title: Effect of pH and bicarbonate on phosphoenolpyruvate carboxykinase and glutaminase mRNA levels in cultured renal epithelial cells.
abstract: A gluconeogenic strain of renal epithelial cells (LLC-PK1-F+) was used to characterize the effect of pH and bicarbonate concentration on the levels of phosphoenolpyruvate carboxykinase (PCK) and glutaminase (GA) mRNAs. The levels of both mRNAs are markedly dependent upon medium glucose concentration. The level of PCK mRNA is increased with increasing glucose concentration from 0 to 40 mM, whereas the level of GA mRNA is maximal between 3 and 5 mM glucose. When LLC-PK1-F+ cells are grown with 5 mM glucose and then subjected to an acute decrease in pH (from 7.4 to 6.9) and bicarbonate concentration (from 25 to 10 mM), the level of PCK mRNA exhibits a biphasic response. The PCK mRNA is initially increased 4-fold within 3 h, then decreases slightly and subsequently increases between 10 and 20 h to a level that is 17-fold greater than normal. Only the initial increase parallels the changes observed in vivo. In contrast, after onset of acidosis, the level of GA mRNA initially remains unchanged, is then increased 8-fold between 10 and 16 h, and then decreases slightly. This response closely mimics the results obtained in vivo. A decrease in media pH at constant bicarbonate causes a marked increase in both mRNAs. However, the levels of the two mRNAs are also elevated by decreasing bicarbonate at a constant pH. Thus, both parameters independently affect the level of the two mRNAs. The use of actinomycin D to measure the half-lives of PCK and GA mRNAs at pH 7.4 and 6.9 indicates that stabilization may fully account for the induction of GA mRNA and contributes to the inductive effects of decreased pH and/or bicarbonate on PCK mRNA. Following recovery from acidic conditions, the two mRNAs exhibit a rapid and coordinate decrease (t1/2 approximately 20 min). Dexamethasone had no effect on the level of either mRNA, whereas cAMP increased only PCK mRNA. The latter effect was additive with the increase caused by decreased pH and/or bicarbonate and was reversed by incubating in alkalotic media. Thus, the induction of PCK and GA mRNAs during acidosis is initiated in direct response to a decrease in extracellular pH and/or bicarbonate.
authors.affiliation: Department of Biochemistry, Colorado State University, Fort Collins 80523.
authors.forename: S
authors.initials: S
authors.lastname: Kaiser
authors.forename: N P
authors.initials: NP
authors.lastname: Curthoys
mesh_terms: D000138:Acidosis / Q000201:enzymology*; D000471:Alkalosis / Q000201:enzymology*; D001639:Bicarbonates / Q000378:metabolism*; D015152:Blotting, Northern; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005972:Glutaminase / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*
chemical_list: D001639:Bicarbonates; D012333:RNA, Messenger; D003907:Dexamethasone; D000242:Cyclic AMP; D005972:Glutaminase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)