Interactions of intracellular mediators of amylase secretion in permeabilized pancreatic acini

Abstract

Mouse pancreatic acini were permeabilized with streptolysin O to investigate amylase secretion stimulated by various intracellular mediators and the kinetics of secretion as a function of temperature. Amylase secretion was temperature dependent in that the initial rate of Ca2+-stimulated secretion increased with increasing temperature. In addition, there was no enhancement of Ca2+-stimulated secretion by GTP[[gamma]S] at 14[deg]C, while enhancement was maximal at 30[deg]C. GTP[[gamma]S]-mediated enhancement of secretion at a given temperature was mostly due to sustained secretion with a small increase in secretory rate. At 30[deg]C Ca2+-stimulated secretion was also enhanced by cAMP and phorbol ester (TPA) to similar extents as by GTP[[gamma]S]. The maximally effective concentration of cAMP was 1-10 [mu]M in the presence of 0.1 mM isobutylmethylxanthine. The enhancements of Ca2+-stimulated amylase secretion by all combinations of cAMP (100 [mu]M plus 0.1 mM isobutylmethylxanthine), TPA (1 [mu]M), and GTP[[gamma]S] (30 [mu]M) were fully additive. In Ca2+-free buffer, cAMP, TPA or GTP[[gamma]S] individually had no effect on amylase secretion. Together, TPA and GTP[[gamma]S] stimulated Ca2+-independent secretion, which was 187 +/- 38% of basal. Cyclic AMP together with TPA and GTP[[gamma]S] in the absence of Ca2+ stimulated 329 +/- 30% of basal secretion. Ca2+-stimulated amylase secretion was decreased about 50% by metabolic inhibition, while the enhancement by cAMP, TPA or GTP[[gamma]S] was totally blocked by metabolic inhibitors. These data demonstrate that amylase secretion in the acinar cell is mediated by multiple intracellular pathways which act in parallel and probably converge at a distal step in the exocytotic process.