The role of cytosolic free Ca2+ and protein kinase C in acetylcholine-induced insulin release in the clonal β-cell line, HIT-T15

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Abstract
We examined the contribution of signal-transduction pathways to acetylcholine-induced insulin release in the clonal .beta.-cell line HIT-T15. To assess the importance of changes in cytosolic free Ca2+ ([Ca2+]i), we studied time courses of the effects of glucose and acetylcholine on [Ca2+]i and insulin release in quin 2-loaded HIT cells. Incubation in the presence of glucose (2 mM) resulted in a sustained increase in [Ca2+]i in HIT cells from 98 .+-. 7 nM to 195 .+-. 12 nM measured after 9 min, whereas subsequent addition of acetylcholine (50 .mu.M) produced a transient increase in [Ca2+]i which reached a peak after 30 s (at 274 .+-. 10 nM), returning to pre-stimulus levels after 3 min. In contrast, incubation of HIT cells with acetylcholine in the presence of glucose produced a sustained increase in insulin release over and above that stimulated by glucose alone; after 10 min acetylcholine had potentiated glucose-stimulated insulin release by an additional increment of 135%. The transient increase in [Ca2+]i induced by acetylcholine was dose-dependent, and was prevented by omission of glucose or extracellular Ca2+ from the incubation medium. It was also inhibited by inclusion of 50 .mu.M-verapamil in the incubation medium (by 87 .+-. 3%) or by decreasing the Na+ concentration in the medium (by 73 .+-. 6%). To evaluate the role of the protein kinase C pathway, we have pretreated HIT cells with the phorbol ester 12-O-tetradecanoylphorbol acetate (TPA), to deplete the protein kinase C activity, and have compared their secretory activity with that of control cells. Protein kinase C activity was decreased by 73% in HIT cells cultured in the presence of 200 nM-TPA for 22-24 h. TPA pre-treatment also significantly decreased the insulin content of HIT cells, but had no effect on cell number or the increases in [Ca2+]i induced by glucose or acetycholine. TPA-pre-treated cells responded comparatively less well to secretagogues than did control cells: glucose-stimulated insulin release was decreased by 40%, whereas potentiation by TPA was significantly decreased by 50% in comparison with control cells (P < 0.05, n = 24). Acetylcholine (50 .mu.M) potentiated glucose-stimulated insulin release by 61% in control cells. This effect was abolished in HIT cells pre-treated with TPA, whereas these cells still retained their normal secretory response to stimulation by forskolin. These data suggest that an early increase in [Ca2+]i may be important for the initial increase in insulin release induced by acetylcholine in HIT cells. However, protein kinase C activation is necessary for sustained cholinergic-induced insulin release. This may involve sensitization of the secretory apparatus to intracellular Ca2+, since insulin release takes place in the absence of any additional increase in [Ca2+]i.