Ca²⁺-free perfusion of rat heart reveals a (Na⁺ + K⁺) ATPase form highly sensitive to ouabain

Abstract

The sensitivities of the cardiac (Na⁺ + K⁺)ATPases to inhibition by ouabain have been shown to correlate with the pharmacologically active digitalis concentrations in the various source species¹. The rat heart enzyme is generally considered to be insensitive to ouabain with half-maximal inhibition at ∼6 × 10⁻⁵ M ouabain^2,3 and a K_D for binding^3–7 of 1–6 × 10⁻⁵ M. However, recent data^5–7 showed that rat cardiac sarcolemma preparations can bind ouabain with high affinity (K_D 1–3 × 10⁻⁷M) with no detectable concomitant enzyme inhibition in vitro but producing a sustained positive inotropic effect in vivo. In the present study, we measured the (Na⁺ + K⁺)ATPase inhibition by ouabain in sarcolemma-enriched fractions obtained from rat hearts relaxed by perfusion with Ca²⁺-free solutions. In these conditions, two enzyme forms having high and low sensitivities to ouabain were found (half-maximal inhibitions with 1.2×10⁻⁸ and 6×10⁻⁵M, respectively). Their relative proportion is close to 1. Confirming previous data^2–7, the highly sensitive form was not detected in preparations from hearts maintained at a physiological calcium level. This heterogeneous population of functional (Na⁺ + K⁺)ATPases suggests that, in rat heart, the positive inotropic effect of low doses of ouabain in vivo^5–9 would act through the inhibition⁹ of the enzyme activity highly sensitive to ouabain as detected in vitro.