Bidirectional Ca2+ coupling of mitochondria with the endoplasmic reticulum and regulation of multimodal Ca2+ entries in rat brown adipocytes

Laboratory of Anatomy and Physiology, School of Nutritional Sciences, Nagoya University of Arts and Sciences, Nissin, Aichi, Japan Submitted 20 December 2005 ; accepted in final form 17 September 2006 How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca 2+ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca 2+ or Mg 2+ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) produced by thapsigargin was abolished by removing extracellular Ca 2+ , depressed by depleting extracellular Na + , and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca 2+ ] i , in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca 2+ -free EGTA solution, and an Na + -free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca 2+ and fully blocked by oligomycin but not by thapsigargin or an Na + -deficient solution, was accompanied by a rise in cytoplasmic Mg 2+ concentration, and was summated with a high pH-induced rise in [Ca 2+ ] i , whereas the extracellular Ca 2+ -independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca 2+ but not by thapsigargin, depressed by decreasing Na + , and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca 2+ ] i in a Ca 2+ -free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca 2+ release, activating Ca 2+ release from the ER and store-operated Ca 2+ entry, and directly elicits a novel plasmalemmal Ca 2+ entry, whereas Ca 2+ release from the ER activates Ca 2+ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP Address for reprint requests and other correspondence: K. Kuba, Laboratory of Anatomy and Physiology, School of Nutritional Sciences, Nagoya Univ. of Arts and Sciences, 57 Takenoyama, Iwasaki-cho, Nissin, Aichi 470-0196, Japan (e-mail: kubak{at}nuas.ac.jp )