OUABAIN RECEPTOR IN ISOLATED ADULT DOG HEART MYOCYTES

Ouabain binding was studied in isolated adult dog heart myocytes. The binding was correlated with the inhibition of K+-activated p-nitrophenylphosphatase (K+-PNPPase) activity and the beating response. The specific binding was dependent upon Mg2+ and was inhibited by K+. The maximal binding capacity (Bmax) was 7.4 .times. 105 ouabain molecules per cell, or 410 pmol ouabain/K+-PNPPase unit (.mu.mol/min). In the presence of Mg2+ (5 mM), there were 2 components in the Scatchard plot, i.e., a high-affinity component with a Kd value of 5.6 .times. 10-8 M and a low-affinity component with a Kd value of 6.7 .times. 10-7 M. The Hill coefficient (n'') for ouabain binding was 0.72 with a S0.5 value of 7.1 .times. 10-7 M. These values were compatible with the values obtained from studies of K+-PNPPase inhibition by ouabain (n = 0.55, S0.5 = 3.6 .times. 10-7 M) and remained unchanged in the presence of physiological concentrations of Na+ plus K+. In the presence of Mg2+ and K+, the high-affinity component tended to conform to the low-affinity component with an apparent decrease in Bmax. In the presence of Mg2+ and p-nitrophenylphosphate, the low-affinity component was changed to the high-affinity component with no change in Bmax. The dissociation rate of the labeled ouabain in the highly dilute medium was not altered in the presence of excess amounts of unlabeled ligand; this eliminated the possibility that the apparent negative cooperativity was due to a site-to-site interaction between receptors. Ouabain increased the number of beating cells and the frequency of beating. Isolated myocytes evidently possess functional receptors for ouabain; the binding of ouabain is associated with its inhibition of K+-PNPPase activity; ouabain receptors in isolated myocytes are of 1 class with at least 2 interconvertible conformational states.