Digitalis receptors in normal and hypertrophied rat hearts. Differential effects of a Ca²⁺ free perfusion

Summary

Recently, it has been demonstrated that more than one receptor type would be responsible for the isotropic effect of ouabain. This effect is linked to an increased availability of Ca_in. In rat heart two classes of ouabain binding sites have been described: a high affinity (H. A.) class with a low capacitiy, and a low affinity (L. A.) class with a high capacity. Although both sites are involved in the inotropic response, these H. A. sites may not be related to inhibition of Na/K-ATPase activity. However, we have reported that a Ca-free perfusion of rat heart revealed a Na/K-ATPase activity highly sensitive to ouabain by modifying the sarcolemmal calcium pool.

The purpose of this study is to further characterize the mechanism(s) by which Ca affects the digitalis receptors. This pharmacodynamical study has been performed on sarcolemmal vesicles isolated from normal and hypertrophied rat hearts. The pressure-overload induced hypertrophy represented a suitable model in that the movements of Ca_in appeared to be slowed.

When the heart was maintained at a physiological calcium level (2.5 mM), hypertrophied and normal cardiac preparations had heterogeneous ouabain binding sites: H. A. (K_D 3 × 10⁻⁸ M) associated with 2–4 pmol/mg protein and L. A. sites (K_D 4–18 × 10⁻⁶ M) associated with 115 ± 25 pmol/mg protein. Although both cardiac preparations had the same total number of sites, in the hypertrophied group the number of H. A. sites is two fold higher (p < 0.02) than in normal group and the dissociation and association rate constants were five times slower. This strongly suggested the apparition of new forms of Na/K-ATPase in hypertrophied rat heart.

As revealed by Ca-free perfusion, the normal heart is greatly affected by the calcium present at the sarcolemmal level: the number of H. A. sites (from 2 to 4 pmol/mg) is doubled, the number of L. A. sites is decreased from 140 to 90 pmol/mg (p < 0.01). As a consequence the relative proportion of H. A. sites reached 22% of the total number of sites. Moreover the pharmacodynamic parameters of the L. A. affinity sites are modified: the apparent affinity was increased by a 4 fold factor. These latter observations were consistent with the fact that the inotropic effect of ouabain is increased at low external Ca (0.25–0.5 mM, compared to 2 mM).

Strikingly, in hypertrophied hearts, a Ca-free perfusion had no apparent effect on the H. A. and L. A. ouabain binding site characteristics (dissociation constant, number of sites, rate constants). This independancy toward Ca is a strong argument in favor of adaptational changes of Na/K-ATPase in hypertrophied rat heart, in relation to the depressed and prolonged effect of the drug in vivo.