Simpson P B, Russell J T
Laboratory of Cellular and Molecular Neurophysiology, NICHD, NIH, Bethesda, MD 20892-4495, USA.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):239-47. doi: 10.1042/bj3250239.
We have characterized the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps in cultured rat cortical type-1 astrocytes, type-2 astrocytes and oligodendrocytes. Perfusion with 10 microM cyclopiazonic acid (CPA) or 1 microM thapsigargin evoked a large and persistent elevation in cytosolic [Ca2+] in normal Ca2+-containing medium and a small and transient increase in nominally Ca2+-free medium. Subtraction of the response in Ca2+-free medium from that in the control revealed a slow-onset Ca2+-entry response to SERCA inhibition, which began after most of the store depletion had occurred. Thapsigargin- and CPA-induced responses propagated as Ca2+ waves, which began in several distinct cellular sites and travelled throughout the cell and through nearby cells, in confluent cultures. Propagation was supported by specialized Ca2+-release sites where the amplitude of the response was significantly higher and the rate of rise steeper. Such higher Ca2+-release kinetics were observed at these sites during Ins(1,4,5)P3-mediated Ca2+ waves in the same cells. Fluorescently tagged thapsigargin labelled SERCA pumps throughout glial cell bodies and processes. In oligodendrocyte processes, multiple domains with elevated SERCA staining were always associated with mitochondria. Our results are consistent with a model in which only a single Ca2+ store, expressing Ins(1,4,5)P3 receptors and SERCAs sensitive to both thapsigargin and CPA, is present in rat cortical glia, and indicate that inhibition of SERCA activates both Ca2+ release as a wavefront and Ca2+ entry via store-operated channels. The spatial relationship between SERCAs and mitochondria is likely to be important for regulating microdomains of elevated Ca2+-release kinetics.
我们已对培养的大鼠皮质1型星形胶质细胞、2型星形胶质细胞和少突胶质细胞中的肌浆网-内质网Ca2+ -ATP酶（SERCA）泵进行了特性描述。在正常含Ca2+的培养基中，用10微摩尔环匹阿尼酸（CPA）或1微摩尔毒胡萝卜素灌注会引起胞质[Ca2+]大幅且持续升高，而在名义上无Ca2+的培养基中则引起小幅且短暂的增加。从对照中的反应减去无Ca2+培养基中的反应，揭示了对SERCA抑制的缓慢起始的Ca2+内流反应，该反应在大部分储存库耗竭后开始。毒胡萝卜素和CPA诱导的反应以Ca2+波的形式传播，在汇合培养物中，这些波从几个不同的细胞位点开始，传遍整个细胞并通过附近的细胞。传播由专门的Ca2+释放位点支持，在这些位点反应的幅度明显更高且上升速率更陡。在同一细胞中，在Ins(1,4,5)P3介导的Ca2+波期间，在这些位点观察到了如此更高的Ca2+释放动力学。荧光标记的毒胡萝卜素标记了整个胶质细胞体和突起中的SERCA泵。在少突胶质细胞突起中，SERCA染色升高的多个区域总是与线粒体相关。我们的结果与以下模型一致：在大鼠皮质胶质细胞中仅存在一个表达对毒胡萝卜素和CPA均敏感的Ins(1,4,5)P3受体和SERCA的单一Ca2+储存库，并且表明SERCA的抑制既激活了作为波前的Ca2+释放，又激活了通过储存库操纵通道的Ca2+内流。SERCA与线粒体之间的空间关系可能对于调节Ca2+释放动力学升高的微区很重要。
