Regulation of Na+,K+-ATPase activity in MDCK kidney epithelial cell cultures: role of growth state, cyclic AMP, and chemical inducers of dome formation and differentiation.

Na+,K+-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me2SO). Ouabain-sensitive Na+,K+-ATPase and 86Rb+ uptake activities, and the number of [3H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 X 10(7) pumps/cell was estimated for subconfluent cultures, declining to 9 X 10(5) pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt2cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me2SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na+ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na+ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me2SO partially inhibited ouabain-sensitive 86Rb+ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na+ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt2cAMP caused a furosemide-sensitive increase in intracellular Na+ content. This class of inducers stimulated ouabain-sensitive 86Rb+ uptake, presumably by substrate effects due to increased Na+ levels. The Na+ or ATP activation of Na+,K+-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb+ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na+ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.