KINETICS OF OUABAIN BINDING AND CHANGES IN CELLULAR SODIUM CONTENT, K-42+ TRANSPORT AND CONTRACTILE STATE DURING OUABAIN EXPOSURE IN CULTURED CHICK HEART-CELLS

Abstract

To define further the mechanism of positive inotropic action of cardiac glycosides, the temporal relationships among ouabain binding, Na pump inhibition and positive inotropy were examined using cultured chick embryo ventricular cells. In K+-free medium, specific [3H]ouabain binding to intact cells followed pseudo 1st-order kinetics with saturation of binding sites occurring at 1 .mu.M ouabain. The KD values calculated from the association and dissociation rate constants were 1.4 and 4.9 .times. 10-7 M, respectively, in K+-free and 4 mM K+ medium. The Scatchard plot of binding in K+-free medium was linear, consistent with the presence of a single class of binding sites (KD = 1.3 .RTM. 10-7 M). In 4 mM K+, 0.1 .mu.M ouabain occupied 10% of the total binding sites and failed to produce an inotropic effect, inhibit 42K+ uptake or alter [Na+]i. Exposure of cells to 1 .mu.M ouabain caused a significant increase in contractile state after 30 s, reaching a plateau after 7 min with 50 .+-. 6% augmentation of the amplitude of cell motion; the 42K+ uptake rate was concurrently inhibited by 36% accompanied by a 35% increase in [Na+]i and occupation of 38% of total ouabain binding sites. The initial rate of 42K+ uptake in cells loaded with Na+ by incubation in K+-free medium was 4 times greater than that observed without Na+ loading. These results indicate that more than 10% of Na pump sites must be inhibited to produce an appreciable change in the rate of monovalent cation transport, [Na+]i or contractile state, due to the reserve capacity of uninhibited Na pumps. At 36% Na pump inhibition, positive inotropy is readily observed, although the reserve capacity of the Na pump permits substantial further increases in turnover rate of individual pump sites in response to elevated [Na+]i.