Properties of calcium-dependent pancreatic hormonsecretion: Evidence for distinct functional pools of secretory granules

Abstract

The patch-clamp technique, fluorimetric measurements of intracellular Ca2+ ([Ca2+]i) and measurements of cell capacitance were used to explore the regulation of hormone secretion in pancreatic A- and B-cells. In both cell types, exocytosis was rapidly initiated when [Ca2+]i was elevated. The magnitude of the secretory responses was found to be influenced by neuronal, hormonal and pharmacological factors. The hypoglycaemic sulphonylureas, previously believed to act solely via inhibition of the ATP-regulated K+-channel, surprisingly also stimulated Ca2+-dependent exocytosis in the B-cell, by a more direct interaction with the exocytotic machinery. This action depends on activity of protein kinase C (PKC) and is likely to involve binding of the drug to intracellular sulphonylurea receptors belonging to the superfamily of ATP-binding cassette proteins. Insulin secretion has long been recognized as a highly temperature-dependent process but the cellular mechanisms have not been established. Here we demonstrate that this is neither the result of reduced Ca2+ influx, nor interference with the exocytotic process itself. Rather, it reflects a high temperature dependence of the process of granule mobilization from a reserve pool to the a release-competent pool of secretory granules. This results in a use-dependent inhibition of secretion during repetitive stimulation. Several hormones elevate cytoplasmic cyclic AMP (cAMP) in the B-cell with resultant potentiation of Ca2+-induced insulin release. The cAMP-mediated stimulation of secretion was exerted by both protein kinase A (PKA)-dependent and PKA-independent mechanisms. The former action was the most important one during protracted secretion and was attributed to enhanced mobilization of granules, such that the number of granules readily available for release was increased. The PKA-independent component of cAMP-stimulated insulin release was only capable of producing a short-lived potentiation of exocytosis. This transiently stimulatory effect of cAMP developed promptly (<100 ms) which suggests that it is exerted at a late stage in the secretory process, possibly exerted immediately before the ultimate Ca2+-dependent reaction. As indicated above, experimental conditions resulting in activation of protein kinases are usually associated with stimulation of insulin secretion. Conversely, conditions leading to the stimulation of protein phosphatases should be predicted to produce suppression of exocytosis. In this study, we demonstate that this is precisely the mechanism by which somatostatin, galanin and a-adrenergic agonists inhibit insulin secretion. The effects of these substances are mediated by G-protein-dependent activation of the Ca2+-dependent protein phosphatase calcineurin. Exocytosis in glucagon-secreting A-cells was demonstrated to be elicited by influx of Ca2+ through voltage-gated N-type and L-type Ca2+ channels. Adrenaline potentiated Ca2+-dependent glucagon secretion by a b-adrenoreceptor-mediated mechanism involving cAMP-dependent activation of PKA. The action of PKA was exerted by dual mechanisms: 1) By phosphorylating L-type Ca2+-channels it increased the whole-cell Ca2+-current by 50%; and 2) More importantly, it selectively mobilized the granules to the vicinity of the L-type Ca2+ channels. The results presented in this study favour the view that the 13 000 secretory vesicles of the B-cell are organized such that only a fraction of the B-cell granules are available for instantaneous (ATP- and temperature-independent) release upon elevation of [Ca2+]i. This readily releasable pool of B-cell granules, was estimated to ~0.5 % of total B-cell granule content. Sustained exocytosis in response to protracted stimulation proceeds at much lower rates which we attribute to the mobilization of granules into the readily releasable pool being rate-limiting under these conditions. The different components of capacitance increase are finally discussed in relationship to the biphasic nature of insulin secretion and against what is known about the pathogenesis and pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM).