Ethanol alters cAMP signal transduction in many cultured cell lines and in cells from actively drinking alcoholics. These changes in cAMP signal transduction have pleiotropic effects on cellular functions since cAMP is an important regulator of many cell processes. The acute and chronic effects of ethanol on cAMP signalling in many cell types are due to an initial inhibition of adenosine uptake by ethanol acting on a specific type of nucleoside transporter. This inhibition leads to an extracellular accumulation of adenosine which then activates adenosine receptors and increases intracellular cAMP levels. After chronic exposure to ethanol, there is an adaptative decrease in receptor-stimulated cAMP production. In addition, after chronic exposure to ethanol, adenosine uptake is no longer inhibited on rechallenge with ethanol. cAMP-dependent protein kinase (PKA) is required for ethanol to inhibit adenosine uptake. Therefore, the loss of ethanol sensitivity of adenosine uptake after chronic exposure to ethanol may be due to decreased cAMP and PKA levels. In this proposal, the role of phosphorylation in reversibly regulating the ethanol sensitivity of nucleoside uptake in the cultured neural cell line, NG108-15, and in human erythrocytes will be determined. The use of NG108-15 cells will allow the determination of whether ethanol- induced changes in extracellular adenosine regulate the ethanol sensitivity of adenosine transport in intact cells. Sealed membranes can be prepared from erythrocytes that exhibit only ethanol-sensitive nucleoside transport. These vesicles can be sequentially phosphorylated or dephosphorylated in vitro allowing the functional consequences of phosphorylation by specific kinases to be determined independent of other kinase activities. Erythrocyte membranes will also be used to determine the differences between ethanol sensitivity of adenosine transport in actively drinking alcoholics and controls. Such studies may provide a test to identify actively drinking alcoholics as well as lead to an understanding of how the transporter is abnormally regulated in cells from alcoholics. In addition, since inhibition of nucleoside uptake by ethanol is the initial step in a cascade of events leading to the chronic effects of ethanol on cAMP signalling, these experiments might lead to new therapies to prevent these chronic effects.
