Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells.

Abstract

Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32PO4, exposure of the chromaffm cells to acetylcholine increased the phosphorylation of a Mr -100,000 protein and a Mr =60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO4/polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins ("100-kDa," "87-kDa," and protein HI) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. "100-kDa" is a Mr =100,000 protein selectively phosphorylated by calcium/calmodulindependent protein kinase Ill, "87-kDa" is a Mr --87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of Mr --74,000 (ma) and Mr -55,000 (hEb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. Furthermore, 100-kDa was shown to be identical to the Mr lO00,000 protein whose phosphorylation was increased by acetylcholine treatment. The acetylcholine-dependent increase in phosphorylation of tyrosine hydroxylase, 100-kDa, 87-kDa, and protein m required extracellular calcium and was mimicked by nicotine, veratridine, elevated K+, and calcium ionophore A23187, but not by muscarine. In addition, forskolin increased the phosphorylation of tyrosine hydroxylase, 100-kDa, and protein Ill, but not that of 87-kDa. Phorbol 12,13-dibutyrate increased the phosphorylation of tyrosine hydroxylase, 87-kDa, and protein Ill, but not that of 100-kDa. The data demonstrate that cholinergic activation ofchromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects.Acetylcholine, released from the splanchnic nerve, causes the exocytosis of catecholamines from bovine adrenal medullary chromaffin cells (1-4). Cholinergic activation of chromaffin cells also increases the phosphorylation of a Mr -100,000 protein and a Mr -60,000 protein (5). The Mr -60,000 protein was shown to be tyrosine hydroxylase (6-8), but the Mr -100,000 protein has remained uncharacterized.We have examined chromaffin cells for the presence and phosphorylation of several phosphoproteins whose phosphorylation state is known to be increased in nerve terminals by treatments that induce transmitter release. In neuronal tissues, such as synaptosomes and brain slices, depolarization produces a calcium-dependent increase in the phosphorylation state of "87-kDa" (9) [an acidic, Mr -87,000 protein present in a wide variety of mammalian tissues but most prominent in brain (10)] and protein III (11-13) [a phosphoprotein doublet of Mr -74,000 (IIIa) and Mr 55,000 (IIIb) associated with synaptic vesicles in brain (14-16)]. In addition, we have evaluated the possible identity of the Mr -100,000 chromaffin cell protein with "100-kDa," a substrate for calcium/calmodulin-...

Synapsin II Phosphorylation and Catecholamine Release in Bovine Adrenal Chromaffin Cells: Additive Effects of Histamine and Nicotine

“…Synapsin I1 is a neuron-specific phosphoprotein found in central and peripheral neurons and in the neural crest cell-derived adrenal chromaffin cells. Its phosphorylation state has been shown to parallel closely exocytotic release of catecholamines in BACC following cholinergic stimulation (Haycock et al, 1988). In this study, we show that histaminergic stimulation also shows a good correspondence between synapsin I1 phosphorylation and catecholamine release.…”

“…Accordingly, we first needed to determine the concentrations of histamine and nicotine that produced maximal effects. We have reported previously that doses of nicotine above 5 X 1 0-5 M produce maximal effects on secretion and phosphorylation (Haycock et al, 1988). The dose-response relationships for histamine-stimulated [3H]NE release (not shown) and synapsin I1 phosphorylation (Fig.…”

“…We have previously shown that nicotinic stimulation of BACC increases the phosphorylation of synapsin I1 (Haycock et al, 1988). Nicotinic receptors activate these cells primarily via a depolarization-dependent influx of extracellular calcium (Burgoyne, 1984).…”

Identification of the phosphorylation sites in elongation factor‐2 from rabbit reticulocytes

“…chain elongation. It is phosphorylated on threonine residues by a specific eEF-2 kinase, (previously termed Caa+/calmodulin.dependent protein kinase III [3,4]) and its phosphorylation is increased in intact ceils in response to stimuli which increase intracellular Ca z÷'ion concentrations [6][7][8][9][10][11][12][13]. Phosphorylation of the endogenous eEF-2 impairs the translation of mRNA in the reticulocyte lysate celt-free system [5] and several other lines of evidence show that phosphorylated eEF-2 is inactive, or has only low activity [3,4,14,15].…”

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen‐Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

“…Although the exocytotic phenomenon is well described, the Ca2-dependent signaling events leading to secretion are not fully characterized. Extensive studies have indicated that protein phosphorylations serve as important signaling intermediates (Amy and Kirschner, 1981;Lee and Holz, 1986;Michener et al, 1986;Gutierrez et al, 1988;Haycock et al, 1988;Firestone and Browning, 1992). Although most phosphorylations occur on serine and threonine residues, a role for tyrosine phosphorylation is suggested from the secretagogue-dependent tyrosine phosphorylation of several proteins, the most prominent of which are the mitogen-activated protein kinases (MAPKs) (Dahmer et al, 1990;Ely et al, 1990).…”