Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K+

Abstract

Long-term upregulation of the sodium pump [Na-K-adenosine triphosphatase (Na-K-ATPase)] entails an increase in the number of enzyme molecules. We incubated Madin-Darby canine kidney (MDCK) cells in low K+ medium and studied the time course and magnitude of change in the relative abundance of the two Na-K-ATPase subunits (alpha and beta), in the synthesis rate of the subunits, and in the relative abundance of alpha- and beta-mRNA. When cells were incubated in medium containing 0.25 mM K+, intracellular Na+ increased from 25.2 +/- 0.9 (SE) mmol/l cell H2O to 69.8 +/- 9.6 at 4 h and 132 +/- 6 at 16 h. Cell K+ fell from 146 +/- 4 mmol/l cell H2O to 105 +/- 9 at 4 h and 42.3 +/- 4.7 at 16 h. The relative abundance of Na-K-ATPase subunits, measured with immunoblots of cell homogenates, increased such that after 24 h alpha was 1.71 +/- 0.33 and beta was 1.67 +/- 0.22 times control. After 8 h of K+ depletion, alpha-synthesis rate, measured by immunoprecipitation of pulse-labeled cells, increased to 2.30 +/- 0.50 and beta increased to 2.07 +/- 0.42 times control. The alpha- and beta-subunit mRNA abundance, measured by hybridizing alpha- and beta-cDNA probes to total RNA, increased within 30 min to 1.93 +/- 0.24 and 2.29 +/- 0.64 times control, respectively. We conclude that regulatory adjustments of Na-K-ATPase abundance involve an increase in translation after a rapid and coordinate increase in the concentrations of alpha- and beta-subunit mRNA.