NA+ K+ PUMP INHIBITION AND POSITIVE INOTROPIC EFFECT OF DIGITOXIGENINAND 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 o f the experiments described below was to identify C-22-substituted der ivatives of digitoxigenin suitable for this purpose. The positive inot ropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy -digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin w as studied in sheep cardiac Purkinje fibres. In addition, the inhibiti on of the Na+/K+ pump by these drugs was investigated by means of simu ltaneous measurements of membrane current and intracellular Na+ concen tration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were perfo rmed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reve rsible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to similar to 5 x 10(-7) M d igitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. Th e EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigen in were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectivel y. From measurements on voltage-clamped Purkinje fibres the concentrat ions required for half maximal Na+/K+ pump inhibition (K-D' value) wer e calculated to be similar to 10(6) M for digitoxigenin, 22-acetoxy-di gitoxigenin or 22-hydroxy-digitoxigenin. The K-D' value for 22-benzoyl oxy-digitoxigenin was 10 times larger. The K-D' value for 22-allyl-dig itoxigenin was even larger and amounted to similar to 4 x 10(-5) M. Th e 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 r ate constant of 22-benzoyloxy-digitoxigenin (similar to 9 x 10(2) s(-1 ) M-1) was smaller than the association rate constant of digitoxigenin (similar to 2 x 10(4) s(-1) M-1), whereas the apparent dissociation r ate constants of both compounds were similar (similar to 4 x 10(-3) s( -1)). Compared ta digitoxigenin 22-allyl-digitoxigenin displayed a low er association rate constant (similar to 3 x 10(3) s(-1) M-1) and a la rger dissociation rate constant (similar to 8 x 10(-2) M-1). The struc ture-activity relationships of the drugs are discussed. We conclude th at esters derived from 22-hydroxy-digitoxigenin might be suitable to l ocalize the binding site(s) of the lactone moiety on the Na+/K+ pump b y affinity labeling.