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アストロサイトにおける硫化水素の細胞内Ca2+シグナルとエネルギー代謝へ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.k14546
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Title: アストロサイトにおける硫化水素の細胞内Ca2+シグナルとエネルギー代謝への作用に関する研究
Other Titles: The effect of hydrogen sulfide on Ca2+ signal and energy metabolism in astrocytes
Authors: 新居, 剛 Browse this author
Issue Date: 25-Mar-2021
Abstract: Hydrogen sulfide (H2S) inhibits the electron transport chain (ETC) in mitochondria. Despite this toxicity, H2S is endogenously produced mainly by astrocytes. In hippocampal astrocytes, H2S increases intracellular Ca2+ concentration ([Ca2+]i) mainly by activating transient receptor potential ankyrin 1 (TRPA1) on the cell membrane through the post-translational modification of protein. It was also shown that Ca2+ release from the intracellular Ca2+ stores was involved in H2S-induced [Ca2+]i increase. However, the precise mechanism and functional role of this Ca2+ response are still unknown. H2S induces not only metabolic changes in mitochondria but also promotes glycolysis. Lactate, a glycolytic product, had been considered a waste product, but it is reevaluated as an energy substrate and signals. Recently, a lot of studies have reported that intracellular metabolic changes mediate Ca2+ signals by influencing concentrations of bioactive substances including ATP and reactive oxygen species (ROS). On the other hand, Ca2+ is a key regulator of energy metabolism. Here, we examined the relationship between H2S-induced metabolic changes and Ca2+ response. [Ca2+]i in cultured rat spinal cord astrocytes was measured using fluorescent Ca2+ indicator Fura-2. The extracellular lactate and intracellular ATP levels were measured by the enzymatic reaction using lactate dehydrogenase and luciferase, respectively. Na2S (150 μM), an H2S donor, induced a nontoxic increase in [Ca2+]i in cultured rat spinal cord astrocytes. This [Ca2+]i increase by Na2S was not inhibited by HC-030031, a TRPA1 inhibitor, and removal of extracellular Ca2+. Ca2+ depletion in the endoplasmic reticulum (ER) after application of thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, inhibited Na2S-induced [Ca2+]i increase. The Na2S-induced [Ca2+]i increase was not suppressed by U73122, a phospholipase C inhibitor, and high concentration of ryanodine, which inhibits ryanodine receptors. ATP also increased [Ca2+]i in spinal cord astrocytes. The ATP-induced [Ca2+]i increase was inhibited by U-73122. Na2S inhibited subsequent ATP-induced [Ca2+]i increase when Na2S clearly increased [Ca2+]i. In the absence of extracellular Ca2+, Na2S-induced [Ca2+]i increase was suppressed after application of ATP. The Na2S-induced [Ca2+]i increase and inhibitory effect of Na2S on ATP-induced [Ca2+]i increase was not inhibited by DTT, a reducing agent. In addition, Na2S had no effect on intracellular cAMP level. The Na2S-induced [Ca2+]i increase was enhanced at a lower concentration of extracellular glucose (0.5 mM). Na2S-induced [Ca2+]i increase was suppressed by rotenone, an ETC inhibitor, but not by pyruvate, an ROS scavenger. Rotenone itself increased [Ca2+]i. Ca2+ release from mitochondria by FCCP, an uncoupler of oxidative phosphorylation, was inhibited by Na2S, while the Na2S-induced [Ca2+]i increase was also inhibited by FCCP. Na2S increased the accumulation of extracellular lactate. Na2S alone did not change intracellular ATP content, but decreased it when glycolysis was inhibited by IA, a glycolytic enzyme inhibitor. Na2S-induced [Ca2+]i increase and accumulation of extracellular lactate were inhibited by emetine, a translocon complex inhibitor, which mediates Ca2+ leak from the ER. AOAA, a Ca2+-sensitive NADH shuttle inhibitor, also decreased Na2S-mediated accumulation of lactate. W7, a calmodulin inhibitor, alone tended to increase the accumulation of extracellular lactate. In the presence of W7, Na2S did not increase accumulation of lactate. In conclusion, inhibition of the ETC by H2S induces Ca2+ release from mitochondria and the ER, which increases lactate production in spinal cord astrocytes. It is suggested that reduction of mitochondria-derived ATP by H2S induces Ca2+ release from the ER through the translocon complex. Lactate is shuttled from astrocytes to neurons as an energy source. In addition, glycolysis is especially important for newborn animals. Therefore, astrocytic metabolic changes by H2S may contribute to the development and/or maintenance of central nervous system function.
Conffering University: 北海道大学
Degree Report Number: 甲第14546号
Degree Level: 博士
Degree Discipline: 獣医学
Examination Committee Members: (主査) 教授 木村 和弘, 教授 乙黒 兼一, 准教授 市居 修, 准教授 山口 聡一郎
Degree Affiliation: 獣医学院(獣医学専攻)
Type: theses (doctoral)
URI: http://hdl.handle.net/2115/81962
Appears in Collections:課程博士 (Doctorate by way of Advanced Course) > 獣医学研究科(Graduate School of Veterinary Medicine)
学位論文 (Theses) > 博士 (獣医学)

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