Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.

Abstract

The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca21 ionophore ionomycin. PMA also enhanced secretion induced by Ba2 . In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion -70% at 0.5 jIM Ca2+ and 30% at 10 jiM Ca2+. PMA enhanced the maximal response to Ca2+ =25% and decreased the Ca2+ concentration required for half-maximal secretion :30 %. The effects of PMA on chromaffin cells were associated with a 2-to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.The exocytotic release of catecholamines from bovine adrenal chromaffin cells is normally triggered by the influx of extracellular Ca2l and the rise in cytosolic Ca" concentration that occur on stimulation of nicotinic receptors (1-4). Membrane depolarization in the presence of Ca2+ or the substitution of Ba2+ for Ca2+ also stimulates exocytosis. Although the mechanism of exocytosis was first described using biochemical techniques in the adrenal medulla, the underlying mechanisms triggered by Ca2+ are not understood.One reaction that could be involved in exocytosis is protein phosphorylation. Previous studies concerning the role of protein phosphorylation in secretion from adrenal chromaffin cells revealed that two proteins, one of 56-60 kDa and one of 100 kDa, were phosphorylated when secretion was stimulated by secretagogues (5, 6). The 56-60-kDa protein was identified as tyrosine hydroxylase (7), the ratelimiting enzyme in catecholamine biosynthesis, and is probably not' directly involved in exocytosis. The 100-kDa protein has not been identified but its phosphorylation is not well correlated with secretion.Protein kinase C is Ca2+-dependent and requires acidic phospholipids for activity (8,9). Diglyceride in the presence of phosphatidylserine increases the Ca2+ sensitivity of the enzyme to micromolar or perhaps submicromolar concentrations (10). The ester phorbol 12-myristate 13-acetate (PMA) can substitute for diglyceride in vitro (11) and similarly increases the Ca2+ sensitivity of the enzyme. Most importantly, PMA activates protein kinase C and protein phosphorylation in intact platelets and enhances serotonin secretion (11,12).In the present study, we have investigated the effects of PMA on catecholamine secretion and protein phosphorylation in bovine adrenal chromaffin cells in monolayer culture. We have used both intact chromaffin cells and cells treated with a low concentration...

Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly

“…Over the physiological Ca 2+ concentration range (0·1-10 µM), PRL release was regulated by the Ca 2+ concentration. Nearly the same pattern of Ca 2+ -dependent exocytosis was observed as in digitonin-permeabilized bovine chromaffin cells (Pocotte et al 1985) and GH3 cells permeabilized by electric field discharge (Ronning & Martin 1985). ATP was not included in Kglu buffer and the addition of ATP to Kglu buffer had no effect on PRL release (data not shown).…”

Protein kinase C activator inhibits voltage‐sensitive Ca2+ channels and catecholamine secretion in adrenal chromaffin cells

“…Although high K ÷ depolarization is not the closest mimick of physiological depolarization, K÷-evoked catecholamine secretion is generally considered to reflect mainly the activation of voltage-sensitive Ca 2+ channels [1]. Other groups have previously shown that phorbol esters enhance both Ca 2+-evoked catecholamine secretion from permeabilized cells and ionomycin-evoked secretion from intact cells [22][23][24][25]. The simplest model to account for these differences is that PKC activation has antagonistic consequences on the early and late steps of secretion, with the inhibition overcoming the stimulation of the exocytotic machinery that occurs downstream the activation of Ca 2+ channels.…”

Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation.

“…Activation of PKC increases evoked-catecholamine release in intact (Pocotte et al, 1985;Morita et al, 1985;TerBush et al, 1988;Bittner and Holz, 1990) as well as in permeabilized chromaflin cells (Knight and Baker, 1983;Brocklehurst et al, 1985;Knight et al, 1988;Bittner and Holz, 1990;Tachikawa et al, 1990;TerBush and Holz, 1990;Isosaki et al, 1991). Moreover, nicotinic stimulation induces translocation of PKC from cytoplasm to membranes (TerBush et al, 1988), a process known to be associated with the activation of the enzyme.…”