Journal of Neuroscience, Vol 9, 507-512, Copyright © 1989 by Society for Neuroscience
Nicotinic and muscarinic agonists stimulate rapid protein kinase C translocation in PC12 cells
RO Messing, AM Stevens, E Kiyasu and AB Sneade
Department of Neurology, University of California, San Francisco 94110.
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)
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