Long‐Term Activation of Protein Kinase C by Nicotine in Bovine Adrenal Chromaffin Cells

Abstract: Previous results from our laboratory suggest that long‐term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12‐myristate 13‐acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine‐synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long‐term effects. Nicotine increased particulate PKC activity in a concentration‐dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1‐dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine‐receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [³H]phorbol 12,13‐dibutyrate ([³H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic‐receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine‐stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [³H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic‐receptor activation increases PKC activity and immunoreactivity in BAM cells. The long‐term PKC activation may serve several functions, such as activation of mRNA production and a negative feedback regulation of either nicotinic receptors or voltage‐dependent Ca²⁺ channels.