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RIN14B細胞の細胞内Ca2+動態に対する硫化水素の作用に関する研究

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Please use this identifier to cite or link to this item:https://doi.org/10.14943/doctoral.k12614
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Title: RIN14B細胞の細胞内Ca2+動態に対する硫化水素の作用に関する研究
Authors: 氏家, 絢子 Browse this author
Issue Date: 23-Mar-2017
Publisher: Hokkaido University
Abstract: Hydrogen sulfide (H2S) is enzymatically synthesized from cysteine in the mammalian cells and acts as a gasotransmitter. I have previously reported that H2S activates transient receptor potential ankyrin 1 (TRPA1) channels and causes the increase of intracellular Ca2+ concentration ([Ca2+]i) in RIN14B cells, the cell line derived from rat pancreatic δ cells. On the other hand, H2S is reported to inhibit Ca2+ influx through the activation of ATP-sensitive K+ (KATP) channels in rat pancreatic β cells. There are no reports how the effects of H2S on Ca2+ signal appear in the cells expressing both TRPA1 and KATP channels. Furthermore, H2S is reported to be involved in pathogenesis of inflammation. Under inflammatory conditions, the changes in the expressions and activities of target channels for H2S and/or intracellular environment may affect the effects of H2S. In this study, the effects of H2S donors, NaHS or Na2S, on Ca2+ signals in RIN14B cells which express both TRPA1 and KATP channels were examined under normal and inflammatory conditions. The [Ca2+]i was measured using fura-2, a fluorescent Ca2+ indicator. NaHS caused three types of [Ca2+]i responses; 1) spontaneous Ca2+ signals were inhibited, 2) after inhibition, the [Ca2+]i increased, 3) the [Ca2+]i rapidly increased without inhibition. The percentage of cells showing each patterns were 37% (19/51 cells), 24% (12/51 cells) and 25% (13/51 cells), respectively. The expressions of Kir6.2/SUR1 subunits of KATP channels were detected in RIN14B cells. In the presence of glibenclamide and tolbutamide, KATP channel blockers, the inhibitory effect by NaHS was abolished, and NaHS induced [Ca2+]i increases in almost cells (87%). Areas under the curves of [Ca2+] responses to NaHS were increased by KATP channel blockers. These results suggest that H2S shows bidirectional effects on Ca2+ signals: excitatory and inhibitory effects through TRPA1 and KATP channels, respectively. Next, the effects of proinflammatory cytokines on Na2S-evoked [Ca2+]i responses were examined. The excitatory effects of Na2S on Ca2+ signals were enhanced by the treatment of IL-1β, not by that of TNF-α. IL-1β failed to affect the inhibitory effect of diazoxide, a KATP channel opener, on Ca2+ signals, suggesting that KATP channels were not involved in the effect of IL-1β. [Ca2+]i increases by Na2S were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blocker in IL-1β-treated cells. However, [Ca2+]i increases by a TRPA1 agonist and a high concentration of KCl were not affected by IL-1β treatment. The effect of polysulfides, the molecules with varying number of sulfane sulfur and more potency against TRPA1, was next examined. Polysulfide Na2S3 increased the [Ca2+]i. Unlike Na2S, the Na2S3-induced [Ca2+]i increases were not enhanced by IL-1β. Intracellular polysulfide was measured using SSP4, a fluorescence indicator of sulfane sulfur. Intracellular polysulfide was increased by Na2S, which was enhanced by IL-1β treatment. These results suggest that IL-1β enhanced the oxidative reaction from H2S to polysulfide, which result in the enhancement of excitatory effect of H2S. The change of balance between H2S and polysulfide by inflammatory factors may affect the role of H2S in inflammation.
Conffering University: 北海道大学
Degree Report Number: 甲第12614号
Degree Level: 博士
Degree Discipline: 獣医学
Examination Committee Members: (主査) 准教授 乙黒 兼一, 特任教授 葉原 芳昭, 教授 木村 和弘, 名誉教授 伊藤 茂男
Degree Affiliation: 獣医学研究科(獣医学専攻)
Type: theses (doctoral)
URI: http://hdl.handle.net/2115/68635
Appears in Collections:課程博士 (Doctorate by way of Advanced Course) > 獣医学院(Graduate School of Veterinary Medicine)
学位論文 (Theses) > 博士 (獣医学)

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