Long-term activation of protein kinase C by nicotine in bovine adrenal chromaffin cells.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Tuominen
R K
RK
Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
McMillian
M K
MK
Ye
H
H
Stachowiak
M K
MK
Hudson
P M
PM
Hong
J S
JS
D000311:Adrenal Glands / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002714:Chlorisondamine / Q000494:pharmacology; D002838:Chromaffin System / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015151:Immunoblotting; D009538:Nicotine / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013997:Time Factors
D004075:Diglycerides; D015240:Phorbol 12,13-Dibutyrate; D004246:Dimethylphenylpiperazinium Iodide; D009538:Nicotine; D001285:Atropine; D011493:Protein Kinase C; D002714:Chlorisondamine