β3-Adrenergic activation of sequential Ca2+ release from mitochondria and the endoplasmic reticulum and the subsequent Ca2+ entry in rodent brown adipocytes

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.