Effect of chronic ethanol exposure on inositol trisphosphate receptors in WB rat liver epithelial cells.

Background: Enhanced agonist-induced Ca 2+ release has been reported in hepatocytes isolated from ethanol-fed rats. Because myo-inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are involved in the mobilization of Ca 2+ , we examined the effects of chronic ethanol treatment on IP 3 R function and levels of IP 3 R protein by using WB rat liver epithelial cells. Methods: WB cells were treated with ethanol (50-150 mM) for 24 to 48 hr and were loaded with Fura-2 to measure agonist-induced Ca 2+ mobilization or saponin permeabilized to measure myo-inositol 1,4,5-trisphosphate (IP 3 )-mediated Ca 2+ release. IP 3 levels were measured in [ 3 H]-inositol labeled cells. Levels of IP 3 R protein were quantitated by immunoblotting with antibodies to IP 3 R isoforms. Lysosomal and proteasomal peptidase activities were assayed in cytosol and membrane fractions using specific fluorogenic peptide substrates. Results: Ethanol treatment enhanced Ca 2+ mobilization in response to angiotensin II, vasopressin, and bradykinin. This effect was not due to an increased production of IP 3 . Chronic ethanol treatment stimulated the mobilization of Ca 2+ from saponin-permeabilized cells in response to subsaturating doses of IP 3 and increased the basal levels of both type I and type III IP 3 Rs by 1.8-fold and 1.6-fold, respectively. Ethanol treatment did not prevent angiotensin II-induced IP 3 R down-regulation or alter lysosomal cathepsin B activity or the trypsin-like and peptidylglutamyl peptidase activities of the proteasome. However, chronic ethanol exposure resulted in a 60% and 41% inhibition of the chymotrypsin-like activity of the proteasome in cytosol and microsomal membranes, respectively. Conclusion: We propose that the enhanced agonist-mediated Ca 2+ mobilization observed in chronic ethanol-treated WB liver epithelial cells results from increased IP 3 R expression caused by an inhibition of IP 3 R degradation pathways by ethanol.