Nicotinic and muscarinic agonists stimulate rapid protein kinase C translocation in PC12 cells.
Phosphoinositide hydrolysis, a major mechanism for signal transduction in neural cells, generates diacylglycerol, which can in turn activate protein kinase C (PKC). Although cholinergic agonists elicit phosphoinositide hydrolysis in neural tissues, little is known about activation of PKC by cholinergic agonists. PKC requires phosphatidylserine for activation, and in intact cells this lipid requirement is satisfied by binding of the enzyme to cell membranes. Therefore, in intact cells, activation of PKC is often associated with a decrease in cytosolic PKC activity accompanied by an increase in membrane-associated activity. We studied cholinergic-induced activation of PKC by examining changes in the subcellular distribution of the enzyme in PC12 cells treated with cholinergic drugs. Carbachol (1 mM) induced large and rapid increases in membrane-associated PKC activity; a maximal increase of 460% occurred after 5 sec of incubation. Carbachol-induced PKC translocation was concentration-dependent, with a biphasic dose-response curve yielding approximate EC50 values of 10(-6) M and 10(-4) M for the high- and low-affinity components, respectively. Experiments with selective cholinergic agents demonstrated that both muscarinic and nicotinic receptors are involved in carbachol-induced PKC translocation, but the response is predominantly mediated by nicotinic receptor stimulation. Muscarinic-induced association of PKC with cell membrane fractions was resistant to extraction by chelators, whereas nicotinic-mediated membrane binding was partially reduced by homogenization of cells in the presence of EGTA. Omission of calcium from the incubation medium or chelation of calcium with EGTA completely blocked muscarinic- and nicotinic-induced translocation. In addition, the calcium channel blocker nifedipine reduced the nicotinic response by 60%. (ABSTRACT TRUNCATED AT 250 WORDS)
Messing
R O
RO
Department of Neurology, University of California, San Francisco 94110.
Stevens
A M
AM
Kiyasu
E
E
Sneade
A B
AB
D000310:Adrenal Gland Neoplasms / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D009116:Muscarine / Q000502:physiology*; D009538:Nicotine / Q000502:physiology*; D010673:Pheochromocytoma / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D013268:Stimulation, Chemical; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000378:metabolism
D004533:Egtazic Acid; D004246:Dimethylphenylpiperazinium Iodide; D009538:Nicotine; D009116:Muscarine; D002217:Carbachol; D011493:Protein Kinase C; D002118:Calcium