IONIC REGULATION OF NA,K-ATPASE IN CELL CULTURE

The Na,K-pump (i.e., Na,K-ATPase) is an intrinsic membrane transport system which is vital to all animal cells, and mediates ATP-dependent transmembrane exchange of Na+ and K+. The crucial functions served by this system include generation of Na+, K+ gradients and resting membrane potentials across plasma membranes, transepithelial transport of salt and water, and Na+ gradient-driven counter-transport of H+ and Ca++, and cotransport of glucose and amino acids. Recent studies indicate that changes in intracellular ion composition, in particular intracellular Na+ and K+ concentrations, regulate the abundance of the Na,K-pump. The proposed studies are designed to analyze the response to low external K+ in terms of the separate roles of intracellular Na+, K+, H+, and Ca++, and of changes in cellular volume, in eliciting the increase in Na,K-ATPase activity in cultured rat hepatocytes. To assess the response, measurements will be made of Na,K-ATPase activity, ouabain-sensitive 86Rb+ influx, and the abundance of the Na,K-pump, as determined by FITC binding, Na+, Mg++-dependent phosphorylation, and ouabain binding. To elucidate the mechanism of the response, estimates will be made of the rates of synthesis and degradation of the individual subunits of Na,K-ATPase by measuring the rates of incorporation of labelled amino acids, and the rates of disappearance of these labels in subunits isolated by immunoprecipitation. If increases in the synthesis of the subunits accounts for a significant fraction of the response to low K+, the activities of their various mRNAs, i.e., mRNAAlpha and mRNABeta, in translational assays will be assessed by immunoprecipitation of nascent Alpha and Beta chains. The abundances of mRNAAlpha and mRNABeta extracted from cells exposed to various external K+ concentrations will also be measured by hybridization to their respective cDNAs. If these mRNAs are increased by exposure to low K+ concentrations, nuclear run-off will be used to assess the role of transcription in the process. To determine the importance of genetic regulatory regions in the low K+ response, cultured cell recipients of the ouabain-resistance gene will also be examined.