Cultured lymphocytes from non-alcoholics will be used to determine how amount and time of exposure to ethanol affect changes in adenosine receptor-dependent cAMP levels. The components of the cAMP signal transduction system (adenosine receptor, GTP-binding proteins, adenylyl cyclase, phosphodiesterase, cAMP-dependent protein kinase (PKA)) that are altered by chronic ethanol treatment will also be determined. Lymphocytes from alcoholics will be used to measure differences in their cAMP responses and which components of the signal transduction system account for the differences. Lymphocytes will be grown in serum-free medium and exposed to 25-200mM ethanol from 6 hours to 7 days. Changes in cAMP will be measured by radioimmunoassay. Adenosine receptor affinity and number will be measured using radioactive ligands. G protein function will be assessed by measuring GDP dissociation and GTPase activity. Changes in amounts of alphas and alphai subunits of G proteins will be assessed by immunoblotting, and alpha subunit mRNA will be quantitated by Northern blots. Changes in catalytic and regulatory components of PKA will be measured by Kemptide phosphorylation and photoaffinity-labelling with a cAMP derivative, respectively. Reduced levels of cAMP in lymphocytes may reflect an acquired abnormality due to alcoholism. On the other hand, if certain individuals are at risk to develop alcoholism because of genetic factors, we might identify genetically-determined differences in the cAMP signal transduction system in lymphocytes from alcoholics grown several generations in culture. In any event, studies with lymphocytes in culture offer a unique opportunity to move from the patient to the laboratory to analyze abnormalities in signal transduction associated with alcoholism.
