Düfer Martina, Krippeit-Drews Peter, Drews Gisela
Pharmazeutisches Institut, Pharmakologie, Auf der Morgenstelle 8, Universität Tübingen, 72076, Germany.
Pflugers Arch. 2002 May;444(1-2):236-43. doi: 10.1007/s00424-002-0799-8. Epub 2002 Mar 2.
The mitochondrial inhibitors NaN(3) and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) were used to study the role of mitochondria in pancreatic B-cell Ca2+ homeostasis. In glucose-stimulated B-cells NaN(3) and FCCP both increased the K(ATP) current and thus hyperpolarized the cell membrane potential, as expected for agents depleting cellular ATP. NaN(3) and FCCP stopped the glucose-induced oscillations in the cytosolic free Ca2+ concentration (Ca2+) and elicited a biphasic response. After a first rapid and transient increase, Ca2+ rose in a second slow phase to a sustained level. In cells pretreated with thapsigargin the first inhibitor-induced rise in Ca2+ was absent, suggesting that it may be due to Ca2+ mobilization from intracellular stores. The glucose-induced oscillations were terminated again by NaN(3) and FCCP, respectively, but the slow increase in Ca2+of the second phase was still present. A minute increase in Ca2+elicited by NaN(3) or FCCP was even visible after the removal of extracellular Ca2+, suggesting that the inhibitors also mobilize Ca2+ from mitochondria. NaN(3) and FCCP induced Ca2+ influx into B-cells treated with low glucose concentrations whose voltage-dependent Ca2+ channels are closed. Experiments with thapsigargin-preincubated cells indicate that disturbance of mitochondrial function stimulates Ca2+ influx through voltage-independent Ca2+ pathways. During the NaN(3)-induced increase in Ca2+, K+-elicited depolarizations of the cells did not further augment Ca2+. Evidently, this is due to a direct inhibitory effect of azide on L-type Ca2+ channels. The data demonstrate that disturbing the mitochondrial function affects cellular Ca2+ homeostasis in B-cells at several sites. Thus, it is concluded that intact mitochondrial function is a prerequisite for regular Ca2+ handling in B-cells.
线粒体抑制剂叠氮化钠（NaN₃）和羰基氰化物对-（三氟甲氧基）苯腙（FCCP）被用于研究线粒体在胰腺β细胞钙稳态中的作用。在葡萄糖刺激的β细胞中，NaN₃和FCCP均增加了ATP敏感性钾电流（K(ATP)电流），从而使细胞膜电位超极化，这与消耗细胞ATP的试剂预期的结果一致。NaN₃和FCCP停止了葡萄糖诱导的胞质游离钙浓度（Ca²⁺）振荡，并引发了双相反应。在第一次快速且短暂的升高之后，Ca²⁺在第二个缓慢阶段上升至持续水平。在用毒胡萝卜素预处理的细胞中，第一种抑制剂诱导的Ca²⁺升高不存在，这表明它可能是由于细胞内钙库释放钙所致。葡萄糖诱导的振荡再次分别被NaN₃和FCCP终止，但第二阶段Ca²⁺的缓慢升高仍然存在。在去除细胞外钙后，仍可见NaN₃或FCCP引起的Ca²⁺微小升高，这表明抑制剂也能从线粒体中释放钙。NaN₃和FCCP诱导钙流入用低葡萄糖浓度处理的β细胞，这些细胞的电压依赖性钙通道是关闭的。用毒胡萝卜素预孵育细胞的实验表明，线粒体功能紊乱会刺激钙通过非电压依赖性钙途径流入。在NaN₃诱导的Ca²⁺升高期间，钾离子引起的细胞去极化并未进一步增加Ca²⁺。显然，这是由于叠氮化物对L型钙通道的直接抑制作用。数据表明，干扰线粒体功能会在多个位点影响β细胞中的细胞钙稳态。因此，可以得出结论，完整的线粒体功能是β细胞中正常钙处理所必需条件。
