Phorbol esters andd-tubocurarine up-regulate α-bungarotoxin sites in chromaffin cells in culture via distinct mechanisms

Abstract

Previous work had shown that nicotinic antagonists resulted in a marked up-regulation of α-bungarotoxin sites in chromaflin cells in culture. The present experiments were done to determine the intracellular mechanism(s) whereby nicotinic antagonists might mediate their effects on these receptors. Chromaffin cells were cultured for three days with various concentrations of 4β-phorbol 12-myristate 13-acetate, an agent which affects protein kinase C by mimicking the actions of diacylglycerol. The phorbol ester resulted in a dose-dependent increase in α-bungarotoxin binding which was maximal with 100 nM 4β-phorbol 12-myristate 13-acetate. This increase in binding appeared to be due to an increase in the maximal number of α-bungarotoxin sites. Time dependence studies showed that the effect of the phorbol was undetectable with incubations of 24 h or less and appeared to plateau by 72–96 h. A similar increase in toxin binding was also observed with 4β-phorbol 12,13-dibutyrate. On the other hand, an inactive analog of 4β-phorbol 12-myristate 13-acetate had no significant effect on binding. d-Sphingosine, an inhibitor of protein kinase C, was able to partially block the phorbol ester-induced increase in toxin binding while polymyxin B, another protein kinase C inhibitor, completely prevented the up-regulation of the α-bungarotoxin sites. Carbachol and nicotine prevented this enhancement of toxin binding in the presence of 4β-phorbol 12-myristate 13-acetate. Although the phorbol ester resulted in an increase in toxin binding, acetylcholine-evoked catecholamine secretion from chromaffin cells in culture was decreased, indicating a dissociation between the functional nicotinic acetylcholine receptor population and the α-bungarotoxin sites. To determine whether agents which affect protein kinase C can alter the up-regulation of α-bungarotoxin sites byd-tubocurarine, 4β-phorbol 12-myristate 13-acetate was added to the cells in combination with the nicotinic antagonist. The up-regulation of toxin binding sites induced byd-tubocurarine was additive with that induced by the phorbol and was not affected by polymyxin B. Thus, the results would suggest that there are at least two mechanisms by which α-bungarotoxin binding sites can be regulated. One is mediated via an interaction at nicotinic receptors, while the other occurs in response to phorbol esters and thus may be mediated by protein kinase C. Interestingly, although the molecular mechanisms resulting in α-bungarotoxin receptor up-regulation differ, both thed-tubocurarine-and the phorbol ester-induced increases were prevented by nicotinic receptor ligands.