Differential expression and acid-base regulation of glutaminase mRNAs in gluconeogenic LLC-PK(1)-FBPase(+) cells.
LLC-PK(1)-FBPase(+) cells, which are a gluconeogenic substrain of porcine renal LLC-PK(1) cells, exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase (PDG) activity. On adaptation to acidic medium (pH 6.9, 9 mM HCO(-)(3)), LLC-PK(1)-FBPase(+) cells also exhibit a greater increase in ammonia production and respond with an increase in assayable PDG activity. The changes in PDG mRNA levels were examined by using confluent cells grown on plastic dishes or on permeable membrane inserts. The latter condition increased the state of differentiation of the LLC-PK(1)-FBPase(+) cells. The levels of the primary porcine PDG mRNAs were analyzed by using probes that are specific for the 5.0-kb PDG mRNA (p2400) or that react equally with both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluent dish- and filter-grown LLC-PK(1)-FBPase(+) cells, the predominant 4.5-kb PDG mRNA is increased threefold after 18 h in acidic media. However, in filter-grown epithelia, which sustain an imposed pH and HCO(-)(3) gradient, this adaptive increase is observed only when acidic medium is applied to both the apical and the basolateral sides of the epithelia. Half-life experiments established 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 of 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 intracellular pH while maintaining normal extracellular pH. LLC-PK(1)-FBPase(+) cells again responded with increased rates of ammonia production and increased levels of the 4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellular acidosis is the initiator of this adaptive response. Because all of the observed responses closely mimic those characterized in vivo, the LLC-PK(1)-FBPase(+) cells represent a valuable tissue culture model to study the molecular mechanisms that regulate renal gene expression in response to changes in acid-base balance.
Gstraunthaler
G
G
Institute of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria. gerhard.gstraunthaler@uibk.ac.at
Holcomb
T
T
Feifel
E
E
Liu
W
W
Spitaler
N
N
Curthoys
N P
NP
D000136:Acid-Base Equilibrium / Q000187:drug effects / Q000502:physiology*; D000138:Acidosis / Q000378:metabolism*; D000818:Animals; D005943:Gluconeogenesis / Q000187:drug effects / Q000502:physiology; D005972:Glutaminase / Q000235:genetics / Q000378:metabolism*; D018374:LLC-PK1 Cells / Q000187:drug effects / Q000378:metabolism*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000008:administration & dosage; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D013552:Swine
D012333:RNA, Messenger; D005972:Glutaminase; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D011188:Potassium