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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
Author(s) -
Kennedy Brian G.,
Lever Julia E.
Publication year - 1984
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041210108
Subject(s) - ouabain , ibmx , intracellular , inducer , biophysics , chemistry , cell , sodium , kidney , cell culture , biochemistry , biology , microbiology and biotechnology , endocrinology , in vitro , genetics , forskolin , organic chemistry , gene
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 (Me 2 SO). Ouabain‐sensitive Na + ,K + ‐ATPase and 86 Rb + uptake activities, and the number of [ 3 H]‐ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 10 7 pumps/cell was estimated for subconfluent cultures, declining to 9 × 10 5 pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt 2 cAMP), 1‐methyl‐3‐isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me 2 SO, 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 Me 2 SO partially inhibited ouabain‐sensitive 86 Rb + 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 Bt 2 cAMP caused a furosemide‐sensitive increase in intracellular Na + content. This class of inducers stimulated ouabain‐sensitive 86 Rb + 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.