Second messengers involved in genetic regulation of the number of calcium channels in bovine adrenal chromaffin cells in culture

Bovine adrenal chromaffin cells in culture show an increased formation of [³H]inositol phosphates (after preloading with [³H]inositol) on depolarisation with increased extracellular K⁺. This increased breakdown of inositol lipid is further increased by the dihydropyridine Ca²⁺ channel activator BAY K 8644 at nM concentrations, implying that proteins which bind dihydropyridines are involved in this mechanism. Further, pretreatment of adrenal cells with pertussis toxin (100 ng ml^-1) prevented the K⁺-induced breakdown of inositol lipids, arguing the involvement of a pertussis toxin-sensitive G protein in the effect. Chronic exposure of bovine adrenal chromaffin cells to a concentration of ethanol which inhibits K⁺-induced breakdown of inositol phospholipid, caused a 70-100% increase in the binding of [³H]DHP sites. In these experiments it was found that excess extracellular Ca²⁺ would considerably reduce this up-regulation, whereas growth of cells in pertussis toxin closely mimicked the up-regulation obtained by growth of cells in ethanol. These experiments suggest that inhibition of membrane Ca²⁺ flux, through a G protein-associated channel, is closely involved in the ethanol-induced regulation of [³H]dihydropyridine binding sites. The inositol lipid-protein kinase C second messenger system is also implicated in this regulation, by experiments in which inhibitors of protein kinase C (chronic treatment with phorbol myristyl acetate, or with sphingosine) up-regulated binding sites for [³H]dihydropyridine to a similar extent as that seen with growth in ethanol.