Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells.
Measurements of internal ion concentrations, amino acid pools, and membrane potential were made across a series of HeLa subclones which are amplified for the genes for the sodium- and potassium-activated ATPase (Na,K-ATPase). These subclones expressed heterogeneous levels of ouabain-binding sites, allowing us to construct a graded amplification series. While [K+]i levels did not vary systematically across the series studied, [Na+]i ranged from 9 to 20 mM as a function of Na,K-ATPase expression. Steady-state accumulation of tetraphenylphosphonium in low versus high potassium was used to measure membrane potential. Values for [Na+]i and the membrane potential were used to calculate the sodium electrochemical potential, which was also found to be a function of Na,K-ATPase expression. Measurements of acid-soluble amino acid pools in cell lysates demonstrated that amino acids which are substrates for sodium-dependent transport systems, or which can potentially exchange through system L for a substrate of a sodium-dependent system, varied as a function of the sodium electrochemical potential. This confirmed our prediction of increased amino acid pool sizes in Na,K-ATPase-amplified lines based on observations of elevated flux through the sodium-independent system L. Finally, we measured lactate production and glycolytic potential in a subset of clones and found that both were reduced in subclones with elevated Na,K-ATPase.
Pauw
P G
PG
Department of Anatomy, University of Utah School of Medicine, Salt Lake City 84132.
Sheck
R N
RN
Ash
J F
JF
D001692:Biological Transport; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006639:Histidine / Q000032:analysis; D006801:Humans; D007773:Lactates / Q000032:analysis; D008564:Membrane Potentials; D010042:Ouabain / Q000378:metabolism; D011188:Potassium / Q000502:physiology; D012964:Sodium / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
D007773:Lactates; D006639:Histidine; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium