Ethanol exposure is known to perturb many signal transduction pathways, including those that involve adenylate cyclase and phosphoinositide turnover. It has been hypothesized that ethanol-mediated alterations in signal transduction pathways may reflect one mechanism by which cells adapt and develop tolerance to the acute effects of ethanol. Previous studies have established that isolated hepatocytes from alcohol-fed animals show an enhanced responsiveness to Ca2+ -mobilizing hormones and that this enhancement is associated with an increased sensitivity of intracellular Ca2+ stores for mobilization, by D-myo-inositol 1,4,5 trisphosphate ( IP3 ). A family of receptor proteins ( IP3R ) serve to recognize IP3 and act as ligand-gated Ca2+ channels that discharge Ca2+ from internal stores. Our principal hypothesis is that an alteration in the regulatory properties and/or expression of IP3 receptors underlies the observed effects of alcohol on Ca2+ signalling. In order to analyze the mechanism of these effects in more detail, we propose to utilize a cultured rat liver epithelial cell line ( WB cells) as an experimental model. These cells mimic the effects of alcohol - exposure on Ca2+ signalling seen in the animal model and also contain high levels of IP3R protein. Preliminary studies in these cells show that IP3R protein can be down-regulated by chronic stimulation with Angiotensin-II or TGF-B. We suggest that these agonists and ethanol may interact with common mechanisms that regulate IP3R expression.
The specific aims of this project are: [1] To further characterize the effects of chronic ethanol exposure on Ca2+ signalling in WB cells in response to hormones and growth factors; [2]. To test the hypothesis that chronic ethanol exposure modifies the regulatory properties of IP3 receptors; [3]. To determine if chronic ethanol, Angiotensin-II and TGF-B change IP3R expression by translational mechanisms; [4]. To determine if chronic ethanol and agonist treatment alter the transcription of IP3R genes. Delineating the molecular mechanism by which chronic ethanol exposure alters hormonal Ca2+ signalling will aid in understanding the effects of alcohol on liver cell growth and injury.
