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Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells.

ABSTRACT: In bovine adrenal chromaffin cells, prostaglandin E2 (PGE2) stimulates the formation of inositol phosphates and Ca2+ mobilization through its specific receptor [Yokohama, Tanaka, Ito, Negishi, Hayashi & Hayaishi (1988) J. Biol. Chem. 263, 1119-1122]. Here we show that PGE2-induced phosphoinositide metabolism was blocked by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using intact cells, we also examined the inhibitory effect of TPA on the individual steps of the activation process of phosphoinositide metabolism. The inhibition was observed within 1 min and complete by 10 min after addition of 1 microM-TPA, and half-maximal inhibition by TPA occurred at 20 nM. TPA prevented Ca2+ mobilization induced by PGE2, but not by the Ca2+ ionophore ionomycin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not inhibit the formation of inositol phosphates and Ca2+ mobilization by PGE2. TPA treatment affected neither the high-affinity binding of [3H]PGE2 to intact cells and membrane fractions nor the ability of guanosine 5'-[gamma-thio]triphosphate to decrease the binding in membrane fractions. TPA also abolished phosphoinositide metabolism induced by muscarinic-receptor activation. NaF plus AlCl3 and ionomycin caused the accumulation of inositol phosphates, probably by directly activating a GTP-binding protein(s) and phospholipase C respectively; neither accumulation was inhibited by TPA treatment. These results suggest that protein kinase C serves as a feedback regulator for PGE2-induced phosphoinositide metabolism. The site of action of TPA appears to be distal to the coupling of the receptor to GTP-binding protein, but on a component(s) specific to the agonist-induced phosphoinositide metabolism.

SUBMITTER: Yokohama H

PROVIDER: S-EPMC1135334 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

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Project description:1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.

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Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells.

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