研究等業績 - その他 - 沼田　朋大

糖尿病性腎症におけるN型Ca²⁺チャネル阻害の腎保護効果

大野祥子, 横井秀基, 笠原正登, 森潔, 桑原宏一郎, 桑原孝成, 今牧博貴, 石井輝, 古賀健一, 沼田朋大, 菅原照, 森泰生, 中尾一和, 柳田素子, 向山政志

高血圧関連疾患モデル学会学術総会抄録集   51st   2015年

J-GLOBAL

オキサリプラチン誘発急性末梢神経におけるTRPA1の関与

三宅崇仁, 中村彩希, 趙萌, 浜野智, 井上圭亮, 沼田朋大, 高橋重成, 白川久志, 森泰生, 中川貴之, 中川貴之, 金子周司

日本薬学会年会要旨集(CD-ROM)   135th   2015年

J-GLOBAL

Transient Receptor Potential Melastatin 2 Protects Mice against Polymicrobial Sepsis by Enhancing Bacterial Clearance

XiaoWei Qian, Tomohiro Numata, Kai Zhang, CaiXia Li, JinChao Hou, Yasuo Mori, XiangMing Fang

ANESTHESIOLOGY ( LIPPINCOTT WILLIAMS & WILKINS )  121 ( 2 ) 336 - 351   2014年08月

Background: Recent studies suggest that the transient receptor potential melastatin 2 (TRPM2) channel plays an important role in inflammation and immune response. However, the role and mechanism of TRPM2 in polymicrobial sepsis remain unclear.
Methods: The authors explored the effects of genetic disruption of TRPM2 on mortality (n = 15), bacterial clearance (n = 6), organ injury, and systemic inflammation during cecal ligation and puncture-induced sepsis. Electrophysiology, immunoblot, bacterial clearance experiment, and quantitative real-time polymerase chain reaction were used to explore the role and mechanism of TRPM2 in sepsis.
Results: After cecal ligation and puncture, Trpm2-knockout mice had increased mortality compared with wild-type mice (73.3 vs. 40%, P = 0.0289). The increased mortality was associated with increased bacterial burden, organ injury, and systemic inflammation. TRPM2-mediated Ca2+ influx plays an important role in lipopolysaccharide or cecal ligation and puncture-induced heme oxygenase-1 (HO-1) expression in macrophage. HO-1 up-regulation decreased bacterial burden both in wildtype bone marrow-derived macrophages and in cecal ligation and puncture-induced septic wild-type mice. Disruption of TRPM2 decreased HO-1 expression and increased bacterial burden in bone marrow-derived macrophages. Pretreatment of Trpm2-knockout bone marrow-derived macrophages with HO-1 inducer markedly increased HO-1 expression and decreased bacterial burden. Pretreatment of Trpm2-knockout mice with HO-1 inducer reversed the susceptibility of Trpm2-knockout mice to sepsis by enhancing the bacterial clearance. In addition, septic patients with lower monocytic TRPM2 and HO-1 messenger RNA levels had a worse outcome compared with septic patients with normal monocytic TRPM2 and HO-1 messenger RNA levels. TRPM2 levels correlated with HO-1 levels in septic patients (r = 0.675, P = 0.001).
Conclusion: The study data demonstrate a protective role of TRPM2 in controlling bacterial clearance during polymicrobial sepsis possibly by regulating HO-1 expression.

DOI PubMed

Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

Kaori Sato-Numata, Tomohiro Numata, Yasunobu Okada

CHANNELS ( LANDES BIOSCIENCE )  8 ( 3 ) 278 - 283   2014年05月

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 degrees C and the Q(10) value of 5.6. After 1 h exposure to acidic solution at 37 degrees C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 degrees C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.

DOI PubMed

糖尿病性腎症におけるN型Ca2+チャネル阻害の意義

大野 祥子, 横井 秀基, 笠原 正登, 森 潔, 桑原 宏一郎, 藤倉 純二, 内藤 雅喜, 桑原 孝成, 今牧 博貴, 石井 輝, 古賀 健一, 沼田 朋大, 菅原 照, 森 泰生, 柳田 素子, 中尾 一和, 向山 政志

糖尿病 ( (一社)日本糖尿病学会 )  57 ( Suppl.1 ) S - 266   2014年04月

Transnitrosylation Directs TRPA1 Selectivity in N-Nitrosamine Activators

Daisuke Kozai, Yoji Kabasawa, Maximilian Ebert, Shigeki Kiyonaka, Firman Yuko Otani, Tomohiro Numata, Nobuaki Takahashi, Yasuo Mori, Tomohiko Ohwada

MOLECULAR PHARMACOLOGY ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS )  85 ( 1 ) 175 - 185   2014年01月

S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca2+-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo, 3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca2+ influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.

DOI PubMed

Design Methodology of Tri-Gate Poly-Si MOSFETs with 10nm Nanowire Channel to Enhance Short-Channel Performance and Reduce V-th & I-d Variability

Masumi Saitoh, Kensuke Ota, Chika Tanaka, Toshinori Numata

2014 SYMPOSIUM ON VLSI TECHNOLOGY (VLSI-TECHNOLOGY): DIGEST OF TECHNICAL PAPERS ( IEEE )    2014年

We present the optimum design of tri-gate poly-Si nanowire transistors (NW Tr.) based on the systematic performance and variability analysis for various NW width (WNW) and thickness (TSi) down to 10nm. Ion difference between poly-Si and crystalline-Si Tr. at short L (down to 25nm) is much smaller than long L due to poly-Si defect-barrier lowering by high lateral field. Ion of poly-Si pFETs is close to nFETs due to smaller interface defects. Both WNW and TSi scaling reduces S factor, SCE and Avt (Vth & Id variations) caused by random grain placement. Avt of thin poly-Si Tr. falls even below doped bulk Tr. Since short-NW Tr. suffers from high RSD and low mu, narrow and tall poly-Si NW Tr. is the best for 3D CMOS.

Development of novel activators for TRPC channels aiming to treatment of neurodegenerative disorders

Seishiro Sawamura, Masahiko Hatano, Yoshinori Takada, Takashi Kuwamura, Tomohiro Numata, Shigeki Kiyonaka, Kyosuke Hino, Tetsuya Kawamura, Jun Tanikawa, Hiroshi Nakagawa, Ryu Nagata, Ryuji Inoue, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  124   181P - 181P   2014年

Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel's Kinetics in a Temperature-Dependent Manner

Yuki Nakamura, Xiuyu Shi, Tomohiro Numata, Yasuo Mori, Ryuji Inoue, Christoph Lossin, Tallie Z. Baram, Shinichi Hirose

PLOS ONE ( PUBLIC LIBRARY SCIENCE )  8 ( 12 ) e80376   2013年12月

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I-h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p. S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I-h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of Ih during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

DOI PubMed

Acid-sensitive outwardly rectifying (ASOR) anion channels in human epithelial cells are highly sensitive to temperature and independent of ClC-3

Kaori Sato-Numata, Tomohiro Numata, Toshiaki Okada, Yasunobu Okada

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  465 ( 11 ) 1535 - 1543   2013年11月

A novel type of anion channel activated by extracellular acidification, called acid-sensitive outwardly rectifying (ASOR) anion channel, was shown to be involved in acidotoxic necrotic death in human epithelial cells. However, its biophysical property and molecular identity have remained elusive. In human epithelial HeLa cells, here, whole-cell currents of ASOR anion channel were found to be augmented by warm temperature, with a threshold temperature of 32 A degrees C. Temperature sensitivity of the conductance was found to be high (with Q (10) of 8.8) in the range of body temperature, suggesting a possible involvement of a non-diffusion-limited process such as a transporter-mediated conduction. In this regard, it is interesting that a Cl-/H+ antiporter ClC-3 has recently been proposed as a candidate for the ASOR channel. However, siRNA-mediated knockdown of hClC-3 failed to suppress ASOR currents in HeLa cells. Also, endogenous ASOR currents in HEK293T cells were not affected by overexpression of human or mouse ClC-3. Furthermore, functional expression of the ASOR channel was virtually absent in the cisplatin-resistant human cancer KCP-4 cell line despite the fact that molecular expression of ClC-3 was indistinguishable between KCP-4 cells and parental cisplatin-sensitive KB-3-1 cells which endogenously exhibit high activity of ASOR anion channels. These results indicate that the ASOR anion channel is highly sensitive to temperature and independent of ClC-3.

DOI PubMed

N型Caチャネルノックアウトマウスを用いた糖尿病性腎症におけるN型カルシウムチャネルの意義

大野 祥子, 横井 秀基, 笠原 正登, 森 潔, 桑原 宏一郎, 藤倉 純二, 内藤 雅喜, 桑原 孝成, 今牧 博貴, 古賀 健一, 沼田 朋大, 菅原 照, 森 泰生, 中尾 一和, 向山 政志

日本高血圧学会総会プログラム・抄録集 ( (NPO)日本高血圧学会 )  36回   282 - 282   2013年10月

TRP channels as sensors of oxygen availability

Tomohiro Numata, Nozomi Ogawa, Nobuaki Takahashi, Yasuo Mori

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  465 ( 8 ) 1075 - 1085   2013年08月

An ability to adapt to changes in oxygen availability is essential for survival in both prokaryotic and eukaryotic organisms. Recently, cation channels encoded by the transient receptor potential (trp) gene superfamily have been recognized as multimodal sensors of a wide variety of factors inside the cells and in the extracellular environment and also as transducers of electrical and chemical signals mediated by ions such as Ca2+. The functional features of TRP channels enable the body to react and adapt to different forms of environmental changes, including oxygen levels. A subclass of TRP channels regulates various cellular processes in response to fluctuations in oxygen. In this article, we describe the physiological and pathological significance of the oxygen-sensitive TRP channels, which are heterogeneous in the cellular responses to acute changes in oxygen, by contrasting their oxygen monitoring function with that of other ion channels, transporters, and enzymes. We also discuss the physiological relevance of oxygen-sensitive TRP channels as a novel class of target proteins for pharmaceutical therapeutics.

DOI PubMed

糖尿病性腎症におけるN型カルシウムチャネルの意義

大野 祥子, 向山 政志, 横井 秀基, 笠原 正登, 森 潔, 藤倉 純二, 桑原 宏一郎, 内藤 雅喜, 桑原 孝成, 今牧 博貴, 石井 輝, 古賀 健一, 沼田 朋大, 菅原 照, 森 泰生, 中尾 一和

日本腎臓学会誌 ( (一社)日本腎臓学会 )  55 ( 3 ) 349 - 349   2013年04月

Targeting TRPs in Neurodegenerative Disorders

Yoshinori Takada, Tomohiro Numata, Yasuo Mori

CURRENT TOPICS IN MEDICINAL CHEMISTRY ( BENTHAM SCIENCE PUBL LTD )  13 ( 3 ) 322 - 334   2013年02月

Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis present a significant medical challenge in the modern world. Recent evidence indicates that perturbation of neuronal Ca2+ homeostasis is involved in the pathogenesis of these neurodegenerative disorders. Transient receptor potential (TRP) channels are non-selective cation channels that are expressed in various cell types and tissues, and play an important role in regulating Ca2+ signaling in both non-neuronal and neuronal cells. TRP channels are related to the onset or progression of several diseases, and defects in the genes encoding TRP channels (so-called "TRP channelopathies") underlie certain neurodegenerative disorders due to their abnormal Ca2+ signaling properties. In this article, we review recent findings regarding the relationship between TRPs and neurodegenerative disorders, and discuss the therapeutic potential of targeting TRP channels pharmacologically.

DOI PubMed

The deltaC splice-variant of TRPM2 is the hypertonicity-induced cation channel (HICC)in HeLa cells, and the ecto-enzyme CD38 mediates its activation

Numata Tomohiro, Sato Kaori, Christmann Jens, Marx Romy, Mori Yasuo, Okada Yasunobu, Wehner Frank

JOURNAL OF PHYSIOLOGICAL SCIENCES   63   S130   2013年

Using physiological temperature variations to perform a functional analysis of mutant HCN channels

Nakamura Yuki, Shi Xiuyu, Numata Tomohiro, Mori Yasuo, Inoue Ryuji, Hirose Shinichi

JOURNAL OF PHYSIOLOGICAL SCIENCES   63   S133   2013年

細胞生死スイッチに関与する細胞容積関連性イオンチャネル

岡田泰伸, 沼田(佐藤)かお理, 沼田朋大, 清水貴浩, 秋田天平, 岡田俊昭

日本Cell Death学会学術集会プログラム抄録集   22nd   28   2013年

J-GLOBAL

Involvements of the ABC protein ABCF2 and α-actinin-4 in regulation of cell volume and anion channels in human epithelial cells.

Ando-Akatsuka Y, Shimizu T, Numata T, Okada Y

Journal of cellular physiology ( WILEY-BLACKWELL )  227 ( 10 ) 3498 - 3510   2012年10月

After osmotic swelling, cell volume is regulated by a process called regulatory volume decrease (RVD). Although actin cytoskeletons are known to play a regulatory role in RVD, it is not clear how actin-binding proteins are involved in the RVD process. In the present study, an involvement of an actin-binding protein, a-actinin-4 (ACTN4), in RVD was examined in human epithelial HEK293T cells. Overexpression of ACTN4 significantly facilitated RVD, whereas siRNA-mediated downregulation of endogenous ACTN4 suppressed RVD. When the cells were subjected to hypotonic stress, the content of ACTN4 increased in a 100,000 x g pellet, which was sensitive to cytochalasin D pretreatment. Protein overlay assays revealed that ABCF2, a cytosolic member of the ABC transporter superfamily, is a binding partner of ACTN4. The ACTN4-ABCF2 interaction was markedly enhanced by hypotonic stimulation and required the NH2-terminal region of ABCF2. Overexpression of ABCF2 suppressed RVD, whereas downregulation of ABCF2 facilitated RVD. We then tested whether ABCF2 has a suppressive effect on the activity of volume-sensitive outwardly rectifying anion channel (VSOR), which is known to mediate Cl- efflux involved in RVD, because another ABC transporter member, CFTR, was shown to suppress VSOR activity. Whole-cell VSOR currents were largely reduced by overexpression of ABCF2 and markedly enhanced by siRNA-mediated depletion of ABCF2. Thus, the present study indicates that ACTN4 acts as an enhancer of RVD, whereas ABCF2 acts as a suppressor of VSOR and RVD, and suggests that a swelling-induced interaction between ACTN4 and ABCF2 prevents ABCF2 from suppressing VSOR activity in the human epithelial cells. J. Cell. Physiol. 227: 34983510, 2012. (C) 2012 Wiley Periodicals, Inc.

DOI PubMed

Active Zone Protein Bassoon Co-Localizes with Presynaptic Calcium Channel, Modifies Channel Function, and Recovers from Aging Related Loss by Exercise

Hiroshi Nishimune, Tomohiro Numata, Jie Chen, Yudai Aoki, Yonghong Wang, Miranda P. Starr, Yasuo Mori, John A. Stanford

PLOS ONE ( PUBLIC LIBRARY SCIENCE )  7 ( 6 ) e38029   2012年06月

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca2+ influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

DOI PubMed

Utilization of Photoinduced Charge-Separated State of Donor-Acceptor-Linked Molecules for Regulation of Cell Membrane Potential and Ion Transport

Tomohiro Numata, Tatsuya Murakami, Fumiaki Kawashima, Nobuhiro Morone, John E. Heuser, Yuta Takano, Kei Ohkubo, Shunichi Fukuzumi, Yasuo Mori, Hiroshi Imahori

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY ( AMER CHEMICAL SOC )  134 ( 14 ) 6092 - 6095   2012年04月

The control of ion transport across cell membranes by light is an attractive strategy that allows targeted, fast control of precisely defined events in the biological membrane, Here we report a novel general strategy for the control of membrane potential and ion transport by using charge-separation molecules and light. Delivery of charge-separation molecules to the plasma membrane of PC12 cells by a membranous nanocarrier and subsequent light irradiation led to depolarization of the membrane potential as well as inhibition of the potassium ion flow across the membrane. Photoregulation of the cell membrane potential and ion transport by using charge-separation molecules is highly promising for control of cell functions.

DOI PubMed