Na+-K+-ATPase that redistributes to apical membrane during ATP depletion remains functional

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Abstract

We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na+-K+-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na+-K+-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na+-K+-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/ filter, P < 0.01). Apical [3H]ouabain binding returned to base-line during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na+-K+-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 ± 0.61 vs. 1.27 ± 0.46 meq·filter-1·30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 ± 0.12 vs. 0.83 ± 0.27 meq·filter-1·30 min-1, P < 0.01), implying that apical Na+-K+-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.

Original languageEnglish
JournalAmerican Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume265
Issue number5 34-5
StatePublished - Nov 1993

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Adenosine Triphosphatases
Adenosine Triphosphate
Ouabain
Membranes
LLC-PK1 Cells
Proximal Kidney Tubule
Tight Junctions
Electric Impedance
Actin Cytoskeleton
Ischemia
Proteins

Keywords

  • Basolateral membrane
  • Ischemia
  • LLC-PK cells
  • Ouabain
  • Sodium transport

ASJC Scopus subject areas

  • Physiology

Cite this

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title = "Na+-K+-ATPase that redistributes to apical membrane during ATP depletion remains functional",
abstract = "We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na+-K+-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na+-K+-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na+-K+-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/ filter, P < 0.01). Apical [3H]ouabain binding returned to base-line during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na+-K+-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 ± 0.61 vs. 1.27 ± 0.46 meq·filter-1·30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 ± 0.12 vs. 0.83 ± 0.27 meq·filter-1·30 min-1, P < 0.01), implying that apical Na+-K+-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.",
keywords = "Basolateral membrane, Ischemia, LLC-PK cells, Ouabain, Sodium transport",
author = "Bruce Molitoris",
year = "1993",
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journal = "American Journal of Physiology",
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T1 - Na+-K+-ATPase that redistributes to apical membrane during ATP depletion remains functional

AU - Molitoris, Bruce

PY - 1993/11

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N2 - We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na+-K+-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na+-K+-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na+-K+-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/ filter, P < 0.01). Apical [3H]ouabain binding returned to base-line during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na+-K+-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 ± 0.61 vs. 1.27 ± 0.46 meq·filter-1·30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 ± 0.12 vs. 0.83 ± 0.27 meq·filter-1·30 min-1, P < 0.01), implying that apical Na+-K+-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.

AB - We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na+-K+-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na+-K+-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na+-K+-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/ filter, P < 0.01). Apical [3H]ouabain binding returned to base-line during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na+-K+-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 ± 0.61 vs. 1.27 ± 0.46 meq·filter-1·30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 ± 0.12 vs. 0.83 ± 0.27 meq·filter-1·30 min-1, P < 0.01), implying that apical Na+-K+-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.

KW - Basolateral membrane

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KW - Sodium transport

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