The Role of Protein Phosphorylation in Amylase Exocytosis from the Parotid Gland.

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Amylase release from parotid acinar cells has been extensively studied as a useful model of cAMP-dependent exocytosis. A crucial role of cAMP-dependent protein kinase in the exocytosis has long been postulated, since the activation of the protein kinase and the phosphorylation of some proteins were observed concurrently with amylase release evoked by beta-adrenergic agonists. However, studies using protein kinase inhibitors (H-8 or heat-stable protein kinase inhibitor) suggested that cAMP-dependent protein phosphorylation is not directly involved in the exocytosis, since the inhibitors mark edly reduced protein phosphorylation without decreasing amylase release. To determine the role of cAMP-dependent protein kinase in the exocytosis, I studied the effects of various cAMP analogues on amylase release and protein phosphorylation in saponin-permeabilized parotid acini. The dose-dependent responses of amylase release and protein phosphorylation evoked by various analogues were closely correlated. The combination of site-selective cAMP analogues synergistically increased amylase release. The cAMP antagonist Rp-cAMPS (Rp-diastereomer of adenosine 3 ; , 5 ; -phosphorothioate) did not stimulate amylase release and inhibited amylase release stimulated by Sp-cAMPS, the Sp-diastereoisomer. These results strongly support the involvement of cAMP-dependent protein kinase in the process of exocytosis. Next I studied the effects of okadaic acid, a potent selective inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation. Okadaic acid by itself weakly stimulated amylase release, but markedly inhibited rather than potentiated amylase release evoked by cAMP. Okadaic acid increased both cAMP-dependent and -independent protein phosphorylation. These results suggest that nonspecific increase in protein phosphorylation does not necessarily enhance amylase release, and rather suppresses full activation of the exocytosis.