Alcoholism is a serious public health problem, and a major challenge in alcohol research is to define the molecular events that underlie ethanol abuse and its neurotoxic consequences. Intracellular Ca2+ ([Ca2+]i) plays a key role in regulating neuronal function. Voltage-dependent Ca2+ channels are important in regulating [CA2+]i, and alterations in Ca2+ channel function may be prominently involved in ethanol's effects on the nervous system. Using the neural cell line PC12, I have found that exposure to 200 mM ethanol for 2-10 days causes a 75-91% increase in 45Ca2+ uptake and a 92% increase in the number of binding sites for Ca2+ channel antagonists. These findings suggest that neuronal adaptation to ethanol involves increased expression of voltage-dependent Ca2+ channels. The biochemical basis for this response appears to involve an interaction between ethanol and protein kinase C (PKC). In preliminary studies, I found that the PKC inhibitor, sphingosine, prevented increases in 45Ca2+ uptake induced by exposure to 200 mM ethanol for 4 days. Moreover, phorbol 12,13-dibutyrate, a potent PKC activator, reversed the effect of sphingosine. These findings suggest that PKC mediates Ca2+ channel up-regulation by ethanol and that PKC-mediated phosphorylation is enhanced by long-term ethanol exposure. This may result from increased levels of PKC in ethanol-treated cells, since the V max for PKC was increased by 66% in cells treated with 200 mM ethanol for 6 days. The main objective of this proposal is to investigate whether PKC mediates ethanol-induced Ca2+ channel up-regulation. K+-stimulated 45Ca2+ uptake and Ca2+ channel antagonist binding will be measured to determine if PKC inhibitors block ethanol-induced Ca2+ channel up-regulation. Phosphorylation of endogeneous PKC substrates will be examined to determine if PKC-mediated phosphorylation is enhanced in ethanol-treated cells. The time course and concentration-dependence of the ethanol-mediated increase in the V max for PKC will be correlated with ethanol's effects on Ca2+ channels. [3H]PDBu binding and immunoblot analyses will be performed to determine if the increase in the V max for PKC in ethanol-treated cells correlates with an increase in PKC polypeptide. Finally, Northern blot analyses with probes for PKC subspecies will be performed to investigate whether ethanol enhances expression of PKC by increasing the abundance of PKC messenger RNA. Since PKC and voltage-dependent Ca2+ channels are key regulators of neuronal function, these studies will provide new information about an important biochemical mechanism by which long-term ethanol exposure could disturb neuronal function. These studies may advance the search for biochemical markers of alcohol abuse and eventually lead to therapeutic approaches to patients with alcoholism.
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