Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion.
Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion.
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受刺激的 Ca2 流入可将胰腺 β 细胞系中的线粒体游离 Ca2 提高至超微摩尔水平。
DOI：
10.1016/s0021-9258(18)41540-2
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
R. Rizzuto
中科院分区：
文献类型：
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作者：
G. Rutter;Jm Theler;M. Murgia;C. Wollheim;T. Pozzan;R. Rizzuto
受刺激的Ca2+内流对细胞质（[Ca2+]c）或线粒体内游离Ca2+ ([Ca2+]m)的影响在新的胰腺β细胞系INS-1中进行了研究。[Ca2+]c通过单个fura-2负载的INS-1细胞或转染非靶向（细胞质）aequorin的细胞群的视频成像进行监测。通过将框架中的基因与细胞色素c氧化酶亚基VIII的信号肽融合，将靶向线粒体的aequorin转染后测定[Ca2+]m。天然β细胞的两种生理刺激，葡萄糖和ATP，主要是通过刺激Ca2+内流来提高INS-1细胞中的[Ca2+]c。因此，葡萄糖（20 mM）诱导[Ca2+]c的重复瞬态增加（0.42 min-1，平均振幅229 nM高于102 nM基线）。这些瞬态很大程度上是由于通过电压敏感的Ca2+通道周期性刺激Ca2+内流，因为它们可以通过外部Ca2+的螯合，通过添加超极化剂二氮氧化物或Ca2+通道阻滞剂SR 7037快速和可逆地阻断。相比之下，ATP引起单次瞬态[Ca2+]c增加，比基础水平高出约300 nM，在外部Ca2+螯合作用下，>可以抑制90%。转染aequorin的细胞受到ATP的攻击后，[Ca2+]m增加至4微米或更高，EGTA阻断了这一作用。此外，用高K+的质膜去极化，作为葡萄糖替代物，以同步的方式模拟在单细胞水平观察到的流入诱导的Ca2+瞬变，也将[Ca2+]m增加到bb0.4微米。在碳醇动员内部Ca2+时，在其他转染了aequorin的胰岛素分泌细胞RINm5F中也测量到了类似的[Ca2+]m的增加。相比之下，葡萄糖诱导的[Ca2+]m的变化低于INS-1细胞群体的检测水平，这与这种营养物质在单细胞水平诱导的[Ca2+]c瞬变的非同步一致，因此[Ca2+]c的平均上升幅度较小。这些数据与受刺激的Ca2+内流进入可兴奋细胞提高[Ca2+]m与不可兴奋细胞内Ca2+动员一样有效的观点一致。就INS-1和胰腺β细胞而言，这可能对增强氧化代谢（从而促进分泌过程）和维持代谢信号分子的产生都很重要。
The effects of stimulated Ca2+ influx on cytosolic ([Ca2+]c) or intramitochondrial free Ca2+ ([Ca2+]m) were examined in the new pancreatic beta-cell line, INS-1. [Ca2+]c was monitored by video imaging of single fura-2-loaded INS-1 cells, or in populations of cells transfected with non-targeted (cytosolic) aequorin. [Ca2+]m was measured after transfection with aequorin targeted to the mitochondria by fusion of the gene in frame with the signal peptide of cytochrome c oxidase subunit VIII. Two physiological stimuli of native beta-cells, glucose and ATP, raised [Ca2+]c in INS-1 cells largely by stimulating Ca2+ influx. Thus, glucose (20 mM) induced repetitive transient increases in [Ca2+]c (0.42 min-1, mean amplitude 229 nM above 102 nM basal). These transients were largely due to periodic stimulation of Ca2+ influx through voltage-sensitive Ca2+ channels, since they could be rapidly and reversibly blocked by chelation of external Ca2+, by addition of the hyperpolarizing agent diazoxide, or with the Ca2+ channel blocker SR 7037. ATP, by contrast, caused single transient [Ca2+]c increases, to about 300 nM above basal levels, which could be inhibited by > 90% upon external Ca2+ chelation. Challenge of aequorin-transfected cells with ATP increased [Ca2+]m to 4 microM or above, an effect blocked by EGTA. Furthermore, plasma membrane depolarization with high K+, used as a glucose surrogate to mimic, in a synchronized fashion, the influx-induced Ca2+ transients observed at the single-cell level, also increased [Ca2+]m to > 4 microM. Similar increases in [Ca2+]m were also measured in other aequorin-transfected insulin-secreting cells, RINm5F, during mobilization of internal Ca2+ with carbachol. In contrast, glucose-induced changes in [Ca2+]m were below the level of detection in INS-1 cell populations, consistent with the asynchrony of the [Ca2+]c transients induced by this nutrient at the single-cell level, and the consequent small average [Ca2+]c rise. These data are in line with the view that stimulated Ca2+ influx into excitable cells raises [Ca2+]m as efficiently as internal Ca2+ mobilization in nonexcitable cells. In the case of INS-1 and pancreatic beta-cells, this may be important both to enhance oxidative metabolism, hence fueling the secretory process, and also to maintain the production of metabolic signaling molecules.