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Activation of membrane protein kinase C by glucagon and Ca(2+)-mobilizing hormones in cultured rat hepatocytes. Role of phosphatidylinositol and phosphatidylcholine hydrolysis.

ABSTRACT: We found that glucagon stimulated membrane protein kinase C (PKC) activity and phosphatidylcholine hydrolysis in 24 h-cultured rat hepatocytes. Phorbol myristate acetate, 8-bromo cyclic AMP, vasopressin, noradrenaline and the Ca2+ ionophore A23187 also stimulated membrane PKC activity. However, only vasopressin and noradrenaline stimulated inositol phosphate accumulation, whereas all agonists stimulated the rate of release of water-soluble choline metabolites into the medium. Choline, and to a much lesser extent phosphocholine, were released, suggesting predominantly phospholipase D activation. This was supported by the finding that the accumulation of phosphatidate and diacylglycerol was enhanced by the agents in [3H]myristate-labelled hepatocytes, as was [32P]phosphatidylethanol formation. Since the time courses for the release of choline into the medium and the accumulation of phosphatidate and diacylglycerol caused by vasopressin and glucagon were similar, the more rapid activation of PKC by vasopressin probably reflects diacylglycerol formation from phosphoinositide breakdown. The inability of glucagon to stimulate inositol phosphate production was not due to the prolonged culture, since similar results were obtained in 4 h cultures. We conclude that the stimulation of membrane PKC activity by glucagon correlates with accumulation of diacylglycerol and phosphatidate derived from the hydrolysis of phosphatidylcholine.

SUBMITTER: Pittner RA

PROVIDER: S-EPMC1151243 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Project description:Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.

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Activation of membrane protein kinase C by glucagon and Ca(2+)-mobilizing hormones in cultured rat hepatocytes. Role of phosphatidylinositol and phosphatidylcholine hydrolysis.

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