The cellular actions of many neurotransmitters involves the adenylyl cyclase system which is responsible for generating cAMP. After chronic ethanol exposure, neurotransmitter stimulation of cAMP production is decreased (i.e. causes desensitization). The present objectives are: 1. to determine the molecular mechanisms responsible for the ethanol-induced desensitization using PC 12 cells as a neuronal model system; and 2. to compare the effects of chronic ethanol treatment on the adenylyl cyclase systems in neurons and in glia. Chronic ethanol exposure decreases the levels of the stimulatory GTP-binding protein Gsalpha (i.e. causes down- regulation). Experiments will determine whether ethanol also decreases activity of the catalytic subunit of adenylyl cyclase, as determined by measuring enzyme activity after reconstitution of PC 12 cell membranes with purified Gsalpha, and/or increases membranes levels of the inhibitory components, beta-gamma subunit and Gialpha3, as determined by immunoblot analysis. Experiments will be carried out to determine whether the ethanol- induced decrease in Gsalpha levels results from a decrease in the rate of Gsalpha synthesis. Other experiments will investigate the effect of ethanol on Gsalpha mRNA levels. The necessity of cAMP-dependent protein kinase (PKA) activation in ethanol-induced desensitization will be confirmed using 123.7 and AB.11 cells which are PC 12 cells that are functional deficient in PKA-mediated responses. The role of PKA in ethanol-induced desensitization will be investigated by determining whether activation of PKA is directly involved in the down-regulation of Gsalpha. Activation of PKA will be carried out using forskolin and by the inducible overexpression of the free, catalytic subunit of PKA. The beta-adrenergic and adenosine- sensitive adenylyl cyclase in cerebellar granule cells and astrocytes will be compared to determine if neurons and glia differ in their response to chronic ethanol exposure. Measurements will be made of: 1. onset of desensitization; 2. timecourse for recovery after chronic ethanol exposure; 3. concentration dependency for ethanol-induced desensitization; and 4. the role of extracellular adenosine in ethanol induced desensitization. Studies in this proposal will provide information critical to understanding the effects of ethanol on the adenylyl cyclase activity in vivo.
Showing the most recent 10 out of 22 publications
