The cellular effects of many neurotransmitters involve the activation of adenylate cyclase with a subsequent increase in intracellular cyclic AMP content. Because of the importance of cyclic AMP in neuronal function, the present proposal will investigate the direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content. Since the direct actions of chronic ethanol exposure can be most readily determined with cells in culture, the present studies will use both clonal cells (PC 12) and cerebellar neurons in culture. It has been observed that chronic ethanol exposure results in a decrease in the stimulation of adenylate cyclase activity by neurotransmitters (i.e. desensitization) and a selective increase in the activation of adenylate cyclase by in vitro ethanol in some brain cells. The specific objectives of the present proposal are; 1. to characterize the ethanol induced alterations in adenylate cyclase activity in cerebellar neurons in culture; and 2. to determine the molecular mechanism responsible for the ethanol-induced desensitization. The beta-adrenergic and adenosine- sensitive adenylate cyclase systems in cerebellar neuronal cultures will be used to determine: 1. the time course for the onset of ethanol-induced changes in adenylate cyclase activity: 2. the effects of withdrawal on enzyme activity (i.e., time course for recovery); and 3. the concentration dependency for the ethanol-induced alterations in enzyme activity. The molecular basis for ethanol-induced desensitization will be determined in PC 12 cells because a homogeneous population of cells will facilitate interpretation of the data. Since adenylate cyclase consists of three subunits, experiments will be carried out to determine whether chronic ethanol exposure alters; 1. the amount of each of the subunits; 2. the properties of the subunits; or 3. the interactions of the subunits. Since PC 12 cells are a transformed cell line, these results will be corroborated the direct effects of chronic ethanol exposure on neuronal adenylate cyclase activity, and permit the determination of the molecular mechanism responsible for ethanol induced desensitization. Results from this project are a necessary first step in understanding the response of adenylate cyclase to chronic ethanol administration in vivo.
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