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

Brian Kennedy, Julia E. Lever

Research output: Contribution to journalArticle

48 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)51-63
Number of pages13
JournalJournal of Cellular Physiology
Volume121
Issue number1
DOIs
StatePublished - 1984
Externally publishedYes

Fingerprint

hexamethylene bisacetamide
Sodium-Potassium-Exchanging ATPase
Domes
Cell culture
Cyclic AMP
Adenosine Triphosphatases
1-Methyl-3-isobutylxanthine
Cell Culture Techniques
Epithelial Cells
Ouabain
Kidney
Monolayers
Growth
Adenosine Triphosphate
Pumps
Bucladesine
Madin Darby Canine Kidney Cells
Dimethyl Sulfoxide
Cell Extracts
Cell Count

ASJC Scopus subject areas

  • Physiology
  • Medicine(all)
  • Clinical Biochemistry
  • Cell Biology

Cite this

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title = "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.",
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T1 - Regulation of Na+, K+‐ATPase activity in MDCK kidney epithelial cell cultures

T2 - Role of growth state, cyclic AMP, and chemical inducers of dome formation and differentiation

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