研究者詳細 - 沼田　朋大

Takahito Miyake, Saki Nakamura, Zhao Meng, Satoshi Hamano, Keisuke Inoue, Tomohiro Numata, Nobuaki Takahashi, Kazuki Nagayasu, Hisashi Shirakawa, Yasuo Mori, Takayuki Nakagawa, Shuji Kaneko

FRONTIERS IN PHYSIOLOGY ( FRONTIERS MEDIA SA ) 8 ( NOV ) 878 - 878 2017年11月

Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1) is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD), which causes sensitization of TRPA1 to reactive oxygen species (ROS). Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1) with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1mM) induced outward-rectifier whole-cell currents and increased the intracellular Ca2+ concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca2+ influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser)-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala)-expressing cells showed similar whole-cell currents and Ca2+ influx. By contrast, a lower concentration of oxaliplatin (100 mu M) did not increase the intracellular Ca2+ concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser)-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower concentration induces cold-induced cysteine oxidation-dependent opening of hTRPA1 via PHD inhibition.

DOI PubMed

糖尿病とがん SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松志保, 野見山崇, 沼田朋大, 川波賢子, 濱口百合子, 高橋貴理子, 田中智子, 井上隆司, 柳瀬敏彦

糖尿病合併症 ( (一社)日本糖尿病合併症学会 ) 31 ( Suppl.1 ) 190 - 190 2017年10月

J-GLOBAL

血管・動脈硬化 SGLT-2阻害薬によるマクロファージ活性抑制を介した糖尿病大血管症進展抑制効果の解析

松村剛, 村上彩子, 瀬ノ口隆文, 石井規夫, 山田沙理恵, 守田雄太郎, 西田周平, 久木留大介, 本島寛之, 近藤龍也, 沼田朋大, 荒木栄一

糖尿病合併症 ( (一社)日本糖尿病合併症学会 ) 31 ( Suppl.1 ) 240 - 240 2017年10月

Integrative Approach with Electrophysiological and Theoretical Methods Reveals a New Role of S4 Positively Charged Residues in PKD2L1 Channel Voltage-Sensing

Tomohiro Numata, Kunichika Tsumoto, Kazunori Yamada, Tatsuki Kurokawa, Shinichi Hirose, Hideki Nomura, Mitsuhiro Kawano, Yoshihisa Kurachi, Ryuji Inoue, Yasuo Mori

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP ) 7 ( 1 ) 9760 2017年08月

Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (P-open)-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, G(max). Subsequent single-channel conductance (gamma) measurements revealed that voltage-dependence of G(max) in WT can be explained by the inward-rectifying property of gamma, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and Na(V)Ab structures suggest that such voltage dependence of P-open and gamma in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.

DOI PubMed

Strategy to Attain Remarkably High Photoinduced Charge-Separation Yield of Donor−Acceptor Linked Molecules in Biological Environment via Modulating Their Cationic Moieties.

Ning Cai, Yuta Takano, Tomohiro Numata, Ryuji Inoue, Yasuo Mori, Tatsuya Murakami, Hiroshi Imahori

Physical Chemistry ( AMER CHEMICAL SOC ) 121 ( 32 ) 17457 - 17465 2017年08月

A series of ferrocene-porphyrin-fullerene linked triads (TC1, TC2, and TC4) possessing different numbers of cationic moieties were designed and prepared to achieve a high photoinduced charge-separation (CS) yield in a biological environment. In a solution, TC1, TC2, and TC4 demonstrated the formation of their nanoaggregates. Among the new triads, TC4 possessing the four cationic moieties exhibited the formation of a long-lived charge-separated state with the highest CS yield (86%) ever reported in cell membrane-like lipid bilayers, which is consistent with the largest change in the cell membrane potential of PC12 cells via the photoinduced CS under green light illumination. The highest CS yield in the biological environment can be rationalized by the well-tailored balance in hydrophobicity and hydrophilicity of TC4. This finding provides a strategy to improve greatly the photoinduced charge-separation yield of donor-acceptor linked molecules in the biological environment and also will be informative for extracting the full potential of the photoinduced charge-separated state toward biological applications.

DOI

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches

Yaopeng Hu, Yubin Duan, Ayako Takeuchi, Lin Hai-Kurahara, Jun Ichikawa, Keizo Hiraishi, Tomohiro Numata, Hiroki Ohara, Gentaro Iribe, Michio Nakaya, Masayuki X. Mori, Satoshi Matsuoka, Genshan Ma, Ryuji Inoue

CARDIOVASCULAR RESEARCH ( OXFORD UNIV PRESS ) 113 ( 10 ) 1243 - 1255 2017年08月

Aims Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4.
Methods and results Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent K-d = similar to 500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+-and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (>= six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced.
Conclusions These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

DOI PubMed

SGLT2 Inhibitor Ipragliflozin Inhibits Breast Cancer via Membrane Hyperpolarization

Shiho Komatsu, Takashi Nomiyama, Tomohiro Numata, Takako Kawanami, Yuriko Hamaguchi, Kiriko Takahashi, Tomoko Tanaka, Ryuji Inoue, Toshihiko Yanase

DIABETES ( AMER DIABETES ASSOC ) 66 A532 - A532 2017年06月

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松志保, 野見山崇, 沼田朋大, 川波賢子, 濱口百合子, 高橋貴理子, 田中智子, 井上隆司, 柳瀬敏彦

日本内分泌学会雑誌 ( (一社)日本内分泌学会 ) 93 ( 1 ) 348 - 348 2017年04月

J-GLOBAL

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松志保, 野見山崇, 沼田朋大, 川波賢子, 濱口百合子, 高橋貴理子, 井上隆司, 柳瀬敏彦

糖尿病 ( (一社)日本糖尿病学会 ) 60 ( Suppl.1 ) S - 394 2017年04月

FUNCTIONAL CHARATERIZATION OF ZEBRAFISH TRANSIENT RECEPTOR POTENTIAL MELASTATIN 2 IN HEK 293 CELL LINE

Ha Nam Tran, Numata Tomohiro, Mori Masayuki X, Maegawa Shingo, Hosokawa Hiroshi, Tanaka Shingo, Mori Yasuo

JOURNAL OF PHARMACOLOGICAL SCIENCES 133 ( 3 ) S126 2017年03月

The regulatory effect of oxaliplatin on human TRPA1 cold sensitivity

Miyake Takahito, Nakamura Saki, Zhao Meng, So Kanako, Inoue Keisuke, Numata Tomohiro, Takahashi Nobuaki, Shirakawa Hisashi, Mori Yasuo, Nakagawa Takayuki, Kaneko Shuji

JOURNAL OF PHARMACOLOGICAL SCIENCES 133 ( 3 ) S85 2017年03月

K562細胞の増殖・赤芽球分化におけるTRPM7を介したイオン流入の役割

高橋貴理子, 沼田朋大, 山浦健, 井上隆司

日本生理学雑誌 ( (一社)日本生理学会 ) 79 ( 1 ) 34 - 34 2017年02月

J-GLOBAL

共培養系を用いた線維芽細胞による心房興奮伝播制御機構の検討

胡耀鵬, 平石敬三, 倉原琳, 市川純, 沼田朋大, 井上隆司

日本生理学雑誌 ( (一社)日本生理学会 ) 79 ( 1 ) 28 - 28 2017年02月

J-GLOBAL

Distinct contributions of LRRC8A and its paralogs to the VSOR anion channel from those of the ASOR anion channel

Kaori Sato-Numata, Tomohiro Numata, Ryuji Inoue, Ravshan Z. Sabirov, Yasunobu Okada

CHANNELS ( TAYLOR & FRANCIS INC ) 11 ( 2 ) 167 - 172 2017年

Volume- and acid-sensitive outwardly rectifying anion channels (VSOR and ASOR) activated by swelling and acidification exhibit voltage-dependent inactivation and activation time courses, respectively. Recently, LRRC8A and some paralogs were shown to be essentially involved in the activity and inactivation kinetics of VSOR currents in human colonic HCT116 cells. In human cervix HeLa cells, here, inactivation of VSOR currents was found to become accelerated by RNA silencing only of LRRC8A but never decelerated by that of any LRRC8 isoform. These data suggest that LRRC8A is associated with the deceleration mechanism of VSOR inactivation, while none of LRRC8 members is related to the acceleration mechanism. Activation kinetics of ASOR currents was unaffected by knockdown of any LRRC8 family member. Double, triple and quadruple gene-silencing studies indicated that combinatory expression of LRRC8A with LRRC8D and LRRC8C is essential for VSOR activity, whereas none of LRRC8 family members is involved in ASOR activity.

DOI PubMed

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