Indeed, levels of two specific isozymes of protein kinase C, d and e, increased dramatically with chronic exposure to ethanol, and it seems likely that one of these isozymes, probably d, is required to mediate the action of ethanol on calcium channel up-regulation. These results indicate that an ethanol-induced increase in gene expression for a particular protein kinase C isozyme may set the stage for the molecular events that lead to alcohol withdrawal seizures. Ongoing studies in several laboratories now suggest that calcium channel blockers can prevent alcohol withdrawal seizures in experimental animals and alcoholics. The point of this illustration is that it would have been very difficult to identify this specific molecular mechanism if whole brain or heterogeneous brain preparations were used initially. Now, however, it is possible to produce transgenic animals with overexpression or alteration of specific protein kinase C isozymes to confirm their roles in specific adaptive responses to ethanol. This work should suggest that it is not possible to explain neural responses to ethanol merely on the basis of membrane perturbation and fluidity changes. Instead, it seems that regulatory mechanisms affecting specific membrane proteins are very special targets for ethanol in the brain.

Another example of a regulatory system affected by alcohol was developed in collaboration with Adrienne Gordon over the past several years. We have been interested in cyclic AMP signal transduction. When a neurotransmitter reacts with its receptor, as in this cartoon, adenylyl cyclase is activated via a G protein, Gas. The result is an increased production of cyclic AMP which then stimulates protein kinase A activity. With long-term exposure to ethanol, however, neural cells adapt by reducing cyclic AMP signal transduction. This desensitization affects all receptors coupled positively to adenylyl cyclase. The advantage of working with cells in culture was that it allowed investigators to determine the molecular mechanism responsible for these changes. We found that long-term exposure to ethanol caused a selective reduction in gene expression for Gas, and thus decreased production of Gas mRNA and protein. This accounts for heterologous desensitization of signal transduction. Interestingly, these changes at a cellular level mimic physical dependence. Receptor-stimulated cyclic AMP levels are abnormally low during alcohol withdrawal and can be restored to normal by adding ethanol back to the cells.

Identification of these short- and long-term neural responses to ethanol made it possible to discover the molecular mechanisms that regulate these events. This slide provides an overview of the pathway we have identified. Ethanol inhibits a specific adenosine transporter to block re-uptake of adenosine into neural cells. As a result, cells exposed to ethanol accumulate extracellular adenosine, which then reacts with adenosine receptors on the cell surface. In this case, adenosine A2 receptors positively coupled to adenylyl cyclase stimulate the production of cyclic AMP to activate protein kinase A. This results in a heterologous desensitization of cyclic AMP production associated with diminished protein kinase A activity at the cellular membrane. As a consequence of reduced



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