Inhibition of voltage-gated Ca2+ channels and insulin secretion in HIT cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca2+ channels.
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.
Li
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Département de Médecine, Centre Médical Universitaire, Geneva, Switzerland.
Hidaka
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H
Wollheim
C B
CB
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives*; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects* / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002463:Cell Membrane Permeability / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D004164:Diphosphonates / Q000494:pharmacology; D004357:Drug Synergism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D007546:Isoquinolines / Q000494:pharmacology*; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D010879:Piperazines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D013268:Stimulation, Chemical; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002147:Calmodulin; D004164:Diphosphonates; D007328:Insulin; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; C061154:belfosdil; C052087:CGS 9343B; D005576:Colforsin; D008345:Manganese; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium