Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

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 (CO2)-C-14 evolved following the hydroxylation and rapid decarboxylation of [C-14]-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 mu M and complete inhibition at 10 mu M. It had no effect on the responses to forskolin. 4 Ro 31-8220 produced a concentration-dependent inhibition of 5 mu M nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 mu M and complete inhibition by 10 mu M. At concentrations up to 10 mu M, 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 mu M 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 mu M and complete inhibition by 3 mu M, 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.