β3-Adrenergic activation of sequential Ca2+ release from mitochondria and the endoplasmic reticulum and the subsequent Ca2+ entry in rodent brown adipocytes
β3-Adrenergic activation of sequential Ca2+ release from mitochondria and the endoplasmic reticulum and the subsequent Ca2+ entry in rodent brown adipocytes
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DOI：
10.1016/j.ceca.2011.02.011
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发表时间：
2011-06-01
期刊：
影响因子：
4
通讯作者：
Kuba, Kenji
中科院分区：
文献类型：
--
作者：
Hayato, Ryotaro;Higure, Yoko;Kuba, Kenji
我们研究了线粒体解偶联的β（3）-肾上腺素能刺激eliminated钙信号在啮齿动物棕色脂肪细胞的钙荧光测定。使用fura-2、rhod-5 N、cameleon和罗丹明123分别测定细胞质、线粒体和内质网（ER）中的[Ca 2 +]（i）、[Ca 2 +]（m）和[Ca 2 +]（ER）浓度，以及线粒体膜电位。免疫印迹显示脂肪细胞中存在α（1A）-和β（3）-肾上腺素能受体和UCP 1，而RT-PCR显示3、7和9型腺苷酸环化酶、UCP 1、UCP 2、UCP 3和1型和2型肌醇三磷酸受体的mRNA。异丙肾上腺素和BRL 37344（β-激动剂）引起脂肪细胞中[Ca 2 +]（i）（β-反应）的三相升高，伴有线粒体去极化。BRL 37344一过性降低[Ca 2 +]（m）。β-反应被普萘洛尔、β-拮抗剂、H-89、蛋白激酶A阻断剂和UCP 1基因敲除阻断。β-反应的晚期被Ca 2+抑制。游离EGTA溶液、U 73122（磷脂酶C阻断剂）和毒胡萝卜素（ER-Ca 2+）。泵阻断剂，以及针对2 IP（3）R的siRNA。BAPTA/AM的细胞内负载抑制晚期比初始阶段更强烈。β-激动剂苯肾上腺素、α-激动剂和环匹阿尼酸、ER-Ca 2+泵阻断剂降低[Ca 2 +]（ER）。因此，通过β（3）-肾上腺素能激活的线粒体解偶联导致Ca 2+从线粒体释放，随后从ER释放，并进一步引起质膜Ca 2+进入，包括钙库操作的Ca 2+。入境(C)2011爱思唯尔有限公司版权所有。
We studied how mitochondrial uncoupling by beta(3)-adrenergic stimulation elicits Ca2+ signals in rodent brown adipocytes by fluorometry of Ca2+. concentrations ([Ca2+](i), [Ca2+](m), and [Ca2+](ER)) in the cytoplasm, mitochondria and the endoplasmic reticulum (ER), respectively, and mitochondrial membrane potential, using fura-2, rhod-5N, cameleon and rhodamine 123. Immunoblotting demonstrated alpha(1A)- and beta(3)-adrenergic receptor and UCP1 in adipocytes, while RT-PCR revealed the mRNA of type 3, 7 and 9 adenylate cyclase, UCP1, UCP2, UCP3 and type 1 and 2 inositoltrisphosphate receptors. Isoproterenol and BRL37344, beta-agonist, caused triphasic rises in [Ca2+](i) (beta-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca2+](m). beta-Responses were blocked by propranolol, beta-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of beta-responses was depressed by a Ca2+. free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca2+. pump blocker, and by transfecting siRNA for type 2IP(3)R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. beta-Agonists, phenylephrine, alpha-agonist, and cyclopiazonic acid, ER-Ca2+ pump blocker, decreased [Ca2+](ER). Thus, the mitochondrial uncoupling by beta(3)-adrenergic activation causes Ca2+ release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca2+ entries, including the store-operated Ca2+. entry. (C) 2011 Elsevier Ltd. All rights reserved.