Cardiac glycoside receptors in cultured heart cells--II. Characterization of a high affinity and a low affinity binding site in heart muscle cells from neonatal rats

Abstract

The binding of [3H]ouabain has been studied in (Na+ + K+)-ATPase enriched cardiac cell membranes, as well as in cardiac muscle and non-muscle cells in culture--all obtained from hearts of neonatal rats. The binding has been correlated with ouabain-induced inhibition of (Na+ + K+)-ATPase (cardiac cell membranes) and the inhibition of active (86Rb+ + K+)-influx (cardiac muscle and non-muscle cells in culture). Furthermore, the effect of ouabain on the amplitude of cell-wall motion and contraction velocity has been studied in electrically driven cardiac muscle cells. In muscle and non-muscle cells, two classes of ouabain binding sites have been identified. In rat heart muscle cells, the high affinity binding site has a dissociation constant (KD) of 3.2 X 10(-8) M and a binding capacity (B) of 0.2 pmole/mg protein (80,000 sites/cell); the values for the low affinity binding site are: KD = 7.1 X 10(-6) M; B = 2.6 pmole/mg protein (10(6) sites/cell). The binding to both types of binding sites is depressed by K+ and abolished after heat denaturation of the cells. The kinetics of [3H]ouabain binding to rat heart muscle cells (association and dissociation rate constants, K+- and temperature-dependence of association and dissociation processes) have been characterized. In rat heart muscle and non-muscle cells, the binding of [3H]ouabain to the low affinity site results in inhibition of the (86Rb+ + K+)-influx (EC50 = 1.3 and 1.5 X 10(-5) M ouabain), a decrease in cell-K+ (EC50 = 1.9 and 1.4 X 10(-5) M) and an increase in cell-Na+ (10(-5)-10(-4) M). The ouabain-induced positive inotropic effect (increase in amplitude of cell-wall motion, increase in contraction velocity) in cardiac muscle cells is observed only at ouabain concentrations greater than or equal to 5 X 10(-6) M, and it is therefore probably attributed to occupation of the low affinity binding site. Coupling of occupation of the low affinity site by ouabain with drug-induced inhibition of the sodium pump and with drug-induced positive inotropic action is further substantiated by kinetic measurements. In contrast, occupation of the high affinity binding site does not produce any measurable inhibition of the sodium pump activity or positive inotropy.(ABSTRACT TRUNCATED AT 400 WORDS)