title: Erythrosin B inhibits high affinity ouabain binding in guinea-pig heart Na+-K+-ATPase without influence on cardiac glycoside 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 X 10(-7) mol 1(-1) (Bmax = 16.8 pmol ouabain mg-1 protein) whereas low affinity ouabain binding occurred at a KD much greater than 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 X 10(-6) mol 1(-1). Contractility of guinea-pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 X 10(-5) mol 1(-1). Only very high concentrations (4 X 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 X 10(-6) mol 1(-1). In a concentration of 1 X 10(-5) mol 1(-1) the dye abolish 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 X 10(-5) mol 1(-1) and ouabain was observed. 5 As there is a coincidence between the high affinity binding (KD = 2.2 x 10-7moll ') and the concentration for half maximum inotropic effects of ouabain (EDIo = 1.6 x 10-7 mol I), the lack of effect oferythrosin B on ouabain-induced inotropy may be caused by an inaccessibility of the dye to the (internal) ATP-site of the Na+-K+-ATPase.
authors.forename: U
authors.initials: U
authors.lastname: Fricke
mesh_terms: D000818:Animals; D002301:Cardiac Glycosides / Q000494:pharmacology*; D004347:Drug Interactions; D004923:Erythrosine / Q000494:pharmacology*; D005260:Female; D005452:Fluoresceins / Q000494:pharmacology*; D006168:Guinea Pigs; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009200:Myocardial Contraction / Q000187:drug effects*; D009206:Myocardium / Q000201:enzymology*; D010042:Ouabain / Q000378:metabolism*; D013268:Stimulation, Chemical
chemical_list: D002301:Cardiac Glycosides; D005452:Fluoresceins; D010042:Ouabain; D008274:Magnesium; D004923:Erythrosine