Dependence of insulin secretion from permeabilized pancreatic β-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 protein 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 CaM kinase II inhibitors to decrease Ca2+-induced insulin secretion from permeabilized β-cells. KN-93 (2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) markedly inhibited both CaM kinase II activation and insulin secretion in parallel in α-toxin-permeabilized β-cells. These effects were specific since they were not mimicked by the inactive analog, KN-92 (2-[N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine). In contrast, KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine), while reported to be similar to KN-93 with respect to mechanism of action, did not inhibit Ca2+-induced activation of CaM kinase II or insulin secretion in these cell preparations. All three agents suppressed Ca2+ influx in intact β-cells induced by depolarization in the presence of elevated extracellular potassium although to different extents. The synthetic peptide inhibitors of CaM kinase II, [Ala286]CaMK 281–302 and AIP (autocamtide-2-related inhibitory peptide), strongly inhibited Ca2+-induced insulin secretion from electropermeabilized islets, an effect that also correlated with an equivalent inhibition of CaM kinase II activation. This re-evaluation (i) explains a lack of effect of KN-62 on insulin secretion from permeabilized cells based on its inability to inhibit CaM kinase II activation in these preparations; (ii) has revealed that CaM inhibitors, either chemical or peptide in nature, that are capable of preventing enzyme activation uniformly suppress Ca2+-sensitive insulin secretion; and (iii) cautions the use of KN-62/93/92 as selective inhibitors of CaM kinase II in intact cell studies. These observations reinforce the suggestion that CaM kinase II plays an important role in insulin exocytosis in the β-cell.