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List of Papers
Motohiro Nishida Last modified date：2023.10.06

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


Papers
1. Iwata Y, Nakade Y, Kitajima S, Yoneda-Nakagawa S, Oshima M, Sakai N, Ogura H, Sato K, Toyama T, Yamamura Y, Miyagawa T, Yamazaki H, Hara A, Shimizu M, Furuichi K, Mita M, Hamase K, Tanaka T, Nishida M, Muramatsu W, Yamamoto H, Shichino S, Ueha S, Matsushima K, Wada T., Protective effect of d-alanine against acute kidney injury., Am J Physiol Renal Physiol., 2023.06.
2. Tokuyama T, Uosaki H, Sugiura A, Nishitai G, Takeda K, Nagashima S, Shiiba I, Ito N, Amo T, Mohri S, Nishimura A, Nishida M, Konno A, Hirai H, Ishido S, Toshizawa T, Shindo T, Takada T, Kinugawa S, Inatome R and Yanagi S, Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation., iScience, 2023.06.
3. Akiko Ogawa, Seiya Ohira, Yuri Kato, Tatsuya Ikuta, Shota Yanagida, Xinya Mi, Yukina Ishii, Yasunari Kanda, Motohiro Nishida, Asuka Inoue, Fan-Yan Wei, Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction., Communications biology, 10.1038/s42003-023-04888-x, 6, 1, 511-511, 2023.05, Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future..
4. Kazuhiro Nishiyama, Kohei Ariyoshi, Akiyuki Nishimura, Yuri Kato, Xinya Mi, Hitoshi Kurose, Sang Geon Kim, Motohiro Nishida, Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis., International journal of molecular sciences, 10.3390/ijms24043800, 24, 4, 2023.02, Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver..
5. Xiaokang Tang, Akiyuki Nishimura, Kohei Ariyoshi, Kazuhiro Nishiyama, Yuri Kato, Elena A Vasileva, Natalia P Mishchenko, Sergey A Fedoreyev, Valentin A Stonik, Hyoung-Kyu Kim, Jin Han, Yasunari Kanda, Keitaro Umezawa, Yasuteru Urano, Takaaki Akaike, Motohiro Nishida, Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice., Marine drugs, 10.3390/md21010052, 21, 1, 2023.01, Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism..
6. Yuri Kato, Kazuhiro Nishiyama, Jae Man Lee, Yuko Ibuki, Yumiko Imai, Takamasa Noda, Noriho Kamiya, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation., International journal of molecular sciences, 10.3390/ijms24010102, 24, 1, 2022.12, Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry..
7. Masahiro Akiyama, Takamitsu Unoki, Hanako Aoki, Akiyuki Nishimura, Yasuhiro Shinkai, Eiji Warabi, Kazuhiro Nishiyama, Yuka Furumoto, Naohiko Anzai, Takaaki Akaike, Motohiro Nishida, Yoshito Kumagai, Cystine-dependent antiporters buffer against excess intracellular reactive sulfur species-induced stress., Redox biology, 10.1016/j.redox.2022.102514, 57, 102514-102514, 2022.10, Reactive sulfur species (RSS) play a role in redox homeostasis; however, adaptive cell responses to excessive intracellular RSS are not well understood. Therefore, in this study, we generated transgenic (Tg) mice overexpressing cystathionine gamma-lyase (CSE) to produce excessive RSS. Contrary to expectations, tissue concentrations of RSS, such as cysteine persulfide (CysSSH), were comparable in both wild-type and CSE Tg mice, but the plasma concentrations of CysSSH were significantly higher in CSE Tg mice than in wild-type mice. This export of surplus intracellular RSS was also observed in primary hepatocytes of CSE Tg mice. Exposure of primary hepatocytes to the RSS generator sodium tetrasulfide (Na2S4) resulted in an initial increase in the intracellular concentration of RSS, which later returned to basal levels after export into the extracellular space. Interestingly, among all amino acids, cystine (CysSSCys) was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during Na2S4 exposure, suggesting that the cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout and overexpression of SLC7A11 and used them to confirm SLC7A11 as the predominant antiporter of CysSSCys and CysSSH. We observed that the poor efflux of excess CysSSH from the cell enhanced cellular stresses induced by Na2S4 exposure, such as polysulfidation of intracellular proteins, mitochondrial damage, and cytotoxicity. These results suggest the presence of a cellular response to excess intracellular RSS that involves the extracellular efflux of excess CysSSH by a cystine-dependent transporter to maintain intracellular redox homeostasis..
8. Sayaka Oda, Kazuhiro Nishiyama, Yuka Furumoto, Yohei Yamaguchi, Akiyuki Nishimura, Xiaokang Tang, Yuri Kato, Takuro Numaga-Tomita, Toshiyuki Kaneko, Supachoke Mangmool, Takuya Kuroda, Reishin Okubo, Makoto Sanbo, Masumi Hirabayashi, Yoji Sato, Yasuaki Nakagawa, Koichiro Kuwahara, Ryu Nagata, Gentaro Iribe, Yasuo Mori, Motohiro Nishida, Myocardial TRPC6-mediated Zn2+ influx induces beneficial positive inotropy through β-adrenoceptors., Nature communications, 10.1038/s41467-022-34194-9, 13, 1, 6374-6374, 2022.10, Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates β-adrenoceptor (βAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated βAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting β-arrestin-mediated βAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure..
9. Takuro Numaga-Tomita, Tsukasa Shimauchi, Yuri Kato, Kazuhiro Nishiyama, Akiyuki Nishimura, Kosuke Sakata, Hiroyuki Inada, Satomi Kita, Takahiro Iwamoto, Junichi Nabekura, Lutz Birnbaumer, Yasuo Mori, Motohiro Nishida, Inhibition of transient receptor potential cation channel 6 promotes capillary arterialization during post-ischaemic blood flow recovery., British journal of pharmacology, 10.1111/bph.15942, 180, 1, 94-110, 2022.09, BACKGROUND AND PURPOSE: Capillary arterialization, characterized by the coverage of pre-existing or nascent capillary vessels with vascular smooth muscle cells (VSMCs), is critical for the development of collateral arterioles to improve post-ischaemic blood flow. We previously demonstrated that the inhibition of transient receptor potential 6 subfamily C, member 6 (TRPC6) channels facilitate contractile differentiation of VSMCs under ischaemic stress. We here investigated whether TRPC6 inhibition promotes post-ischaemic blood flow recovery through capillary arterialization in vivo. EXPERIMENTAL APPROACH: Mice were subjected to hindlimb ischaemia by ligating left femoral artery. The recovery rate of peripheral blood flow was calculated by the ratio of ischaemic left leg to non-ischaemic right one. The number and diameter of blood vessels were analysed by immunohistochemistry. Expression and phosphorylation levels of TRPC6 proteins were determined by western blotting and immunohistochemistry. KEY RESULTS: Although the post-ischaemic blood flow recovery is reportedly dependent on endothelium-dependent relaxing factors, systemic TRPC6 deletion significantly promoted blood flow recovery under the condition that nitric oxide or prostacyclin production were inhibited, accompanying capillary arterialization. Cilostazol, a clinically approved drug for peripheral arterial disease, facilitates blood flow recovery by inactivating TRPC6 via phosphorylation at Thr69 in VSMCs. Furthermore, inhibition of TRPC6 channel activity by pyrazole-2 (Pyr2; BTP2; YM-58483) promoted post-ischaemic blood flow recovery in Apolipoprotein E-knockout mice. CONCLUSION AND IMPLICATIONS: Suppression of TRPC6 channel activity in VSMCs could be a new strategy for the improvement of post-ischaemic peripheral blood circulation..
10. Yuri Kato, Kazuhiro Nishiyama, Akiyuki Nishimura, Takamasa Noda, Kaori Okabe, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, Drug repurposing for the treatment of COVID-19., Journal of pharmacological sciences, 10.1016/j.jphs.2022.04.007, 149, 3, 108-114, 2022.07, Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure..
11. Yamada S, Noda T, Okabe K, Yanagida S, Nishida M, Kanda Y., SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium., J Pharmacol Sci. 2022 Jul;149(3):139-146., 10.1016/j.jphs.2022.04.010., 2022.07.
12. Tsukasa Shimauchi, Takuro Numaga-Tomita, Yuri Kato, Hiroyuki Morimoto, Kosuke Sakata, Ryosuke Matsukane, Akiyuki Nishimura, Kazuhiro Nishiyama, Atsushi Shibuta, Yutoku Horiuchi, Hitoshi Kurose, Sang Geon Kim, Yasuteru Urano, Takashi Ohshima, Motohiro Nishida, A TRPC3/6 Channel Inhibitor Promotes Arteriogenesis after Hind-Limb Ischemia., Cells, 10.3390/cells11132041, 11, 13, 2022.06, Retarded revascularization after progressive occlusion of large conductance arteries is a major cause of bad prognosis for peripheral artery disease (PAD). However, pharmacological treatment for PAD is still limited. We previously reported that suppression of transient receptor potential canonical (TRPC) 6 channel activity in vascular smooth muscle cells (VSMCs) facilitates VSMC differentiation without affecting proliferation and migration. In this study, we found that 1-benzilpiperadine derivative (1-BP), a selective inhibitor for TRPC3 and TRPC6 channel activities, induced VSMC differentiation. 1-BP-treated mice showed increased capillary arterialization and improvement of peripheral circulation and skeletal muscle mass after hind-limb ischemia (HLI) in mice. 1-BP had no additive effect on the facilitation of blood flow recovery after HLI in TRPC6-deficient mice, suggesting that suppression of TRPC6 underlies facilitation of the blood flow recovery by 1-BP. 1-BP also improved vascular nitric oxide bioavailability and blood flow recovery after HLI in hypercholesterolemic mice with endothelial dysfunction, suggesting the retrograde interaction from VSMCs to endothelium. These results suggest that 1-BP becomes a potential seed for PAD treatments that target vascular TRPC6 channels..
13. Yagi-Utsumi M, Tanaka T, Otsubo Y, Yamashita A, Yoshimura S, Nishida M, Kato K., Atmospheric Plasma Modification of Amyloid β, Int J Mol Sci. , 10.3390, 2022.03.
14. Ryota Murata, Hiroshi Watanabe, Hiroto Nosaki, Kento Nishida, Hitoshi Maeda, Motohiro Nishida, Toru Maruyama, Long-Acting Thioredoxin Ameliorates Doxorubicin-Induced Cardiomyopathy via Its Anti-Oxidative and Anti-Inflammatory Action., Pharmaceutics, 10.3390/pharmaceutics14030562, 14, 3, 2022.03, Although the number of patients with heart failure is increasing, a sufficient treatment agent has not been established. Oxidative stress and inflammation play important roles in the development of myocardial remodeling. When thioredoxin (Trx), an endogenous anti-oxidative and inflammatory modulator with a molecular weight of 12 kDa, is exogenously administered, it disappears rapidly from the blood circulation. In this study, we prepared a long-acting Trx, by fusing human Trx (HSA-Trx) with human serum albumin (HSA) and evaluated its efficacy in treating drug-induced heart failure. Drug-induced cardiomyopathy was created by intraperitoneally administering doxorubicin (Dox) to mice three times per week. A decrease in heart weight, increased myocardial fibrosis and markers for myocardial damage that were observed in the Dox group were suppressed by HSA-Trx administration. HSA-Trx also suppressed the expression of atrogin-1 and myostatin, myocardial atrophy factors in addition to suppressing oxidative stress and inflammation. In the Dox group, a decreased expression of endogenous Trx in cardiac tissue and an increased expression of macrophage migration inhibitory factor were observed, but these changes were restored to normal levels by HSA-Trx administration. These findings suggest that HSA-Trx improves the pathological condition associated with Dox-induced cardiomyopathy by its anti-oxidative/anti-inflammatory and myocardial atrophy inhibitory action..
15. Naoya Hirata, Shigeru Yamada, Shota Yanagida, Atsushi Ono, Yukuto Yasuhiko, Motohiro Nishida, Yasunari Kanda, Lysophosphatidic Acid Promotes the Expansion of Cancer Stem Cells via TRPC3 Channels in Triple-Negative Breast Cancer., International journal of molecular sciences, 10.3390/ijms23041967, 23, 4, 2022.02, Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC..
16. Akiyuki Nishimura, Tomohiro Tanaka, Yuri Kato, Kazuhiro Nishiyama, Motohiro Nishida, Cardiac robustness regulated by reactive sulfur species., Journal of clinical biochemistry and nutrition, 10.3164/jcbn.21-84, 70, 1, 1-6, 2022.01, The human myocardium contains robust cells that constantly beat from birth to death without being replaced, even when exposed to various environmental stresses. Myocardial robustness is thought to depend primarily on the strength of the reducing power to protect the heart from oxidative stress. Myocardial antioxidant systems are controlled by redox reactions, primarily via the redox reaction of Cys sulfhydryl groups, such as found in thioredoxin and glutathione. However, the specific molecular entities that regulate myocardial reducing power have long been debated. Recently, reactive sulfide species, with excellent electron transfer ability, consisting of a series of multiple sulfur atoms, i.e., Cys persulfide and Cys polysulfides, have been found to play an essential role in maintaining mitochondrial quality and function, as well as myocardial robustness. This review presents the latest findings on the molecular mechanisms underlying mitochondrial energy metabolism and the maintenance of quality control by reactive sulfide species and provides a new insight for the prevention of chronic heart failure..
17. Kazuhiro Nishiyama, Akiyuki Nishimura, Kakeru Shimoda, Tomohiro Tanaka, Yuri Kato, Takahiro Shibata, Hiroshi Tanaka, Hitoshi Kurose, Yasu-Taka Azuma, Hideshi Ihara, Yoshito Kumagai, Takaaki Akaike, Philip Eaton, Koji Uchida, Motohiro Nishida, Redox-dependent internalization of the purinergic P2Y6 receptor limits colitis progression., Science signaling, 10.1126/scisignal.abj0644, 15, 716, eabj0644, 2022.01, After ligand stimulation, many G protein–coupled receptors (GPCRs) undergo β-arrestin–dependent desensitization, during which they are internalized and either degraded or recycled to the plasma membrane. Some GPCRs are not subject to this type of desensitization because they lack the residues required to interact with β-arrestins. We identified a mechanism of redox-dependent alternative internalization (REDAI) that promotes the internalization and degradation of the purinergic P2Y6 receptor (P2Y6R). Synthetic and natural compounds containing electrophilic isothiocyanate groups covalently modified P2Y6R at Cys220, which promoted the ubiquitylation of Lys137 and receptor internalization and degradation in various mouse and human cultured cell lines. Endogenous electrophiles also promoted ligand-dependent P2Y6R internalization and degradation. P2Y6R is highly abundant in inflammatory cells and promotes the pathogenesis of colitis. Deficiency in P2Y6R protected mice against experimentally induced colitis, and mice expressing a form of P2Y6R in which Cys220 was mutated to nonmodifiable serine were more sensitive to the induction of colitis. Several other GPCRs, including A2BAR, contain cysteine and lysine residues at the appropriate positions to mediate REDAI, and isothiocyanate stimulated the internalization of A2BAR and of a form of P2Y2R with insertions of the appropriate residues. Thus, endogenous and exogenous electrophiles may limit colitis progression through cysteine modification of P2Y6R and may also mediate internalization of other GPCRs..
18. Masuda A, Lee J, Miyata T, Mon H, Sato K, Oyama K, Sakurai Y, Yasuda J, Takahashi D, Ueda T, Kato Y, Nishida M, Karasaki N, Kakino K, Ebihara T, Nagasato T, Hino M, Nakashima A, Suzuki K, Tonooka Y, Tanaka M, Moriyama T, Nakatake H, Fujita R, Kusakabe T., Optimization of SARS-CoV-2 Spike protein expression in the silkworm and induction of efficient protective immunity by inoculation with alum adjuvants., Front. Immunol., 2021.12.
19. Yuta Matsuoka, Masatomo Takahashi, Yuki Sugiura, Yoshihiro Izumi, Kazuhiro Nishiyama, Motohiro Nishida, Makoto Suematsu, Takeshi Bamba, Ken-Ichi Yamada, Structural library and visualization of endogenously oxidized phosphatidylcholines using mass spectrometry-based techniques., Nature communications, 10.1038/s41467-021-26633-w, 12, 1, 6339-6339, 2021.11, Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases..
20. Eizo Marutani, Masanobu Morita, Shuichi Hirai, Shinichi Kai, Robert M H Grange, Yusuke Miyazaki, Fumiaki Nagashima, Lisa Traeger, Aurora Magliocca, Tomoaki Ida, Tetsuro Matsunaga, Daniel R Flicker, Benjamin Corman, Naohiro Mori, Yumiko Yamazaki, Annabelle Batten, Rebecca Li, Tomohiro Tanaka, Takamitsu Ikeda, Akito Nakagawa, Dmitriy N Atochin, Hideshi Ihara, Benjamin A Olenchock, Xinggui Shen, Motohiro Nishida, Kenjiro Hanaoka, Christopher G Kevil, Ming Xian, Donald B Bloch, Takaaki Akaike, Allyson G Hindle, Hozumi Motohashi, Fumito Ichinose, Sulfide catabolism ameliorates hypoxic brain injury., Nature communications, 10.1038/s41467-021-23363-x, 12, 1, 3108-3108, 2021.05, The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury..
21. 4. Eco-pharma Research Aimed at Therapeutic Agents for Amyotrophic Diseases.
22. Tomohiro Sawa, Motohiro Nishida, Introduction to serial reviews: Recent developments in research of reactive sulfur species., Journal of clinical biochemistry and nutrition, 10.3164/jcbn.68-1-Intro, 68, 1, 4-4, 2021.01.
23. Kakeru Shimoda, Akiyuki Nishimura, Caroline Sunggip, Tomoya Ito, Kazuhiro Nishiyama, Yuri Kato, Tomohiro Tanaka, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Motohiro Nishida, Modulation of P2Y6R expression exacerbates pressure overload-induced cardiac remodeling in mice., Scientific reports, 10.1038/s41598-020-70956-5, 10, 1, 13926-13926, 2020.08, Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y6 receptor (P2Y6R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y6R prevents or promotes heart failure. We demonstrate that inhibition of P2Y6R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y6R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y6R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y6R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y6R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y6R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y6R exacerbates pressure overload-induced heart failure in mice, although P2Y6R in cardiomyocytes contributes to the progression of cardiac fibrosis..
24. Miho Shiratori, Chiharu Yamaguchi, Kazushi Eguchi, Yuto Shiraishi, Keita Kohno, Katsuhiko Mikoshiba, Kazuhide Inoue, Motohiro Nishida, Makoto Tsuda, Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca2+ signals in mice, Journal of Allergy and Clinical Immunology, 10.1016/j.jaci.2020.06.039, 2020.08, Background: Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism. Objective: We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions. Methods: To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch. Results: IP3 receptor type 1 (IP3R1) knockdown in astrocytes suppressed IL-6–induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IP3R1-dependent astrocytic Ca2+ responses involved Ca2+ influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron–specific IL-6 knockdown, spinal astrocyte–specific IP3R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch. Conclusion: Our findings suggest that IP3R1/TRPC channel–mediated Ca2+ signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention..
25. Kazuhiro Nishiyama, Tomohiro Tanaka, Akiyuki Nishimura, Motohiro Nishida, TRPC3-based protein signaling complex as a therapeutic target of myocardial atrophy., Current molecular pharmacology, 10.2174/1874467213666200407090121, 2020.04, BACKGROUND: Transient receptor potential (TRP) channels, especially canonical TRP channel subfamily members 3 (TRPC3) and 6 (TRPC6), have attracted attention as a putative therapeutic target of heart | 1 failure. Moreover, TRPC3 and TRPC6 channels are physiologically important for maintaining cellular homeostasis. How TRPC3/C6 channels alter intracellular signaling from adaptation to maladaptation has been discussed for many years. We recently showed that formation of a protein signal complex between TRPC3 and NADPH oxidase (Nox) 2 caused by environmental stresses (e.g., hypoxia, nutritional deficiency, and anticancer drug treatment) promotes Nox2-dependent reactive oxygen species production and cardiac stiffness, including myocardial atrophy and interstitial fibrosis, in rodents. In fact, pharmacological prevention of the TRPC3-Nox2 protein complex can maintain cardiac flexibility in mice after anti-cancer drug treatment. CONCLUSION: In this mini-review, we discuss the relationship between TRPC3/C6 channels and cardiovascular disease, and propose a new therapeutic strategy by focusing on pathology-specific protein- protein interactions..
26. Nishiyama K, Toyama C, Kato Y, Tanaka T, Nishimura A, Nagata R, Mori Y, Nishida M, Deletion of TRPC3 or TRPC6 fails to attenuate the formation of inflammation and fibrosis in non-alcoholic steatohepatitis., Biol. Pharm. Bull. (in press)., 10.1248/bpb.b20-00903, 44, 3, 431-436, 2020.01, Non-alcoholic steatohepatitis (NASH) is a disease that has progressed from non-alcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. Two transient receptor potential canonical (TRPC) subfamily members, TRPC3 and TRPC6 (TRPC3/6), reportedly participate in the development of fibrosis in cardiovascular and renal systems. We hypothesized that TRPC3/6 may also participate in NASH fibrosis. We evaluated the effects of TRPC3 or TRPC6 functional deficiency in a NASH mouse model using choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Wild-type (WT) and TRPC3 or TRPC6 gene-deficient (KO) mice were fed with CDAHFD or standard diet for 6 weeks. The CDAHFD-induced body weight loss in TRPC6 KO mice was significantly lower compared with WT mice with CDAHFD. CDAHFD treatment significantly increased TRPC3 mRNA expression level and tissue weight in WT liver, which were suppressed in TRPC3 KO mice. However, either systemic deletion of TRPC3 or TRPC6 failed to attenuate liver steatosis, inflammation and fibrosis. These results imply that TRPC3 and TRPC6 are unlikely to be involved in liver dysfunction and fibrosis of NASH model mice..
27. 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, 9, 1, 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..
28. Nishiyama K, Numaga-Tomita T, Fujimoto Y, Tanaka T, Toyama C, Nishimura A, Yamashita T, Matsunaga N, Koyanagi S, Azuma YT, Ibuki Y, Uchida K, Ohdo S, Nishida M, Ibudilast attenuates doxorubicin-induced cytotoxicity by suppressing formation of TRPC3-Nox2 protein complex., British journal of pharmacology, 10.1111/bph.14777, 176, 18, 3723-3738, 2019.09.
29. 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 journal : official publication of the Federation of American Societies for Experimental Biology, 10.1096/fj.201802811R, 33, 9, 9785-9796, 2019.09.
30. Sudi SB, Tanaka T, Oda S, Nishiyama K, Nishimura A, Sunggip C, Mangmool S, Numaga-Tomita T, Nishida M, TRPC3-Nox2 axis mediates nutritional deficiency-induced cardiomyocyte atrophy., Scientific reports, 10.1038/s41598-019-46252-2, 9, 1, 9785-9785, 2019.07.
31. Nishimura A, Shimoda K, Tanaka T, Toyama T, Nishiyama K, Shinkai Y, Numaga-Tomita T, Yamazaki D, Kanda Y, Akaike T, Kumagai Y, Nishida M, Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload., Science signaling, 10.1126/scisignal.aaw1920, 12, 587, Jun 25;12(587). pii: eaaw1920. doi: 10.1126/scisignal.aaw1920, 2019.06.
32. Nishiyama K, Aono K, Fujimoto Y, Kuwamura M, Okada T, Tokumoto H, Izawa T, Okano R, Nakajima H, Takeuchi T, Azuma YT., Chronic kidney disease after 5/6 nephrectomy disturbs the intestinal microbiota and alters intestinal motility., 2019.05.
33. Numaga-Tomita T, Oda S, Nishiyama K, Tanaka T, Nishimura A, Nishida M, TRPC channels in exercise-mimetic therapy., Pflugers Archiv : European journal of physiology, 10.1007/s00424-018-2211-3, 471, 3, 507-517, 2019.03.
34. Tanaka T, Nishimura A, Nishiyama K, Goto T, Numaga-Tomita T, Nishida M, Mitochondrial dynamics in exercise physiology., Pflugers Archiv : European journal of physiology, 10.1007/s00424-019-02258-3, 2019.02.
35. Hideshi Ihara, Yuki Kakihana, Akane Yamakage, Kenji Kai, Takahiro Shibata, Motohiro Nishida, Ken-Ichi Yamada, Koji Uchida, 2-Oxo-histidine-containing dipeptides are functional oxidation products, Journal of Biological Chemistry, 10.1074/jbc.RA118.006111, 294, 4, 1279-1289, 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, H
2
O
2
exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H
2
O
2
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..
36. Tomohiro Tanaka, Akiyuki Nishimura, Kazuhiro Nishiyama, Takumi Goto, Takuro Numaga-Tomita, Motohiro Nishida, Mitochondrial dynamics in exercise physiology, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-019-02258-3, 2019.01, A growing body of evidence suggests that exercise shows pleiotropic effects on the maintenance of systemic homeostasis through mitochondria. Dysregulation of mitochondrial dynamism is associated with metabolic inflexibility, resulting in many of the metabolic diseases and aging. Studies have suggested that exercise prevents and delays the progression of mitochondrial dysfunction by improving mitochondrial metabolism, biogenesis, and quality control. Exercise modulates functions of mitochondrial dynamics-regulating proteins through post-translational modification mechanisms. In this review, we discuss the putative mechanisms underlying maintenance of mitochondrial homeostasis by exercise, especially focusing on the post-translational modifications of several signaling proteins contributing to mitochondrial biogenesis, autophagy or mitophagy flux, and fission/fusion cycle. We also introduce novel small molecules that can potentially mimic exercise therapy through preserving mitochondrial dynamism. These recent advancements in the field of mitochondrial biology may lead to a greater understanding of exercise signaling..
37. Nishimura A, Shimoda K, Tanaka T, Toyama T, Nishiyama K, Shinkai Y, Numaga-Tomita T, Yamazaki D, Kanda Y, Akaike T, Kumagai Y, Nishida M., Depolysulfidation of Drp1 induced by low-dose methylmercury exposure increases cardiac vulnerability to hemodynamic overload., Science Signaling, 2019.01.
38. Nishimura A, Shimauchi T, Tanaka T, Shimoda K, Toyama T, Kitajima N, Ishikawa T, Shindo N, Numaga-Tomita T, Yasuda S, Sato Y, Kuwahara K, Kumagai Y, Akaike T, Ide T, Ojida A, Mori Y, Nishida M, Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence., Science Signaling, 11, eaat5185, 2018.11.
39. Parichatikanond W, Nishimura A, Nishida M, Mangmool S, Prolonged stimulation of β2-adrenergic receptor with β2-agonists impairs insulin actions in H9c2 cells., J. Pharmacol. Sci., 138, 3, in press-191, 2018.11.
40. Morita Masanobu, Ida Tomoaki, Tanaka Tomohiro, Matsunaga Tetsuro, Nishimura Akira, Fujii Shigemoto, Nishida Motohiro, Motohashi Hozumi, Akaike Takaaki, Mitochondria-specific SQR deficiency in mice causes lethal impairment of sulfur respiration, FREE RADICAL BIOLOGY AND MEDICINE, 10.1016/j.freeradbiomed.2018.10.209, 128, S90-S90, 2018.11.
41. 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, 9, MAY, 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..
42. 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, 495, 3, 2165-2170, 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..
43. Licht Miyamoto, Motohiro Nishida, Lifestyle inspires future pharmacotherapy and drug discovery, Yakugaku Zasshi, 10.1248/yakushi.18-00091-F, 138, 10, 1255-1256, 2018.01.
44. Sayaka Oda, Takuro Numaga-Tomita, Motohiro Nishida, New strategies for exercise-mimetic medication, Yakugaku Zasshi, 10.1248/yakushi.18-00091-1, 138, 10, 1257-1262, 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 (Ca
2

＋
) signaling as well as production of reactive oxygen species (ROS). However, crosstalk between Ca
2

＋
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..
45. Tomohiro Sawa, Katsuhiko Ono, Hiroyasu Tsutsuki, Tianli Zhang, Tomoaki Ida, Motohiro Nishida, Takaaki Akaike, Reactive Cysteine Persulphides
Occurrence, Biosynthesis, Antioxidant Activity, Methodologies, and Bacterial Persulphide Signalling, Nitric Oxide and Other Small Signalling Molecules, 10.1016/bs.ampbs.2018.01.002, 1-28, 2018.01, Cysteine hydropersulphide (CysSSH) is a cysteine derivative having one additional sulphur atom bound to a cysteinyl thiol group. Recent advances in the development of analytical methods for detection and quantification of persulphides and polysulphides have revealed the biological presence, in both prokaryotes and eukaryotes, of hydropersulphides in diverse forms such as CysSSH, homocysteine hydropersulphide, glutathione hydropersulphide, bacillithiol hydropersulphide, coenzyme A hydropersulphide, and protein hydropersulphides. Owing to the chemical reactivity of the persulphide moiety, biological systems utilize persulphides as important intermediates in the synthesis of various sulphur-containing biomolecules. Accumulating evidence has revealed another important feature of persulphides: their potent reducing activity, which implies that they are implicated in the regulation of redox signalling and antioxidant functions. In this chapter, we discuss the biological occurrence and possible biosynthetic mechanisms of CysSSH and related persulphides, and we include descriptions of recent advances in the analytical methods that have been used to detect and quantitate persulphide species. We also discuss the antioxidant activity of persulphide species that contributes to protecting cells from reactive oxygen species-associated damage, and we examine the signalling roles of CysSSH in bacteria..
46. 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 & THERAPEUTICS, 10.1016/j.pharmthera.2017.06.010, 180, 113-128, 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 P2Y(6)R and angiotensin (Ang) II typel receptor (AT1R) signaling, and introduce our recent finding that the P2Y(6)R antagonist MRS2578 interrupts heterodimerization between P2Y(6)R and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2Y(6)R oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases. (C) 2017 The Author(s). Published by Elsevier Inc..
47. Role of TRPC3 and TRPC6 channels in the myocardial response to stretch: Linking physiology and pathophysiology..
48. 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, 8, 1, 1177-1177, 2017.10, 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..
49. 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, 135, 2, 81-88, 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. (C) 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society..
50. Hideshi Ihara, Shingo Kasamatsu, Atsushi Kitamura, Akira Nishimura, Hiroyasu Tsutsuki, Tomoaki Ida, Kento Ishizaki, Takashi Toyama, Elko Yoshida, Hisyam Abdul Hamid, Minkyung Jung, Tetsuro Matsunaga, Shigemoto Fuji, 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, 30, 9, 1673-1684, 2017.09, 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-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP 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..
51. 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, 4, 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..
52. Shimauchi T, Numaga-Tomita T, Ito T, Nishimura A, Matsukane R, Oda S, Hoka S, Ide T, Koitabashi N, Uchida K, Sumimoto H, Mori Y, Nishida M, TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy, JCI Insight, 10.1172/jci.insight.93358, 2, 15, pii: 93358, 2017.08.
53. 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, 7, 1, 7511, 2017.08, 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..
54. 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, 109, 132-140, 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..
55. Caroline Sunggip, Akiyuki Nishimura, Kakeru Shimoda, Takuro Numaga-Tomita, Makoto Tsuda, Motohiro Nishida, Purinergic P2Y(6) receptors: A new therapeutic target of age-dependent hypertension, PHARMACOLOGICAL RESEARCH, 10.1016/j.phrs.2017.03.013, 120, 51-59, 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 P2Y(12) receptor (P2Y(12)R), 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 P2Y(6)R 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 P2Y(6)R was disrupted by MRS2578, a P2Y(6)R-selective inhibitor. These findings suggest that P2Y(6)R is a therapeutic target to prevent age-related hypertension. (C) 2017 Published by Elsevier Ltd..
56. Phosri S, Arieyawong A, Boonrukchai K, Parichatikanond W, Nishimura A, Nishida M, Mangmool S, 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, Front. Pharmacol., 10.3389/fphar.2017.00428, 8, 428, 2017.06.
57. 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, 6, 2, pii: e003601, 2017.02, 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..
58. Akiyuki Nishimura, Motohiro Nishida, Purinergic signaling in cardiovascular system, Folia Pharmacologica Japonica, 10.1254/fpj.149.84, 149, 2, 84-90, 2017.01.
59. Yamaguchi Y, Iribe G, Kaneko T, Takahashi K, Numaga-Tomita T, Nishida M, Birnbaumer L, Naruse K, TRPC3 participates in angiotensin II type 1 receptor-dependent stress-induced slow increase in intracellular Ca2+ concentration in mouse cardiomyocytes, J. Physiol. Sci., doi: 10.1007/s12576-016-0519-3, 2017.01.
60. 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, 149, 6, 269-273, 2017.
61. 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, 6, 39383, 2016.12, Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ 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 Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ 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)beta stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubuleassociated 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..
62. 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, 6, 37001, 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 Ca2+-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 Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ 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 Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities..
63. Takamitsu Unoki, Yumi Abiko, Takashi Toyama, Takashi Uehara, Koji Tsuboi, Motohiro Nishida, Toshiyuki Kaji, Yoshito Kumagai, Methylmercury, an environmental electrophile capable of activation and disruption of the Akt/CREB/Bcl-2 signal transduction pathway in SH-SY5Y cells, SCIENTIFIC REPORTS, 10.1038/srep28944, 6, 28944, 2016.06, Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as "S-mercuration", potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 mu M MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death..
64. 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, 595, 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..
65. 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, 58, 2, 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..
66. Seishiro Sawamura, Masahiko Hatano, Yoshinori Takada, Kyosuke Hino, Tetsuya Kawamura, Jun Tanikawa, Hiroshi Nakagawa, Hideharu Hase, Akito Nakao, Mitsuru Hirano, Rachapun Rotrattanadumrong, Shigeki Kiyonaka, Masayuki X. Mori, Motohiro Nishida, Yaopeng Hu, Ryuji Inoue, Ryu Nagata, Yasuo Mori, Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds, MOLECULAR PHARMACOLOGY, 10.1124/mol.115.102863, 89, 3, 348-363, 2016.03, Transient receptor potential canonical (TRPC) proteins form Ca2+-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl) piperazin-1-yl](3-fluorophenyl) methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)-piperazin-1-yl]-N-(2-ethoxyphenyl) acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca2+ influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca2+ signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca2+-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca2+ signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs..
67. 西田 基宏, The purinergic P2Y6 receptor heterodimerizes with the angiotensin AT1 receptor to promote angiotensin II-induced hypertension, Science Signaling, doi: 10.1126/scisignal.aac9187., Vol. 9, Issue 411, ra7, 2016.01, The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR), promoted Ang II–induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II–induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein–dependent vascular hypertrophy but reduced β-arrestin–dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II–induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascular smooth muscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin–dependent proliferation to G protein–dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II..
68. Akiyuki Nishimura, Caroline Sunggip, Hidetoshi Tozaki-Saitoh, Tsukasa Shimauchi, Takuro Numaga-Tomita, Katsuya Hirano, Tomomi Ide, Jean-Marie Boeynaems, Hitoshi Kurose, Makoto Tsuda, Bernard Robaye, Kazuhide Inoue, Motohiro Nishida, Purinergic P2Y(6) receptors heterodimerize with angiotensin AT1 receptors to promote angiotensin II-induced hypertension, SCIENCE SIGNALING, 10.1126/scisignal.aac9187, 9, 411, ra7, 2016.01, The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y(6) receptor (P2Y(6)R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR), promoted Ang II-induced hypertension in mice. In mice, deletion of P2Y(6)R attenuated Ang II-induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y(6)R formed stable heterodimers, which enhanced G protein-dependent vascular hypertrophy but reduced beta-arrestin-dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y(6)R heterodimers by the P2Y(6)R antagonist MRS2578 suppressed Ang II-induced hypertension in mice. Furthermore, P2Y(6)R abundance increased with age in vascularsmoothmuscle cells. The increased abundance of P2Y(6)R converted AT1R-stimulated signaling in vascular smooth muscle cells from beta-arrestin-dependent proliferation to G protein-dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y(6)R heterodimers with age may increase the likelihood of hypertension induced by Ang II..
69. Supachoke Mangmool, Tananat Denkaew, Sarawuth Phosri, Darawan Pinthong, Warisara Parichatikanond, Tsukasa Shimauchi, Motohiro Nishida, Sustained beta AR Stimulation Mediates Cardiac Insulin Resistance in a PKA-Dependent Manner, MOLECULAR ENDOCRINOLOGY, 10.1210/me.2015-1201, 30, 1, 118-132, 2016.01, Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of beta-adrenergic receptors (beta ARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained beta AR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained beta AR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT) 4 expression that were mediated by the beta(2)AR subtype in cardiomyocytes and heart tissue. Overstimulation of beta(2)AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, beta AR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with beta-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in beta AR-mediated insulin resistance..
70. Masayori Hagimori, Takahiro Murakami, Kinue Shimizu, Motohiro Nishida, Takashi Ohshima, Takahiro Mukai, Synthesis of radioiodinated probes to evaluate the biodistribution of a potent TRPC3 inhibitor, MedChemComm, 10.1039/c6md00023a, 7, 5, 1003-1006, 2016.01, The transient receptor potential canonical 3 (TRPC3) channel is a member of the TRPC family that contributes to the entry of Ca2+ through the plasma membrane or modulates the driving force for Ca2+ entry channels. The pyrazole compound Pyr3 has recently been reported to be a selective TRPC3 inhibitor and has become an attractive research tool and therapeutic agent for the treatment of heart failure. However, the in vivo characteristics of Pyr3 have not been investigated. To monitor the fate of Pyr3 in vivo, we designed and synthesized a radioiodinated Pyr3 probe ([125I]I-Pyr3) by introducing radioiodine at the 2-position of the central phenyl ring of Pyr3. I-Pyr3 was shown to have direct TRPC3 inhibition activity similar to that of Pyr3 in TRPC3-overexpressing HEK293 cells. Using the tributyltin derivative as a radioiodination precursor, [125I]I-Pyr3 was successfully prepared with high radiochemical purity. Biodistribution studies of [125I]I-Pyr3 and [125I]I-Pyr8 (the esterolysis product of [125I]I-Pyr3) indicated high uptake of intact [125I]I-Pyr3 in the lung and rapid metabolism to [125I]I-Pyr8. These findings provide useful information about the in vivo kinetics of the selective TRPC inhibitor Pyr3..
71. Takuro Numaga-Tomita, Motohiro Nishida, James W. Putney, Yasuo Mori, TRPC3 amplifies B-cell receptor-induced ERK signalling via protein kinase D-dependent Rap1 activation, BIOCHEMICAL JOURNAL, 10.1042/BJ20150596, 473, 2, 201-210, 2016.01, Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca2+ influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes..
72. 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, 290, 37, 22851-22861, 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 carboxyl-terminal 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..
73. 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, 28, 5, 838-847, 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-S-n-H(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH(2)-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH(2)-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-NQH(2)-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-NQH(2)-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH(2) adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH(2)-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-NQH(2)-SAc activated Nrf(2) 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-NQH(2)-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress..
74. Establishment of a Novel Therapeutic Strategy for Heart Failure Based on the Mechanism Underlying Maintenance of Redox Homeostasis by Reactive Sulfur Species
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 modification (S-guanylation) and activation of H-Ras. We also found that hydrogen sulfide-related reactive sulfur species (RSS) function as potent nucleophiles to eliminate electrophilic modification of H-Ras and suppress the onset of chronic heart failure after myocardial infarction. Our results strongly suggest a new concept of redox biolog.
75. 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, 104, 1, 183-193, 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 beta-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 alpha 1 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..
76. 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, 73, 10-17, 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"..
77. Motohiro Nishida, [Regulation of redox homeostasis by hydrogen sulfide anion and its clinical application], Seikagaku. The Journal of Japanese Biochemical Society, 85, 11, 996-999, 2013.11.
78. 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, 33, 9, 2121-2129, 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 [Ca2+](i) in vitro and of vascular leakage in vivo were markedly attenuated by the Ca2+-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 Thr(69) 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..
79. 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, 8, 7, 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..
80. 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, 434, 2, 210-216, 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. © 2013 Elsevier Inc..
81. 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, 4, 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..
82. 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, 4, 1532, 2013.02, 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..
83. Takaaki Akaike, Motohiro Nishida, Shigemoto Fujii, Regulation of redox signalling by an electrophilic cyclic nucleotide, Journal of Biochemistry, 10.1093/jb/mvs145, 153, 2, 131-138, 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. © The Authors 2012. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved..
84. Nakaya M, Chikura S, Watari K, Mizuno N, Mochinaga K, Mangmool S, Koyanagi S, Ohdo S, Sato Y, Ide T, Nishida M, Kurose H, Induction of cardiac fibrosis by β-blocker in G protein-independent and G protein-coupled receptor kinase 5/β-arrestin2-dependent Signaling pathways., The Journal of biological chemistry, 10.1074/jbc.M112.357871, 287, 42, 35669-35677, 2012.10.
85. Meikun Kan-o, Ryu Takeya, Takaya Abe, Naoyuki Kitajima, Motohiro Nishida, Ryuji Tominaga, Hitoshi Kurose, Hideki Sumimoto, Mammalian formin Fhod3 plays an essential role in cardiogenesis by organizing myofibrillogenesis, BIOLOGY OPEN, 10.1242/bio.20121370, 1, 9, 889-896, 2012.09, Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development. (C) 2012. Published by The Company of Biologists Ltd..
86. 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, 8, 8, 714-724, 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..
87. Fujino T, Ide T, Yoshida M, Onitsuka K, Tanaka A, Hata Y, Nishida M, Takehara T, Kanemaru T, Kitajima N, Takazaki S, Kurose H, Kang D, Sunagawa K, 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, 12, 4, 449-458, 2012.07.
88. Kinue Nishioka, Motohiro Nishida, Marina Ariyoshi, Zhong Jian, Shota Saiki, Mayumi Hirano, Michio Nakaya, Yoji Sato, Satomi Kita, Takahiro Iwamoto, Katsuya Hirano, Ryuji Inoue, Hitoshi Kurose, Cilostazol Suppresses Angiotensin II-Induced Vasoconstriction via Protein Kinase A-Mediated Phosphorylation of the Transient Receptor Potential Canonical 6 Channel, ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY, 10.1161/ATVBAHA.110.221010, 31, 10, 2278-U304, 2011.10, Objective-The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)-induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition.
Methods and Results-Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II-induced contraction was largely dependent on C(a2+) influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)-dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II-induced Ca(2+) influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II-induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6.
Conclusion-PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II. (Arterioscler Thromb Vasc Biol. 2011;31:2278-2286.).
89. Motohiro Nishida, Roles of heterotrimeric GTP-binding proteins in the progression of heart failure, Journal of Pharmacological Sciences, 10.1254/jphs.11R05CP, 117, 1, 1-5, 2011.09, Heart failure is a major cause of death in developed countries, and the development of an epoch-making cure is desired from the viewpoint for improving the quality of life and reducing the medical cost of the patient. The importance of neurohumoral factors, such as angiotensin (Ang) II and catecholamine, for the progression of heart failure has been supported by a variety of evidence. These agonists stimulate seven transmembrane-spanning receptors that are coupled to heterotrimeric GTP-binding proteins (G proteins). Using specific pharmacological tools to assess the involvement of G protein signaling pathways, we have revealed that á subunit of G q (Gα q) activates Ca 2+-dependent hypertrophic signaling through diacylglycerol-activated transient receptor potential canonical (TRPC) channels (TRPC3 and TRPC6: TRPC3/6). In contrast, activation of Gα 12 family proteins in cardiomyocytes confers pressure overload-induced cardiac fibrosis via stimulation of purinergic P2Y 6 receptors induced by extracellular nucleotides released from cardiomyocytes. In fact, direct or indirect inhibition of TRPC3/6 or P2Y 6 receptors attenuates pressure overload-induced cardiac dysfunction. These findings will provide a new insight into the molecular mechanisms underlying pathogenesis of heart failure..
90. Nishioka K, Nishida M, Ariyoshi M, Saiki S, Jian Z, Hirano M, Nakaya M, Sato Y, Kita S, Iwamoto T, Hirano K, Inoue R and Kurose H, Protein kinase A-mediated phosphorylation of TRPC6 channels underlies suppression of angiotensin II-induced vasoconstriction., Arterioscler. Thromb. Vasc. Biol., 10.1161/ATVBAHA.110.221010, 31, 2278-2286, 2011.08.
91. Nishida M, Kitajima N, Saiki S, Nakaya M, Kurose H, Regulation of Angiotensin II receptor signaling by cysteine modification of NF-κB., Nitric oxide : biology and chemistry, 10.1016/j.niox.2010.10.003, 25, 2, 112-117, 2011.08.
92. Naoyuki Kitajima, Kunihiro Watanabe, Sachio Morimoto, Yoji Sato, Shigeki Kiyonaka, Masahiko Hoshijima, Yasuhiro Ikeda, Michio Nakaya, Tomomi Ide, Yasuo Mori, Hitoshi Kurose, Motohiro Nishida, TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2011.04.124, 409, 1, 108-113, 2011.05, Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca2+ signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (-/-)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca2+/calmodulin-dependent kinase II (CaMKII) were increased in MLP (-/-) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (-/-) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (-/-) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM. (C) 2011 Elsevier Inc. All rights reserved..
93. Kitajima N, Watanabe K, Morimoto S, Sato Y, Kiyonaka S, Hoshijima M, Ikeda Y, Nakaya M, Ide T, Mori Y, Kurose H and Nishida M, TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts., Arterioscler. Thromb. Vasc. Biol., 10.1016/j.bbrc.2011.04.124, 409, 108-113, 2011.05.
94. Motohiro Nishida, Mariko Ogushi, Reiko Suda, Miyuki Toyotaka, Shota Saiki, Naoyuki Kitajima, Michio Nakaya, Kyeong-Man Kim, Tomomi Ide, Yoji Sato, Kazuhide Inoue, Hitoshi Kurose, Heterologous down-regulation of angiotensin type 1 receptors by purinergic P2Y(2) receptor stimulation through S-nitrosylation of NF-kappa B, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1017640108, 108, 16, 6662-6667, 2011.04, Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT(1)R) density through NO-mediated S-nitrosylation of nuclear factor kappa B (NF-kappa B) in rat cardiac fibroblasts. Stimulation of purinergic P2Y(2) receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-kappa B in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. beta-Arrestins anchored the formation of p65/I kappa B alpha/beta-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-kappa B transcriptional activity and AT(1)R density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT(1)R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles..
95. Nishida M, Ogushi M, Suda R, Toyotaka M, Saiki S, Kitajima N, Nakaya M, Kim K-M, Ide T, Sato Y, Inoue K and Kurose H, Heterologous down-regulation of angiotensin type1 receptors by purinergic P2Y2 receptor stimulation through S-nitrosylation of NF-kB., Proc. Natl. Acad. Sci. USA., 10.1073/pnas.1017640108, 108, 6662-6667, 2011.04.
96. 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, 115, 3, 336-345, 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 DOG relieves ADR-induced cardiotoxicity. In this study, we investigated the influence of DOG on the pharmacokinetics and pharmacodyamics of ADR in order to clarify the mechanism by which DOG pre-treatment relieves ADR-induced cardiotoxicity. When ADR and/or DOG was intravenously administered, the DOG pre-treatment (DOG-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 DOG-ADR group. To elucidate the mechanism by which DOG 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 DOG-ADR groups. On the other hand, the DOG-ADR group showed significantly inhibited lipid peroxidation in the heart compared with the ADR group. It was considered that DOG pre-administration inhibited ADR-induced free radicals and decreased cardiotoxicity..
97. 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, 115, 3, 293-308, 2011.03.
98. Nakaya Michio, Mina Ohba, Motohiro Nishida, Hitoshi Kurose, Determining the activation of rho as an index of receptor coupling to G12/13 proteins, Methods in Molecular Biology, 10.1007/978-1-61779-126-0_17, 746, 317-327, 2011.01, Heterotrimeric G proteins are composed of α, β, and γ subunits. G proteins can be activated by a large number of cell-surface hepathelical receptors and can transduce signals from these receptors to various intracellular signaling molecules. When G protein-coupled receptors are bound by their cognate ligand, interaction with specific subtypes of G protein leads to dissociation of the α subunit of the heterotrimeric G protein from the βγ dimer, and both Gα-GTP and Gβγ are capable of initiating their own signal transduction pathways. G proteins are functionally divided into four groups based on the nature of α subunit into G s, Gi, Gq, and G12 families. The members of the G12 subfamily are G12 and G13. Increasing evidence indicates that G12/13 proteins play critical roles in various physiological functions. G12 and G13 regulate the small GTPase Rho through modulation of guanine nucleotide exchange factor (RhoGEF) activity to regulate various cellular responses, such as cytoskeletal changes and cell growth. Therefore, Rho activity can often represent a sensitive marker of G12/13 activity. Here, we describe the Rho activation assay to monitor the activity of G12/13 proteins..
99. Natriuretic Peptides-Guanylyl Cyclase-A Signaling Pathway Inhibits TRPC6-Mediated Pro-Hypertrophic Signaling in the Hearts.
100. Hideyuki Kinoshita, Koichiro Kuwahara, Motohiro Nishida, Hitoshi Kurose, Shigeki Kiyonaka, Yasuo Mori, Satoru Usami, Yasuaki Nakagawa, Kenji Ueshima, Kazuwa Nakao, Blockade of TRPC6 is a Novel Therapeutic Approach Against Pathological Cardiac Remodeling, JOURNAL OF CARDIAC FAILURE, 16, 9, 2010.09.
101. Kinoshita H, Kuwahara K, Nishida M, Jiang Z, Rong X, Kiyonaka S, Kuwabara Y, Kurose H, Inoue R, Mori Y, Li Y, Nakagawa Y, Usami S, Fujiwara M, Yamada Y, Minami T, Ueshima K and Nakao K., Inhibition of TRPC6 channel activity contributes to the anti-hypertrophic effects of natriuretic peptides-guanylyl cyclase-A signaling in the heart., Circulation Research, in press, 2010.06.
102. 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, 285, 20, 15268-15277, 2010.05, Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from G(i) 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)-1 beta among cytokines. Inhibition of IL-1 beta suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1 beta and AT1R through NF-kappa B, and a small GTP-binding protein, Rac, mediated PTX-induced NF-kappa B 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-1 beta siRNA, suggesting that IL-1 beta-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 G(i)/G(o) 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 G(i)/G(o)..
103. [Molecular mechanism underlying the development of heart failure mediated by heterotrimeric G protein signaling]..
104. Motohiro Nishida, Kenta Watanabe, Yoji Sato, Michio Nakaya, Naoyuki Kitajima, Tomomi Ide, Ryuji Inoue, Hitoshi Kurose, Phosphorylation of TRPC6 Channels at Thr(69) Is Required for Anti-hypertrophic Effects of Phosphodiesterase 5 Inhibition, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M109.074104, 285, 17, 13244-13253, 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 Thr(69) and prevented TRPC6-mediated Ca2+ influx. Substitution of Ala for Thr(69) 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 G alpha(q)-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..
105. Takuro Numaga, Motohiro Nishida, Shigeki Kiyonaka, Kenta Kato, Masahiro Katano, Emiko Mori, Tomohiro Kurosaki, Ryuji Inoue, Masaki Hikida, James W, Jr. Putney, Yasuo Mori, Ca2+ influx and protein scaffolding via TRPC3 sustain PKC beta and ERK activation in B cells, JOURNAL OF CELL SCIENCE, 10.1242/jcs.061051, 123, 6, 927-938, 2010.03.
106. Hideyuki Kinoshita, Koichiro Kuwahara, Motohiro Nishida, Zhong Jiang, Xianglu Rong, Shigeki Kiyonaka, Yoshihiro Kuwabara, Hitoshi Kurose, Ryuji Inoue, Yasuo Mori, Yuhao Li, Yasuaki Nakagawa, Satoru Usami, Kenji Ueshima, Kazuwa Nakao, Atrial and brain natriuretic peptides-guanylyl cyclase-A signaling pathway inhibits TRPC6-mediated pro-hypertrophic signaling in the hearts, ENDOCRINE JOURNAL, 57, 2010.03.
107. [Mechanism of cardiac hypertrophy via diacylglycerol-sensitive TRPC channels].
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 Ca2+ 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 Ca2+ transients evoked by Ca2+ influx-induced Ca2+ 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.
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108. Hideyuki Kinoshita, Koichiro Kuwahara, Ryuji Inoue, Motohiro Nishida, Hitoshi Kurose, Shigeki Kiyonaka, Yasuo Mori, Masaki Harada, Masao Murakami, Yasuaki Nakagawa, Shinji Yasuno, Satoru Usami, Masataka Fujiwara, Yoshihiro Kuwabara, Takeya Minami, Yuko Yamada, Kenji Ueshima, Kazuwa Nakao, Cardiac Natriuretic Peptides Inhibit TRPC6-Mediated Prohypertrophic Signaling, CIRCULATION RESEARCH, 105, 7, E47, 2009.09.
109. Regulation of cardiac hypertrophy by the formation of G protein-coupled receptor--TRPC channel protein complex..
110. Yano Takahisa, 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 subsequentactivation of mitogen-activated protein kinases and elevation of intracellular Ca2+ concentration., Antimicrobial agents and chemotherapy, 10.1128/AAC.01137-08, 53, 4, 1420-6, Vol. 53, No.4, 1420-1426, 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-PK1 cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, theleakage of lactate dehydrogenase, and tissue ATP depletion. Propidium 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-beta-cyclodextrin, which lowered the extracellularNa(+) concentration or the chelation of intracellular Ca(2+). The rise in the intracellular Ca(2+) concentration may be mediated through the activation of the ryanodine receptor (RyR) on the endoplasmic reticulum and the mitochondrial Na(+)-Ca(2+) exchanger, since cell injury was attenuated by dantrolene (an RyR antagonist) and CGP37157 (an Na(+)-Ca(2+) exchanger.
111. Takahisa Yano, Yoshinori Itoh, Eiko Kawamura, Asuka Maeda, Nobuaki Egashira, Motohiro Nishida, Hitoshi Kurose, Ryozo Oishi, 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, 53, 4, 1420-1426, 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-PK1 cells) to AMB caused cell injury, as assessed by mitochondrial enzyme activity, the leakage of lactate dehydrogenase, and tissue ATP depletion. Propidium 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-beta-cyclodextrin, which lowered the extracellular Na+ concentration or the chelation of intracellular Ca2+. 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+-Ca2+ 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..
112. 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, 106, 13, 5400-5405, Vol. 106, No.13, 5400-5405, 2009.03, Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) 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 Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-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..
113. Shibata,Takahiro, Nakahara,Hiroko, Kita,Narumi, Matsubara,Yui, Han,Chunguang, Morimitsu,Yasujiro, Iwamoto,Noriko, Kumagai,Yoshito, Nishida,Motohiro, Kurose,Hitoshi, Aoki,Naohito, Ojika,Makoto, Uchida,Koji, 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, 107, 5, 1248-1260, Vol. 107, 1248-1260, 2008.12.
114. Motohiro Nishida, Yoji Sato, Aya Uemura, Yusuke Narita, Hidetoshi Tozaki-Saitoh, Michio Nakaya, Tomomi Ide, Kazuhiro Suzuki, Kazuhide Inoue, Taku Nagao, Hitoshi Kurose, P2Y(6) receptor-G alpha(12/13) signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis, EMBO JOURNAL, 10.1038/emboj.2008.237, 27, 23, 3104-3115, Vol27, No.23, 3104-3115, 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)-beta. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (G alpha(12/13)) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF-beta, connective tissue growth factor, and periostin) and Ang-converting enzyme (ACE) was suppressed by the functional inhibition of G alpha(12/13). The expression of these fibrogenic genes through G alpha(12/13) 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 G alpha(12/13) in cardiomyocytes by the extracellular nucleotides-stimulated P2Y(6) receptor triggers fibrosis in pressure overload-induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF-beta..
115. Motohiro Nishida, Hitoshi Kurose, Roles of TRP channels in the development of cardiac hypertrophy, Naunyn-Schmiedeberg's Archives of Pharmacology, 10.1007/s00210-008-0321-8, 378, 4, 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 Ca2+ ([Ca2+]i) plays an important role in the development of hypertrophy. It is generally thought that the increase in [Ca2+]i for hypertrophy occurs via Gq-stimulated production of inositol-1,4,5-trisphosphate (IP 3) and IP3-mediated release of Ca2+ from intracellular store. However, several groups recently reported that canonical transient receptor potential (TRPC) channels are responsible for the increase in [Ca2+]i. Among them, three TRPC subtypes (TRPC3/TRPC6/TRPC7) are activated by another Gq-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 Ca2+ 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..
116. 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, 47, 23, 6169-6177, Vol. 47, 6169-7760, 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 beta-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 A1 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..
117. Motohiro Nishida, Naoya Onohara, Yoji Sato, Reiko Suda, Mariko Ogushi, Shihori Tanabe, Ryuji Inoue, Yasuo Mori, Hitoshi Kurose, G alpha(12/13)-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, 282, 32, 23117-23128, 282, 23117-23128, 2007.08, Sustained elevation of [Ca2+](i) has been implicated in many cellular events. We previously reported that alpha subunits of G(12) family G proteins (G alpha(12/13)) 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 G alpha(12/13)-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 G alpha(12) or G alpha(13) increased the expression of TRPC6 proteins and basal Ca2+ influx activity. The treatment with ET-1 increased TRPC6 protein levels through G alpha(12/13), 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 G alpha(12/13)-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 G alpha(12/13)-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 G alpha(12/13) in cardiac fibroblasts. First, G alpha(12/13) mediate ET-1- induced myofibroblast formation. Second, G alpha(12/13) 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..
118. Shinichiro Yamamoto, Teruaki Wajima, Yuji Hara, Motohiro Nishida, Yasuo Mori, Transient receptor potential channels in Alzheimer's disease, Biochimica et Biophysica Acta - Molecular Basis of Disease, 10.1016/j.bbadis.2007.03.006, 1772, 8, 958-967, 2007.08, Cognitive impairment and emotional disturbances in Alzheimer's disease (AD) result from the degeneration of synapses and neuronal death in the limbic system and associated regions of the cerebral cortex. An alteration in the proteolytic processing of the amyloid precursor protein (APP) results in increased production and accumulation of amyloid β-peptide (Aβ) in the brain. Aβ can render neurons vulnerable to excitotoxicity and apoptosis by disruption of cellular Ca2+ homeostasis and neurotoxic factors including reactive oxygen species (ROS), nitric oxide (NO), and cytokines. Many lines of evidence have suggested that transient receptor potential (TRP) channels consisting of six main subfamilies termed the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) are involved in Ca2+ homeostasis disruption. Thus, emerging evidence of the pathophysiological role of TRP channels has yielded promising candidates for molecular entities mediating Ca2+ homeostasis disruption in AD. In this review, we focus on the TRP channels in AD and highlight some TRP "suspects" for which a role in AD can be anticipated. An understanding of the involvement of TRP channels in AD may lead to the development of new target therapies..
119. 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, 25, 22, 5305-5316, 25, 5305-5316, 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 Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C(PLC)-mediated production of inositol-1,4,5-trisphosphate (IP3) is responsible for Ca(2+) 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 Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) 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 Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy..
120. Mangmool S, Haga T, Kobayashi H, Kim K-M, Nakata H, Nishida M & Kurose H., Clathrin regulates the activation of G protein-coupled receptor kinase 2 (GRK2) in cells but not in vitro., The Journal of Biological Chemistry, 281(42), 31940-31949, 2006.10.
121. Supachoke Mangmool, Tatsuya Haga, Hiroyuki Kobayashi, Kyeong-Man Kim, Hiroyasu Nakata, Motohiro Nishida, Hitoshi Kurose, Clathrin required for phosphorylation and internalization of beta(2)-adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2), JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M602832200, 281, 42, 31940-31949, 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 beta(2)-adrenergic receptor (beta(2)AR). However, the interaction of beta(2)AR 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 beta(2)AR. 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..
122. Suzuki T, Delgado-Escueta AV, Aguan K, Shi J, Alonso ME, Hara Y, Nishida M, Numata T, Takeuchi T, Morita R, Medina MT, Bai D, Ganesh S, Sugimoto Y, Inazawa J, Bailey JN, Ochoa A, Jara-Prado A, Rasmussen A, Ramos-Peek J, Cordova S, Rubio-Donnadieu F, Inoue, A gene mutated in juvenile myoclonic epilepsy encodes a protein regulating Cav2.3 channel., Nature Genetics, 36, 842-849, 2004.07.
123. H Kobayashi, Y Narita, M Nishida, H Kurose, beta-Arrestin2 enhances beta(2)-adrenergic receptor-mediated nuclear translocation of ERK, CELLULAR SIGNALLING, 10.1016/j.cellsig.2004.12.014, 17, 10, 1248-1253, 17, 1248-1253, 2005.10, beta-Arrestin mediates desensitization and internalization of beta-adrenergic receptors (beta ARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, we have examined the role of beta-arrestin2 in the beta AR-mediated ERK signaling pathways. Isoproterenol stimulation equally activated cytoplasmic and nuclear ERK in COS-7 cells expressing beta(1)AR or beta(2)AR. However, the activity of nuclear ERK was enhanced by co-expression of beta-arrestin2 in beta(2)AR-but not beta(1)AR-expressing cells. Pertussis toxin treatment and blockade of G beta gamma action inhibited beta-arrestin2-enhanced nuclear activation of ERK, suggesting that beta-arrestin2 promotes nuclear ERK localization in a G beta gamma dependent mechanism upon receptor stimulation. beta(2)AR containing the carboxyl terminal region of beta(1)AR lost the beta-arrestin2-promoted nuclear translocation. As the carboxyl terminal region is important for beta-arrestin binding, these results demonstrate that recruitment of beta-arrestin2 to carboxyl terminal region of beta(2)AR is important for ERK localization to the nucleus. (c) 2005 Elsevier Inc. All rights reserved..
124. M Nishida, S Tanabe, Y Maruyama, S Mangmool, K Urayama, Y Nagamatsu, S Takagahara, JH Turner, T Kozasa, H Kobayashi, Y Sato, T Kawanishi, R Inoue, T Nagao, H Kurose, G alpha(12/13)- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M409710200, 280, 18, 18434-18441, 280, 18434-18441, 2005.05, In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH2-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the G alpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of G alpha(12/13), but not by an RGS domain specific for G alpha(q). Constitutively active G alpha(12)- or G alpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated G alpha(13), which was completely inhibited by the G alpha(12/13)-specific RGS domain. Furthermore, the G alpha(12/13)-specific but not the G alpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by G alpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that G alpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation..
125. Okamura Yasushi, Nishino Atsuo, Murata Yoshimichi, Nakajo Koichi, Iwasaki Hirohide, Ohtsuka Yukio, Tanaka-Kunishima Motoko, Takahashi Nobuyuki, Hara Yuji, Yoshida Takashi, Nishida Motohiro, Okado Haruo, Watari Hirofumi, Meinertzhagen Ian A, Satoh Nori, Takahashi Kunitaro, Satou Yutaka, Okada Yasunobu, Mori Yasuo, Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes., Physiol Genomics, 10.1152/physiolgenomics.00229.2004, 22, 3, 269-282, 22, 269-282, 2005.08.
126. Fujii T, Onohara N, Maruyama Y, Tanabe S, Kobayashi H, Fukutomi M, Nagamatsu Y, Nishihara N, Inoue R, Sumimoto H, Shibasaki F, Nagao T, Nishida M & Kurose H., Ga12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts., The Journal of Biological Chemistry, 10.1074/jcb.M409397200, 280, 24, 23041-23047, 280, 23041-23047, 2005.06.
127. M Fukutomi, M Nishida, Y Maruyama, H Kobayashi, H Kurose, Caveolae-independent activation of protein kinase a in rat neonatal myocytes, JOURNAL OF PHARMACOLOGICAL SCIENCES, 98, 2, 168-174, 98, 168-174, 2005.06, Cardiomyocytes express both beta(1)- and beta(2)-adrenergic receptors, and these receptors play a differential role in chronotropic and inotropic effects of the heart. Caveolae are known as an important regulator of G-protein-coupled receptor signaling. In the present report, we examined whether caveolae have a role in beta-adrenergic receptor-stimulated cAMP production and protein kinase A activation in neonatal myocytes. Isoproterenol-stimulated cAMP production was mediated by beta(1)- and beta(2)-subtypes, which depends on the receptor number of each subtype. However, protein kinase A activation was exclusively mediated by the beta(1)-subtype. Disruption of caveolae by methyl-beta-cyclodextrin treatment did not affect the relative contribution of subtypes to isoproterenol-stimulated cAMP production. beta(1)-Subtype-mediated protein kinase A activation was also not affected by the disruption of caveolae. These results suggest that beta(1)-adrenergic receptor-mediated protein kinase A activation is compartmentalized and independent of caveolae..
128. Y Nagamatsu, M Nishida, N Onohara, M Fukutomi, Y Maruyama, H Kobayashi, Y Sato, H Kurose, Heterotrimeric G protein Ga-13-induced induction of cytokine mRNAs through two distinct pathways in cardiac fibroblasts, JOURNAL OF PHARMACOLOGICAL SCIENCES, 10.1254/jphs.FP0051036, 101, 2, 144-150, 101, 144-150, 2006.06, Overexpression of constitutively active (CA)-G alpha(13) significantly increased the expression of interleukin (IL)-1 beta and IL-6 mRNAs and proteins in rat cardiac fibroblasts. IL-1 beta mRNA induction by CA-G alpha(13) 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-G alpha(13) was suppressed by BAPTA-AM but not by DPI. However, both IL-1 beta and IL-6 mRNA induction was suppressed by nuclear factor kappa B (NF-kappa B) inhibitors. The CA-G alpha(13)-induced NF-kappa B activation was suppressed by DPI and BAPTA-AM, but not C3 toxin and the Rho-kinase inhibitor Y27632. IL-6 mRNA induction by CA-G alpha(13) was suppressed by SK&F96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), an inhibitor of receptor-activated nonselective cation channels, and the expression of CA-G alpha(13) increased basal Ca2+ influx. These results suggest that G alpha(13) regulates IL-1 beta mRNA induction through the reactive oxygen species-NF-kappa B pathway, while it regulates IL-6 mRNA induction through the Ca2+-NF-kappa B pathway..
129. Yamazaki K, Shigetomi E, Ikeda R, Nishida M, Kiyonaka S, Mori Y & Kato F, Blocker-resistant presynaptic voltage-dependent Ca2+ channels underlying glutamate release from the visceral primary afferents in the nucleus tractus solitarii of the mice., Brain Research, 1104, 103-113, 2006.06.
130. Koji Yamazaki, Eiji Shigetomi, Ryo Ikeda, Motohiro Nishida, Shiqeki 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, 1104, 103-113, 2006.08.
131. Motohiro Nishida, Yuji Hara, Takashi Yoshida, Ryuji Inoue, Yasuo Mori, TRP channels: Molecular diversity and physiological function, MICROCIRCULATION, 10.1080/10739680600885111, 13, 7, 535-550, 2006.10.
132. Ryuji Inoue, Lars Jorn 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, 99, 2, 119-131, 2006.07, Sustained elevation in the intracellular Ca2+ concentration via Ca2+ 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 Ca2+-entry channel molecules involved in this Ca2+ 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 Ca2+ entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca2+ 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 Ca2+-overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg2+ entry channel, and TRPP2 as a Ca2+-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..
133. T Suzuki, AV Delgado-Escueta, K Aguan, ME Alonso, J Shi, Y Hara, M Nishida, T Numata, MT Medina, T Takeuchi, R Morita, DS Bai, S Ganesh, Y Sugimoto, J Inazawa, JN Bailey, A Ochoa, A Jara-Prado, A Rasmussen, J Ramos-Peek, S Cordova, F Rubio-Donnadieu, Y Inoue, M Osawa, S Kaneko, H Oguni, Y Mori, K Yamakawa, Mutations in EFHC1 cause juvenile myoclonic epilepsy, NATURE GENETICS, 10.1038/ng1393, 36, 8, 842-849, 2004.08, Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures(1,2). 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 (Ca(v)2.3). EFHC1 and Ca(v)2.3 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..
134. Sugimoto K, Nishida M, Otsuka M, Ohkubo K, Mori Y & Morii T, Novel real time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells., Chemistry and Biology, 11, 475-485, 2004.04.
135. 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, 22, 18, 4677-4688, 22, 4677-4688, 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..
136. Arai K, Maruyama Y, Nishida M, Tanabe S, Kozasa T, Mori Y, Nagao T & Kurose H, Endothelin-1-induced MAPK activation and cardiomyocyte hypertrophy are mediated by Ga12 and Ga13 as well as Gaq and Gbg subunits., Molecular Pharmacology, 63(3), 478-488, 2003.07.
137. Kitano J, Nishida M, Itsukaichi Y, Minami I, Ogawa M, Hirano T, Mori Y & Nakanishi S, Direct interaction and functional coupling between metabotropic glutamate receptor subtype 1 and voltage-sensitive Cav2.1 Ca2+ channel., The Journal of Biological Chemistry, 278, 25101-25108, 2003.06.
138. S Shimizu, K Shiota, S Yamamoto, Y Miyasaka, M Ishii, T Watabe, M Nishida, Y Mori, T Yamamoto, Y Kiuchi, Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxidesynthase activity in vascular endothelial cells, FREE RADICAL BIOLOGY AND MEDICINE, 10.1016/S0891-5849(03)00172-2, 34, 10, 1343-1352, 34(10), 1343-1352, 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. (C) 2003 Elsevier Inc..
139. Y Maruyama, M Nishida, Y Sugimoto, S Tanabe, JH Tumer, T Kozasa, T Wada, T Nagao, H Kurose, G alpha(12/13) mediates alpha(1)-adrenergic receptor-induced cardiac hypertrophy, CIRCULATION RESEARCH, 10.1161/01.RES.0000043282.39776.7C, 91, 10, 961-969, 91, 961-969, 2002.11, In neonatal cardiomyocytes, activation of the G(q)-coupled alpha(1)-adrenergic receptor (alpha(1)AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK). Here, we show that JNK activation is essential for alpha(1)AR-induced hypertrophy, in that alpha(1)AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate alpha(1)AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. alpha(1)AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Galpha(q), Galpha(12), and Galpha(13)- JNK activation was also inhibited by the Galpha(q/11)- or Galpha(12/13)-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters Gbetagamma. alpha(1)AR-induced hypertrophic responses were inhibited by Galpha(q/11)- and Galpha(12/13)-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Galpha(12) and Galpha(13) but not by Gaq. Our findings suggest that alpha(1)AR-induced hypertrophic responses are mediated in part by a Galpha(12/13)-Rho-JNK pathway, in part by a G(q/11)-JNK pathway that is Rho independent, and in part by a Gbetagamma pathway that is JNK independent..
140. Y Hara, M Wakamori, M Ishii, E Maeno, M Nishida, T Yoshida, H Yamada, S Shimizu, E Mori, J Kudoh, N Shimizu, H Kurose, Y Okada, K Imoto, Y 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, 9, 1, 163-173, 9, 163-173, 2002.01, Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Call-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 (beta-NAD(+)) to the MutT motif. Arachidonic acid and Call 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 TNFalpha-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..
141. M Nishida, S Takagahara, Y Maruyama, Y Sugimoto, T Nagao, H Kurose, G beta gamma counteracts G alpha(q) signaling upon alpha(1)-adrenergic receptor stimulation, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1006/bbrc.2002.6553, 291, 4, 995-1000, 291, 995-1000, 2002.03, In rat neonatal myocytes, a constitutively active Galpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the Gbetagamma-sequestering peptide betaARK-ct increases the phenylephrine-induced cardiac injury, indicating that Gbetagamma released from G(q) counteracts the Galpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by betaARK-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 Gbetagamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the Galpha(q)-induced cellular injury and apoptosis. (C) 2002 Elsevier Science (USA)..
142. Y Mori, M Wakamori, T Miyakawa, M Hermosura, Y Hara, M Nishida, K Hirose, A Mizushima, M Kurosaki, E Mori, K Gotoh, T Okada, A Fleig, R Penner, M Iino, T 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, 195, 6, 673-681, 195, 673-681, 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..
143. M Nishida, KL Schey, S Takagahara, K Kontani, T Katada, Y Urano, T Nagano, T Nagao, H Kurose, Activation mechanism of G(i) and G(o) by reactive oxygen species, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M107392200, 277, 11, 9036-9042, 277, 9036-9042, 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 (Galpha(i) and Galpha(o)), leading to activation. H2O2 is one of the reactive oxygen species and activates purified Galpha(i2). 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 (Cys(66), Cys(112), Cys(140), cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with .OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in .OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) 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 Gai2 but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Gai2 by the mechanism similar to .OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses..
144. Nishida M, Sakamoto K, Urushidani T & Nagao T, Treatment with l-cis diltiazem before reperfusion reduces infarct size in the ischemic rabbit heart in vivo., The Journal of Pharmaceutical Sciences, 80, 319-325, 1999.07.
145. Nishida M, Nagao T & Kurose H, 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 Communication, 262, 350-354, 1999.08.
146. Nishida M, Urushidani T, Sakamoto K & Nagao T, l-cis Diltiazem attenuates intracellular Ca2+ overload by metabolic inhibition in guinea pig myocytes., The European Journal of Pharmacology, 385, 225-230, 1999.12.
147. Nishida M, Maruyama Y, Tanaka R, Kontani K, Nagao T, & Kurose H, Gai and Gao are target proteins of reactive oxygen species., Nature, 408, 492-495, 2000.12.
148. 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, 408, 6811, 492-495, 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..


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