Differential expression and acid-base regulation of glutaminase mRNAs in gluconeogenic LLC-PK1-FBPase(+) cells

LLC-PK1-FBPase(+) cells, which are a gluconeogenic substrain of porcine ren al LLC-PK1 cells, exhibit enhanced oxidative metabolism and increased level s of phosphate-dependent glutaminase (PDG) activity. On adaptation to acidi c medium (pH 6.9, 9 mM HCO3-), LLC-PK1-FBPase(+) cells also exhibit a great er increase in ammonia production and respond with an increase in assayable PDG activity. The changes in PDG mRNA levels were examined by using conflu ent cells grown on plastic dishes or on permeable membrane inserts. The lat ter condition increased the state of differentiation of the LLC-PK1-FBPase( +) cells. The levels of the primary porcine PDG mRNAs were analyzed by usin g probes that are specific for the 5.0-kb PDG mRNA (p2400) or that react eq ually with both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluen t dish- and filter-grown LLC-PK1 FBPase(+) cells, the predominant 4.5-kb PD G mRNA is increased threefold after 18 h in acidic media. However, in filte r-grown epithelia, which sustain an imposed pH and HCO3- gradient, this ada ptive increase is observed only when acidic medium is applied to both the a pical and the basolateral sides of the epithelia. Half-life experiments est ablished that induction of the 4.5-kb PDG mRNA was due to its stabilization . An identical pattern of adaptive increases was observed for the cytosolic PEPCK mRNA. In contrast, no adaptive changes were observed in the levels o f the 5.0-kb PDG mRNA in either cell culture system. Furthermore, cultures were incubated in low-potassium (0.7 mM) media for 24-72 h to decrease intr acellular pH while maintaining normal extracellular pH. LLC-PK1-FBPase(+) c ells again responded with increased rates of ammonia production and increas ed levels of the 4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellul ar acidosis is the initiator of this adaptive response. Because all of the observed responses closely mimic those characterized in vivo, the LLC-PK1-F BPase(+) cells represent a valuable tissue culture model to study the molec ular mechanisms that regulate renal gene expression in response to changes in acid-base balance.