The effect of ouabain on the electrolyte and water transport in kidney cortex and liver slices

Kleinzeller, A.; Knotková, A.

doi:10.1113/jphysiol.1964.sp007510

Cited by 110 publications

(36 citation statements)

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“…Changes in the oxygen consumption of kidney cortex slices have been used to investigate interactions betweeen cellular metabolism, and water and ion regulation. Such studies have shown that tissue water content is inversely proportional to oxygen consumption when the latter is depressed by cyanide [Robinson, 1950], that ouabain in high concentrations produces a 30 45o% decrease in oxygen consumption, and in lower concentrations a fall in oxygen consumption that is proportional to potassium loss, and that replacement of extracellular sodium by another cation produces a fall in oxygen consumption similar to that found with high concentrations of ouabain [Whittam and Willis, 1963;Kleinzeller and Knotkova, 1964;Allison, 1975].A number of authors have claimed that renal cortical and hepatic cells possess an ouabain-insensitive volume regulating mechanism [Kleinzeller and Knotkova, 1964;Macknight, 1968a;Whittembury, 1968;Macknight, Pilgrim and Robinson, 1974]. In order to demonstrate rigorously the existence of a ouabain-insensitive cell volume regulating mechanism, cells must continue to regulate their volume after the conventional sodium pump has been completely inhibited.…”

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“…Black and White rats. Slices were then either incubated in a large volume of oxygenated ordinary medium at 25°C until all the slices were cut and then for a further 20 minutes, hereafter called 'equilibrated' slices [Macknight, 1968a] or were placed in approximately 30 ml of air-equilibrated medium at room temperature until all the slices were cut and then for a further 5 minutes with occasional gentle shaking, 'freshly prepared' slices [Whittam and Willis, 1963;Kleinzeller and Knotkova, 1964], before being incubated under the different experimental conditions described with each experiment. …”

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Oxygen Consumptions and Potassium Contents of Slices of Rat Renal Cortex

Cooke

1979

Exp Physiol

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An oxygen electrode respirometer for determining the oxygen consumption of slices of mammalian renal cortex is described and assessed.Though rat renal cortical slices incubated in potassium-free medium for one hour lost 102 + 14 mmol of potassium/kg dry weight, there was only a small, nonsignificant fall in oxygen consumption. In contrast the oxygen consumption of slices incubated in potassium-free medium with 10 mmol.1 1 ouabain was markedly reduced (by 32 + 6, while such slices lost 180+15 mmol of potassium/kg dry weight. These disproportionate effects on potassium loss and inhibition of oxygen consumption suggest that in renal cortical slices the loss of potassium in low potassium medium is not primarily due to inhibition of the conventional sodium pump.Tissue oxygen consumption appears to consist of both a relatively constant basal rate found in the resting cell, and a suprabasal component that varies in relation to demands for energy during activity [Barcroft, 1938;Whittam and Willis, 1963]. In slices from rabbit renal cortex, variations in the rates of metabolically-dependent ion transport are associated with proportional alterations in oxygen consumption [Whittam and Willis, 1963]. Changes in the oxygen consumption of kidney cortex slices have been used to investigate interactions betweeen cellular metabolism, and water and ion regulation. Such studies have shown that tissue water content is inversely proportional to oxygen consumption when the latter is depressed by cyanide [Robinson, 1950], that ouabain in high concentrations produces a 30 45o% decrease in oxygen consumption, and in lower concentrations a fall in oxygen consumption that is proportional to potassium loss, and that replacement of extracellular sodium by another cation produces a fall in oxygen consumption similar to that found with high concentrations of ouabain [Whittam and Willis, 1963;Kleinzeller and Knotkova, 1964;Allison, 1975].A number of authors have claimed that renal cortical and hepatic cells possess an ouabain-insensitive volume regulating mechanism [Kleinzeller and Knotkova, 1964;Macknight, 1968a;Whittembury, 1968;Macknight, Pilgrim and Robinson, 1974]. In order to demonstrate rigorously the existence of a ouabain-insensitive cell volume regulating mechanism, cells must continue to regulate their volume after the conventional sodium pump has been completely inhibited. Macknight [1968a] and Whittembury and Proverbio [1970] used the magnitude of potassium loss from renal cortical slices incubated in potassium-69 B 70 K. R. COOKE free media with different ouabain concentrations to assess the degree of inhibition of the sodium pump. In this paper, the oxygen consumptions and ionic contents of rat renal cortical slices incubated in potassium-free media were measured to see whether there was a decline in oxygen consumption commensurate with the potassium loss, as would be expected if potassium loss in low potassium media was primarily due to inhibition of the conventional sodium pump. MethodsProcedure. Slices 0.2-0.3 mm thick were ...

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Oxygen Consumptions and Potassium Contents of Slices of Rat Renal Cortex

Cooke

1979

Exp Physiol

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“…Over the last decade evidence has been presented from studies in several animal species, indicating that isosmotic transporting epithelia such as those of the proximal tubule and mammalian gall-bladder possess two pumps handling sodium (Kleinzeller, 1961;Kleinzeller & Knotkova, 1964;MacKnight, 1968;Whittembury, 1968;Whittembury & Fishman, 1969;Whittembury & Proverbio, 1970;Giebisch, Sullivan & Whittembury, 1973). One is the classical ATPase-dependent Na-K pump maintaining high K and low Na concentrations in the cytosol; this pump is inhibited by ouabain or absence of extracellular K. The other is a pump handling the extrusion of NaCl and water in isosmotic proportion across the baso-lateral cell border, responsible probably for both transepithelial isosmotic salt and water transport and cell volume regulation; this pump is inhibited by the diuretic ethacrynic acid (ETCA).…”

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Functional distinction between two transport mechanisms in rabbit gall‐bladder epithelium by use of ouabain, ethacrynic acid and metabolic inhibitors.

Frederiksen¹

1978

The Journal of Physiology

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SUMMARY1. Net fluid transport rate, transepithelial p.d. and resistance, and unidirectional Na+-fluxes were measured in rabbit gall-bladder preparations exposed on both sides to bicarbonate-Ringer solution in vitro.2. Both ouabain and ethacrynic acid (ETCA) caused dose-dependent decreases of net fluid transport rate; ouabain inhibited fluid transport predominantly from the serosal side, whereas the inhibitory effect of ETCA was elicited mainly from the mucosal (luminal) side. Applied bilaterally, the ID50 for ouabain was 2-5 x 10-6 M, and for ETCA 2-3 x 104 M. After maximal inhibition at each concentration level of the two inhibitors fluid transport could not be reversed.3. 2,4-Dinitrophenol (2,4-DNP) (2 x 104 M) or substitution of 02 by N2 caused an 80% reversible decrease of net fluid transport.4. The spontaneous p.d. across the rabbit gall-bladder was about 2-7 mV, mucosal side positive. 2,4-DNP, N2 and serosal application of ouabain depressed the p.d. after an initial hyperpolarization. This decrease was reversible during recovery from 2,4-DNP and N2, but irreversible after removal of ouabain at concentrations > 10-4 M. Mucosal application of ETCA (10-3 M) caused no decrease in p.d., which actually increased slightly.5. Calculated passive serosal-to-mucosal Na+-fluxes changed in the same direction as did changes in conductance.6. It is concluded that ETCA does not interfere primarily with the Na-K-ATPase or cellular oxidative metabolism. The data support the proposal that the pump responsible for isosmotic transepithelial fluid transfer is located in the luminal end of the cells. This pump is ETCA-sensitive. The ATPase-dependent Na-K pump, which can be inhibited by ouabain, is localized in the serosa-facing cell membrane. The data suggest that the inhibition of net fluid transport by ouabain is indirect and mediated by changes in intracellular ion concentrations.7. The results support the concept that the transepithelial fluid transport mechanism is electroneutral, and suggest that the mucosa positive transepithelial p.d. is due to differences in electromotive forces arising from ion (mainly K+) diffusion across the mucosal and serosal cell membranes. FREDERIKSEN

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“…Thus, it has been shown in studies on isosmotic fluid transport by rabbit gall-bladder in vitro that marked changes in net sodium transport can occur acutely without changes in water transfer when sodium concentration (and osmolality) is changed abruptly and identically in the media bathing the serosal as well as the mucosal side; and suprabasal energy consumption was strictly correlated to fluid volume (water) transport rate independent of the actual amounts of sodium chloride transferred (Frederiksen & Leyssac, 1969). Cell volume regulation by kidney cortical cells depends on an ethacrynic acidsensitive pump distinct from the ouabain-sensitive Na+-K+ pump, and evidence has been provided suggesting that cell volume regulation involves a mechanochemical process (Kleinzeller & Knotkova, 1964). Similarly, transepithelial isosmotic solute and water transfer involves an ethacrynic acid-sensitive pump mechanism distinct from the ouabainsensitive Na+-K+ pump (Leyssac & Frederiksen, 1974a 105 Frederiksen (1974a) proposed a mechanochemical pump mechanism for transcellular isosmotic fluid transport.…”

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Effects of cytochalasin B and dimethylsulphoxide on isosmotic fluid transport by rabbit gall‐bladder in vitro.

Frederiksen¹,

Leyssac²

1977

The Journal of Physiology

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SUMMARY1. Net fluid transport rate, transepithelial ohmic resistance and potential difference (p.d.), and unidirectional fluxes of Na+ were measured in rabbit gall-bladder preparations in vitro exposed on both sides to Ringer solutions of identical electrolyte composition. cytochalasin B on the active transcellular pump mechanism and to 0. FREDERIKSEN AND P. P. LEYSSAC suggest a cytochalasin B-mediated progressive increase in cell membrane permeability to sodium resulting ultimately in a highly leaky epithelium. The results are compatible with the concept that a mechanochemical process is involved in isosmotic transcellular transport of fluid across lowresistance epithelia.

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The effect of ouabain on the electrolyte and water transport in kidney cortex and liver slices

Cited by 110 publications

References 26 publications

Oxygen Consumptions and Potassium Contents of Slices of Rat Renal Cortex

Oxygen Consumptions and Potassium Contents of Slices of Rat Renal Cortex

Functional distinction between two transport mechanisms in rabbit gall‐bladder epithelium by use of ouabain, ethacrynic acid and metabolic inhibitors.

Effects of cytochalasin B and dimethylsulphoxide on isosmotic fluid transport by rabbit gall‐bladder in vitro.

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