Two ouabain binding sites in guinea pig heart Na+-K+- ATPase. Differentiation by sodium and erythrosin B

3H-ouabain binding to guinea pig heart NaK-ATPase resulted in two different cardiac glycoside binding sites: A high affinity, low capacity binding was obtained at a KD = 2.6 x 10−7 mol/1 (about 40% of the binding sites) whereas a low affinity, high capacity binding was established at a KD = 1.3 x 10−6 mol/1 (about 60% of the binding sites). Similar results were obtained when studying the effect of increasing Na+-concentrations on ouabain binding: again two distinct processes involved in the Na+/ouabain interaction could be demonstrated. Whereas one ouabain binding process was activated at rather low Na+-concentrations (K0.5 = 4.5 mmol/1), the other ouabain binding process was predominant at high Na+- concentrations only (KQ 5 = 69 mmol/1). Comparing these data with the ouabain action on contractile force and with NaK-ATPase activity in guinea pig heart, the high affinity binding of ouabain seems to correlate with the inotropic action, whereas the low affinity ouabain binding is more related to NaK-ATPase inhibition. To further discriminate the two cardiac glycoside binding sites operative in guinea pig heart muscle, erythrosin B, shown to be an inhibitor of the high affinity ouabain binding in this preparation, was applied to isolated guinea pig left atria in the presence of increasing ouabain concentrations. There was no change in the ouabain induced inotropy or toxicity by erythrosin B. These results could indicate that the low affinity ouabain binding is responsible for both inotropic and toxic actions in guinea pig heart. However, the functional significance of the high affinity ouabain binding would then remain obscure. Therefore, an alternate, and more plausible explanation for the lack of effect of erythrosin B on ouabain action in guinea pig atria would be an interaction of the dye with the ATP-site of the NaK-ATPase, which would suppose a cellular uptake of this drug, as yet not established.