Li G, Hidaka H, Wollheim C B
Département de Médecine, Centre Médical Universitaire, Geneva, Switzerland.
Mol Pharmacol. 1992 Sep;42(3):489-8.
To probe for the involvement of Ca2+/calmodulin-dependent protein kinase II in the regulation of insulin secretion, the effects of a specific inhibitor of this enzyme, KN-62, on secretagogue-stimulated insulin secretion, cytosolic Ca2+ concentration ([Ca2+]i) rise, membrane depolarization, and nutrient metabolism were examined in HIT-T15 cells. KN-62 dose-dependently inhibited insulin secretion induced by a nutrient mixture (10 mM glucose, 5 mM leucine, and 5 mM glutamine) alone or combined with either the Ca(2+)-mobilizing receptor agonist bombesin or the cAMP-raising agent forskolin in intact cells. KN-62 did not affect Ca(2+)- or GTP analogue-induced insulin secretion from permeabilized cells, indicating an action at a step before exocytosis. The stimulating effects of nutrients on insulin secretion, [Ca2+]i, and membrane depolarization were potentiated by bombesin. Similarly, bombesin promoted a larger depolarization and [Ca2+]i rise in the presence of nutrients. This was associated with enhanced Ca2+ mobilization and the appearance of sustained [Ca2+]i elevation. The bombesin-induced membrane depolarization, like the nutrient effect, was inhibited by diazoxide, suggesting that this is due to closure of ATP-sensitive K+ channels. Bombesin elicited Ca2+ influx by both membrane potential-sensitive and -insensitive conductance pathways. KN-62 did not affect Ca2+ mobilization and only partially reduced Ca2+ entry during the sustained [Ca2+]i rise in bombesin-stimulated cells. When added before or during the stimulation, KN-62 dose-dependently inhibited nutrient- and KCl-stimulated [Ca2+]i elevation and Mn2+ influx (reflecting Ca2+ entry). The calmodulin antagonist CGS 9343B and the L-type Ca2+ channel blocker SR-7037 mimicked the inhibitory effect of KN-62 on stimulated insulin secretion and [Ca2+]i elevation. Membrane depolarization and nutrient metabolism (reduction of a tetrazolium derivative), however, were not altered by KN-62 treatment, indicating that the early coupling events from nutrient metabolism to closure of ATP-sensitive K+ channels remain operative. These results suggest that KN-62 and the calmodulin antagonist CGS 9343B inhibit Ca2+ influx by means of direct interaction with L-type Ca2+ channels, which, in turn, causes inhibition of stimulated insulin secretion. Thus, it appears that Ca2+/calmodulin-dependent protein kinase II is not involved in the regulation of insulin secretion.
为探究钙/钙调蛋白依赖性蛋白激酶II是否参与胰岛素分泌的调节，研究了该酶的特异性抑制剂KN-62对HIT-T15细胞中促分泌剂刺激的胰岛素分泌、胞质钙浓度（[Ca2+]i）升高、膜去极化及营养物质代谢的影响。在完整细胞中，KN-62呈剂量依赖性地抑制单独由营养混合物（10 mM葡萄糖、5 mM亮氨酸和5 mM谷氨酰胺）诱导的胰岛素分泌，以及与钙动员受体激动剂蛙皮素或cAMP升高剂福斯可林联合诱导的胰岛素分泌。KN-62不影响通透细胞中钙或GTP类似物诱导的胰岛素分泌，表明其作用于胞吐作用之前的步骤。蛙皮素可增强营养物质对胰岛素分泌、[Ca2+]i和膜去极化的刺激作用。同样，在存在营养物质的情况下，蛙皮素可促进更大程度的去极化和[Ca2+]i升高。这与增强的钙动员及持续性[Ca2+]i升高的出现有关。与营养物质的作用类似，蛙皮素诱导的膜去极化受到二氮嗪的抑制，提示这是由于ATP敏感性钾通道关闭所致。蛙皮素通过膜电位敏感性和非敏感性电导途径引起钙内流。在蛙皮素刺激的细胞中，KN-62不影响钙动员，仅部分降低持续性[Ca2+]i升高期间的钙内流。在刺激前或刺激期间添加时，KN-62呈剂量依赖性地抑制营养物质和氯化钾刺激的[Ca2+]i升高及锰内流（反映钙内流）。钙调蛋白拮抗剂CGS 9343B和L型钙通道阻滞剂SR-7037模拟了KN-62对刺激的胰岛素分泌和[Ca2+]i升高的抑制作用。然而，KN-6治疗并未改变膜去极化和营养物质代谢（四氮唑衍生物的还原），表明从营养物质代谢到ATP敏感性钾通道关闭的早期偶联事件仍然有效。这些结果提示，KN-62和钙调蛋白拮抗剂CGS 9343B通过与L型钙通道直接相互作用抑制钙内流，进而导致刺激的胰岛素分泌受到抑制。因此，似乎钙/钙调蛋白依赖性蛋白激酶II不参与胰岛素分泌的调节。
