Effects of altered calcium homeostasis on the expression of glutathione S-transferase isozymes in primary cultured rat hepatocytes.
The effects of altered Ca2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class alpha and mu specific antibodies showed an approximately 1.6- and 1.5-fold increase in the class alpha, Ya and Yc subunits, respectively, whereas no significant increase (approximately 1.2-fold) in class mu GST expression was observed following A23187 treatment. Northern blot analysis revealed an approximately 5-fold increase in GST class alpha and an approximately 7-fold increase in class mu GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Immunoblot analysis showed a significant increase in the expression of GST class alpha, Ya and Yc subunits, approximately 1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class mu GSTs. Northern blot analysis showed approximately 3- and 2-fold increases, respectively, in class alpha and mu GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca2+ homeostasis produced by either Ca2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.
Dwivedi
R S
RS
Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48201.
Gruebele
A
A
Novak
R F
RF
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006706:Homeostasis / Q000187:drug effects*; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010545:Peroxides / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate; D020122:tert-Butylhydroperoxide
D007527:Isoenzymes; D010545:Peroxides; D012967:Sodium Dodecyl Sulfate; D000001:Calcimycin; D020122:tert-Butylhydroperoxide; D005982:Glutathione Transferase; D002118:Calcium