Phosphorylation of MARCKS is involved in amylase release in parotid acinar cells

Abstract

Myristoylated alanine-rich C kinase substrate (MARCKS) is a major cellular substrate for protein kinase C (PKC). Involvement of phosphorylation of MARCKS in secretory function has been reported in pancreatic islets and chromaffin cells. In rat parotid acinar cells, the activation of β-adrenergic receptor provokes exocytotic amylase release. We investigated the involvement of phosphorylation of MARCKS in β-adrenergic agonist-induced amylase release in rat parotid acinar cells. Rat parotid acinar cells were prepared using trypsin and collagenase. Membrane and cytosolic fractions were isolated by ultracentrifugation. Secretory granules were purified using Percoll gradient. Protein expression was determined by western blotting using anti-MARCKS and anti-phosphorylated MARCKS (p-MARCKS) antibodies. Amylase activity in the medium was measured by Bernfeld's methods. In western blotting analysis, MARCKS and p-MARCKS were detected in membrane and cytosolic fractions and secretory granule membrane. The β-adrenergic agonist isoproterenol (IPR) stimulated phosphorylation of MARCKS time-dependently. This agonist had no effect on the total amount of MARCKS. Myristoylated PKC peptide inhibitor partially inhibited IPR-induced phosphorylation of MARCKS and amylase release. These results suggest that phosphorylation of MARCKS is involved in regulation of amylase release in rat parotid acinar cells. [J Physiol Sci. 2007;57 Suppl:S129]