Yada T, Russo L L, Sharp G W
Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401.
J Biol Chem. 1989 Feb 15;264(5):2455-62.
In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.
在研究佛波酯对胰岛素分泌的调节作用时，我们使用钙离子指示剂fura - 2在大鼠胰岛素分泌β细胞系RINm5F中检测了它们对胞质游离钙离子浓度（[Ca2+]i）的影响。[Ca2+]i与胰岛素释放速率同时进行测定。50 nM的12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）可能通过蛋白激酶C起作用，它刺激胰岛素释放并导致[Ca2+]i升高。无钙条件消除了[Ca2+]i的升高，并导致TPA对胰岛素释放的刺激作用减弱。钙离子通道阻滞剂尼群地平（300 nM）抑制了[Ca2+]i的升高以及胰岛素分泌速率的增加。另一种佛波酯，4β - 佛波醇12,13 - 二癸酸酯，可激活蛋白激酶C，也诱导了[Ca2+]i和胰岛素释放速率的增加，而不能刺激蛋白激酶C的4α - 佛波醇12,13 - 二癸酸酯则没有作用。用双苯甲酰羟肟酸作为膜电位指示剂进行的进一步研究表明，TPA使β细胞质膜去极化。从这些结果可以得出结论，TPA使质膜去极化，诱导RINm5Fβ细胞质膜上钙离子通道开放，增加[Ca2+]i，并导致胰岛素分泌。接下来将TPA的作用与去极化浓度的氯化钾（25 mM）的作用进行了比较，氯化钾仅通过打开钙离子通道来刺激胰岛素分泌。TPA始终引起较小的去极化、较小的[Ca2+]i升高，但比氯化钾释放更多的胰岛素。因此提示TPA有额外的作用，即增强升高的[Ca2+]i对胰岛素分泌的作用。
