Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations.

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na+/K+-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na+/K+ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na+ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to approximately 5 x 10(-7) M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na+/K+ pump inhibition (K'D value) were calculated to be approximately 10(-6) M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K'D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K'D value for 22-allyl-digitoxigenin was even larger and amounted to approximately 4 x 10(-5) M. The K'D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (approximately 9 x 10(2) s(-1) M[-1]) was smaller than the association rate constant of digitoxigenin (approximately 2 x 10(4) s(-1) M[-1]), whereas the apparent dissociation rate constants of both compounds were similar (approximately 4 x 10(-3) s[-1]). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (approximately 3 x 10(3) s(-1) M[-1]) and a larger dissociation rate constant (approximately 8 x 10(-2) M[-1]). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na+/K+ pump by affinity labeling.