Ethanol's actions on cAMP-mediated signaling in cells transfected with type VII adenylyl cyclase

Background: Adenylyl cyclase (AC) activity is increased in the presence of ethanol. The magnitude of ethanol's action on AC depends on the isoform of AC expressed in a particular cell type. Type VII AC demonstrates the greate st potentiation of activity in the presence of ethanol, but questions have arisen regarding the effects of pharmacologically relevant (similar to 50 m M) concentrations of ethanol on type VII AC activity. Questions also remain as to whether the potentiation of AC activity by ethanol initiates downstr eam effects on protein kinase A activity. Methods: HEK293 (human embryonic kidney 293) cells overexpressing type VII AC were used to study the dose-dependent actions of ethanol on cyclic adeno sine monophosphate (cAMP) production. Studies were performed in the presenc e and absence of phosphodiesterase inhibitors. Protein kinase A activity wa s assessed under conditions similar to those used to measure ethanol's acti ons on AC. Results: A significantly greater percent stimulation of prostaglandin E-1-m ediated cAMP accumulation was evident in the absence of the phosphodiestera se inhibitors 3-isobutyl-1-methylxanthine and Ro 20-1724 than in the presen ce of the phosphodiesterase inhibitors. We also showed that ethanol was not , itself, acting as a phosphodiesterase inhibitor. The calculated percent s timulation of AC activity by ethanol depended on the baseline levels of cAM P production in the absence of ethanol. In the absence of 3-isobutyl-1-meth ylxanthine (or other phosphodiesterase inhibitors), a 50 mM concentration o f ethanol produced a 56% increase in prostaglandin E-1-stimulated cAMP prod uction in the type VII AC transfected HEK293 cells. This concentration of e thanol also produced a significant activation of protein kinase A beyond th at produced by prostaglandin E-1, alone. Conclusions: The present study indicates that, in the presence of a particu lar isoform of AC, moderately intoxicating concentrations of ethanol will s ignificantly potentiate the transmitter-mediated activation of the cAMP sig naling cascade. Activation of this signaling cascade may have important imp lications for the mechanisms by which ethanol produces intoxication and/or in the mechanisms of neuroadaptation leading to tolerance to, and physical dependence on, ethanol.