title: β₃-Adrenergic activation of sequential Ca(2+) release from mitochondria and the endoplasmic reticulum and the subsequent Ca(2+) entry in rodent brown adipocytes.
abstract: We studied how mitochondrial uncoupling by β(3)-adrenergic stimulation elicits Ca(2+) signals in rodent brown adipocytes by fluorometry of Ca(2+) concentrations ([Ca(2+)](i), [Ca(2+)](m) and [Ca(2+)](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 α(1A)- and β(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, β-agonist, caused triphasic rises in [Ca(2+)](i) (β-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca(2+)](m). β-Responses were blocked by propranolol, β-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of β-responses was depressed by a Ca(2+) free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca(2+) pump blocker, and by transfecting siRNA for type 2 IP(3)R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. β-Agonists, phenylephrine, α-agonist, and cyclopiazonic acid, ER-Ca(2+) pump blocker, decreased [Ca(2+)](ER). Thus, the mitochondrial uncoupling by β(3)-adrenergic activation causes Ca(2+) release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca(2+) entries, including the store-operated Ca(2+) entry.
authors.affiliation: Laboratory of Anatomy and Physiology, School of Nutritional Sciences, Nagoya University of Arts and Sciences, Nissin, Aichi, Japan.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Hayato
authors.forename: Yoko
authors.initials: Y
authors.lastname: Higure
authors.forename: Masako
authors.initials: M
authors.lastname: Kuba
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nagai
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Yamashita
authors.forename: Kenji
authors.initials: K
authors.lastname: Kuba
mesh_terms: D052437:Adipocytes, Brown / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D058667:Adrenergic beta-3 Receptor Agonists / Q000494:pharmacology; D058673:Adrenergic beta-3 Receptor Antagonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000378:metabolism; D022702:Receptors, Adrenergic, beta-3 / Q000737:chemistry / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism; D000071256:Uncoupling Protein 1
chemical_list: D058667:Adrenergic beta-3 Receptor Agonists; D058673:Adrenergic beta-3 Receptor Antagonists; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D018340:Receptors, Adrenergic, alpha-1; D022702:Receptors, Adrenergic, beta-3; C000604372:Ucp1 protein, mouse; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1; D011433:Propranolol; D010738:Type C Phospholipases; D002118:Calcium