Summary
Na+, K+ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10−4 M produced maximal inhibition (95%) of K+ uptake and enhanced intracellular Na+ accumulation, showing that active fluxes account for a very large proportion of Na+ and K+ exchanges. Inhibition of the Na−K pump by ouabain was significant at low concentrations (10−8 M). When external K+ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC50) of K+ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [3H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K+ concentration in the medium, the other linear and independent of external K+. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·105 per cell) and independent of the external K+ concentration (7, 0.5 or 0 mM), while the dissociation constant (KD) decreased from 4.2 μM to 7.3 nM when K+ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min−1), as estimated from ouabain-sensitive K+ flux, in comparison to those described in other cell types of higher vertebrates. At each external K+ concentration studied, the inhibition of K+ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.
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References
Baker PF, Willis JS (1970) Potassium ions and the binding of cardiac glycosides to mammalian cells. Nature 226:521–523
Baker PF, Willis JS (1972) Binding of the cardiac glycoside ouabain to intact cells. J Physiol 224:441–462
Bianchini L, Fossat B, Porthé-Nibelle J, Ellory JC, Lahlou B (1988) Effects of hypoosmotic shock on ion exchange in isolated trout hepatocytes. J Exp Biol 137:303–318
Borgese F, Garcia Roumeu F, Motais R (1987) Control of cell volume and ion transport by β-adrenergic catecholamines in erythrocytes of rainbow trout Salmo gairdneri. J Physiol 382:123–144
Bourne PK, Cossins AR (1984) Sodium and potassium transport in trout Salmo gairdneri erythrocytes. J Physiol 347:361–375
Erdmann E, Hasse W (1975) Quantitative aspects of ouabain binding to human erythrocyte and cardiac membranes. J Physiol 251:671–682
Fehlmann M, Freychet P (1981) Insulin and glucagon stimulation of (Na+−K+)-ATPase transport activity in isolated rat hepatocytes. J Biol Chem 256:7449–7453
Furukawa H, Bilezikian JP, Loeb JN (1980) Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte. Quantitative relation to ligand binding and cyclic AMP generation. Biochim Biophys Acta 598:345–356
Hoffman JF (1966) The red cell membrane and the transport of sodium and potassium. Am J Med 41:666–680
Joiner CH, Lauf PK (1978) The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes. J Physiol 283:155–175
Lahlou B, Crenesse D, Bensahla-Talet A, Porthé-Nibelle J (1975) Adaptation de la truite d'élevage à l'eau de mer. Effects sur les concentrations plasmatiques, les échanges branchiaux et le transport intestinal du sodium. J de Physiologie 70:593–603
Lobaugh LA, Lieberman M (1987) Na−K pump site density and ouabain binding affinity in cultured chick heart cells. Am J Physiol 253:C731–C743
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Porthé-Nibelle J, Lahlou B (1981) Mechanisms of glucocorticoid uptake by isolated hepatocytes of the trout. Comp Biochem Physiol 69B:425–433
Sachs JR, Dunham PB, Kropp DL, Ellory JC, Hoffman JF (1974) Interaction of HK and LK goat red blood cells with ouabain. J Physiol 64:536–550
Walton MJ, Cowey CB (1979) Gluconeogenesis by isolated hepatocytes from rainbow trout Salmo gairdneri. Comp Biochem Physiol 62B:75–81
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Bianchini, L., Fossat, B., Porthé-Nibelle, J. et al. Characteristics of ouabain binding to isolated trout hepatocytes. J Comp Physiol B 160, 93–98 (1990). https://doi.org/10.1007/BF00258767
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DOI: https://doi.org/10.1007/BF00258767