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

Open Access
Publisher Website
Google Scholar
Abstract
1 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. 2 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%). 3 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. 4 In contrast, in guinea-pig isolated atria, no functional antagonism between erythrosin B (5 × 10−5 mol 1−1) and ouabain was observed. 5 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.