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List of Reports
Motohiro Nishida Last modified date：2024.04.18

Professor / Department of Physiology / Department of Pharmaceutical Health Care and Sciences / Faculty of Pharmaceutical Sciences


Reports
1. Barayeu U, Sawa T, Nishida M, Wei F-Y, Motohashi H, Akaike T , Supersulfide biology and translational medicine for disease control. , https://doi.org/10.1111/bph.16271, 2023.10.
2. Nishimura A, Tanaka T, Kato Y, Nishiyama K, Nishida M*., Cardiac robustness regulated by reactive sulfur species. , J Clin Biochem Nutr. , 10.3164, 2022.01.
3. K. Nishiyama, T. Tanaka, A. Nishimura, M. Nishida, TRPC3-based protein signaling complex as a therapeutic target of myocardial atrophy, Curr. Mol.r Pharmacol., in press, 2020.04.
4. T. Numaga-Tomita, M. Nishida, TRPC channels in cardiac plasticity, 2020.02.
5. M. Nishid. Tanaka, S. Mangmool, K. Nishiyama, A. Nishimura, Canonical Transient Receptor Potential Channels and Vascular Smooth Muscle Cell Plasticity, J. Lipid Atheroscler., 9(1):e13, 2020.01.
6. Suhaini Binti Sudi, Tomohiro Tanaka, Sayaka Oda, Kazuhiro Nishiyama, Akiyuki Nishimura, Caroline Sunggip, Supachoke Mangmool, Takuro Numaga-Tomita, Motohiro Nishida, TRPC3-Nox2 axis mediates nutritional deficiency-induced cardiomyocyte atrophy, Scientific reports, 10.1038/s41598-019-46252-2, 2019.12, Myocardial atrophy, characterized by the decreases in size and contractility of cardiomyocytes, is caused by severe malnutrition and/or mechanical unloading. Extracellular adenosine 5′-triphosphate (ATP), known as a danger signal, is recognized to negatively regulate cell volume. However, it is obscure whether extracellular ATP contributes to cardiomyocyte atrophy. Here, we report that ATP induces atrophy of neonatal rat cardiomyocytes (NRCMs) without cell death through P2Y2 receptors. ATP led to overproduction of reactive oxygen species (ROS) through increased amount of NADPH oxidase (Nox) 2 proteins, due to increased physical interaction between Nox2 and canonical transient receptor potential 3 (TRPC3). This ATP-mediated formation of TRPC3-Nox2 complex was also pathophysiologically involved in nutritional deficiency-induced NRCM atrophy. Strikingly, knockdown of either TRPC3 or Nox2 suppressed nutritional deficiency-induced ATP release, as well as ROS production and NRCM atrophy. Taken together, we propose that TRPC3-Nox2 axis, activated by extracellular ATP, is the key component that mediates nutritional deficiency-induced cardiomyocyte atrophy..
7. Kazuhiro Nishiyama, Takuro Numaga-Tomita, Yasuyuki Fujimoto, Tomohiro Tanaka, Chiemi Toyama, Akiyuki Nishimura, Tomohiro Yamashita, Matsunaga Naoya, Koyanagi Satoru, Yasu Taka Azuma, Yuko Ibuki, Koji Uchida, Shigehiro Ohdo, Motohiro Nishida, Ibudilast attenuates doxorubicin-induced cytotoxicity by suppressing formation of TRPC3 channel and NADPH oxidase 2 protein complexes, British Journal of Pharmacology, 10.1111/bph.14777, 2019.09, Background and Purpose: Doxorubicin is a highly effective anticancer agent but eventually induces cardiotoxicity associated with increased production of ROS. We previously reported that a pathological protein interaction between TRPC3 channels and NADPH oxidase 2 (Nox2) contributed to doxorubicin-induced cardiac atrophy in mice. Here we have investigated the effects of ibudilast, a drug already approved for clinical use and known to block doxorubicin-induced cytotoxicity, on the TRPC3-Nox2 complex. We specifically sought evidence that this drug attenuated doxorubicin-induced systemic tissue wasting in mice. Experimental Approach: We used the RAW264.7 macrophage cell line to screen 1,271 clinically approved chemical compounds, evaluating functional interactions between TRPC3 channels and Nox2, by measuring Nox2 protein stability and ROS production, with and without exposure to doxorubicin. In male C57BL/6 mice, samples of cardiac and gastrocnemius muscle were taken and analysed with morphometric, immunohistochemical, RT-PCR and western blot methods. In the passive smoking model, cells were exposed to DMEM containing cigarette sidestream smoke. Key Results: Ibudilast, an anti-asthmatic drug, attenuated ROS-mediated muscle toxicity induced by doxorubicin treatment or passive smoking, by inhibiting the functional interactions between TRPC3 channels and Nox2, without reducing TRPC3 channel activity. Conclusions and Implications: These results indicate a common mechanism underlying induction of systemic tissue wasting by doxorubicin. They also suggest that ibudilast could be repurposed to prevent muscle toxicity caused by anticancer drugs or passive smoking..
8. Takuro Numaga-Tomita, Tsukasa Shimauchi, Sayaka Oda, Tomohiro Tanaka, Kazuhiro Nishiyama, Akiyuki Nishimura, Lutz Birnbaumer, Yasuo Mori, Motohiro Nishida, TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN, FASEB Journal, 10.1096/fj.201802811R, 2019.09, Vascular smooth muscle cells (VSMCs) play critical roles in the stability and tonic regulation of vascular homeostasis. VSMCs can switch back and forth between highly proliferative synthetic and fully differentiated contractile phenotypes in response to changes in the vessel environment. Although abnormal phenotypic switching of VSMCs is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty, how control of VSMC phenotypic switching is dysregulated in pathologic conditions remains obscure. We found that inhibition of canonical transient receptor potential 6 (TRPC6) channels facilitated contractile differentiation of VSMCs through plasma membrane hyperpolarization. TRPC6-deficient VSMCs exhibited more polarized resting membrane potentials and higher protein kinase B (Akt) activity than wild-type VSMCs in response to TGF-β1 stimulation. Ischemic stress elicited by oxygen-glucose deprivation suppressed TGF-β1-induced hyperpolarization and VSMC differentiation, but this effect was abolished by TRPC6 deletion. TRPC6-mediated Ca2+ influx and depolarization coordinately promoted the interaction of TRPC6 with lipid phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of Akt activation. Given the marked up-regulation of TRPC6 observed in vascular disorders, our findings suggest that attenuation of TRPC6 channel activity in pathologic VSMCs could be a rational strategy to maintain vascular quality control by fine-tuning of VSMC phenotypic switching.—Numaga-Tomita, T., Shimauchi, T., Oda, S., Tanaka, T., Nishiyama, K., Nishimura, A., Birnbaumer, L., Mori, Y., Nishida, M. TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN. FASEB J. 33, 9785–9796 (2019). www.fasebj.org..
9. Takuro Numaga-Tomita, Sayaka Oda, Kazuhiro Nishiyama, Tomohiro Tanaka, Akiyuki Nishimura, Motohiro Nishida, TRPC channels in exercise-mimetic therapy, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-018-2211-3, 2019.03, Physical exercise yields beneficial effects on all types of muscle cells, which are essential for the maintenance of cardiovascular homeostasis and good blood circulation. Daily moderate exercise increases systemic antioxidative capacity, which can lead to the prevention of the onset and progression of oxidative stress-related diseases. Therefore, exercise is now widely accepted as one of the best therapeutic strategies for the treatment of ischemic (hypoxic) diseases. Canonical transient receptor potential (TRPC) proteins are non-selective cation channels activated by mechanical stress and/or stimulation of phospholipase C-coupled surface receptors. TRPC channels, especially diacylglycerol-activated TRPC channels (TRPC3 and TRPC6; TRPC3/6), play a key role in the development of cardiovascular remodeling. We have recently found that physical interaction between TRPC3 and NADPH oxidase (Nox) 2 under hypoxic stress promotes Nox2-dependent reactive oxygen species (ROS) production and mediates rodent cardiac plasticity, and inhibition of the TRPC3-Nox2 protein complex results in enhancement of myocardial compliance and flexibility similar to that observed in exercise-treated hearts. In this review, we describe current understanding of the roles of TRPC channels in striated muscle (patho)physiology and propose that targeting TRPC-based protein complexes could be a new strategy to imitate exercise therapy..
10. T. Tanaka, A. Nishimura, K. Nishiyama, T. Goto,　T. Numaga-Tomita, M. Nishida, Mitochondrial dynamics in exercise physiology, Pflügers Archiv., 472(2):137-153, 2019.02.
11. Hideshi Ihara, Yuki Kakihana, Akane Yamakage, Kenji Kai, Takahiro Shibata, Motohiro Nishida, Yamada Kenichi, Koji Uchida, 2-Oxo-histidine-containing dipeptides are functional oxidation products, Journal of Biological Chemistry, 10.1074/jbc.RA118.006111, 2019.01, Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC- electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H2O2 exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H2O2 cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs..
12. Akiyuki Nishimura, Kakeru Shimoda, Tomohiro Tanaka, Takashi Toyama, Kazuhiro Nishiyama, Yasuhiro Shinkai, Takuro Numaga-Tomita, Daiju Yamazaki, Yasunari Kanda, Takaaki Akaike, Yoshito Kumagai, Motohiro Nishida, Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload, Science Signaling, 10.1126/scisignal.aaw1920, 2019.01, Chronic exposure to methylmercury (MeHg), an environmental electrophilic pollutant, reportedly increases the risk of human cardiac events. We report that exposure to a low, non-neurotoxic dose of MeHg precipitated heart failure induced by pressure overload in mice. Exposure to MeHg at 10 ppm did not induce weight loss typical of higher doses but caused mitochondrial hyperfission in myocardium through the activation of Drp1 by its guanine nucleotide exchange factor filamin-A. Treatment of neonatal rat cardiomyocytes with cilnidipine, an inhibitor of the interaction between Drp1 and filamin-A, suppressed mitochondrial hyperfission caused by low-dose MeHg exposure. Modification of cysteine residues in proteins with polysulfides is important for redox signaling and mitochondrial homeostasis in mammalian cells. We found that MeHg targeted rat Drp1 at Cys624, a redox-sensitive residue whose SH side chain forms a bulky and nucleophilic polysulfide (Cys624-S(n)H). MeHg exposure induced the depolysulfidation of Cys624-S(n)H in Drp1, which led to filamin-dependent activation of Drp1 and mitochondrial hyperfission. Treatment with NaHS, which acts as a donor for reactive polysulfides, reversed MeHg-evoked Drp1 depolysulfidation and vulnerability to mechanical load in rodent and human cardiomyocytes and mouse hearts. These results suggest that depolysulfidation of Drp1 at Cys624-S(n)H by low-dose MeHg increases cardiac fragility to mechanical load through filamin-dependent mitochondrial hyperfission..
13. Akiyuki Nishimura, Tsukasa Shimauchi, Tomohiro Tanaka, Kakeru Shimoda, Takashi Toyama, Naoyuki Kitajima, Tatsuya Ishikawa, Naoya Shindo, Takuro Numaga-Tomita, Satoshi Yasuda, Yoji Sato, Koichiro Kuwahara, Yoshito Kumagai, Takaaki Akaike, Tomomi Ide, Akio Ojida, Yasuo Mori, Motohiro Nishida, Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence, Science Signaling, 10.1126/scisignal.aat5185, 2018.11, Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission-associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding-dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1-filamin A interaction by cilnidipine suppressed mitochondrial hyperfission-associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development..
14. Warisara Parichatikanond, Akiyuki Nishimura, Motohiro Nishida, Supachoke Mangmool, Prolonged stimulation of β2-adrenergic receptor with β2-agonists impairs insulin actions in H9c2 cells, Journal of Pharmacological Sciences, 10.1016/j.jphs.2018.09.007, 2018.11, Insulin resistance is a condition in which there is a defect in insulin actions to induce glucose uptake into the cells. Overstimulation of β2-adrenergic receptors (β2ARs) is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which β2-agonists affect insulin resistance in the heart are incompletely understood. The β2-agonists are used for treatment of asthma due to bronchodilating effects. We also investigated the effects of β2-agonists in human bronchial smooth muscle (HBSM) cells. In this study, we demonstrate that chronic treatment with salbutamol, salmeterol, and formoterol inhibited insulin-induced glucose uptake and GLUT4 synthesis in H9c2 myoblast cells. Sustained β2AR stimulation also attenuated GLUT4 translocation to the plasma membrane, whereas short-term stimulation had no effect. In HBSM cells, prolonged treatment with β2-agonists had no effect on insulin-induced glucose uptake and did not alter insulin-induced expressions of GLUT1, GLUT4, and GLUT10. In addition, genetic polymorphisms at amino acid positions 16 and 27 of β2AR are linked to insulin resistance by significant suppression of GLUT4 translocation compared to wild-type. Thus, prolonged β2AR stimulation by β2-agonists impairs insulin actions through suppression of GLUT synthesis and translocation only in H9c2 cells..
15. Caroline Sunggip, Kakeru Shimoda, Sayaka Oda, Tomohiro Tanaka, Kazuhiro Nishiyama, Supachoke Mangmool, Akiyuki Nishimura, Takuro Numaga-Tomita, Motohiro Nishida, TRPC5-eNOS axis negatively regulates ATP-induced cardiomyocyte hypertrophy, Frontiers in Pharmacology, 10.3389/fphar.2018.00523, 2018.05, Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2+-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation..
16. Mitochondrial quality control and cardiac risk regulation by reactive persulfide species.
17. Yohei Yamaguchi, Gentaro Iribe, Toshiyuki Kaneko, Ken Takahashi, Takuro Numaga-Tomita, Motohiro Nishida, Lutz Birnbaumer, Keiji Naruse, TRPC3 participates in angiotensin II type 1 receptor-dependent stress-induced slow increase in intracellular Ca2+ concentration in mouse cardiomyocytes, Journal of Physiological Sciences, 10.1007/s12576-016-0519-3, 2018.03, When a cardiac muscle is held in a stretched position, its [Ca2+] transient increases slowly over several minutes in a process known as stress-induced slow increase in intracellular Ca2+ concentration ([Ca2+]i) (SSC). Transient receptor potential canonical (TRPC) 3 forms a non-selective cation channel regulated by the angiotensin II type 1 receptor (AT1R). In this study, we investigated the role of TRPC3 in the SSC. Isolated mouse ventricular myocytes were electrically stimulated and subjected to sustained stretch. An AT1R blocker, a phospholipase C inhibitor, and a TRPC3 inhibitor suppressed the SSC. These inhibitors also abolished the observed SSC-like slow increase in [Ca2+]i induced by angiotensin II, instead of stretch. Furthermore, the SSC was not observed in TRPC3 knockout mice. Simulation and immunohistochemical studies suggest that sarcolemmal TRPC3 is responsible for the SSC. These results indicate that sarcolemmal TRPC3, regulated by AT1R, causes the SSC..
18. Kumiko Masuda, Hiroyasu Tsutsuki, Shingo Kasamatsu, Tomoaki Ida, Tsuyoshi Takata, Kikuya Sugiura, Motohiro Nishida, Yasuo Watanabe, Tomohiro Sawa, Takaaki Akaike, Hideshi Ihara, Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.12.088, 2018.01, To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP+) treatment (a model of Parkinson's disease). We show that MPP+-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12 cells (NPC12 cells) and rat cerebellar granule neurons (CGNs). Following MPP+ treatment, we found production of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12 cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP+ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP+-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP+-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP+-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease..
19. Licht Miyamoto, Motohiro Nishida, Lifestyle inspires future pharmacotherapy and drug discovery, 10.1248/yakushi.18-00091-F, 2018.01.
20. Sayaka Oda, Takuro Numaga-Tomita, Motohiro Nishida, New strategies for exercise-mimetic medication, Yakugaku Zasshi, 10.1248/yakushi.18-00091-1, 2018.01, Moderate exercise has been reported to combat several diseases, including cardiovascular diseases and depressants. However, many patients do not have ability to undergo exercise therapy due to aging and severity of the symptoms. Therefore development of new drugs that can imitate exercise therapy is desired and actually studied worldwide. The heart is one of the physical load-responsive target organs such as skeletal muscles and vascular smooth muscles. The heart can adapt from environmental stress by changing its structure and morphology (i.e., remodeling). Physiological remodeling, caused by exercise or pregnancy, can be defined by compensative and reversible changes to the heart, whereas pathological remodeling can be defined by irreversible changes of the heart, through aberrant calcium ion (Ca2 ＋ ) signaling as well as production of reactive oxygen species (ROS). However, crosstalk between Ca2 ＋ and ROS remains obscure. In this review we will introduce our recent findings on the functional crosstalk between transient receptor potential canonical (TRPC) 3 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) 2 as a novel molecular target to mimic exercise therapy..
21. Takaaki Akaike, Tomoaki Ida, Fan Yan Wei, Motohiro Nishida, Yoshito Kumagai, Md Morshedul Alam, Hideshi Ihara, Tomohiro Sawa, Tetsuro Matsunaga, Shingo Kasamatsu, Akiyuki Nishimura, Masanobu Morita, Kazuhito Tomizawa, Akira Nishimura, Satoshi Watanabe, Kenji Inaba, Hiroshi Shima, Nobuhiro Tanuma, Minkyung Jung, Shigemoto Fujii, Yasuo Watanabe, Masaki Ohmuraya, Péter Nagy, Martin Feelisch, Jon M. Fukuto, Hozumi Motohashi, Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics, Nature communications, 10.1038/s41467-017-01311-y, 2017.12, Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction..
22. Akiyuki Nishimura, Caroline Sunggip, Sayaka Oda, Takuro Numaga-Tomita, Makoto Tsuda, Motohiro Nishida, Purinergic P2Y receptors
Molecular diversity and implications for treatment of cardiovascular diseases, Pharmacology and Therapeutics, 10.1016/j.pharmthera.2017.06.010, 2017.12, Purinergic signaling, mediated mainly by G protein-coupled P2Y receptors (P2YRs), is now attracting attention as a new therapeutic target for preventing or treating cardiovascular diseases. Observations using mice with genetically modified P2YRs and/or treated with a pharmacological P2YR inhibitor have helped us understand the physiological and pathological significance of P2YRs in the cardiovascular system. P2YR-mediated biological functions are predominantly activated by mononucleotides released from non-adrenergic, non-cholinergic nerve endings or non-secretory tissues in response to physical stress or cell injury, though recent studies have suggested the occurrence of ligand-independent P2YR function through receptor-receptor interactions (oligomerization) in several biological processes. In this review, we introduce the functions of P2YRs and possible dimerization with G protein-coupled receptors (GPCRs) in the cardiovascular system. We focus especially on the crosstalk between uridine nucleotide-responsive P2Y6R and angiotensin (Ang) II type1 receptor (AT1R) signaling, and introduce our recent finding that the P2Y6R antagonist MRS2578 interrupts heterodimerization between P2Y6R and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2Y6R oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases..
23. Sayaka Oda, Takuro Numaga-Tomita, Naoyuki Kitajima, Takashi Toyama, Eri Harada, Tsukasa Shimauchi, Akiyuki Nishimura, Tatsuya Ishikawa, Yoshito Kumagai, Lutz Birnbaumer, Motohiro Nishida, TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice, Scientific reports, 10.1038/s41598-017-07903-4, 2017.12, Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6..
24. Yohei Yamaguchi, Gentaro Iribe, Motohiro Nishida, Keiji Naruse, Role of TRPC3 and TRPC6 channels in the myocardial response to stretch
Linking physiology and pathophysiology, Progress in Biophysics and Molecular Biology, 10.1016/j.pbiomolbio.2017.06.010, 2017.11, Transient receptor potential (TRP) channels constitute a large family of versatile multi-signal transducers. In particular, TRP canonical (TRPC) channels are known as receptor-operated, non-selective cation channels. TRPC3 and TRPC6, two members in the TRPC family, are highly expressed in the heart, and participate in the pathogenesis of cardiac hypertrophy and heart failure as a pathological response to chronic mechanical stress. In the pathological response, myocardial stretch increases intracellular Ca2+ levels and activates nuclear factor of activated T cells to induce cardiac hypertrophy. Recent studies have revealed that TRPC3 and TRPC6 also contribute to the physiological stretch-induced slow force response (SFR), a slow increase in the Ca2+ transient and twitch force during stretch. In the physiological response, a stretch-induced increase in intracellular Ca2+ mediated by TRPC3 and TRPC6 causes the SFR. We here overview experimental evidence of the involvement of TRPC3 and TRPC6 in cardiac physiology and pathophysiology in response to stretch..
25. Yu Guan, Daisuke Nakano, Yifan Zhang, Lei Li, Wenhua Liu, Motohiro Nishida, Takashige Kuwabara, Asahiro Morishita, Hirofumi Hitomi, Kiyoshi Mori, Masashi Mukoyama, Tsutomu Masaki, Katsuya Hirano, Akira Nishiyama, A protease-activated receptor-1 antagonist protects against podocyte injury in a mouse model of nephropathy, Journal of Pharmacological Sciences, 10.1016/j.jphs.2017.09.002, 2017.10, The kidney expresses protease-activated receptor-1 (PAR-1). PAR-1 is known as a thrombin receptor, but its role in kidney injury is not well understood. In this study, we examined the contribution of PAR-1 to kidney glomerular injury and the effects of its inhibition on development of nephropathy. Mice were divided into 3 groups: control, doxorubicin + vehicle (15 mg/kg doxorubicin and saline) and doxorubicin + Q94 (doxorubicin at 15 mg/kg and the PAR-1 antagonist Q94 at 5 mg/kg/d) groups. Where indicated, doxorubicin was administered intravenously and PAR-1 antagonist or saline vehicle by subcutaneous osmotic mini-pump. PAR-1 expression was increased in glomeruli of mice treated with doxorubicin. Q94 treatment significantly suppressed the increased albuminuria in these nephropathic mice. Pathological analysis showed that Q94 treatment significantly attenuated periodic acid–Schiff and desmin staining, indicators of podocyte injury, and also decreased glomerular levels of podocin and nephrin. Furthermore, thrombin increased intracellular calcium levels in podocytes. This increase was suppressed by Q94 and Rox4560, a transient receptor potential cation channel (TRPC)3/6 antagonist. In addition, both Q94 and Rox4560 suppressed the doxorubicin-induced increase in activities of caspase-9 and caspase-3 in podocytes. These data suggested that PAR-1 contributes to development of podocyte and glomerular injury and that PAR-1 antagonists have therapeutic potential..
26. Takuro Numaga-Tomita, Sayaka Oda, Tsukasa Shimauchi, Akiyuki Nishimura, Supachoke Mangmool, Motohiro Nishida, TRPC3 Channels in Cardiac Fibrosis, Frontiers in Cardiovascular Medicine, 10.3389/fcvm.2017.00056, 2017.09, Cardiac stiffness, caused by interstitial fibrosis due to deposition of extracellular matrix proteins, is thought as a major clinical outcome of heart failure with preserved ejection fraction (HFpEF). Canonical transient receptor potential (TRPC) subfamily proteins are components of Ca2+-permeable non-selective cation channels activated by receptor stimulation and mechanical stress, and have been attracted attention as a key mediator of maladaptive cardiac remodeling. How TRPC-mediated local Ca2+ influx encodes a specific signal to induce maladaptive cardiac remodeling has been long obscure, but our recent studies suggest a pathophysiological significance of channel activity-independent function of TRPC proteins for amplifying redox signaling in heart. This review introduces the current understanding of the physiological and pathophysiological roles of TRPCs, especially focuses on the role of TRPC3 as a positive regulator of reactive oxygen species (PRROS) in heart. We have revealed that TRPC3 stabilizes NADPH oxidase 2 (Nox2), a membrane-bound reactive oxygen species (ROS)-generating enzyme, by forming stable protein complex with Nox2, which leads to amplification of mechanical stress-induced ROS signaling in cardiomyocytes, resulting in induction of fibrotic responses in cardiomyocytes and cardiac fibroblasts. Thus, the TRPC3 function as PRROS will offer a new therapeutic strategy for the prevention or treatment of HFpEF..
27. Tsukasa Shimauchi, Takuro Numaga-Tomita, Tomoya Ito, Akiyuki Nishimura, Ryosuke Matsukane, Sayaka Oda, Sumio Hoka, Tomomi Ide, Norimichi Koitabashi, Koji Uchida, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy, JCI Insight, 10.1172/jci.insight.93358, 2017.08, Myocardial atrophy is a wasting of cardiac muscle due to hemodynamic unloading. Doxorubicin is a highly effective anticancer agent but also induces myocardial atrophy through a largely unknown mechanism. Here, we demonstrate that inhibiting transient receptor potential canonical 3 (TRPC3) channels abolishes doxorubicin-induced myocardial atrophy in mice. Doxorubicin increased production of ROS in rodent cardiomyocytes through hypoxic stress-mediated upregulation of NADPH oxidase 2 (Nox2), which formed a stable complex with TRPC3. Cardiomyocyte-specific expression of TRPC3 C-terminal minipeptide inhibited TRPC3-Nox2 coupling and suppressed doxorubicin-induced reduction of myocardial cell size and left ventricular (LV) dysfunction, along with its upregulation of Nox2 and oxidative stress, without reducing hypoxic stress. Voluntary exercise, an effective treatment to prevent doxorubicin-induced cardiotoxicity, also downregulated the TRPC3-Nox2 complex and promoted volume load-induced LV compliance, as demonstrated in TRPC3-deficient hearts. These results illustrate the impact of TRPC3 on LV compliance and flexibility and, focusing on the TRPC3-Nox2 complex, provide a strategy for prevention of doxorubicin-induced cardiomyopathy..
28. Motohiro Nishida, Akiyuki Nishimura, Tetsuro Matsunaga, Hozumi Motohashi, Shingo Kasamatsu, Takaaki Akaike, Redox regulation of electrophilic signaling by reactive persulfides in cardiac cells, Free Radical Biology and Medicine, 10.1016/j.freeradbiomed.2017.01.024, 2017.08, Maintaining a redox balance by means of precisely controlled systems that regulate production, and elimination, and metabolism of electrophilic substances (electrophiles) is essential for normal cardiovascular function. Electrophilic signaling is mainly regulated by endogenous electrophiles that are generated from reactive oxygen species, nitric oxide, and the derivative reactive species of nitric oxide during stress responses, as well as by exogenous electrophiles including compounds in foods and environmental pollutants. Among electrophiles formed endogenously, 8-nitroguanosine 3’,5’-cyclic monophosphate (8-nitro-cGMP) has unique cell signaling functions, and pathways for its biosynthesis, signaling mechanism, and metabolism in cells have been clarified. Reactive persulfide species such as cysteine persulfides and polysulfides that are endogenously produced in cells are likely to be involved in 8-nitro-cGMP metabolism. These new aspects of redox biology may stimulate innovative and multidisciplinary research in cardiovascular physiology and pathophysiology. In our review, we focus on the redox-dependent regulation of electrophilic signaling via reduction and metabolism of electrophiles by reactive persulfides in cardiac cells, and we include suggestions for a new therapeutic strategy for cardiovascular disease..
29. Tsukasa Shimauchi, Takuro Numaga-Tomita, Tomoya Ito, Akiyuki Nishimura, Ryosuke Matsukane, Sayaka Oda, Sumio Hoka, Tomomi Ide, Norimichi Koitabashi, Koji Uchida, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy, JCI Insight, 10.1172/jci.insight.93358, 2017.08, Myocardial atrophy is a wasting of cardiac muscle due to hemodynamic unloading. Doxorubicin is a highly effective anticancer agent but also induces myocardial atrophy through a largely unknown mechanism. Here, we demonstrate that inhibiting transient receptor potential canonical 3 (TRPC3) channels abolishes doxorubicin-induced myocardial atrophy in mice. Doxorubicin increased production of ROS in rodent cardiomyocytes through hypoxic stress-mediated upregulation of NADPH oxidase 2 (Nox2), which formed a stable complex with TRPC3. Cardiomyocyte-specific expression of TRPC3 C-terminal minipeptide inhibited TRPC3-Nox2 coupling and suppressed doxorubicin-induced reduction of myocardial cell size and left ventricular (LV) dysfunction, along with its upregulation of Nox2 and oxidative stress, without reducing hypoxic stress. Voluntary exercise, an effective treatment to prevent doxorubicin-induced cardiotoxicity, also downregulated the TRPC3-Nox2 complex and promoted volume load-induced LV compliance, as demonstrated in TRPC3-deficient hearts. These results illustrate the impact of TRPC3 on LV compliance and flexibility and, focusing on the TRPC3-Nox2 complex, provide a strategy for prevention of doxorubicin-induced cardiomyopathy..
30. Caroline Sunggip, Akiyuki Nishimura, Kakeru Shimoda, Takuro Numaga-Tomita, Makoto Tsuda, Motohiro Nishida, Purinergic P2Y6 receptors
A new therapeutic target of age-dependent hypertension, Pharmacological Research, 10.1016/j.phrs.2017.03.013, 2017.06, Aging has a remarkable effect on cardiovascular homeostasis and it is known as the major non-modifiable risk factor in the development of hypertension. Medications targeting sympathetic nerve system and/or renin-angiotensin-aldosterone system are widely accepted as a powerful therapeutic strategy to improve hypertension, although the control rates remain unsatisfactory especially in the elder patients with hypertension. Purinergic receptors, activated by adenine, uridine nucleotides and nucleotide sugars, play pivotal roles in many biological processes, including platelet aggregation, neurotransmission and hormone release, and regulation of cardiovascular contractility. Since clopidogrel, a selective inhibitor of G protein-coupled purinergic P2Y12 receptor (P2Y12R), achieved clinical success as an anti-platelet drug, P2YRs has been attracted more attention as new therapeutic targets of cardiovascular diseases. We have revealed that UDP-responsive P2Y6R promoted angiotensin type 1 receptor (AT1R)-stimulated vascular remodeling in mice, in an age-dependent manner. Moreover, the age-related formation of heterodimer between AT1R and P2Y6R was disrupted by MRS2578, a P2Y6R-selective inhibitor. These findings suggest that P2Y6R is a therapeutic target to prevent age-related hypertension..
31. Sarawuth Phosri, Ajaree Arieyawong, Kwanchai Bunrukchai, Warisara Parichatikanond, Akiyuki Nishimura, Motohiro Nishida, Supachoke Mangmool, Stimulation of adenosine A2B receptor inhibits endothelin-1-induced cardiac Fibroblast proliferation and α-smooth muscle actin synthesis through the cAMP/Epac/PI3K/Akt-signaling pathway, Frontiers in Pharmacology, 10.3389/fphar.2017.00428, 2017.06, Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express α-smooth muscle actin (α-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and α-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A2 receptor inhibit ET-1-induced fibroblast proliferation and α-SMA expression in cardiac fibroblasts are not clearly identified. Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of α-SMA. Key results: Stimulation of adenosine subtype 2B (A2B) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced α-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A2B receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation. Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A2B receptor. Thus, A2B receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis..
32. 抗酸化レドックスと活性イオウによる解毒代謝機構の新展開 環境親電子ストレスの新規分子メカニズム 生体内パースルフィド解毒制御系の破綻.
33. 細菌性アミノアシルtRNA合成酵素のパースルフィド産生能に関する研究.
34. Regulation of Cardiovascular Risk by Environmental Electrophiles.
35. Tsukasa Shimauchi, Akiyuki Nishimura, Tatsuya Ishikawa, Motohiro Nishida, Eco-pharma of approved drug focused on mitochondria fission, Folia Pharmacologica Japonica, 10.1254/fpj.149.269, 2017.01.
36. Hideshi Ihara, Shingo Kasamatsu, Atsushi Kitamura, Akira Nishimura, Hiroyasu Tsutsuki, Tomoaki Ida, Kento Ishizaki, Takashi Toyama, Eiko Yoshida, Hisyam Abdul Hamid, Minkyung Jung, Tetsuro Matsunaga, Shigemoto Fujii, Tomohiro Sawa, Motohiro Nishida, Yoshito Kumagai, Takaaki Akaike, Exposure to electrophiles impairs reactive persulfide-dependent redox signaling in neuronal cells, Chemical Research in Toxicology, 10.1021/acs.chemrestox.7b00120, 2017.01, Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitrocGMP), accompanied by depletion of reactive persulfide species and 8-SHcGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles..
37. Yasuaki Nakagawa, Toshio Nishikimi, Koichiro Kuwahara, Aoi Fujishima, Shogo Oka, Takayoshi Tsutamoto, Hideyuki Kinoshita, Kazuhiro Nakao, Kosai Cho, Hideaki Inazumi, Hiroyuki Okamoto, Motohiro Nishida, Takao Kato, Hiroyuki Fukushima, Jun K. Yamashita, Wino J. Wijnen, Esther E. Creemers, Kenji Kangawa, Naoto Minamino, Kazuwa Nakao, Takeshi Kimura, MiR30-GALNT1/2 axis-mediated glycosylation contributes to the increased secretion of inactive human prohormone for brain natriuretic peptide (proBNP) from failing hearts, Journal of the American Heart Association, 10.1161/JAHA.116.003601, 2017.01, Background-Recent studies have shown that plasma levels of the biologically inactive prohormone for brain natriuretic peptide (proBNP) are increased in patients with heart failure. This can contribute to a reduction in the effectiveness of circulating BNP and exacerbate heart failure progression. The precise mechanisms governing the increase in proBNP remain unclear, however. Methods and Results-We used our recently developed, highly sensitive human proBNP assay system to investigate the mechanisms underlying the increase in plasma proBNP levels. We divided 53 consecutive patients hospitalized with heart failure into 2 groups based on their aortic plasma levels of immunoreactive BNP. Patients with higher levels exhibited more severe heart failure, a higher proportion of proBNP among the immunoreactive BNP forms secreted from failing hearts, and a weaker effect of BNP as estimated from the ratio of plasma cyclic guanosine monophosphate levels to log-transformed plasma BNP levels. Glycosylation at threonines 48 and 71 of human proBNP contributed to the increased secretion of proBNP by attenuating its processing, and GalNAc-transferase (GALNT) 1 and 2 mediated the glycosylation-regulated increase in cardiac human proBNP secretion. Cardiac GALNT1 and 2 expression was suppressed by microRNA (miR)-30, which is abundantly expressed in the myocardium of healthy hearts, but is suppressed in failing hearts. Conclusions-We have elucidated a novel miR-30-GALNT1/2 axis whose dysregulation increases the proportion of inactive proBNP secreted by the heart and impairs the compensatory actions of BNP during the progression of heart failure..
38. Akiyuki Nishimura, Motohiro Nishida, Purinergic signaling in cardiovascular system, Folia Pharmacologica Japonica, 10.1254/fpj.149.84, 2017.01.
39. Takuro Numaga-Tomita, Naoyuki Kitajima, Takuya Kuroda, Akiyuki Nishimura, Kei Miyano, Satoshi Yasuda, Koichiro Kuwahara, Yoji Sato, Tomomi Ide, Lutz Birnbaumer, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis, Scientific reports, 10.1038/srep39383, 2016.12, Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca 2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca 2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca 2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-Associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-Associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation..
40. Naoyuki Kitajima, Takuro Numaga-Tomita, Masahiko Watanabe, Takuya Kuroda, Akiyuki Nishimura, Kei Miyano, Satoshi Yasuda, Koichiro Kuwahara, Yoji Sato, Tomomi Ide, Lutz Birnbaumer, Hideki Sumimoto, Yasuo Mori, Motohiro Nishida, TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling, Scientific reports, 10.1038/srep37001, 2016.11, Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca 2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca 2+-dependent Nox2 activation through TRPC3-mediated background Ca 2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca 2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca 2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities..
41. 新しいシステインパースルフィド合成酵素の発見とパースルフィドによるミトコンドリア機能制御機構の解明.
42. Shigemoto Fujii, Tomohiro Sawa, Motohiro Nishida, Hideshi Ihara, Tomoaki Ida, Hozumi Motohashi, Takaaki Akaike, Redox signaling regulated by an electrophilic cyclic nucleotide and reactive cysteine persulfides, Archives of Biochemistry and Biophysics, 10.1016/j.abb.2015.11.008, Vol.595, pp.140-146, 2016.04, Reactive oxygen (oxidant) and free radical species are known to cause nonspecific damage of various biological molecules. The oxidant toxicology is developing an emerging concept of the physiological functions of reactive oxygen species in cell signaling regulation. Redox signaling is precisely modulated by endogenous electrophilic substances that are generated from reactive oxygen species during cellular oxidative stress responses. Among diverse electrophilic molecular species that are endogenously generated, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a unique second messenger whose formation, signaling, and metabolism in cells was recently clarified. Most important, our current studies revealed that reactive cysteine persulfides that are formed abundantly in cells are critically involved in the metabolism of 8-nitro-cGMP. Modern redox biology involves frontiers of cell research and stem cell research
medical and clinical investigations of infections, cancer, metabolic syndrome, aging, and neurodegenerative diseases
and other fields. 8-Nitro-cGMP-mediated signaling and metabolism in cells may therefore be potential targets for drug development, which may lead to discovery of new therapeutic agents for many diseases..
43. Motohiro Nishida, Yoshito Kumagai, Hideshi Ihara, Shigemoto Fujii, Hozumi Motohashi, Takaaki Akaike, Redox signaling regulated by electrophiles and reactive sulfur species, Journal of Clinical Biochemistry and Nutrition, 10.3164/jcbn.15-111, Vol.58, No.2, pp.91-98, 2016.03, Redox signaling is a key modulator of oxidative stress induced by nonspecific insults of biological molecules generated by reactive oxygen species. Current redox biology is revisiting the traditional concept of oxidative stress, such that toxic effects of reactive oxygen species are protected by diverse antioxidant systems upregulated by oxidative stress responses that are physiologically mediated by redox-dependent cell signaling pathways. Redox signaling is thus precisely regulated by endogenous electrophilic substances that are generated from reactive oxygen species and nitric oxide and its derivative reactive species during stress responses. Among electrophiles formed endogenously, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) has unique cell signaling functions, and pathways for its biosynthesis, signaling mechanism, and metabolism in cells have been clarified. Reactive sulfur species such as cysteine hydropersulfides that are abundant in cells are likely involved in 8-nitro-cGMP metabolism. These new aspects of redox biology may stimulate innovative and multidisciplinary research in cell and stem cell biology
infectious diseases, cancer, metabolic syndrome, ageing, and neurodegenerative diseases
and other oxidative stress-related disorders. This review focuses on the most recent progress in the biosynthesis, cell signaling, and metabolism of 8-nitro-cGMP, which is a likely target for drug development and lead to discovery of novel therapeutics for many diseases..
44. Leanna R. Gentry, Akiyuki Nishimura, Adrienne D. Cox, Timothy D. Martin, Denis Tsygankov, Motohiro Nishida, Timothy C. Elston, Channing J. Der, Divergent roles of CAAX motif-signaled posttranslational modifications in the regulation and subcellular localization of Ral GTPases, Journal of Biological Chemistry, 10.1074/jbc.M115.656710, 2015.09, The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxylterminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB..
45. Yasuhiro Shinkai, Yumi Abiko, Tomoaki Ida, Takashi Miura, Hidenao Kakehashi, Isao Ishii, Motohiro Nishida, Tomohiro Sawa, Takaaki Akaike, Yoshito Kumagai, Reactive Sulfur Species-Mediated Activation of the Keap1-Nrf2 Pathway by 1,2-Naphthoquinone through Sulfenic Acids Formation under Oxidative Stress, Chemical Research in Toxicology, 10.1021/tx500416y, 2015.05, Sulfhydration by a hydrogen sulfide anion and electrophile thiolation by reactive sulfur species (RSS) such as persulfides/polysulfides (e.g., R-S-SH/R-S-Sn-H(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH2-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH2-SH) and a generator of reactive oxygen species (ROS), we demonstrate that protein thiols can be modified by a reactive sulfenic acid to form disulfide adducts that undergo rapid cleavage in the presence of glutathione (GSH). As expected, 1,2-NQH2-SAc is rapidly hydrolyzed and partially oxidized to yield 1,2-NQ-SH, resulting in a redox cycling reaction that produces ROS through a chemical disproportionation reaction. The sulfenic acid forms of 1,2-NQ-SH and 1,2-NQH2-SH were detected by derivatization experiments with dimedone. 1,2-NQH2-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH2 adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH2-SAc caused an extensive chemical modification of cellular proteins in both cases. Protein adduction by 1,2-NQ through a thio ether (C-S-C) bond slowly declined through a GSH-dependent S-transarylation reaction, whereas that originating from 1,2-NQH2-SAc through a disulfide (C-S-S-C) bond was rapidly restored to the free protein thiol in the cells. Under these conditions, 1,2-NQH2-SAc activated Nrf2 and upregulated its target genes, which were enhanced by pretreatment with buthionine sulfoximine (BSO), to deplete cellular GSH. Pretreatment of catalase conjugated with poly(ethylene glycol) suppressed Nrf2 activation by 1,2-NQH2-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress..
46. Yasuo Mori, Masayuki X. Mori, Motohiro Nishida, Bone and Calcium Research Update 2015. Basic mechanisms underlying calcium signaling and their biological potentiality, 2015.01, Calcium ion (Ca2+) is the only second messenger well recognized for the established biological role among various inorganic ions. When cellular Ca2+ controls diverse biological phenomena, it is regulated under exquisitely precise mechanisms. Many proteins have been identified as Ca2+ signal-regulating factors, and still new "players" are added to the repertory. In this review, we will focus on Ca2+ channels, Ca2+-binding proteins,and signaling pathways controlled by these Ca2+ signal-regulating proteins, in order to discuss on molecular bases, biological significance, and possible future developments of Ca2+ signaling..
47. Yuko Yamada, Hideyuki Kinoshita, Koichiro Kuwahara, Yasuaki Nakagawa, Yoshihiro Kuwabara, Takeya Minami, Chinatsu Yamada, Junko Shibata, Kazuhiro Nakao, Kosai Cho, Yuji Arai, Shinji Yasuno, Toshio Nishikimi, Kenji Ueshima, Shiro Kamakura, Motohiro Nishida, Shigeki Kiyonaka, Yasuo Mori, Takeshi Kimura, Kenji Kangawa, Kazuwa Nakao, Inhibition of N-type Ca2+ channels ameliorates an imbalance in cardiac autonomic nerve activity and prevents lethal arrhythmias in mice with heart failure, Cardiovascular research, 10.1093/cvr/cvu185, 2014.10, Aims Dysregulation of autonomic nervous system activity can trigger ventricular arrhythmias and sudden death in patients with heart failure. N-type Ca2+ channels (NCCs) play an important role in sympathetic nervous system activation by regulating the calcium entry that triggers release of neurotransmitters from peripheral sympathetic nerve terminals. We have investigated the ability of NCC blockade to prevent lethal arrhythmias associated with heart failure. Methods and results We compared the effects of cilnidipine, a dual N- and L-type Ca2+ channel blocker, with those of nitrendipine, a selective L-type Ca2+ channel blocker, in transgenic mice expressing a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). In this mouse model of dilated cardiomyopathy leading to sudden arrhythmic death, cardiac structure and function did not significantly differ among the control, cilnidipine, and nitrendipine groups. However, cilnidipine dramatically reduced arrhythmias in dnNRSF-Tg mice, significantly improving their survival rate and correcting the imbalance between cardiac sympathetic and parasympathetic nervous system activity. A β-blocker, bisoprolol, showed similar effects in these mice. Genetic titration of NCCs, achieved by crossing dnNRSF-Tg mice with mice lacking CACNA1B, which encodes the α1 subunit of NCCs, improved the survival rate. With restoration of cardiac autonomic balance, dnNRSF-Tg;CACNA1B+/- mice showed fewer malignant arrhythmias than dnNRSF-Tg;CACNA1B+/+ mice. Conclusions Both pharmacological blockade of NCCs and their genetic titration improved cardiac autonomic balance and prevented lethal arrhythmias in a mouse model of dilated cardiomyopathy and sudden arrhythmic death. Our findings suggest that NCC blockade is a potentially useful approach to preventing sudden death in patients with heart failure..
48. Motohiro Nishida, Takashi Toyama, Takaaki Akaike, Role of 8-nitro-cGMP and its redox regulation in cardiovascular electrophilic signaling, Journal of Molecular and Cellular Cardiology, 10.1016/j.yjmcc.2014.02.003, 2014.08, Structural and morphological changes of the cardiovascular systems (cardiovascular remodeling) are a major clinical outcome of cardiovascular diseases. Many lines of evidences have implied that transfiguration of reduction/oxidation (redox) homeostasis due to excess production of reactive oxygen species (ROS) and/or ROS-derived electrophilic metabolites (electrophiles) is the main cause of cardiovascular remodeling. Gasotransmitters, such as nitric oxide (NO) and endogenous electrophiles, are considered major bioactive species and have been extensively studied in the context of physiological and pathological cardiovascular events. We have recently found that hydrogen sulfide-related reactive species function as potent nucleophiles to eliminate electrophilic modification of signaling proteins induced by NO-derived electrophilic byproducts (e.g., 8-nitroguanosine 3',5'-cyclic monophosphate and nitro-oleic acid). In this review, we discuss the current understanding of redox control of cardiovascular pathophysiology by electrophiles and nucleophiles. We propose that modulation of electrophile-mediated post-translational modification of protein cysteine thiols may be a new therapeutic strategy of cardiovascular diseases. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System"..
49. Motohiro Nishida, Takashi Toyama, Yoshito Kumagai, Takuro Numaga-Tomita, Establishment of a novel therapeutic strategy for heart failure based on the mechanism underlying maintenance of redox homeostasis by reactive sulfur species, Yakugaku Zasshi, 10.1248/yakushi.14-00209-1, 2014.01, Cardiac redox homeostasis is precisely regulated by reactive oxygen species (ROS) or electrophilic molecules that are formed by ROS reacting with intracellular substrates, and their eliminating systems. We have focused on the role of nitric oxide (NO) generated from inducible NO synthase (iNOS) that is continuously upregulated from early stage of heart failure, and revealed that iNOS-derived NO acts as a protective factor in the early stage of heart failure, whereas it contributes to induction of cardiac early senescence in later stages. The switching mechanism of NO-mediated signaling includes formation of endogenous NO-derived electrophilic byproducts such as 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), which selectively targets an oncogenic small GTPase H-Ras at Cys-184, leading to cardiac cell senescence via covalent modiˆcation (S-guanylation) and activation of H-Ras. We also found that hydrogen sulde-related reactive sulfur species (RSS) function as potent nucleophiles to eliminate electrophilic modiˆcation of HRas and suppress the onset of chronic heart failure after myocardial infarction. Our results strongly suggest a new concept of redox biology in which suppression of electrophilic irreversible modiˆcation of protein cysteine thiols by RSS may be a new therapeutic strategy of cardiovascular diseases..
50. [Regulation of redox homeostasis by hydrogen sulfide anion and its clinical application]..
51. Wen Chen, Heike Oberwinkler, Franziska Werner, Birgit Ganer, Hitoshi Nakagawa, Robert Feil, Franz Hofmann, Jens Schlossmann, Alexander Dietrich, Thomas Gudermann, Motohiro Nishida, Sabrina Del Galdo, Thomas Wieland, Michaela Kuhn, Atrial natriuretic peptide-mediated inhibition of microcirculatory endothelial Ca2+ and permeability response to histamine involves cGMP-dependent protein kinase i and TRPC6 channels, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/ATVBAHA.113.001974, 2013.09, Objective-Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and-independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI). Approach and Results-We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial- restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion. Conclusions-ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP..
52. Kenji Watari, Nakaya Michio, Motohiro Nishida, Kyeong Man Kim, Hitoshi Kurose, β-arrestin2 in Infiltrated Macrophages Inhibits Excessive Inflammation after Myocardial Infarction, PloS one, 10.1371/journal.pone.0068351, 2013.07, Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation..
53. Caroline Sunggip, Naoyuki Kitajima, Motohiro Nishida, Redox control of cardiovascular homeostasis by angiotensin II, Current Pharmaceutical Design, 10.2174/1381612811319170008, 2013.06, Covalent modification of sulfur-containing amino acids in proteins by reactive oxygen species (ROS) has been attracting attention as a major post-translational modification regulating intracellular signal transduction pathways. Angiotensin II (Ang II), a major physiologically active substrate in renin-angiotensin (RAS) system, plays a central role in the pathophysiology of cardiovascular systems. Many evidences show that Ang II activates several signaling pathways via an oxidative modification of proteins by Ang II-induced ROS. Ang II induced ROS production is predominantly regulated by three enzymes: NADPH oxidase, mitochondrial respiratory complex, and nitric oxide synthase (NOS), and each enzyme-generating ROS are found to activate appropriate signaling pathways via selective oxidation of specific proteins. These reactions are negatively regulated by ROS-scavenging enzymes or disulfide bridge reducing enzymes, and functional disorders of these enzymes are found to cause cardiovascular dysfunctions. Thus, the spatial and temporal regulation of oxidative modification of signaling proteins by ROS is essential to maintain cardiovascular homeostasis by Ang II. This review brings in the new aspect in understanding ROS-mediated regulation of cardiovascular homeostasis by Ang II, and provides the possible mechanisms underlying metamorphosis of cardiovascular homeostasis by ROS..
54. Motohiro Nishida, Tatsuya Ishikawa, Shota Saiki, Caroline Sunggip, Shizuka Aritomi, Eri Harada, Koichiro Kuwahara, Katsuya Hirano, Yasuo Mori, Shokei Kim-Mitsuyama, Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.03.040, 2013.05, N-type voltage-dependent Ca2+channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca2+ influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice..
55. Motohiro Nishida, Masaaki Sumita, Naoyuki Kitajima, [Function and role of transient receptor potential channels]., 2013.04, Transient receptor potential (TRP) proteins are components of Ca(2 +) -permeable non-selective cation channels activated by physical and chemical stimulus except for membrane depolarization. The pathophysiological role of TRP channels is documented in heart failure. Especially, canonical TRP subfamily C (TRPC) channels activated by neurohumoral factors have been implicated in the structural remodeling of the heart. TRPC proteins act not only as components of receptor-activated cation channels, but also as protein scaffold to form protein complex with intracellular signaling proteins, leading to amplification of receptor signaling. Recently, selective inhibitors of TRPC channels have been continuously identified, anticipating the possibility of drug discovery targeting TRPC channels for the treatment of heart failure..
56. Michio Nakaya, Mitsuru Tajima, Hidetaka Kosako, Takeo Nakaya, Akiko Hashimoto, Kenji Watari, Hiroaki Nishihara, Mina Ohba, Shiori Komiya, Naoki Tani, Motohiro Nishida, Hisaaki Taniguchi, Yoji Sato, Mitsuru Matsumoto, Makoto Tsuda, Masahiko Kuroda, Kazuhide Inoue, Hitoshi Kurose, GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance, Nature communications, 10.1038/ncomms2540, 2013.03, Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80 + macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis..
57. Takaaki Akaike, Motohiro Nishida, Shigemoto Fujii, Regulation of redox signalling by an electrophilic cyclic nucleotide, Journal of Biochemistry, 10.1093/jb/mvs145, 2013.02, Reactive oxygen species (ROS) have been believed to be toxic substances that induce nonspecific damage in various biological molecules. ROS toxicology is now developing an emerging concept for physiological functions of ROS in the regulation of cell signal transductions. ROS signalling functions and their mechanisms are precisely regulated by several endogenous moderate electrophiles that are themselves generated from ROS during diverse physiological and pathophysiological cellular responses. The chemical biology of electrophiles is an emerging scientific area involving molecular mechanisms that conduct ROS cell signals through receptors to effector molecules at molecular, cellular and organism levels. The formation, signalling and metabolism of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) in cells are probably precisely regulated, and nonselective ROS reactions can be converted into stable, well-controlled electrophilic signal transduction via 8-nitro-cGMP. Modern redox biology is today advancing its frontier of basic research and clinical medicine, including infection, cancer biology, metabolic syndromes, ageing and even stem cell research. As one aspect of this advance, the 8-nitro-cGMP-mediated signalling that may be integrated into cells as a major redox signalling pathway may be a potential target in drug development and may lead to discovery of new therapeutic agents for various diseases..
58. 北島 直幸, 西田 基宏, 硫化水素のケミカルバイオロジー：心不全抑制効果の新しいメカニズム, 日本薬理学雑誌, 10.1254/fpj.141.350, Vol.141, No.6, pp.350-351, 2013.01.
59. Tomohiro Sawa, Hideshi Ihara, Tomoaki Ida, Shigemoto Fujii, Motohiro Nishida, Takaaki Akaike, Formation, signaling functions, and metabolisms of nitrated cyclic nucleotide, Nitric Oxide - Biology and Chemistry, 10.1016/j.niox.2013.04.004, 2013.01, 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a unique derivative of guanosine 3',5'- cyclic monophosphate (cGMP) formed in mammalian and plant cells in response to production of nitric oxide and reactive oxygen species. 8-Nitro-cGMP possesses signaling activity inherited from parental cGMP, including induction of vasorelaxation through activation of cGMP-dependent protein kinase. On the other hand, 8-nitro-cGMP mediates cellular signaling that is not observed for native cGMP, e.g., it behaves as an electrophile and reacts with protein sulfhydryls, which results in cGMP adduction to protein sulfhydryls (protein S-guanylation). Several proteins have been identified as targets for endogenous protein S-guanylation, including Kelch-like ECH-associated protein 1 (Keap1), H-Ras, and mitochondrial heat shock proteins. 8-Nitro-cGMP signaling via protein S-guanylation of those proteins may have evolved to convey adaptive cellular stress responses. 8-Nitro-cGMP may not undergo conventional cGMP metabolism because of its resistance to phosphodiesterases. Hydrogen sulfide has recently been identified as a potent regulator for metabolisms of electrophiles including 8-nitro-cGMP, through sulfhydration of electrophiles, e.g., leading to the formation of 8-SH-cGMP. Better understanding of the molecular basis for the formation, signaling functions, and metabolisms of 8-nitro-cGMP would be useful for the development of new diagnostic approaches and treatment of diseases related to oxidative stress and redox metabolisms..
60. Caroline Sunggip, Naoyuki Kitajima, Motohiro Nishida, Redox control of cardiovascular homeostasis by angiotensin II, Current Pharmaceutical Design, 10.2174/1381612811319170008, 2013, Covalent modification of sulfur-containing amino acids in proteins by reactive oxygen species (ROS) has been attracting attention as a major post-translational modification regulating intracellular signal transduction pathways. Angiotensin II (Ang II), a major physiologically active substrate in renin-angiotensin (RAS) system, plays a central role in the pathophysiology of cardiovascular systems. Many evidences show that Ang II activates several signaling pathways via an oxidative modification of proteins by Ang II-induced ROS. Ang II induced ROS production is predominantly regulated by three enzymes: NADPH oxidase, mitochondrial respiratory complex, and nitric oxide synthase (NOS), and each enzyme-generating ROS are found to activate appropriate signaling pathways via selective oxidation of specific proteins. These reactions are negatively regulated by ROS-scavenging enzymes or disulfide bridge reducing enzymes, and functional disorders of these enzymes are found to cause cardiovascular dysfunctions. Thus, the spatial and temporal regulation of oxidative modification of signaling proteins by ROS is essential to maintain cardiovascular homeostasis by Ang II. This review brings in the new aspect in understanding ROS-mediated regulation of cardiovascular homeostasis by Ang II, and provides the possible mechanisms underlying metamorphosis of cardiovascular homeostasis by ROS..
61. Motohiro Nishida, Tomohiro Sawa, Naoyuki Kitajima, Katsuhiko Ono, Hirofumi Inoue, Hideshi Ihara, Hozumi Motohashi, Masayuki Yamamoto, Makoto Suematsu, Hitoshi Kurose, Albert Van Der Vliet, Bruce A. Freeman, Takahiro Shibata, Koji Uchida, Yoshito Kumagai, Takaaki Akaike, Hydrogen sulfide anion regulates redox signaling via electrophile sulfhydration, Nature Chemical Biology, 10.1038/nchembio.1018, 2012.08, An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS-) regulates the metabolism and signaling actions of various electrophiles. HS- reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS- biosynthesis. Cardiac HS -, in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS - on myocardial infarction-associated heart failure. Thus, this study reveals HS--induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling..
62. Takeo Fujino, Tomomi Ide, Masayoshi Yoshida, Ken Onitsuka, Atsushi Tanaka, Yuko Hata, Motohiro Nishida, Takako Takehara, Takaaki Kanemaru, Naoyuki Kitajima, Shinya Takazaki, Hitoshi Kurose, Dongchon Kang, Kenji Sunagawa, Recombinant mitochondrial transcription factor A protein inhibits nuclear factor of activated T cells signaling and attenuates pathological hypertrophy of cardiac myocytes, Mitochondrion, 10.1016/j.mito.2012.06.002, 2012.07, The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy..
63. MPP+神経細胞毒性における一酸化窒素‐活性酸素シグナル.
64. Takeo Fujino, Tomomi Ide, Masayoshi Yoshida, Ken Onitsuka, Atsushi Tanaka, Yuko Hata, Takako Takehara, Kazuya Hosokawa, Takafumi Sakamoto, Motohiro Nishida, Kenji Sunagawa, Recombinant Mitochondrial Transcriptional Factor A Protein Attenuates Pathological Remodeling in Cardiac Myocytes, CIRCULATION, Vol.124, No.21, 2011.11.
65. 赤池 孝章, 澤 智裕, 西田 基宏, 硫化水素により調節されるROSと求電子性細胞内シグナル伝達(ROS and electrophilic cellular signaling regulated by hydrogen sulfide), 日本生化学会大会プログラム・講演要旨集, Vol.84回, pp.1S10p-1, 2011.09.
66. Nishida M, Kitajima N, Saiki S, Nakaya M & Kurose H, Regulation of angiotensin II receptor signaling by cysteine modification of NF-kB., Nitric Oxide, 2011.08.
67. Motohiro Nishida, Naoyuki Kitajima, Shota Saiki, Michio Nakaya, Hitoshi Kurose, Regulation of Angiotensin II receptor signaling by cysteine modification of NF-κB, Nitric Oxide - Biology and Chemistry, 10.1016/j.niox.2010.10.003, 2011.08, Angiotensin II (Ang II) is a major vasoactive peptide of the renin-angiotensin system. Ang II is originally found as one of potent vasoconstrictors, but is now attracted attention as an essential mediator of many cardiovascular problems, including endothelial dysfunction, arrhythmia and structural remodeling of cardiovascular systems. Most of the known pathophysiological effects of Ang II are mediated through Ang type1 receptors (AT 1Rs), and the up-regulation of AT 1Rs is one of important causes by which Ang II can contribute to cardiovascular diseases. A growing body of evidence has suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in the regulation of AT 1R signaling. In cardiac fibroblasts, stimulation with cytokines or bacterial toxins induces AT 1R up-regulation through NADPH oxidase-dependent ROS production. In contrast, nitric oxide (NO) decreases AT 1R density through cysteine modification (S-nitrosylation) of a transcriptional factor, nuclear factor κB (NF-κB). The difference between the effects of ROS and NO on AT 1R expression may be caused by the difference between intracellular location of ROS signaling and that of NO signaling, as the agonist-induced S-nitrosylation of NF-κB requires a local interaction between NO synthase (NOS) and NF-κB in the perinuclear region. Thus, the spatial and temporal regulation of cysteine modification by ROS or RNS may underlie the resultant changes of AT 1R signaling induced by agonist stimulation..
68. H. Kinoshita, K. Kuwahara, M. Nishida, H. Kurose, S. Kiyonaka, Y. Mori, Y. Kuwabara, Y. Nakagawa, K. Ueshima, K. Nakao, Blockade of TRPC6/3 is a novel therapeutic approach for preventing pathological cardiac hypertrophy, EUROPEAN HEART JOURNAL, Vol.32, pp.1093-1094, 2011.08.
69. Nishida M, Roles of heterotrimetric GTP-binding proteins in the progression of heart failure., J. Pharmacol. Sci., 2011.06.
70. Takeo Fujino, Tomomi Ide, Yuko Hata, Takako Takehara, Masayoshi Yoshida, Ken Onitsuka, Atsushi Tanaka, Shinya Takazaki, Motohiro Nishida, Donchon Kang, Kenji Sunagawa, Recombinant TFAM attenuates pathological hypertrophy of cardiac myocytes via inhibiting NFAT signaling, FASEB JOURNAL, Vol.25, 2011.04.
71. Megumi Sugihara, Hiromitsu Morita, Miho Matsuda, Hisanori Umebayashi, Shunichi Kajioka, Shinichi Ito, Motohiro Nishida, Ryosuke Inoue, Toshiko Futatsuki, Jun Yamazaki, Yasuo Mori, Ryuji Inoue, Yushi Ito, Kihachiro Abe, Masato Hirata, Dual signaling pathways of arterial constriction by extracellular uridine 5′-triphosphate in the rat, Journal of Pharmacological Sciences, 10.1254/jphs.10281FP, 2011.03, We investigated actions of uridine 5′-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X1 receptor, and a similar current was also induced by α,β-methylene adenosine 5′-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X 1-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 ≥M) provoked a dose-dependent contraction of deendothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca2+ or bath-applied 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca2+]i were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X1 receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X1-like and P2Y receptors, but not TRPC3..
72. Mari Tomonari, Hideto To, Motohiro Nishida, Takashi Mishima, Hitoshi Sasaki, Hitoshi Kurose, Mechanism of the cardioprotective effects of docetaxel pre-administration against adriamycin-induced cardiotoxicity, Journal of Pharmacological Sciences, 10.1254/jphs.10279FP, 2011.03, We revealed that pre-treatment with docetaxel (DOC) 12 h before adriamycin (ADR) administration significantly reduced ADR-induced toxic death compared with the simultaneous dosing schedule that was commonly used in previous studies. We considered that pre-treatment with DOC relieves ADR-induced cardiotoxicity. In this study, we investigated the influence of DOC on the pharmacokinetics and pharmacodynamics of ADR in order to clarify the mechanism by which DOC pre-treatment relieves ADR-induced cardiotoxicity. When ADR and/or DOC was intravenously administered, the DOC pre-treatment (DOC-ADR) group showed significantly less toxic death than the ADR-alone group. We examined hepatopathy, nephropathy, leukopenia, and cardiotoxicity, all of which can cause toxic death. Of these toxicities, ADR-induced cardiotoxicity was significantly relieved in the DOC-ADR group. To elucidate the mechanism by which DOC pre-treatment relieved ADR-induced cardiotoxicity, lipid peroxidation as a proxy for the free radical level and the pharmacokinetics of ADR were measured. There was no difference in the pharmacokinetics of ADR between the ADR and DOC-ADR groups. On the other hand, the DOC-ADR group showed significantly inhibited lipid peroxidation in the heart compared with the ADR group. It was considered that DOC pre-administration inhibited ADR-induced free radicals and decreased cardiotoxicity..
73. Nakaya M, Ohba M, Nishida M & Kurose H., Determining the activation of Rho as an index of coupling to G12/13., Methods in Molecular Biology, 2010.12.
74. Motohiro Nishida, Shota Saiki, Naoyuki Kitajima, Michio Nakaya, Yoji Sato, Hitoshi Kurose, Regulation of cardiovascular functions by the phosphorylation of TRPC channels, Yakugaku Zasshi, 10.1248/yakushi.130.1427, 2010.11, Calcium ions (Ca2+) play an essential role in homeostasis and the activity of cardiovascular cells. Ca2+ influx across the plasma membrane induced by neurohumoral factors or mechanical stress elicits physiologically relevant timing and spatial patterns of Ca2+ signaling, which leads to the activation of various cardiovascular functions, such as muscle contraction, gene expression, and hypertrophic growth of myocytes. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca2+ signaling pathway required for pathological hypertrophy. We have recently found that the inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppresses agonist- and mechanical stretch-induced hypertrophy through inhibition of Ca2+ influx in rat cardiomyocytes. The inhibition of PDE5 suppressed the increase in frequency of Ca2+ spikes induced by receptor stimulation or mechanical stretch. Activation of protein kinase G by PDE5 inhibition phosphorylated TRPC6 proteins at Thr69 and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr69 in TRPC6 abolished the antihypertrophic effects of PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlies the prevention of cardiac hypertrophy by PDE5 inhibition. As TRPC6 proteins are also expressed in vascular smooth muscle cells and reportedly participate in vascular remodeling, TRPC6 blockade may be an effective therapeutic strategy for preventing pathologic cardiovascular remodeling..
75. Motohiro Nishida, Signal transduction in the development of vascular disease and new therapeutic strategy, Yakugaku Zasshi, 10.1248/yakushi.130.1397, 2010.11.
76. Motohiro Nishida, Mina Ohba, Michio Nakaya, Hitoshi Kurose, Molecular mechanism underlying the development of heart failure mediated by heterotrimeric G protein signaling, Folia Pharmacologica Japonica, 10.1254/fpj.135.179, 2010.07.
77. Hideyuki Kinoshita, Koichiro Kuwahara, Motohiro Nishida, Zhong Jian, Xianglu Rong, Shigeki Kiyonaka, Yoshihiro Kuwabara, Hitoshi Kurose, Ryuji Inoue, Yasuo Mori, Yuhao Li, Yasuaki Nakagawa, Satoru Usami, Masataka Fujiwara, Yuko Yamada, Takeya Minami, Kenji Ueshima, Kazuwa Nakao, Inhibition of TRPC6 channel activity contributes to the antihypertrophic effects of natriuretic peptides-guanylyl cyclase-a signaling in the heart, Circulation research, 10.1161/CIRCRESAHA.109.208314, 2010.06, Rationale: Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized. Objective: We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca channel known to positively regulate prohypertrophic calcineurin-nuclear factor of activated T cells (NFAT) signaling. Methods and results: In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca2+ influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II-induced cardiac hypertrophy in mice. Conclusions: Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling..
78. Motohiro Nishida, Mariko Ogushi, Reiko Suda, Miyuki Toyotaka, Michio Nakaya, Hitoshi Kurose, ATP decreases angiotensin type 1 receptor expression through S-nitrosylation of nuclear factor kappa B, NITRIC OXIDE-BIOLOGY AND CHEMISTRY, 10.1016/j.niox.2010.05.087, Vol.22, p.S31, 2010.06.
79. Motohiro Nishida, Reiko Suda, Yuichi Nagamatsu, Shihori Tanabe, Naoya Onohara, Michio Nakaya, Yasunori Kanaho, Takahiro Shibata, Koji Uchida, Hideki Sumimoto, Yoji Sato, Hitoshi Kurose, Pertussis toxin up-regulates angiotensin type 1 receptors through toll-like receptor 4-mediated Rac activation, Journal of Biological Chemistry, 10.1074/jbc.M109.076232, 2010.05, Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from Gi and Go through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1β among cytokines. Inhibition of IL-1β suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1β and AT1R through NF-κB, and a small GTP-binding protein, Rac, mediated PTX-induced NF-κB activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1β siRNA, suggesting that IL-1β-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of Gi/Go by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of Gi/Go..
80. Motohiro Nishida, Kenta Watanabe, Yoji Sato, Michio Nakaya, Naoyuki Kitajima, Tomomi Ide, Ryuji Inoue, Hitoshi Kurose, Phosphorylation of TRPC6 channels at Thr69 is required for anti-hypertrophic effects of phosphodiesterase 5 inhibition, Journal of Biological Chemistry, 10.1074/jbc.M109.074104, 2010.04, Activation of Ca2+ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca2+ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca2+ influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca2+ spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca2+ influx itself or molecule(s) upstream of Ca2+ influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr69 and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr69 in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce Gαq-mediated signaling, did not affect the suppression of receptor-activated Ca2+ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition..
81. Takuro Numaga, Motohiro Nishida, Shigeki Kiyonaka, Kenta Kato, Masahiro Katano, Emiko Mori, Tomohiro Kurosaki, Ryuji Inoue, Masaki Hikida, James W. Putney, Yasuo Mori, Ca2+ influx and protein scaffolding via TRPC3 sustain PKCβ and ERK activation in B cells, Journal of Cell Science, 10.1242/jcs.061051, 2010.03, Ca2+ signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca2+ entry activated by Ins(1,4,5)P3-induced Ca2+ release from endoplasmic reticulum, the importance of DAG-activated Ca2+ entry remains elusive. Here, we describe the physiological role of DAG-activated Ca2+ entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cβ (PKCβ) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCβ that maintained localization of PKCβ at the PM. Thus, TRPC3 functions as both a Ca2+-permeable channel and a protein scaffold at the PM for downstream PKCβ activation in B cells..
82. Motohiro Nishida, Kenta Watanabe, Nakaya Michio, Hitoshi Kurose, Mechanism of cardiac hypertrophy via diacylglycerol-sensitive TRPC channels, Yakugaku Zasshi, 10.1248/yakushi.130.295, 2010.03, Activation of Ca2+ signaling in cardiomyocytes induced by receptor stimulation or mechanical stress has been implicated in the development of cardiac hypertrophy. However, it is still unclear how intracellular Ca 2+ targets specifically decode the alteration of intracellular Ca2+ concentration ([Ca2+]i) on the background of the rhythmic Ca2+ increases required for muscle contraction. In excitable cardiomyocytes, changes in the frequency or amplitude of Ca 2+ transients evoked by Ca2+ influx-induced Ca 2+ release have been suggested to encode signals for induction of hypertrophy, and a partial depolarization of plasma membrane by receptor stimulation will increase the frequency of Ca2+ oscillations. We found that activation of diacylglycerol (DAG)-responsive canonical transient receptor potential (TRPC) subfamily channels (TRPC3 and TRPC6) mediate membrane depolarization induced by Gq protein-coupled receptor stimulation. DAG-mediated membrane depolarization through activation of TRPC3/TRPC6 channels increases the frequency of Ca2+ spikes, leading to activation of calcineurin-dependent signaling pathways. Inhibition of either TRPC3 or TRPC6 completely suppressed agonist-induced hypertrophic responses, suggesting that TRPC3 and TRPC6 form heterotetramer channels. Furthermore, we found that hypertrophic agonists increase the expression of TRPC6 proteins through activation of G12 family proteins, leading to amplification of DAG-mediated hypertrophic signaling in cardiomyocytes. As heart failure proceeds through cardiac hypertrophy, TRPC3/TRPC6 channels may be a new therapeutic target for heart failure..
83. Shigeki Kiyonaka, Kenta Kato, Motohiro Nishida, Yasuo Mori, Pharmacological properties of novel TRPC channel inhibitors, Yakugaku Zasshi, 10.1248/yakushi.130.303, 2010.03, Ca2+ signals control diverse cellular processes, ranging from ubiquitous activities like gene expression to tissue specific responses such as lymphocyte activation and cardiac diseases. TRPC channels control Ca 2+ influxes that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a novel pyrazole compound (Pyr3) which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In B lymphocytes, Pyr3 eliminated the B cell receptor-induced Ca2+ oscillation regulated by TRPC3-mediated Ca2+ influx. In the cardiac system, Pyr3 attenuates activation of nuclear factor of activated T cells and hypertrophic growth in myocytes and pressure overload-induced hypertrophy in vivo. Thus, the TRPC3-selective inhibitor Pyr3 is useful for treatments of TRPC3-mediated diseases and for clarification of crucial and widespread functions of TRPC3 as well..
84. Motohiro Nishida, Yoji Sato, Michio Nakaya, Hitoshi Kurose, Regulation of cardiac hypertrophy by the formation of G protein-coupled receptor-TRPC channel protein complex, Folia Pharmacologica Japonica, 10.1254/fpj.134.131, 2009.12.
85. Yano Takahisatyano, Itoh Yoshinori, Kawamura Eiko, Maeda Asuka, Egashira Nobuaki, Nishida Motohiro, Kurose Hitoshi, Oishi Ryozo, Amphotericin B-induced renal tubular cell injury is mediated by Na+ influx through ion-permeable pores and subsequent activation of mitogen-activated protein kinases and elevation of intracellular Ca2+ concentration, Antimicrobial Agents and Chemotherapy, 10.1128/AAC.01137-08, 2009.04, Amphotericin B (AMB) is one of the most effective antifungal agents; however, its use is often limited by the occurrence of adverse events, especially nephrotoxicity. The present study was designed to determine the possible mechanisms underlying the nephrotoxic action of AMB. The exposure of a porcine proximal renal tubular cell line (LLC-PKl cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, the leakage of lactate dehydrogenase, and tissue ATP depletion. Propldium iodide uptake was enhanced, while terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was not affected by AMB, suggesting a lack of involvement of apoptosis in AMB-induced cell injury. The cell injury was inhibited by the depletion of membrane cholesterol with methyl-ß-cyclodextrin, which lowered the extracellular Na+ concentration or the chelation of intracellular Ca. The rise in the intracellular Ca2+ concentration may be mediated through the activation of the ryanodine receptor (RyR) on the endoplasmic reticulum and the mitochondrial Na+-Ca2+ exchanger, since cell injury was attenuated by dantrolene (an RyR antagonist) and CGP37157 (an Na+-Ca exchanger inhibitor). Moreover, AMB-induced cell injury was reversed by PD169316 (a p38 mitogen-activated protein [MAP] kinase inhibitor), c-Jun N-terminal kinase inhibitor II, and PD98059 (a MEK1/2 inhibitor). The phosphorylations of these MAP kinases were enhanced by AMB in a calcium-independent manner, suggesting the involvement of MAP kinases in AMB-induced cell injury. These findings suggest that Na+ entry through membrane pores formed by the association of AMB with membrane cholesterol leads to the activation of MAP kinases and the elevation of the intracellular Ca2+concentration, leading to renal tubular cell injury..
86. Shigeki Kiyonaka, Kenta Kato, Motohiro Nishida, Kazuhiro Mio, Takuro Numaga, Yuichi Sawaguchi, Takashi Yoshida, Minoru Wakamori, Emiko Mori, Tomohiro Numata, Masakazu Ishii, Hiroki Takemoto, Akio Ojida, Kenta Watanabe, Aya Uemura, Hitoshi Kurose, Takashi Morii, Tsutomu Kobayashi, Yoji Sato, Chikara Sato, Itaru Hamachi, Yasuo Mori, Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0808793106, 2009.03, Canonical transient receptor potential (TRPC) channels control influxes of Ca2+ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca2+ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca2+ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca2+-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3- selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy..
87. Kinoshita Hideyuki, Kuwahara Koichiro, Inoue Ryuji, Nishida Motohiro, Kurose Hitoshi, Kiyonaka Shigeki, Mori Yasuo, Li Yuhao, Rong Xiang Lu, Murakami Masao, Nakagawa Yasuaki, Yasuno Shinji, Usami Satoru, Fujiwara Masataka, Kuwabara Yoshihiro, Yamada Yuko, Minami Takeshi, Harada Masaki, Ueshima Kenji, Nakao Kazuwa, DPE-007 Protein Kinase G-mediated Inhibition of TRPC6 Channel Activity Participates in Anti-hypertrophic Effect of Cardiac Natriuretic Peptides(DPE02,Heart Failure, Basic (M),Digital Poster Session (English),The 73rd Annual Scientific Meeting of The Japanese Circulation Society), Circulation journal : official journal of the Japanese Circulation Society, Vol.73, p.357, 2009.03.
88. Takahiro Shibata, Hiroko Nakahara, Narumi Kita, Yui Matsubara, Chunguang Han, Yasujiro Morimitsu, Noriko Iwamoto, Yoshito Kumagai, Motohiro Nishida, Hitoshi Kurose, Naohito Aoki, Makoto Ojika, Koji Uchida, A food-derived synergist of NGF signaling
Identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor-initiated signal transduction, Journal of Neurochemistry, 10.1111/j.1471-4159.2008.05686.x, 2008.12, Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic- promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates..
89. Motohiro Nishida, Yoji Sato, Aya Uemura, Yusuke Narita, Hidetoshi Tozaki-Saitoh, Michio Nakaya, Tomomi Ide, Kazuhiro Suzuki, Kazuhide Inoue, Taku Nagao, Hitoshi Kurose, P2Y6 receptor-Gα12/13 signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis, EMBO Journal, 10.1038/emboj.2008.237, 2008.12, Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)-β. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Gα12/13) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF-β, connective tissue growth factor, and periostin) and Ang-converting enzyme (ACE) was suppressed by the functional inhibition of Gα12/13. The expression of these fibrogenic genes through Gα12/13 by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G-protein-coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Gα12/13 in cardiomyocytes by the extracellular nucleotides-stimulated P2Y6 receptor triggers fibrosis in pressure overload-induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF-β..
90. Motohiro Nishida, Hitoshi Kurose, Roles of TRP channels in the development of cardiac hypertrophy, NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 10.1007/s00210-008-0321-8, Vol.378, No.4, pp.395-406, 2008.10, Cardiac hypertrophy is induced by various stresses such as hypertension and myocardial infarction. It is believed that hypertrophy is adaptive in the early phase but becomes maladaptive in the late phase. Cardiac hypertrophy develops heart failure when the heart is exposed persistently to the stresses. The increase in intracellular Ca(2+) ([Ca(2+)](i)) plays an important role in the development of hypertrophy. It is generally thought that the increase in [Ca(2+)](i) for hypertrophy occurs via G(q)-stimulated production of inositol-1,4,5-trisphosphate (IP(3)) and IP(3)-mediated release of Ca(2+) from intracellular store. However, several groups recently reported that canonical transient receptor potential (TRPC) channels are responsible for the increase in [Ca(2+)](i). Among them, three TRPC subtypes (TRPC3/TRPC6/TRPC7) are activated by another G(q)-mediated second messenger, diacylglycerol. Although several groups independently demonstrated that TRPC channels mediate receptor-stimulated and pressure overload-induced hypertrophy, there is discrepancy of which subtypes of TRPC channels predominantly mediate hypertrophy. However, there is consensus that TRPC-mediated Ca(2+) influx is essential for hypertrophy. As TRPC channels participate in pathological hypertrophy, but not physiological contraction and the relaxation cycle, TPRC channels are a new target for the treatment of hypertrophy..
91. Motohiro Nishida, TRPC, 2008.08.
92. Yuri Kusano, Shunsuke Horie, Takahiro Shibata, Hideo Satsu, Makoto Shimizu, Eri Hitomi, Motohiro Nishida, Hitoshi Kurose, Ken Itoh, Akira Kobayashi, Masayuki Yamamoto, Koji Uchida, Keap1 regulates the constitutive expression of GST A1 during differentiation of caco-2 cells, Biochemistry, 10.1021/bi800199z, 2008.06, Kelch-like ECH-associated protein 1 (Keap1), a BTB-Kelch substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex, regulates the induction of the phase 2 enzymes, such as glutathione S-transferase (GST), by repressing the transcription factor Nrf2. It is known that, in the human gastrointestinal tract, both GST A1 and P1 are constitutively expressed as the major GST isozymes. In the present study, using the Keap1-overexpressing derivatives of Caco-2 cells, human carcinoma cell line of colonic origin, by stable transfection of wild type Keap1, we investigated the molecular mechanism underlying the constitutive expression of these GST isozymes during differentiation. It was revealed that the overexpression of Keap1 completely repressed the constitutive expression of GST A1, but not GST P1. In Keap1-overexpressed cells, dome formation disappeared, and the formation of the intact actin cytoskeletal organization at cell-cell contact sites and the recruitment of E-cadherin and β-catenin to adherens junctions were inhibited. The constitutive GST A1 expression in Caco-2 cells was repressed by disruption of E-cadherin-mediated cell-cell adhesion, suggesting the correlation between epithelial cell polarization and induction of the basal GST A1 expressions during Caco-2 differentiation. Keap1 overexpression indeed inhibited the activation of the small guanosine triphosphatase Rac1 on the formation of E-cadherin-mediated cell-cell adhesion. The transfection of V12Rac1, the constitutively active Rac1 mutant, into Keap1-overexpressed cells promoted the basal GST A1 expression, suggesting that Keap1 regulated the basal GST Al1 expression during Caco-2 differentiation via Rac1 activation on the formation of E-cadherin-mediated cell-cell adhesion. The results of this study suggest the involvement of a novel Keap1-dependent signaling pathway for the induction of the constitutive GST A1 expression during epithelial cell differentiation..
93. Yamamoto S, Wajima T, Hara Y, Nishida M & Mori Y., Transient receptor potential channels in Alzheimer's disease., 2007.08.
94. Motohiro Nishida, Naoya Onohara, Yoji Sato, Reiko Suda, Mariko Ogushi, Shihori Tanabe, Ryuji Inoue, Yasuo Mori, Hitoshi Kurose, Gα12/13-mediated up-regulation of TRPC6 negatively regulates endothelin-1-induced cardiac myofibroblast formation and collagen synthesis through nuclear factor of activated T cells activation, Journal of Biological Chemistry, 10.1074/jbc.M611780200, 2007.08, Sustained elevation of [Ca2+]i has been implicated in many cellular events. We previously reported that α subunits of G 12 family G proteins (Gα12/13) participate in sustained Ca2+ influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that Gα12/13-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca2+ influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active Gα12 or Gα13 increased the expression of TRPC6 proteins and basal Ca2+ influx activity. The treatment with ET-1 increased TRPC6 protein levels through Gα12/13, reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca2+]i through up-regulated TRPC6. A Gα12/13-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a Gα12/13-reactive oxygen species-JNK pathway. The ET-1-induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of Gα12/13 in cardiac fibroblasts. First, Gα12/13 mediate ET-1-induced myofibroblast formation. Second, Gα12/13 mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts..
95. Motohiro Nishida, Naoya Onohara, Hitoshi Kurose, G alpha(13)-TRPC6-NFAT signaling pathway negatively regulates cardiac myofibroblast formation, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 10.1016/j.yjmcc.2007.03.117, Vol.42, pp.S41-S42, 2007.06.
96. M. Nishida, R. Sudal, Y. Sato, H. Kurose, RAC-ROS-NF-kappa B-dependent up-regulation of angiotensin type 1 receptors in rat cardiac fibroblasts, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 10.1016/j.yjmcc.2007.03.083, Vol.42, pp.S29-S30, 2007.06.
97. Motohiro Nishida, Naoya Onohara, Hitoshi Kurose, Diacylglycerol-mediated Ca2+ influx through TRPC3/6 is essential for Angiotensin II-induced cardiac hypertrophy, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 10.1016/j.yjmcc.2006.08.039, Vol.41, No.6, p.1046, 2006.12.
98. Naoya Onohara, Motohiro Nishida, Ryuji Inoue, Hiroyuki Kobayashi, Hideki Sumimoto, Yoji Sato, Yasuo Mori, Taku Nagao, Hitoshi Kurose, TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy, EMBO Journal, 10.1038/sj.emboj.7601417, 2006.11, Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP3) is responsible for Ca2+ increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca2+ oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca2+ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca2+ signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy..
99. Supachoke Mangmool, Tatsuya Haga, Hiroyuki Kobayashi, Kyeong Man Kim, Hiroyasu Nakata, Motohiro Nishida, Hitoshi Kurose, Clathrin required for phosphorylation and internalization of β2-adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2), Journal of Biological Chemistry, 10.1074/jbc.M602832200, 2006.10, Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the β2-adrenergic receptor (β2AR). However, the interaction of β2AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of β2AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis..
100. Motohiro Nishida, Yuji Hara, Takashi Yoshida, Ryuji Inoue, Yasuo Mori, TRP channels
Molecular diversity and physiological function, Microcirculation, 10.1080/10739680600885111, 2006.10, Calcium ions (Ca2+) are particularly important in cellular homeostasis and activity. To elicit physiologically relevant timing and spatial patterns of Ca2+ signaling, ion channels in the surface of each cell precisely control Ca2+ influx across the plasma membrane. A group of surface membrane ion channels called receptor-activated cation/Ca2+ channels (RACCs) are activated by diverse cellular stimuli from the surrounding extracellular environment via receptors and other pathways such as heat, osmotic pressure, and mechanical and oxidative stress. An important clue to understanding the molecular mechanisms underlying the functional diversity of RACCs was first attained by molecular identification of the transient receptor potential (trp) protein (TRP), which mediates light-induced depolarization in Drosophila photoreceptor cells, and its homologues from various biological species. Recent studies have revealed that respective TRP channels are indeed activated by characteristic cellular stimuli. Furthermore, the involvement of TRP channels has been demonstrated in the signaling pathways essential for tissue-specific functions as well as ubiquitous biological responses, such as cell proliferation, differentiation, and death. These findings encourage the usage of TRP channels and their signalplexes as powerful tools for developing novel pharmaceutical targets..
101. Hitoshi Kurose, Reiko Suda, Naoya Onohara, Supachoke Mangmool, Yuichi Nagamatsu, Yoji Sato, Taku Nagao, Motohiro Nishida, Rac up-regulates angiotensin II type 1 receptors through ROS and NF-kappa B-dependent interleukin-1 beta production in rat cardiac fibroblasts, CIRCULATION, Vol.114, No.18, pp.86-87, 2006.10.
102. Nishida M, Hara Y, Yoshida T, Inoue R & Mori Y., TRP channels: formation of signal complex and regulation of cellular functions, 2006.09.
103. Koji Yamazaki, Eiji Shigetomi, Ryo Ikeda, Motohiro Nishida, Shigeki Kiyonaka, Yasuo Mori, Fusao Kato, Blocker-resistant presynaptic voltage-dependent Ca2+ channels underlying glutamate release in mice nucleus tractus solitarii, Brain Research, 10.1016/j.brainres.2006.05.077, 2006.08, The visceral sensory information from the internal organs is conveyed via the vagus and glossopharyngeal primary afferent fibers and transmitted to the second-order neurons in the nucleus of the solitary tract (NTS). The glutamate release from the solitary tract (TS) axons to the second-order NTS neurons remains even in the presence of toxins that block N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs). The presynaptic VDCC playing the major role at this synapse remains unidentified. To address this issue, we examined two hypotheses in this study. First, we examined whether the remaining large component occurs through activation of a ω-conotoxin GVIA (ω-CgTX)-insensitive variant of N-type VDCC by using the mice genetically lacking its pore-forming subunit α1B. Second, we examined whether R-type VDCCs are involved in transmitter release at the TS-NTS synapse. The EPSCs evoked by stimulation of the TS were recorded in medullary slices from young mice. ω-Agatoxin IVA (ω-AgaIVA; 200 nM) did not significantly affect the EPSC amplitude in the mice genetically lacking N-type VDCC. SNX-482 (500 nM) and Ni2+ (100 μM) did not significantly reduce EPSC amplitude in ICR mice. These results indicate that, unlike in most of the brain synapses identified to date, the largest part of the glutamate release at the TS-NTS synapse in mice occurs through activation of non-L, non-P/Q, non-R, non-T and non-N (including its posttranslational variants) VDCCs at least according to their pharmacological properties identified to date..
104. Ryuji Inoue, Lars Jørn Jensen, Juan Shi, Hiromitsu Morita, Motohiro Nishida, Akira Honda, Yushi Ito, Transient receptor potential channels in cardiovascular function and disease, Circulation research, 10.1161/01.RES.0000233356.10630.8a, 2006.07, Sustained elevation in the intracellular Ca concentration via Ca influx, which is activated by a variety of mechanisms, plays a central regulatory role for cardiovascular functions. Recent molecular biological research has disclosed an unexpectedly diverse array of Ca-entry channel molecules involved in this Ca influx. These include more than ten transient receptor potential (TRP) superfamily members such as TRPC1, TRPC3-6, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and polycystin (TRPP2). Most of them appear to be multimodally activated or modulated and show relevant features to both acute hemodynamic control and long-term remodeling of the cardiovascular system, and many of them have been found to respond not only to receptor stimulation but also to various forms of stimuli. There is good evidence to implicate TRPC1 in neointimal hyperplasia after vascular injury via store-depletion-operated Ca entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca mobilizations, being involved in vasoconstrictor and myogenic responses and pulmonary arterial proliferation and its associated disease (idiopathic pulmonary arterial hypertension). Considerable evidence has also been accumulated for unique involvement of TRPV1 in blood flow/pressure regulation via sensory vasoactive neuropeptide release. New lines of evidence suggest that TRPV2 may act as a Ca-overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg entry channel, and TRPP2 as a Ca-entry channel requisite for vascular integrity. This review attempts to provide an overview of the current knowledge on TRP proteins and discuss their possible roles in cardiovascular functions and diseases..
105. Inoue R, Jensen LJ, Shi J, Morita H, Nishida M, Honda A & Ito Y, Transient receptor potential (TRP) channels in cardiovascular function and disease, Circ. Res, 99, 119-131, 2006.06.
106. Yuichi Nagamatsu, Motohiro Nishida, Naoya Onohara, Masashi Fukutomi, Yoshiko Maruyama, Hiroyuki Kobayashi, Yoji Sato, Hitoshi Kurose, Heterotrimeric G protein Gα13-induced induction of cytokine mRNAs through two distinct pathways in cardiac fibroblasts, Journal of Pharmacological Sciences, 10.1254/jphs.FP0051036, 2006.06, Overexpression of constitutively active (CA)-Gα13 significantly increased the expression of interleukin (IL)-1β and IL-6 mRNAs and proteins in rat cardiac fibroblasts. IL-1β mRNA induction by CA-Gα13 was suppressed by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, but not by BAPTA-AM, an intracellular Ca2+ chelator. In contrast, IL-6 mRNA induction by CA-Gα13 was suppressed by BAPTA-AM but not by DPI. However, both IL-1β and IL-6 mRNA induction was suppressed by nuclear factor κB (NF-κB) inhibitors. The CA-Gα13-induced NF-κB activation was suppressed by DPI and BAPTA-AM, but not C3 toxin and the Rho-kinase inhibitor Y27632. IL-6 mRNA induction by CA-Gα13 was suppressed by SK&F96365 (1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), an inhibitor of receptor-activated nonselective cation channels, and the expression of CA-Gα13 increased basal Ca2+ influx. These results suggest that Gα13 regulates IL-1β mRNA induction through the reactive oxygen species-NF-κB pathway, while it regulates IL-6 mRNA induction through the Ca2+-NF-κB pathway..
107. Yasushi Okamura, Atsuo Nishino, Yoshimichi Murata, Koichi Nakajo, Hirohide Iwasaki, Yukio Ohtsuka, Motoko Tanaka-Kunishima, Nobuyuki Takahashi, Yuji Hara, Takashi Yoshida, Motohiro Nishida, Haruo Okado, Hirofumi Watari, Ian A. Meinertzhagen, Nori Satoh, Kunitaro Takahashi, Yutaka Satou, Yasunobu Okada, Yasuo Mori, Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes, Physiological Genomics, 10.1152/physiolgenomics.00229.2004, 2005.10, Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment..
108. Tomomi Fujii, Naoya Onohara, Yoshiko Maruyama, Shihori Tanabe, Hiroyuki Kobayashi, Masashi Fukutomi, Yuichi Nagamatsu, Naoki Nishihara, Ryuji Inoue, Hideki Sumimoto, Futoshi Shibasaki, Taku Nagao, Motohiro Nishida, Hitoshi Kurose, Gα12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts, Journal of Biological Chemistry, 10.1074/jbc.M409397200, Vol.280, No.24, pp.23041-23047, 2005.06, Angiotensin II (Ang II) activates multiple signaling pathways leading to hyperplasia of cardiac fibroblasts. Reactive oxygen species (ROS) produced by Ang II stimulation are assumed to play pivotal roles in this process. Here, we show that ROS mediate Ang II-induced activation of nuclear factor of activated T cells (NFAT) in rat cardiac fibroblasts. Ang II-induced NFAT activation was suppressed by diphenyleneiodonium (an NADPH oxidase inhibitor), dominant negative (DN)-Rac, DN-p47phox, and an inhibitor of Gα12/13 (Gα12/13-specific regulator of G protein signaling domain of p115RhoGEF, p115-regulator of G protein signaling (RGS)). Stimulation of Ang II receptor increased the intracellular ROS level in a Rac- and p47phox-dependent manner. Because p115-RGS suppressed Ang II-induced Rac activation, Ang II receptor-coupled Gα12/13 mediated NFAT activation through ROS production by Rac activation. Ang II-induced nuclear translocation of the green fluorescent protein (GFP)-tagged amino-terminal region of NFAT4 (GFP-NFAT4) was suppressed by p115-RGS or BAPTA but not by diphenyleneiodonium. The expression of constitutively active (CA)-Gα12, CA-Gα13, or CA-Rac increased the nuclear translocation of GFP-NFAT4. These results suggest that NFAT activity is regulated by both Ca2+-dependent and ROS-dependent pathways. Furthermore, activation of c-Jun NH2-terminal kinase (JNK) induced by Ang II stimulation is required for NFAT activation because Ang II-induced NFAT activation was inhibited by SP600125, a selective JNK inhibitor. These results indicate that Ang II stimulates the nuclear translocation and activation of NFAT by integrated pathways including the activation of Gα12/13, Rac, NADPH oxidase, and JNK and that Gα12/13-mediated ROS production is essential for NFAT transcriptional activation. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc..
109. 若年性ミオクロニーてんかん(JME)責任遺伝子の同定.
110. M Nishida, T Fujii, N Onohara, M Fukutomi, Y Nagamatsu, Y Maruyama, H Kobayashi, F Shibasaki, T Nagao, H Kurose, G alpha(13) regulates NFAT activity through ROS production in cardiac fibroblasts, CIRCULATION, Vol.110, No.17, p.257, 2004.10.
111. Toshimits Suzuki, Antonio Delgado-Escueta V, Kripamoy Aguan, Maria E. Alonso, Jun Shi, Yuji Haras, Motohiro Nishidas, Tomohiro Numata, Marco T. Medina, Tamaki Takeuchi, Ryoji Morita, Dongsheng Bai, Subramaniam Ganesh, Yoshihisa Sugimoto, Johji Inazawa, Julia N. Bailey, Adriana Ochoa, Aurelio Jara-Prado, Astrid Rasmussen, Jaime Ramos-Peek, Sergio Cordova, Francisco Rubio-Donnadieu, Yushi Inoue, Makiko Osawa, Sunao Kaneko, Hirokazu Oguni, Yasuo Mori, Kazuhiro Yamakawa, Mutations in EFHC1 cause juvenile myoclonic epilepsy, Nature genetics, 10.1038/ng1393, 2004.08, Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures1,2. We previously mapped and narrowed a region associated with JME on chromosome 6p12-p11 (EJM1)3-5. Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca2+ channel (Cav2.3). EFHC1 and Cav2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Cav2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca2+ currents that were reversed by the mutations associated with JME..
112. 鈴木 俊光, Delgado-Escueta Av, Aguan K, 原 雄二, 西田 基宏, Numata T, 竹内 環, Bai D, 井上 有史, 大沢 真木子, 兼子 直, 小国 弘量, 森 泰生, 山川 和弘, 若年性ミオクローヌスてんかん原因遺伝子の単離に向けて(Towards the identification of genes responsible for juvenile myoclonic epilepsy), 神経化学, Vol.43, No.2-3, p.512, 2004.08.
113. 繁冨 英治, 山崎 弘二, 西田 基宏, 森 泰生, 加藤 総夫, N型電位依存性カルシウムチャネル欠損マウスにおける孤束核シナプス伝達のGタンパク質共役型受容体による制御(Altered mechanism of transmitter release regulation by G-protein-coupled receptors in the autonomic sensory synapse in mice lacking N-type calcium channels), 神経化学, Vol.43, No.2-3, p.463, 2004.08.
114. Mori Y, Itsukaichi Y, Nishida M & Oka H, Ca2+ channel mutations and associated diseases., 2004.06.
115. Kenji Sugimoto, Motohiro Nishida, Masami Otsuka, Keisuke Makino, Katsutoshi Ohkubo, Yasuo Mori, Takashi Morii, Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells, 10.1016/j.chembiol.2004.03.019, 2004.04, Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP3) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) δ. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP3 binding pocket for enhanced IP3 selectivity and for rapid and direct visualization of intracellular IP3 ≥ 0.5 μM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP3 production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP3 probe was extremely effective to quantitatively assess real-time physiological IP3 production via those pathways formed only in the intact cellular configuration..
116. U937におけるH2O2刺激によるTRPM2を介したCa2+流入のサイトカイン産生及びアポトーシス誘導に及ぼす影響.
117. Motohiro Nishida, Kenji Sugimoto, Yuji Hara, Emiko Mori, Takashi Morii, Tomohiro Kurosaki, Yasuo Mori, Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCγ2 in B lymphocytes, EMBO Journal, 10.1093/emboj/cdg457, 2003.09, In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cγ2 (PLCγ2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCγ2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCγ2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCγ2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCγ2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCγ2. Strikingly, Ca2+ entry-activated PLCγ2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca 2+ entry with PLCγ2 translocation and activation controls the amplification and co-ordination of receptor signalling..
118. Jun Kitano, Motohiro Nishida, Yuko Itsukaichi, Itsunari Minami, Masaaki Ogawa, Tomoo Hirano, Yasuo Mori, Shigetada Nakanishi, Direct Interaction and Functional Coupling between Metabotropic Glutamate Receptor Subtype 1 and Voltage-sensitive Cav2.1 Ca2+ Channel, 10.1074/jbc.M303266200, 2003.07, Intracellular Ca2+ concentrations ([Ca2+] i) are regulated in a spatiotemporal manner via both entry of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Metabotropic glutamate receptor subtype 1 (mGluR1) is a G protein-coupled receptor that stimulates the inositol 1,4,5-trisphosphate-Ca2+ signaling cascade, whereas Cav2. 1 is a pore-forming channel protein of P/Q-type voltage-sensitive Ca 2+ channels. In this investigation, we showed that mGluR1 and Ca v2.1 are colocalized at dendrites of cerebellar Purkinje neurons and form the heteromeric assembly in both the brain and heterologously expressing COS-7 cells. This assembly occurs through the direct interaction between their carboxyl-terminal intracellular domains. Calcium imaging and whole-cell recording showed that mGluR1 inhibits Cav2.1-mediated [Ca 2+]i increases and Ba2+ currents in HEK 293 cells expressing Cav2.1 with auxiliary α2/δ and β1 subunits, respectively. This inhibition occurred in a ligand-independent manner and was enhanced by pre-activation of mGluR1 in a ligand-dependent manner. In contrast, simultaneous stimulation of mGluR1 and Cav2.1 induced large [Ca2+]i increases. Furthermore, the temporally regulated inhibition and stimulation of [Ca2+]i increases by mGluR1 and Cav2.1 were observed at dendrites but not soma of cultured Purkinje neurons. These data suggest that the assembly of mGluR1 and Cav2.1 provides the mechanism that ensures spatiotemporal regulation of [Ca2+]i in glutamatergic neurotransmission..
119. Shunichi Shimizu, Kazuhiro Shiota, Shinichiro Yamamoto, Yoshiyuki Miyasaka, Masakazu Ishii, Tatsuya Watabe, Motohiro Nishida, Yasuo Mori, Toshinori Yamamoto, Yuji Kiuchi, Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells, Free Radical Biology and Medicine, 10.1016/S0891-5849(03)00172-2, 2003.05, Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H2O2), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H2O2 to ECs markedly increased the BH4 level, but not its oxidized forms. The H2O2-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H2O2 induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H2O2-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H2O2. Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H2O2, and the H2O2-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H2O2 induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H2O2 appears to be one of the important signaling molecules to regulate the BH4-NOS system..
120. TRP channels : formation of signal complex and regulation of cellular functions
Cellular stimulation from the surrounding extracellular environment via receptors and other pathways evoke activation of Ca2+-permeable cation channels. An important clue to understand the molecular mechanisms underlying these receptor-activated cation channels (RACC) was first provided through molecular studies of the transient receptor potential (trp) protein (TRP), which controls light-induced deporlarization in Drosophila photoreceptor cells. Recent studies have revealed that these TRP channels are also activated by diverse stimuli such as heat, osmotic stress, and oxidative stress. Furthermore, involvement of TRP channels has been demonstrated in signaling pathways essential for biological responses, such as proliferation, differentiation, and cell death. These findings encourage usage of TRP channels and their signalplexes as powerful tools for the development of novel pharmaceutical targets.
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121. Motohiro Nishida, Yuji Hara, Ryuji Inoue, Yasuo Mori, TRP channels
Formation of signal complex and regulation of cellular functions, Folia Pharmacologica Japonica, 10.1254/fpj.121.223, 2003.04, Cellular stimulation from the surrounding extracellular environment via receptors and other pathways evoke activation of Ca2+-permeable cation channels. An important clue to understand the molecular mechanisms underlying these receptor-activated cation channels (RACC) was first provided through molecular studies of the transient receptor potential (trp) protein (TRP), which controls light-induced deporlarization in Drosophila photoreceptor cells. Recent studies have revealed that these TRP channels are also activated by diverse stimuli such as heat, osmotic stress, and oxidative stress. Furthermore, involvement of TRP channels has been demonstrated in signaling pathways essential for biological responses, such as proliferation, differentiation, and cell death. These findings encourage usage of TRP channels and their signalplexes as powerful tools for the development of novel pharmaceutical targets..
122. Ken Arai, Yoshiko Maruyama, Motohiro Nishida, Shihori Tanabe, Shuichi Takagahara, Tohru Kozasa, Yasuo Mori, Taku Nagao, Hitoshi Kurose, Differential requirement of Gα12, Gα13, Gα13, and Gβγ for endothelin-1-induced c-Jun NH2-terminal kinase and extracellular signal-regulated kinase activation, Molecular Pharmacology, 10.1124/mol.63.3.478, Vol.63, No.3, pp.478-488, 2003.03, In the present study, we examined the roles of G12, G13, Gq, and Gi in endothelin-1-induced hypertrophic responses. Endothelin-1 stimulation activated extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in cultured rat neonatal myocytes. The activation of JNK, but not ERK, was inhibited by the expression of carboxyl terminal regions of Gα12 and Gα13. JNK activation was also inhibited by expression of the Gα12/Gα13-specific inhibitor regulator of G protein signaling (RGS) domain of p115RhoGEF and the Gαq-specific inhibitor RGS domain of the G protein-coupled receptor kinase 2 (GRK2-RGS). JNK activation was not, however, inhibited by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2 (GRK2-ct), which is a Gβγ-sequestering polypeptide. Additionally, JNK activation but not ERK activation was inhibited by the expression of C3 exoenzyme that inactivates small GTPase Rho. These results suggest that JNK activation by Gα12, Gα13, and Gαq is involved in Rho. On the other hand, ERK activation was inhibited by pertussis toxin treatment, the receptor-Gi uncoupler, and GRK2-ct. Thus, ERK was activated by Gαi- and Gβγ-dependent pathways. These results clearly demonstrate that differential pathways activate JNK and ERK..
123. Mori Y, Nishida M, Shimizu S, Ishii M, Yoshinaga T, Ino M, Sawada K & Niidome T, Mice lacking the a1B subunit (CaV 2.2) reveals a predominant role of N-type Ca2+ channels in the sympathetic regulation of circulatory system., 2002.12.
124. Y Mori, M Nishida, S Shimizu, M Ishii, T Yoshinaga, M Ino, K Sawada, T Niidome, Ca2+ channel alpha(1B) subunit (Ca-V 2.2) knockout mouse reveals a predominant role of N-type channels in the sympathetic regulation of the circulatory system, TRENDS IN CARDIOVASCULAR MEDICINE, Vol.12, No.6, pp.270-275, 2002.08, N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been proposed to play vital roles in a variety of neuronal functions such as neurotransmitter release at sympathetic nerve terminals. To directly approach the elucidation of the physiological significance of N-type VDCCs in the autonomic nervous system, alpha(1B) subunit (Ca-V 2.2)-deficient mice were generated, in which peripheral neurons showed a complete and selective elimination of N-type channel currents sensitive to omega-conotoxin GVIA (the peptide toxin from the fish-hunting cone snail Conus geographus), without a significant effect on the activity of other VDCC types. In isolated left atria prepared from N-type-deficient mice, the positive inotropic response mediated by the sympathetic nervous system was dramatically decreased, whereas the negative inotropic response mediated by parasympathetic neurons was nearly intact compared with those of normal mice. The baroreflex response to bilateral carotid occlusion was markedly reduced in the mutant mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct in vivo evidence for an essential role of N-type VDCCs in maintaining the normal function of the sympathetic nervous system in circulatory regulation, demonstrating a potential of N-type VDCC-deficient mice as a useful model for studying disorders attributable to sympathetic nerve dysfunction. (C) 2002, Elsevier Science Inc..
125. Motohiro Nishida, Kevin L. Schey, Shuichi Takagahara, Kenji Kontani, Toshiaki Katada, Yasuteru Urano, Tetsuo Nagano, Taku Nagao, Hitoshi Kurose, Activation mechanism of Gi and Go by reactive oxygen species, Journal of Biological Chemistry, 10.1074/jbc.M107392200, 2002.03, Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the a subunit of heterotrimeric GTP-binding proteins (Gαi and Gαo), leading to activation. H2O2 is one of the reactive oxygen species and activates purified Gαi2. However, the activation requires the presence of Fe2+, suggesting that H2O2 is converted to more reactive species such as ·OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys66, Cys112, Cys140, Cys255, Cys287, Cys326, and Cys352) of Gαi2 are modified by the treatment with ·OH. Among these cysteine residues, Cys66, Cys112, Cys140, Cys255, and Cys352 are not involved in ·OH-induced activation of Gαi2. Although the modification of Cys287 but not Cys326 is required for subunit dissociation, the modification of both Cys287 and Cys326 is necessary for the activation of Gαi2 as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5′.3-O.(thio)triphosphate binding. Wild type Gαi2 but not Cys287- or Cys326-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Gαi2 by the mechanism similar to ·OH-induced activation. Because Cys287 exists only in Gi family, this study explains the selective activation of Gi/Go by oxidative stresses..
126. Motohiro Nishida, Shuichi Takagahara, Yoshiko Maruyama, Yoshiyuki Sugimoto, Taku Nagao, Hitoshi Kurose, Gβγ counteracts Gαq signaling upon α1-adrenergic receptor stimulation, Biochemical and Biophysical Research Communications, 10.1006/bbrc.2002.6553, 2002.03, In rat neonatal myocytes, a constitutively active Gαq causes cellular injury and apoptosis. However, stimulation of the α1-adrenergic receptor, one of the Gq protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the Gβγ-sequestering peptide βARK-ct increases the phenylephrine-induced cardiac injury, indicating that Gβγ released from Gq counteracts the Gαq-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by βARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that Gβγ released from Gq upon α1-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the Gαq-induced cellular injury and apoptosis..
127. Yasuo Mori, Minoru Wakamori, Tomoya Miyakawa, Meredith Hermosura, Yuji Hara, Motohiro Nishida, Kenzo Hirose, Akiko Mizushima, Mari Kurosaki, Emiko Mori, Kumiko Gotoh, Takaharu Okada, Andrea Fleig, Reinhold Penner, Masamitsu Iino, Tomohiro Kurosaki, Transient receptor potential 1 regulates capacitative Ca2+ entry and Ca2+ release from endoplasmic reticulum in B lymphocytes, Journal of Experimental Medicine, 10.1084/jem.20011758, 2002.03, Capacitative Ca2+ entry (CCE) activated by release/depletion of Ca2+ from internal stores represents a major Ca2+ influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca2+ release-activated Ca2+ currents and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca2+ oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP3 receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca2+ signaling in B lymphocytes..
128. Yuji Hara, Minoru Wakamori, Masakazu Ishii, Emi Maeno, Motohiro Nishida, Takashi Yoshida, Hisanobu Yamada, Shunichi Shimizu, Emiko Mori, Jun Kudoh, Nobuyoshi Shimizu, Hitoshi Kurose, Yasunobu Okada, Keiji Imoto, Yasuo Mori, LTRPC2 Ca2+-Permeable channel activated by changes in redox status confers susceptibility to cell death, Molecular Cell, 10.1016/S1097-2765(01)00438-5, 2002.01, Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (β-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFα-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death..
129. M Nishida, KL Schey, S Takagahara, T Nagao, H Kurose, Activation of G(i)alpha-2 by reactive oxygen species, CIRCULATION, Vol.104, No.17, p.230, 2001.10.
130. K Arai, Y Maruyama, M Nishida, T Kozasa, T Nagao, H Kurose, G alpha(12) and G alpha(13) mediate endothelin-1-induded cardiomyocyte hypertrophy, CIRCULATION, Vol.104, No.17, p.111, 2001.10.
131. Y Maruyama, M Nishida, T Kozasa, T Nagao, H Kurose, alpha(1)-adrenergic receptor-induced cardiomyocyte hypertrophy is mediated by G alpha(12/13) as well as G alpha(Q)., CIRCULATION, Vol.104, No.17, p.198, 2001.10.
132. Motohiro Nishida, Yoshiko Maruyama, Rie Tanaka, Kenji Kontani, Taku Nagao, Hitoshi Kurose, Gα(i) and Gα(o) are target proteins of reactive oxygen species, Nature, 10.1038/35044120, 2000.11, Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the βγ-subunit of G protein (Gβγ) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The Gβγ-responsive ERK activation induced by H2O2 is independent of ligands binding to G(i)-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [35S]GTPγS binding to cardiac membranes and directly activates purified heterotrimeric G(i) and G(o) but not G(s). Analysis using heterotrimeric G(o) and its individual subunits indicates that H2O2 modifies Gα(o) but not Gβγ, which leads to subunit dissociation. We conclude that Gα(i) and Gα(o) are critical targets of oxidative stress for activation of ERK..
133. M Nishida, Y Maruyama, R Tanaka, K Kontani, T Nagao, H Kurose, G alpha(i) and G alpha(o) are target proteins of reactive oxygen species, NATURE, 10.1038/35044120, Vol.408, No.6811, pp.492-495, 2000.11, Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events(1-4). In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury(5,6) and in cytokine-stimulated hypertrophy(7). Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS4,8; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the beta gamma -subunit of G protein (G beta gamma) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The G beta gamma -responsive ERK activation induced by H2O2 is independent of ligands binding to G(i)-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [S-35]GTP-gammaS binding to cardiac membranes and directly activates purified heterotrimeric G(i) and G(o) but not G(s). Analysis using heterotrimeric G(o) and its individual subunits indicates that H2O2 modifies G alpha (o) but not G beta gamma, which leads to subunit dissociation. We conclude that G alpha (i) and G alpha (o) are critical targets of oxidative stress for activation of ERK..
134. M Nishida, T Nagao, H Kurose, G(i)alpha and G(o)alpha are target proteins of reactive oxygen intermediates leading to activation of extracellular signal-regulated kinase, CIRCULATION, Vol.102, No.18, p.352, 2000.10.
135. M Nishida, T Urushidani, K Sakamoto, T Nagao, L-cis diltiazem attenuates intracellular Ca2+ overload by metabolic inhibition in guinea pig myocytes, EUROPEAN JOURNAL OF PHARMACOLOGY, 10.1016/S0014-2999(99)00709-8, Vol.385, No.2-3, pp.225-230, 1999.12, We have previously demonstrated that treatment with L-cis diltiazem reduced cardiac infarct size in vivo. To examine the effect of L-cis diltiazem on Ca2+ overload induced by ischemia/reperfusion, we used a model for Ca2+ overload produced by metabolic inhibition in isolated guinea pig myocytes. Intracellular Ca2+ concentration ([Ca2+](i)) was quantified by fura-2 fluorescence microscopy and Ca2+ overload was induced by inclusion of 1 mu M of carbonyl cyanide m-chrolophenylhydrazone (CCCP) for 40 min treatment followed by washout for 30 min. This treatment caused a large [Ca2+], elevation as well as a sustained contracture of the cardiomyocytes. The increase was suppressed by 10 mu M of 2-[2-[4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea methanesulphonate (KB-R7943), a specific inhibitor of the Na+/Ca2+ exchanger, but not by nitrendipine (10 mu M). L-cis Diltiazem (10 mu M) attenuated the [Ca2+](i) increase, suggesting that L-cis diltiazem elicits a cardioprotective effect via attenuation of the [Ca2+](i) increase induced by metabolic inhibition and energy repletion. (C) 1999 Elsevier Science B.V. All rights reserved..
136. Hitoshi Kurose, Motohiro Nishida, Taku Nagao, βγ subunit of heterotrimeric G protein as a new target molecule for drug development, Folia Pharmacologica Japonica, 1999.11, Although ischemia-reperfusion produces reactive oxygen species and induces injury of the heart, the mechanism leading to injury is largely unknown. Hydrogen peroxide (H2O2) is widely used for a reagent to mimic the action of reactive oxygen species produced by ischemia-reperfusion. Treatment of the rat neonatal myocytes with H2O2 resulted in activation of mitogen- activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38. To study the involvement of βγ subunit of heterotrimeric G protein in H2O2-induced activation of MAPKs, we expressed the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2-ct) which can bind βγ subunit and inhibit the interaction with various effector proteins. Expression of GRK2-ct inhibited the H2O2-induced activation of ERK by 70% and also inhibited the activation of Akt by 30%. In contrast with H2O2-induced activation of ERK, the activation of ERK induced by phorbol ester PMA and the activation of JNK and p38 induced by H2O2 were not affected by expression of GRK2-ct, indicating that the activation of ERK but not JNK and p38 is dependent on βγ subunit. Among several inhibitors for analyzing intracellular signaling pathways, wortmannin inhibited the activation of ERK by H2O2 treatment. These data suggest that treatment of the rat neonatal myocytes with H2O2 releases βγ subunit from heterotrimeric G protein, and leads to activation of ERK in part by phosphatidylinositol-3 kinase dependent pathway. Thus βγ subunit may be a novel target molecule to selectively modulate the intracellular signaling cascade..
137. M Nishida, K Sakamoto, T Urushidani, T Nagao, Treatment with l-cis diltiazem before reperfusion reduces infarct size in the ischemic rabbit heart in vivo, JAPANESE JOURNAL OF PHARMACOLOGY, 10.1254/jjp.80.319, Vol.80, No.4, pp.319-325, 1999.08, l-cis Diltiazem, an optical isomer of diItiazem, protects against myocardial dysfunction in vitro, whereas its Ca2+ channel blocking activity is about 100 times less potent than that of diltiazem. However, there is no evidence that I-cis diItiazem actually protects against ischemia/reperfusion injury in vivo. To assess this, we employed an anesthetized rabbit model, where the left circumflex artery was occluded for 15 min and reperfused for 360 min. Treatment with diltiazem before and during ischemia (bolus 200 mu g/kg and 15 mu g/kg per minute for 25 min, i.v.; 575 mu g/kg total) showed slightly depressed hemodynamic parameters, while I-cis diltiazem (1150 mu g/kg) had no effect. Treatment with I-cis diItiazem produced a high recovery of the thickening fraction and limited the infarct size in a dose-dependent manner. Furthermore, the treatment with l-cis diltiazem (1150 mu g/kg) or diltiazem (575 mu g/kg) 5 min before reperfusion also limited the infarct size, but not after reperfusion. These results suggest that l-cis diltiazem affects some events in the onset of reperfusion, independently of Ca2+-channel-blocking action. Our observations are the first to show that I-cis diltiazem demonstrated its cardioprotective action in the ischemic rabbit heart in vivo..
138. M Nishida, T Nagao, H Kurose, Activation of Rac1 increases c-Jun NH2-terminal kinase activity and DNA fragmentation in a calcium-dependent manner in rat myoblast cell line H9c2, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1006/bbrc.1999.1218, Vol.262, No.2, pp.350-354, 1999.08, We examined the role of intracellular Ca2+ in c-Jun NH2-terminal kinase (JNK) activation and DNA fragmentation in the rat myoblast cell line H9c2 using small GTP-binding protein Rad. A constitutively active mutant of Rad (V12-Rac1) increased JNK-responsive gene expression 6-fold, although this increase was attenuated by the intracellular Ca2+ chelator BAPTA-AM. V12-Rac1 also increased the number of DNA fragmentated cells. However, V12-Rac1-mediated JNK activation was not affected by BAPTA-AM as determined by direct measurement of active forms, and V12-Rac1 did not affect intracellular Ca2+ concentration. These results suggest that Rad can activate JNK and induces cell injury, but [Ca2+](i) is necessary for V12-Rac1 to induce DNA fragmentation downstream Of JNK activation. (C) 1999 Academic Press..
139. Nishida M. , Heterotrimetric G protein signaling in Heart Failure. , J. Pharmacol. Sci., in press..


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