Kuba Masako, Higure Yoko, Susaki Hisashi, Hayato Ryotaro, Kuba Kenji
Laboratory of Anatomy and Physiology, School of Nutritional Sciences, Nagoya Univ. of Arts and Sciences, 57 Takenoyama, Iwasaki-cho, Nissin, Aichi 470-0196, Japan.
Am J Physiol Cell Physiol. 2007 Feb;292(2):C896-908. doi: 10.1152/ajpcell.00649.2005. Epub 2006 Sep 20.
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+)) 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+), 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+), 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+) 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.
我们在培养的大鼠棕色脂肪细胞中研究了内质网（ER）与线粒体如何相互通讯以及它们如何调节质膜Ca(2+)内流。通过荧光测定法测量细胞质Ca(2+)或Mg(2+)以及线粒体膜电位。毒胡萝卜素引起的细胞质Ca(2+)浓度（Ca(2+)）升高的持续成分在去除细胞外Ca(2+)后消失，在耗尽细胞外Na(+)后降低，在提高细胞外pH后增强。FCCP、二硝基苯酚和鱼藤酮导致Ca(2+)出现双相或三相升高，其中第一相伴随着线粒体去极化。FCCP诱导的第一相部分被寡霉素抑制，但不被钌红、环孢素A、U-73122、无Ca(2+)的EGTA溶液和无Na(+)的溶液抑制。FCCP诱导的与线粒体复极化平行的第二相在去除细胞外Ca(2+)后部分受阻，被寡霉素完全阻断，但不被毒胡萝卜素或缺乏Na(+)的溶液阻断，伴随着细胞质Mg(2+)浓度升高，并与高pH诱导的Ca(2+)升高相加，而不依赖细胞外Ca(2+)的成分被U-73122和环匹阿尼酸阻断。FCCP诱导的第三相被去除Ca(2+)阻断，但不被毒胡萝卜素阻断，在降低Na(+)后降低，在提高pH后增强。在FCCP作用后，环匹阿尼酸在无Ca(2+)溶液中引起的Ca(2+)升高受到抑制。因此，线粒体解偶联导致Ca(2+)释放，激活内质网Ca(2+)释放和储存-操作性Ca(2+)内流，并直接引发一种新的质膜Ca(2+)内流，而内质网Ca(2+)释放激活线粒体Ca(2+)积累或释放，表明大鼠棕色脂肪细胞中线粒体-内质网存在双向偶联。
