Dependence of insulin secretion from permeabilized pancreatic beta-cells on the activation of Ca2+/calmodulin-dependent protein kinase II - A re-evaluation of inhibitor studies

Previous studies utilizing inhibitors of the Ca2+/calmodulin-dependent prot ein kinase II (CaM kinase II) to address the role of this enzyme in insulin secretion have produced contradictory results. In the current study, these inconsistencies have been addressed by evaluating the effect of various Ca M kinase II inhibitors to decrease Ca2+-induced insulin secretion from perm eabilized beta -cells. KN-93 (2-[N-(2-hydroxyethyl)-N-(dr-methoxy-benzenesu lfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) markedly inhibited both CaM kinase II activation and insulin secretion in parallel in alpha - toxin-permeabilized beta -cells. These effects were specific since they wer e not mimicked by the inactive analog, KN-92 (2-[N-(4-methoxybenzenesulfony l)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine). In contrast, KN-62 (1- [N,O-bis(5-isoquinolinesulfonyl)-N-methyl-1-tyrosyl]-4-phenylpiperazine), w hile reported to be similar to KN-93 with respect to mechanism of action, d id not inhibit Ca2+-induced activation of CaM kinase II or insulin secretio n in these cell preparations. All three agents suppressed Ca2+ influx in in tact beta -cells induced by depolarization in the presence of elevated extr acellular potassium although to different extents. The synthetic peptide in hibitors of CaM kinase II, [Ala(286)]CaMK 281-302 and AIP (autocamtide-2-re lated inhibitory peptide), strongly inhibited Ca2+-induced insulin secretio n from electropermeabilized islets, an effect that also correlated with an equivalent inhibition of CaM kinase II activation. This re-evaluation (i) e xplains a lack of effect of KN-62 on insulin secretion from permeabilized c ells based on its inability to inhibit CaM kinase II activation in these pr eparations; (ii) has revealed that CaM inhibitors, either chemical or pepti de in nature, that are capable of preventing enzyme activation uniformly su ppress Ca2+-sensitive insulin secretion; and (iii) cautions the use of KN-6 2/93/92 as selective inhibitors of CaM kinase II in intact cell studies. Th ese observations reinforce the suggestion that CaM kinase II plays an impor tant role in insulin exocytosis in the beta -cell. (C) 2000 Elsevier Scienc e Inc.