Effect of chronic ethanol exposure on inositol trisphosphate receptors in WB rat liver epithelial cells.
BACKGROUND
Enhanced agonist-induced Ca2+ release has been reported in hepatocytes isolated from ethanol-fed rats. Because myo-inositol 1,4,5-trisphosphate receptors (IP3Rs) are involved in the mobilization of Ca2+, we examined the effects of chronic ethanol treatment on IP3R function and levels of IP3R 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 Ca2+ mobilization or saponin permeabilized to measure myo-inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release. IP3 levels were measured in [3H]-inositol labeled cells. Levels of IP3R protein were quantitated by immunoblotting with antibodies to IP3R isoforms. Lysosomal and proteasomal peptidase activities were assayed in cytosol and membrane fractions using specific fluorogenic peptide substrates.


RESULTS
Ethanol treatment enhanced Ca2+ mobilization in response to angiotensin II, vasopressin, and bradykinin. This effect was not due to an increased production of IP3. Chronic ethanol treatment stimulated the mobilization of Ca2+ from saponin-permeabilized cells in response to subsaturating doses of IP3 and increased the basal levels of both type I and type III IP3Rs by 1.8-fold and 1.6-fold, respectively. Ethanol treatment did not prevent angiotensin II-induced IP3R 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.


CONCLUSIONS
We propose that the enhanced agonist-mediated Ca2+ mobilization observed in chronic ethanol-treated WB liver epithelial cells results from increased IP3R expression caused by an inhibition of IP3R degradation pathways by ethanol.
Bokkala
S
S
Department of Pathology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107, USA.
Rubin
E
E
Joseph
S K
SK
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D002401:Cathepsin B / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects* / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D015220:Calcium Channels; D002492:Central Nervous System Depressants; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D000431:Ethanol; D002401:Cathepsin B; D002118:Calcium