Role of Ca 2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells

Abstract

The role of Ca 2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca 2+. 12- O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca 2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca 2+-evoked secretion. Polymyxin B at a low concentration (1 μM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca 2+. However, polymyxin B at higher concentrations (10–100 μM) greatly reduced Ca 2+-evoked catecholamine secretion. Sphingosine (10μM-1 mM), staurosporine (100nM-1 μM), and H-7 (100–500 μM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca 2+-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [ 3H]phorbol-12, 13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca 2+-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca 2+-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.