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

Abstract

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 × 10⁷ pumps/cell was estimated for subconfluent cultures, declining to 9 × 10⁵ 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.