Involvement of MARCKS phosphorylation in amylase release in parotid

Objectives: Myristoylated alanine-rich C kinase substrate (MARCKS) is a major cellular substrate for protein kinase C (PKC). Phosphorylated-MARCKS (p-MARCKS) translocates from membrane to cytosol. The MARCKS phosphorylation has been reported to be involved in Ca²⁺-mediated exocytosis. In rat parotid acinar cells, stimulation of β-adrenergic receptors induces an increase in intracellular cAMP levels, which causes exocytotic amylase release. We investigated the involvement of MARCKS phosphorylation in β-adrenergic agonist-induced amylase release in rat parotid acinar cells.

Methods: Rat parotid acinar cells were prepared using trypsin, collagenase and hyaluronidase. Membrane and cytosolic fractions of the dispersed cells were isolated by ultracentrifugation. MARCKS and p-MARCKS were detected by Western blotting analysis using anti-MARCKS and anti-p-MARCKS antibodies, respectively. MARCKS translocation was detected by immunohistchemistry with confocal microscopy. Amylase activity in the medium and the cell lysate was measured by Bernfeld's method.

Results: MARCKS and p-MARCKS were detected in membrane and cytosolic fractions. Isoproterenol (IPR), a β-adrenergic agonist, stimulated MARCKS phosphorylation in a time-dependent manner. This agonist had no effect on the total amount of MARCKS. IPR stimulated MARCKS translocation from membrane to cytosol by immunohistochemistry and Western blotting analysis. Myristoylated PKC peptide inhibitor (Myr-PKC peptide) and Calphostin C, PKC inhibitors, inhibited IPR-induced MARCKS phosphorylation. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited IPR-induced MARCKS phosphorylation. Myr-PKC peptide partially inhibited IPR-induced amylase release.

Conclusions: These results suggest that MARCKS phosphorylation is regulated by PKA activation and is involved in β-adrenergic agonist-induced amylase release in rat parotid acinar cells.