivity to alphabeta-methylene ATP and ecto-ATPase-dependent recovery from endogenous desensitization; these two functions were localized to the N- and C-terminal halves of the P2X(3) extracellular loop, respectively. The chimeric P2X(2a)R/X(3)ex receptors also desensitized with accelerated rates compared with native P2X(2a)R, and the introduction of P2X(2) C-terminal splicing into the chimeric subunit (P2X(2b)/X(3)ex) further increased the rate of desensitization. Physical and functional heteromerization of native P2X(2a) and P2X(2b) subunits was also demonstrated. In heteromeric receptors, the ectodomain of P2X(3) was a structural determinant for ligand selectivity and recovery from desensitization, and the C terminus of P2X(2) was an important factor for the desensitization rate. Furthermore, [gamma-P-32]8-azido ATP, a photoreactive agonist, was effectively cross-linked to P2X(3) subunit in homomeric receptors but not in heteromeric P2X(2) + P2X(3)Rs. These results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.
  • ML He, TA Koshimizu, M Tomic, SS Stojilkovic
    MOLECULAR PHARMACOLOGY 62 5 1187 - 1197 2002年11月 [査読有り][通常論文]
     
    The wild-type P2X(2) purinergic receptor (P2X(2a)R) and its splice form lacking the intracellular Val(370)-Gln(438) C-terminal sequence (P2X(2b)R) respond to ATP stimulation with comparable EC50 values and peak current/calcium responses but desensitize in a receptor-specific manner. P2X(2a)R desensitizes slowly and P2X(2b)R desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X(2a)R and P2X(2b)R. Both receptors showed similar EC50 values (estimated from the peak calcium response) and IC50 values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP less than or equal to ATP less than or equal to ATPgammaS < BzATP << αβ-meATP, and the IC50 values for agonists were shifted to the right compared with their EC50 values. Furthermore, the ATP-induced receptor-subtype specific pattern of desensitization was mimicked by high- but not by low-efficacy agonists, suggesting a ligand-specific desensitization pattern. To test this hypothesis, we generated chimeric P2X(2a)R and P2X(2b)R containing the Val(60)-Phe(301) ectodomain sequence of P2X(3)R and Val(61)-Phe(313) ectodomain sequence of P2X(7)R instead the native Ile(66)-Tyr(310) sequence. The mutated P2X(2a)+X3R and P2X(2b)+X3R exhibited comparable EC50 values for ATP, BzATP, and αβ-meATP in the submicromolar concentration range and desensitized in a receptor-specific and ligand-nonspecific manner. On the other hand, the chimeric P2X(2)+X7R exhibited decreased sensitivity for ATP and desensitized in a receptor-nonspecific manner. These results suggest that efficacy of agonists for the ligand-binding domain of P2X(2)Rs reflects the strength of desensitization controlled by their C-terminal structures.
  • A Tanoue, T Koshimizu, M Tsuchiya, K Ishii, M Osawa, M Saeki, G Tsujimoto
    JOURNAL OF BIOLOGICAL CHEMISTRY 277 36 33205 - 33212 2002年09月 [査読有り][通常論文]
     
    Hirschsprung disease is a heterogeneous genetic disorder, causative genes of which include the endothelin B receptor (ETB). To investigate the mutations of ETB in Hirschsprung disease, expression of the ETB gene in lymphoblastoid cells from patients and normal healthy adults was examined, and novel mutant transcripts were found. The mutant ETB gene transcripts lacked a 134-bp nucleotide sequence corresponding to exon 5, and some also contained a substitution from A to G at position 950 in exon 4, resulting in an amino acid substitution from glutamine (Q) to arginine (R). This substitution was suspected to be the result of RNA editing because it was not present in the genomic sequence. Transfection experiments using ETB minigenes containing the editing site with or without the gene for double-strand RNA deaminases (ADAR1 and ADAR2) revealed that the deaminases were involved in RNA editing. Furthermore, a c-Myc-tagged mutant ETB protein was not detected by Western blot analysis. The present results show that the mutant ETB transcripts were novel splice variants, which might not be translated, or that the products translated from splice variants might be quickly degraded, presumably because of their instability. The preferential production of this null function ETB by RNA editing/splicing could be involved in the etiology of some cases of Hirschsprung disease.
  • A Tanoue, T Koshimizu, G Tsujimoto
    LIFE SCIENCES 71 19 2207 - 2215 2002年09月 [査読有り][通常論文]
     
    Mice with altered alpha(1)-adrenergic receptor (AR) genes have become important tools in elucidating the subtype-specific functions of the three alpha(1)-AR subtypes because of the lack of sufficiently subtype-selective pharmacological agents. Mice with a deletion (knockout, KO) or an overexpression (transgenic, TG) of the alpha(1A)-, alpha(1B)-, or alpha(1D)-AR subtypes have been generated. The alpha(1)-ARs are the principal mediators of the hypertensive response to alpha(1)-agonists in the cardiovascular system. Studies with these mice indicate that alpha(1A)-AR and alpha(1B)-AR subtypes play an important role in cardiac development and/or function as well as in blood pressure (BP) response to alpha(1)-agonists via vasoconstriction. The alpha(1B)- and alpha(1D)-subtypes also appear to be involved in central nervous system (CNS) processes such as nociceptive responses, modulation of memory consolidation and working memory The ability to study subtype-specific functions in different mouse strains by altering the same alpha(1)-AR in different ways strengthens the conclusions drawn from these studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(1)-AR subtypes. (C) 2002 Elsevier Science Inc. All rights reserved.
  • A Tanoue, M Koba, S Miyawaki, T Koshimizu, C Hosoda, S Oshikawa, G Tsujimoto
    HYPERTENSION 40 1 101 - 106 2002年07月 [査読有り][通常論文]
     
    In an attempt to elucidate whether there is a specific alpha(1)-adrenergic receptor (alpha(1)-AR) subtype involved in the genesis or maintenance of hypertension, the alpha(ID)-AR subtype was evaluated in a model of salt-induced hypertension. The alpha(1D)-AR-deficient (alpha(1D)) and control (alpha(1D)(+/+)) mice (n=8 to 14 in each group) were submitted to subtotal nephrectomy and given 1% saline as drinking water for 35 days. Blood pressure (BP) was monitored by tail-cuff readings and confirmed at the end point by direct intraarterial BP recording. The alpha(1D)(-/-) mice had a significantly (P=0.0004) attenuated increase in BP response in this protocol (baseline 94.6 +/- 2.8 versus end point 107.4 +/- 4.5 mm Hg) compared with that of their wild-type counterparts (alpha(1D)(+/+)), from a baseline 97.4 +/- 2.9 to an end point 139.4 +/- 4.5 mm Hg. Seven of 15 alpha(1D)(+/+) mice died with edema, probably owing to renal failure, whereas 14 of 15 alpha(1D)(-/-) mice were maintained for 35 days. Body weight, renal remnant weight, and residual renal function were similar in the 2 groups, whereas the values of plasma catecholamines (epinephrine, norepinephrine, and dopamine) were higher in alpha(1D)(+/+) than in the alpha(1D)(-/-) mice. These data suggest that aID-AR plays an important role in developing a high BP in response to dietary salt-loading, and that agents having selective alpha(1D)-AR antagonism could have significant therapeutic potential in the treatment of hypertension.
  • TA Koshimizu, S Komori, T Ishihara, Kohno, I, K Umetani, T Sawanobori, H Ijiri, K Tamura
    PACE-PACING AND CLINICAL ELECTROPHYSIOLOGY 25 2 217 - 219 2002年02月 [査読有り][通常論文]
     
    Atrial standstill is electrophysiologically characterized by the loss of spontaneous excitation in atrial muscle and the inability to cause action potential firing upon electrical stimulation. Clinical diagnosis of transient standstill of the right atrium was made in a patient with acute occlusion of the right coronary artery and acute renal failure. Percutaneous coronary intervention, performed 5 days after the onset, restored the coronary blood flow and resulted in full recovery of electrical activity and regular sinus rhythm.

作品等

  • ノックアウトマウスを用いた薬理学研究
    2002年 - 現在
  • Physiological analysis using knockout mouse
    2002年 -2005年
  • 受容体の分子生物学的解析
    2000年 -2004年
  • Receptor molecular biology
    2000年 -2004年

MISC

  • T Koshimizu, G Tsujimoto, A Hirasawa, Y Kitagawa, A Tanoue CARDIOVASCULAR RESEARCH 63 (4) 662 -672 2004年09月 [査読無し][通常論文]
     
    Background: Increasing evidence from clinical trials indicates that carvedilol, an antagonist of alpha1- and beta-adrenergic receptors (ARs), provides an effective treatment for chronic heart failure, whereas nonselective alpha1-AR blockade has an adverse outcome in this disease. It is, however, not clear whether carvedilol exhibits a subtype-dependent impact on three distinct alpha1-adrenergic receptors (alpha1-ARs). Methods and results: We determined binding properties of human ARs for carvedilol using HEK293 human embryonic kidney cells expressing a single AR subtype. Our results showed that the affinities of alpha1D-AR and alpha1B-AR for carvedilol are higher than that of the beta1-AR subtype, a major target in heart failure treatment. The affinity rank order and pKi values of ARs for carvedilol were as follows: alpha1D-AR (8.9) > alpha1B-AR (8.6) > 1-AR (8.4) > beta2-AR (8.0) > alpha1A-AR (7.9)much greater thanalpha2C-AR (5.9) > alpha2B-AR (5.5) > alpha2A-AR (5.3). Furthermore, temporal kinetics of intracellular calcium signaling mediated via alpha1D- and alpha1B-ARs, but not via alpha1A-AR (P < 0.01), showed oscillatory patterns with frequencies ranging from 0.3 to 3 per minute in human smooth muscle and HEK293 cells, which were inhibited by the therapeutic concentrations of carvedilol (10 nM) in a subtype-dependent manner. When oscillatory alpha1B-AR and non-oscillatory alpha1A-AR were co-expressed and heteromer receptors were detected with bioluminescence resonance energy transfer and co-immunoprecipitation, carvedilol suppressed only oscillatory component of global cytosolic free calcium change. Conclusions: These results indicate that in addition to beta-ARs, receptor inhibition by carvedilol is directed to alpha1-ARs, preferably to alpha1D- and alpha1B-AR-mediated signaling events, including intracellular calcium oscillations in vascular smooth muscle. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
  • T Koshimizu, S Ueno, A Tanoue, N Yanagihara, SS Stojilkovic, G Tsujimoto JOURNAL OF BIOLOGICAL CHEMISTRY 277 (49) 46891 -46899 2002年12月 [査読無し][通常論文]
     
    P2X purinergic receptors (P2XRs) differ among themselves with respect to their ligand preferences and channel kinetics during activation, desensitization, and recovery. However, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. Here we show that homomeric receptors having the extracellular domain of the P2X(3) subunit in the P2X(2a)-based backbone (P2X(2a)/X(3)ex) mimicked two intrinsic functions of P2X(3)R, sensitivity to alphabeta-methylene ATP and ecto-ATPase-dependent recovery from endogenous desensitization; these two functions were localized to the N- and C-terminal halves of the P2X(3) extracellular loop, respectively. The chimeric P2X(2a)R/X(3)ex receptors also desensitized with accelerated rates compared with native P2X(2a)R, and the introduction of P2X(2) C-terminal splicing into the chimeric subunit (P2X(2b)/X(3)ex) further increased the rate of desensitization. Physical and functional heteromerization of native P2X(2a) and P2X(2b) subunits was also demonstrated. In heteromeric receptors, the ectodomain of P2X(3) was a structural determinant for ligand selectivity and recovery from desensitization, and the C terminus of P2X(2) was an important factor for the desensitization rate. Furthermore, [gamma-P-32]8-azido ATP, a photoreactive agonist, was effectively cross-linked to P2X(3) subunit in homomeric receptors but not in heteromeric P2X(2) + P2X(3)Rs. These results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.

受賞

  • 2009年11月 臨床薬理研究財団研究大賞
     
    受賞者: 輿水 崇鏡
  • 2009年03月 日本薬理学会優秀発表賞(共同演者)
     
    受賞者: 輿水 崇鏡
  • 2008年01月 バゾプレッシン研究会 バゾプレッシン研究会研究奨励賞
     
    受賞者: 輿水 崇鏡
  • 2004年11月 日本循環薬理学会若手研究者賞
     JPN
  • 2004年 2004 Young investigator award, Japanese cardiovascular pharmacology

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2012年 -2014年 
    代表者 : 輿水 崇鏡
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2009年 -2011年 
    代表者 : 輿水崇鏡
     
    ステロイドホルモンの作用は多岐に渡り副腎皮質機能不全症あるいは亢進症の影響は全身性及ぶ。最近では本態性高血圧症と思われていた患者の中にサブクリニカルな副腎皮質機能亢進症が存在することが明らかになっている。しかし機能亢進とは反対に、副腎皮質の予備能のみが低下した場合の病態についての詳細は未だ明らかにされていない。一方バゾプレッシンが作用する3種類のサブタイプ受容体は体内に広く分布しバゾプレッシンの多様な機能の発現を司る。本研究ではVla受容体遺伝子を特異的に欠失させた遺伝子改変マウス(Vla-/-)を用い、これまで明らかにされていなかった副腎皮質細胞の加齢色素沈着におけるVla受容体の役割を解明する。本年度は細胞並びに動物個体での検討を進め以下の成果を得た。副腎由来細胞株でのVasopressinの作用;Y1細胞をACTHとVasopressin(100nM)の存在化で刺激するとACTH単独よりもステロイドホルモンの分泌が亢進することが示された。この亢進は3時間後には有意な上昇となり6時間まで持続した。発現解析によりこの細胞にはVla受容体が発現し、他のバゾプレッシンファミリー受容体であるVlb,V2,ごキシトシン受容体は存在しないことが示された。よって副腎皮質細胞に存在するVla受容体は単独で、或はMC2メフノコルチン受容体と共同で糖質ステロイドの分泌を亢進させる能力を有する...
  • 文部科学省:科学研究費補助金(基盤研究(S))
    研究期間 : 2007年 -2011年 
    代表者 : 辻本豪三, 奥野恭史, 輿水崇鏡, 足達哲也, 寺澤和哉, 平澤明, 木村郁夫
     
    (1)近年トランスクリプトーム中には従来考えられていたよりも複雑多様な転写物が存在し、それら転写物が生物学的機能を有している可能性があり、中でも機能調節には蛋白質をコードしないRNA(non-protein-coding RNA ; ncRNA)の関与が最も注目されている。バイオインフォマティクス解析によりncRNAはヒト、マウスで数千から一万以上存在することが示されつつあり、ncRNAは蛋白質をコードするRNA(coding RNA ; cRNA)に対してアンチセンスDNAやsiRNAとして機能し得る構造を持つ場合が多い。ncRNAを含む包括的トランスクリプトーム解析研究は疾病の分子メカニズムや医薬品の作用機構に新たな次元の理解を導くことが予測される。本研究では、バイオインフォマティクスにより生物学的に機能が予測されるncRNA(特にmiRNA)にcRNAを含めた包括的トランスクリプトーム解析用マイクロアレイDNAチップを用い、特にがん関連機能RNA(群)、中でもmiRNAを抽出するとともにその生物学的意義を検証することを目的とする。(2)国内外で、臨床トランスクリプトーム解析がなされており、一部の疾患では個人化医療の実現に臨床応用されようとしている。しかしながら、すべての研究はcRNAのみを対象とするDNAチップ解析研究であり、生物学的機能がほとんど未知であるncRNA、...
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2007年 -2008年 
    代表者 : 輿水崇鏡
     
    本研究では、ヘテロマーG蛋白質共役型受容体蛋白質に対して特異的に作用する薬物を効率的かつ迅速にスクリーニングする方法を開発することを目的とした。方針としては「蛍光標識リガンドとヘテロマー受容体の間にのみ起る光共鳴エネルギー移動を検出し、受容体と化合物の間の相互作用を特異的に検出する」系を確立することを目標とし、これまで副作用や特異性が十分出ないために開発が中止されていた化合物ライブラリーをヘテロマー受容体に対して再度検討し直すことにより新たなリソースとして利用出来ることを示し得ると考えられる。昨年度にモデルとなる受容体遺伝子共発現細胞を樹立し受容体ホモマー、ヘテロマーの検出条件を確立し、相互作用が報告されたオピオイド受容体サブタイプmuとdeltaオピオイド受容体をモデルとして系を構築した。今年度はホモマー受容体のアミノ基末端の細胞外部分に抗原配列を付加した受容体を発現する細胞に対し、蛍光標識抗体間で光エネルギー移動を観察し、受容体二量体の状態を検出することに成功した。蛍光リガンドと受容体との光共鳴エネルギー移動を検出する条件検討の段階で研究期間を終了することとなったが、細胞表面の蛍光観察がリガンドのスクリーニングに有効な方法論を示すことができ、今後も研究を進めることでヘテロマーリガンドをスクリーニングは可能と考えられた。さらに、mu受容体がdelta受容体以外のGタンパク質...
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2006年 -2007年 
    代表者 : 平澤明, 足達哲也, 輿水崇鏡
     
    オーファン受容体のリガンド探索研究の成果として、我々は、GPR120が長鎖不飽和脂肪酸をリガンドとする受容体であり、腸内分泌物質に発現しGLP-1の分泌に関与する重要な代謝調節受容体であることを見いだした。またCCKの分泌にも関与する事を見いだした。このGPR120が各種脂肪組織に発現していることをmRNAレベルで確認した。さらに、GPR120の脂肪細胞におけるmRNA量をリアルタイムPCRで検討したところ、分化過程に対応して発現量が増加することを確認できた。また、分化過程の様々な時期にGPR120を活性化する脂肪酸(α-リノレン酸、ドコサヘキサエン酸、エイコサペンタエン酸)または活性化しない脂肪酸(オクタン酸など)を添加して、脂肪細胞の分化に及ぼす作用を検討した。その結果、GPR120を活性化する脂肪酸のみが細胞内への脂肪の蓄積及び増殖の抑制の両方に強く関与していることが明らかになった。一方で、GPR120に対するsiRNAを投与したところ、脂肪細胞への分化の抑制が認められた。このことから、GPR120は、脂肪細胞の分化過程に重要な役割を果たす受容体であることが示唆された。また、脂肪細胞からのアディポネクチンの分泌にも作用する事を見いだした。GPR120に特異的なアゴニスト、アンタゴニストの探索のため、化合物スクリーニングを行った結果、GPR120に特異性を有する天然物由来...
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2006年 -2007年 
    代表者 : 辻本豪三, 奥野恭史, 足達哲也, 輿水崇鏡, 平澤明
     
    本研究では、患者の遺伝的体質に合わせた処方、治療計画を実践するテーラーメイド医療の実現のために、我々が開発した超高感度マイクロアレイ技術を極微量の臨床試料-特に食道や大腸癌の生検試料-に対して応用し、癌の診断並びに治療に役立てることを目標とする。今年度我々は、生検試料に対して高感受性チップを使用し、データの再現性を確認後、チップ搭載コンテンツを決定し癌判別アルゴリズムを改善することに成功した。まず(1)既に株化されている培養食道がん細胞株(約50種)において、化学療法や放射線療法の感受性と発現プロファイルの関係を統合し、これら治療に対する感受性判定に重要な遺伝子群を絞り込んだ。次に(2)これらの遺伝子群によるマイクロアレイを作製し、生検試料によるプロスペクティブ研究を進めた。さらに、食道癌検体について京都大学腫瘍外科、その他の病院施設において倫理審査委員会の承認を得て研究を実施できる体制を構築した。また上記各機関から実験実施箇所である京都大学薬学部までの検体収集搬体制を確立し、品質良好なRNAを抽出可能な状態で検体を収集できることを確認した。具体的には、食道癌生検検体計54組(生検判断によるガン・非ガン)を収集した。癌判別アルゴリズムによってモデル実験を行ったところ、癌組織の割合が6割以上の検体を癌と判別できることが確認された。このアルゴリズムで新規検体を判別したところ、食道...
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 2005年 -2006年 
    代表者 : 辻本豪三, 門脇正史, 平澤明, 輿水崇鏡, 奥野恭史, 足達哲也
     
    当初計画した以下の研究項目を実施した。(1)遊離脂肪酸受容体ファミリーのクローニングゲノム情報よりオーファンG蛋白質共役型受容体GPR120を抽出、クローニングし、更にその天然リガンドが遊離脂肪酸であることを発見した。本研究では、GPR120を含む近年見出された遊離脂肪酸を天然リガンドとする一群の受容体群(GPR40,GPR41,GPR43)について、ゲノムデータベース(ヒト、マウス、ラット)を最大限活用しIn Silicoクローニングを行った。(2)in vivo/in vitro機能解析用ツール開発それぞれの受容体に特異的な抗体の作成に成功した。また、各受容体の詳細な組織発現解析情報を収集した。更に、高効率の細胞内情報伝達解析プラットフォームを用いて各種化合物をスクリーニングし、一部の受容体に特異的な天然物を見出した。更に、各受容体のマウスゲノムをクローニングし、その構造を決定するとともに、遺伝子改変動物(特にノックアウトマウス)作出を試み、一部の受容体でその作出に成功した。(3)GPR120機能解析GPR120に関しては天然発現細胞株STC-1細胞を用いてin vitro機能解析を行い、その受容体機構がインスリン分泌調節の重要な因子であるインクレチンホルモンGLP-1分泌機構に極めて重要な機能を有する事を明らかにした。また、その細胞内情報伝達機構にはカルシウムシグナル伝...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2005年 -2006年 
    代表者 : 輿水崇鏡
     
    [1]細胞膜蛋白質cDNA入手-ヒト及びマウスのゲノム情報からG-タンパク質共役型受容体遺伝子の塩基配列を獲得し、理化学研究所、国立遺伝学研究所、FLJクローン(NEDO)の中から迅速に完全長cDNAクローンを入手した。[2]融合蛋白質作成-次に発現用受容体一蛍光蛋白質と受容体一発光蛋白質の作成した。塩基配列を確認し作成した発現用コンストラクトが受容体として機能することは、受容体の内在化や細胞内メッセンジャーの変化により確認した。[3]発現量の滴定-蛍光蛋白質が過剰に存在する場合擬陽性率が高くなるため、適当な発現量が得られるよう発現実験に用いるDNA量を決定した。蛍光蛋白質から得られる蛍光について蛍光異方性を計測し、光共鳴エネルギー移動と配向性の変化の関係を確かめることにより擬陽性や見落としを最小限にしたアッセイ系を確立した。また、ポジテイブコントロールとなる受容体ホモオリゴマー形成の有無を確かめた。受容体の中にはこのホモオリゴマー形成がBRETで検出できないものがあることが判明し、その場合免疫沈降法による評価のみ行う方針とした。[4]G蛋白質共役型受容体の相互作用検出-アゴニストに依存的な受容体機能とホモダーマー形成が確認された受容体はヘテロダイマーの形成についてBRETと免疫沈降法により総当たりにてスクリーニングした。[5]個体レベルでの解析-細胞レベルで見いだした受容体...
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2005年 -2005年 
    代表者 : 辻本豪三, 平澤明, 輿水崇鏡
     
    本年度は、DNAチップ基板をこれまでの平板から凹凸形状にする画期的なアイデアと、解析のための検体との反応を強制的に促進させる技術を開発し、従来のDNAチップに比べ最高で100倍の高感度化が達成できる技術の確立に成功した。多数の遺伝子情報を一斉に解析できるDNAチップは、今後テーラーメイド医療や創薬開発のキーツールとして期待され、多くの研究機関やメーカーで種々のDNAチップが開発されるが感度が充分でないため、患者に負担が少ない低侵襲の採取方法により得られた微量検体の解析や、極僅かにしか存在しない遺伝子に関する高い精度の解析が難しいという課題があった。我々は、世界のデファクトスタンダードである米国アフィメトリクス社のジーンチップと比して更に高感度のチップ開発に成功した。基礎検討より通常のチップの約100倍の高感度検出が可能となり、微量臨床検体(例えば、臨床生検試料など)によるテーラーメード医療に大いに活用できる基盤が世界に先駈けて構築できた。このことはメディアなどでも大いに喧伝されたが、更に基礎研究、特に創薬科学研究等の治療目的の医療科学分野にも広く用いる可能性を示した。高感度化は上述のようにむしろ予測を超えた成功に繋がった。一方で、その検証できる数は現在256と数が少なくまだ網羅性に乏しい。このプラットフォームでその数を多くする努力も継続的に必要である。今後は更にその高感度性を...
  • ノックアウトマウスを用いた受容体研究
    科学技術振興調整費
    研究期間 : 2004年 -2005年
  • receptor biology using knockout mouse
    Special Coordination Funds for Promoting Science and Technology
    研究期間 : 2004年 -2005年
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2003年 -2004年 
    代表者 : 辻本豪三, 三輪嘉尚, 松本治, 田上昭人, 輿水崇鏡
     
    この研究ではマウス下垂体より新たなP2X2受容体スプライシングバリアントをトランスクリプトームの解析より同定した。P2X2には野生型のP2X2aとカルボキシ末端スプライシングバリアントのP2X2bに加え、新たなバリアントP2X2eを発見した。このP2X2eは、ATPの刺激に対し、P2X2bなどこれまで知られているどのP2X2スプライシングバリアントよりもはるかに早い脱感作を起こすことが分かった。これはATP受容体の活性を担う、重要な受容体部分を同定する際に、手がかりとなる有用な知見である。P2X2eはバリアントの中ではカルボキシル末端が最も短く、スプライシングにより、受容体の活性化時間が短くなり、細胞内カルシウム濃度の過度な上昇が押さえられる。下垂体細胞内ではP2X2aとP2X2bがほぼ等量共存し、P2X2eの発現量はこれらの約10%であった。P2X2aによる細胞内カルシウム濃度の上昇が、P2X2b及びP2X2eと共存することで適度に保たれている可能性が示唆された。これより、細胞内カルシウム動態を通じ、成長ホルモンや、ゴナドトロピン分泌ホルモンの分泌が調節を受けている事が示唆された。さらに、3種類のスプライシングバリアント受容体は全ての組み合わせでチャネルを作成しうることを、光エネルギーの共振による移動を利用して同定した。すなわち、ルシフェラーゼまたはGreen flores...
  • 新規受容体作用薬の開発
  • Drug development research for pain management

担当経験のある科目

  • 薬理学自治医科大学
  • ゲノム創薬科学演習京都大学大学院薬学研究科
  • 病態生理学京都大学大学院薬学研究科


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