THE ROLE OF PROTEIN-KINASE-C IN CARBACHOL-INDUCED AND OF CAMP-DEPENDENT PROTEIN-KINASE IN ISOPROTERENOL-INDUCED SECRETION IN PRIMARY CULTURED GUINEA-PIG PAROTID ACINAR-CELLS

Stimulation of secretion by muscarinic agonists in guinea pig parotid or pancreatic acini is accompanied by a translocation of protein kinas e C (PKC) from the cytosol to the particulate fraction [Machado-De Dom enech and Soling (1987) Biochem, J. 242, 749-754] and by a PKC-mediate d phosphorylation of the ribosomal protein S6 [Padel and Soling (1985) Eur. J. Biochem. 151, 1-10]. In order to decide whether PKC is direct ly involved in the secretory process, the effect of down regulation of PKC by phorbol 12-myristate 13-acetate (PMA) was studied in primary c ultured guinea pig parotid acinar cells. These cells secrete in respon se to carbachol and isoproterenol. Only the carbachol response is asso ciated with an increase in cytosolic calcium. Carbachol plus isoproter enol lead to an over-additive stimulation of secretion, an effect whic h depends completely on the presence of external calcium. Down regulat ion of PKC by about 90% did not significantly affect carbachol-induced exocytosis, whereas isoproterenol-stimulated secretion was almost dou bled. The secretory response to permeable cAMP analogues was also enha nced in PKC-down-regulated acini, indicating a post-receptor effect, T he increased response to isoproterenol was also observed in the absenc e of external calcium. The isoproterenol effect was significantly inhi bited by the relatively specific cAMP-dependent protein kinase inhibit or H-89, which had only a minor effect on carbachol-induced exocytosis . Although down regulation of total PKC by up to 90% did not significa ntly affect the secretory response to carbachol, RO 31-8220, a relativ ely specific inhibitor of PKC, abolished carbachol-induced secretion i n normal as well as in PMA-down-regulated cells. This indicates that a PKC isoform resistant to down regulation by PMA is involved in carbac hol- but not in cAMP-mediated secretion.