Erythrosin B inhibits high affinity ouabain binding in guinea‐pig heart Na⁺‐K⁺‐ATPase without influence on cardiac glyoside induced contractility

Abstract

Binding of [3H]‐ouabain to guinea‐pig heart membranes enriched in Na+‐K+‐ATPase revealed two different cardiac glycoside binding sites. High affinity binding was obtained at a KD = 2.2 × 10−7 mol 1−1 (Bmax = 16.8 pmol ouabain mg−1 protein) whereas low affinity ouabain binding occurred at a KD > 10−6 mol 1−1. To discover whether the two ouabain binding sites are functional in guinea‐pig heart muscle, erythrosin B, an inhibitor of the high affinity ouabain binding in rat brain tissue, was tested in guinea‐pig isolated heart muscle preparations. Erythrosin B proved to be a potent inhibitor of the Mg2+(Na+)‐dependent‐, as well as Na+‐K+‐activated ATPase (ID50 = 9 × 10−6 mol 1−1). Contractility of guinea‐pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 × 10−5 mol 1−1. Only very high concentrations (4 × 10−4 mol 1−1) resulted in a slightly negative inotropic effect (about 20%). Erythrosin B dose‐dependently inhibited [3H]‐ouabain binding to the Na+‐K+‐ATPase (KD = − 3.6 × 10−6 mol 1−1). In a concentration of 1 × 10−5 mol 1−1 the dye abolished high affinity [3H]‐ouabain binding without affecting the low affinity binding sites. In contrast, in guinea‐pig isolated atria, no functional antagonism between erythrosin B (5 × 10−5 mol 1−1) and ouabain was observed. As there is a coincidence between the high affinity binding (KD = 2.2 × 10−7 mol 1−1) and the concentration for half maximum inotropic effects of ouabain (ED50 = 1.6 × 10−7 mol 1−1), the lack of effect of erythrosin B on ouabain‐induced inotropy may be caused by an inaccessibility of the dye to the (internal) ATP‐site of the Na+‐K+‐ATPase.