Kinetic Analysis of the Positive Inotropic Action (PIA) of Ouabain in Isolated Perfused Rabbit Heart. Slow Onset of PIA and Slow Binding to Na^+, K^+-Adenosine Triphosphatase

The positive inotropic action (PIA) of ouabain was analyzed kinetically using isolated perfused rabbit heart. The input function of the ouabain concentration in the perfusate (C_i) into the heart was controlled by changing the volume of the reservoir and the rate of ouabain infusion into the reservoir fixed in front of the heart. The time courses of PIA were measured continuously with different infusion rates. The relationship between C_i and PIA clearly depended on the infusion rate in isolated perfused rabbit heart. The binding kinetics of ouabain to Na^+, K^+-adenosine triphosphatase (ATPase) in the cardiac homogenate showed two kinds of binding sites. The association rate constant (k_1), the dissociation rate constant (K_1) and the binding capacity of each site was estimated by the simultaneous fitting method. The occupation curve of the high affinity site corresponded well with the PIA measured in the isolated perfused heart at steady state. These results indicate that ouabain binding to the high affinity site is related to the PIA, and the slow binding process of ouabain to Na^+, K^+-ATPase may be one of the principal reasons for the infusion-rate dependence of ouabain PIA.