BINDING-PROPERTIES AND BIOLOGICAL EFFECTS OF OXIDIZED-OUABAIN ON CULTURED NEONATAL-RAT CARDIAC MYOCYTES - IMPLICATIONS ON THE MECHANISM OF ACTION OF THE DIGITALIS-GLYCOSIDES

Mild oxidation of ouabain with NaIO4, causes the cleavage of the bond between C2' and C3' of the rhamnose ring, leaving the steroid moiety intact. The oxidized ouabain (ox-ouabain) was examined on spontaneously contracting cultured rat-cardiac myocytes. Two classes of binding sites, with high and low affinities, were detected for both ox-ouabain and unmodified oubain. The dissociation constants (K(D)) were found to be similar for both compounds, but the rate constants of association (k(a)) and dissociation (k(d)) of the low affinity sites were higher for ox-ouabain as compared with ouabain. Displacement experiments showed that ox-ouabain and ouabain bind to the same sites. The effects of ox-ouabain and ouabain on the activity of Na+, K+-ATPase were determined in microsomal preparations. Similar dose-response curves for the inhibition of the enzyme activity were determined for both drugs. Inhibition was observed only at concentrations above 10(-6) M. The biological effects of the drugs were examined by their capacity to induce positive inotropic or toxic effects. Concentrations of ox-ouabain which induced positive inotropic effects (increase in amplitude of systolic cell motion), ranged from 5 x 10(-8) M to 5 x 10(-6) M, as compared with 10(-7) M to 5 x 10(-7) M with ouabain. "Toxic" effects (decrease in the amplitude of systolic motion, increased beating frequencies and elevation in the position of maximal relaxation) was observed only with 10(-5) M ox-ouabain as compared with 10(-6) M ouabain. The mechanism of the inotropic action of ox-ouabain at the lower concentration range was investigated by measuring the effect of the drugs on Rb-86+ (analogue of K+) influx. Dose-response curves of effects of ouabain and ox-ouabain on Rb-86+ influx were bi-phasic. At low concentrations stimulation was observed, whereas at high concentrations Rb-86+ influx was inhibited. Ox-ouabain stimulated Rb-86+ influx by lower concentrations and to a greater extent than ouabain. A part of Rb-86+ influx into cardiac myocytes is mediated by the K+/Na+/Cl- cotransporter, which can be inhibited by loop diuretic drugs such as bumetanide. We have previously shown that ouabain, at low concentrations, stimulates the activity of the cotransporter. It is shown in the present work that ox-ouabain stimulates the activity of the cotransporter by lower concentrations and to a greater extent than ouabain. The inotropic effects at the lower concentration range of ox-ouabain, as compared to ouabain, seem to be caused by the greater stimulation of the cotransporter, and not by the inhibition of the Na+, K+-ATPase activity, which was found to be similar for both drugs. The lower toxicity at the higher concentration range seems to be linked with a lower steady-state level of [Ca2+]i and a higher rate-constant of dissociation of the low affinity site.