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

<jats:p> How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca<jats:sup>2+</jats:sup> entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca<jats:sup>2+</jats:sup> or Mg<jats:sup>2+</jats:sup> and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca<jats:sup>2+</jats:sup> concentration ([Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>) produced by thapsigargin was abolished by removing extracellular Ca<jats:sup>2+</jats:sup>, depressed by depleting extracellular Na<jats:sup>+</jats:sup>, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>, 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<jats:sup>2+</jats:sup>-free EGTA solution, and an Na<jats:sup>+</jats:sup>-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca<jats:sup>2+</jats:sup> and fully blocked by oligomycin but not by thapsigargin or an Na<jats:sup>+</jats:sup>-deficient solution, was accompanied by a rise in cytoplasmic Mg<jats:sup>2+</jats:sup> concentration, and was summated with a high pH-induced rise in [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub>, whereas the extracellular Ca<jats:sup>2+</jats:sup>-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca<jats:sup>2+</jats:sup> but not by thapsigargin, depressed by decreasing Na<jats:sup>+</jats:sup>, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca<jats:sup>2+</jats:sup>]<jats:sub>i</jats:sub> in a Ca<jats:sup>2+</jats:sup>-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca<jats:sup>2+</jats:sup> release, activating Ca<jats:sup>2+</jats:sup> release from the ER and store-operated Ca<jats:sup>2+</jats:sup> entry, and directly elicits a novel plasmalemmal Ca<jats:sup>2+</jats:sup> entry, whereas Ca<jats:sup>2+</jats:sup> release from the ER activates Ca<jats:sup>2+</jats:sup> accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. </jats:p>