Characteristics of ouabain binding to isolated trout hepatocytes.
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 x 10(5) per cell) and independent of the external K+ concentration (7, 0.5 or 0 mM), while the dissociation constant (KD) decreased from 4.2 microM 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.
Bianchini
L
L
Laboratoire de Physiologie Cellulaire et Comparée et UA CNRS 651, Faculté des Sciences, Université de Nice-Sophia Antipolis, France.
Fossat
B
B
Porthé-Nibelle
J
J
Lahlou
B
B
D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D066298:In Vitro Techniques; D007474:Ion Exchange; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism* / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism; D012487:Salmonidae / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D014337:Trout / Q000378:metabolism*
D015221:Potassium Channels; D015222:Sodium Channels; D010042:Ouabain; D012964:Sodium; D011188:Potassium