Metabolic support of Na+ pump in apically permeabilized A6 kidney cell epithelia: role of creatine kinase.
The contribution of ATP-generating systems to Na+ pump (Na+-K+-ATPase) function was studied in Xenopus laevis A6 kidney epithelia apically permeabilized with digitonin. The ouabain-inhibitable Na+ pump current (I(P)) was measured in the presence of otherwise impermeant inhibitors and/or substrates at Na+ and K+ concentrations that allowed near-maximal pump function. Confocal fluorescence microscopy after apical addition of sulfosuccinimidobiotin (molecular weight of 443) showed that all cells were permeabilized. Less than 15% of the endogenous lactate dehydrogenase and creatine kinase (CK) were released into the apical medium. The I(P) was approximately 5 microA/cm2 in the presence of D-glucose. Blocking glycolysis with 2-deoxy-D-glucose or oxidative phosphorylation with antimycin A decreased it by > or = 50%. Exogenously added ATP prevented these decreases fully or partially, respectively. Two CK isoforms were detected, one likely being mitochondrial and the other corresponding to mammalian B isoform of CK. Phosphocreatine partially restored Na+ pump activity during inhibition of either ATP synthesis pathway. In conclusion, the ATP used by Na+ pumps of apically digitonin-permeabilized A6 epithelia is generated to a similar extent by glycolysis and oxidative phosphorylation. The CK system can partially support the ATP supply to the Na+ pumps.
Guerrero
M L
ML
Institute for Cell Biology, Swiss Federal Institute of Technology, Zurich.
Beron
J
J
Spindler
B
B
Groscurth
P
P
Wallimann
T
T
Verrey
F
F
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism / Q000502:physiology*; D004072:Digitonin / Q000494:pharmacology; D004553:Electric Conductivity; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology; D014982:Xenopus laevis
D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D000254:Sodium-Potassium-Exchanging ATPase; D004072:Digitonin