Carbachol-induced [Ca2+]i increase, but not activation of protein kinase C, stimulates exocytosis in rat parotid acini.

1. A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)-induced amylase secretion. 2. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose-response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i). 3. The CCh-induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3-sensitive stores was relatively ATP independent. 4. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca(2+)-free medium evoked a second rapid and large peak of amylase secretion. 5. Phorbol 12,13-dibutyrate (PDBu) potentiated the CCh-induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh-induced [Ca2+]i changes. 6. PKC inhibitors such as Ro 31-8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone. 7. These results suggest that a CCh-induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP-dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh-induced sustained plateau of amylase secretion. PDBu-induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.