Ouabain and regulation of cellular volume in freshly prepared slices of rabbit renal cortex.

Abstract

SUMMARY1. Changes in the water and ion contents of rabbit renal cortical slices, which had been bathed, immediately after slicing, in air-equilibrated media at room temperature ('freshly prepared slices'), were followed during subsequent incubation at 25 TC in oxygenated media of the same composition as the initial bathing medium.2. In comparison with conventional 'equilibrated' slices (slices incubated at 25 TC in oxygenated ordinary medium immediately after slicing) these 'freshly prepared' slices had increased tissue water, sodium and chloride contents and low tissue potassium contents.3. Control freshly prepared slices incubated at 25 TC in oxygenated ordinary medium recoveredwithin 4min to the tissue water content that is usual forrabbitrenal cortical slices incubated in oxygenated ordinary medium at 25 'C. Freshly prepared slices incubated at 25 'C in oxygenated media containing 1 mM-ouabain took 75 min or more to recover to this usual tissue water content. Thus the presence of 1 mmouabain in both bathing and incubation media produced a marked inhibition of the volume recovery observed when freshly prepared slices are incubated in oxygenated media at 25 'C.4. Reduction of the ouabain concentration reduced the inhibition of cell volume recovery.5. Replacement of medium glucose by 3-0-methylglucose did not inhibit cell volume recovery in the absence of ouabain.6. The oxygen consumptions of slices that were bathed and incubated in 1 mMouabain media were similar to those of slices initially bathed and incubated in ouabain-free media and then incubated in ouabain media. Thus the effect of ouabain in inhibiting cell volume recovery was unlikely to be secondary to inhibition of cellular energy production.7. The tissue potassium content of slices incubated aerobically in 1 or 10 mM ouabain fell to an apparently stable value of approximately 100 m-mole/kg dry wt., which corresponds to a calculated concentration ratio of 10: 1 across the cellular membrane, suggesting that some residual potassium uptake may still have been occurring.8. These results indicate that in freshly prepared rabbit renal cortical slices ouabainsensitive mechanisms play a major role in cell volume recovery. They are not in accord with the postulate that renal cortical cells possess a separate ouabaininsensitive mechanism regulating cell volume.

Age-Dependent Brain Tissue Hydration, Ca Exchange and their Dose- Dependent Ouabain Sensitivity

“…These data are in harmony with literature data that in early postnatal period of development the Na + /K + pump expression is rather weak and it increases during periods of maturation [12]. Therefore, a more pronounced ouabain-induced hydration of brain tissues in adult and old animals as compared to young ones can be explained by more expressed Na + /K + pump in maturated animals [36,37]. Observed differences of brain tissues hydration in three groups of animals can be more clearly seen on right colon of Figure 2D-2F where the differences (∆) between water content of sham and ouabain-poisoned animals are presented.…”

“…In prenatal and early postnatal periods the expression proteins of Na + /Ca 2+ exchanger prevails, while by maturating the expression Na + /K + pump protein increases, reaching its maximum in adults and then intervenes its dysfunction in aging [12]. From this point of view the high water content in brain tissues of young animals can be explained by higher activity of electrogenic Na + / Ca 2+ exchange, working in stechiometry of 3 Na + /1Ca 2+ [10] while the brain tissue dehydration in adult animals can be explained by activation of electrogenic Na + /K + pump [36,37]. Therefore, it is predictable that 10 -4 M ouabain induced inhibition of Na + /K + pump leads to lower hydration in brain tissues of young than maturated animals ( Figure 2).…”

Primary mechanism responsible for age-dependent neuronal dehydration

“…It is known that inhibition of ion-transporting function of electrogenic Na + /K + -pump leads to neuronal hydration [17], [18], [19]. As the dysfunction of Na + /K + -pump is a consequence of aging, it is predicted that pump inhibition could cause more pronounced brain cortex tissue hydration in young animals than in old ones.…”

“…Because of the existence of special water channels (aqua pores) in membrane, water transport through membrane takes place much faster than predicted by simple osmotic diffusion [16]. Therefore, the osmotic gradient on membrane generated by extracellular or intracellular factors has a transient character and the factorinduced changes of intracellular water content are compensated by cell metabolism [17,18,19,20], the dysfunction of which leads to cell pathology. Na + /K + -pump, the dysfunction of which is a consequence of aging, has a key role in metabolic regulation of cell hydration [13], [10], [21], [22].…”

“…Na + /K + -pump, the dysfunction of which is a consequence of aging, has a key role in metabolic regulation of cell hydration [13], [10], [21], [22]. The latter is dictated by the facts that Na + /K + -pump has electrogenic character [23], [24] and generates Na + gradient on membrane which serves as an energy source for a number of sec-ondary ionic transporters in membrane, such as Na , Na + /sugars, amino acids and osmolytes [17], [18], [21], [22], [25]. On the other hand Na + /K + -pump, being the highest adenosine triphosphate (ATP) energy utilizing mechanism, determines the rate of metabolic cascades of ATP synthesis.…”