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

Abstract

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca2+entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca2+or Mg2+and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca2+concentration ([Ca2+]i) produced by thapsigargin was abolished by removing extracellular Ca2+, depressed by depleting extracellular Na+, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca2+]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 Ca2+-free EGTA solution, and an Na+-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca2+and fully blocked by oligomycin but not by thapsigargin or an Na+-deficient solution, was accompanied by a rise in cytoplasmic Mg2+concentration, and was summated with a high pH-induced rise in [Ca2+]i, whereas the extracellular Ca2+-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca2+but not by thapsigargin, depressed by decreasing Na+, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca2+]iin a Ca2+-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca2+release, activating Ca2+release from the ER and store-operated Ca2+entry, and directly elicits a novel plasmalemmal Ca2+entry, whereas Ca2+release from the ER activates Ca2+accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes.