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

Abstract

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° C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC₅₀ value) amounted to ∼ 5 × 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 × 10^–6 M and 1.1 × 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 ∼ 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 ∼ 4 × 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 (∼ 9 × 10² s^–1 M^–1) was smaller than the association rate constant of digitoxigenin (∼ 2 × 10⁴ s^–1 M^–1), whereas the apparent dissociation rate constants of both compounds were similar (∼ 4 × 10^–3 s^–1). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (∼ 3 × 10³ s^–1 M^–1) and a larger dissociation rate constant (∼ 8 × 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.