title: Phorbol esters and d-tubocurarine up-regulate alpha-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 alpha-bungarotoxin sites in chromaffin 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 4 beta-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 alpha-bungarotoxin binding which was maximal with 100 nM 4 beta-phorbol 12-myristate 13-acetate. This increase in binding appeared to be due to an increase in the maximal number of alpha-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 4 beta-phorbol 12,13-dibutyrate. On the other hand, an inactive analog of 4 beta-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 alpha-bungarotoxin sites. Carbachol and nicotine prevented this enhancement of toxin binding in the presence of 4 beta-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 alpha-bungarotoxin sites. To determine whether agents which affect protein kinase C can alter the up-regulation of alpha-bungarotoxin sites by d-tubocurarine, 4 beta-phorbol 12-myristate 13-acetate was added to the cells in combination with the nicotinic antagonist. The up-regulation of toxin binding sites induced by d-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 alpha-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 alpha-bungarotoxin receptor up-regulation differ, both the d-tubocurarine- and the phorbol ester-induced increases were prevented by nicotinic receptor ligands.
authors.affiliation: Department of Pharmacology, McGill University, Montreal Quebec, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Geertsen
authors.forename: R
authors.initials: R
authors.lastname: Afar
authors.forename: J M
authors.initials: JM
authors.lastname: Trifaró
authors.forename: M
authors.initials: M
authors.lastname: Quik
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D009538:Nicotine / Q000494:pharmacology; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014403:Tubocurarine / Q000494:pharmacology*
chemical_list: D002038:Bungarotoxins; D011950:Receptors, Cholinergic; D009538:Nicotine; D002217:Carbachol; D013755:Tetradecanoylphorbol Acetate; D014403:Tubocurarine