The Binding of 3H-Ouabain to Na+-K+ ATPase Sites in Arterial Smooth Muscle

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Abstract
Binding of ¾ouabain to Na+-K+ ATPase sites in strips of rabbit aorta was measured by kinetic analysis of washout (efflux) data. This approach allowed the separation of specific binding fron nonspecific binding including unbound extracellular glycoside. Lowering efflux temperature to 2 °C caused the rate of loss of 3H-ouabain from specific sites to be slowed (T½ = 293 min) while loss from other sites was not affected. Specificity of binding was established by: (1) a reduction of such binding in the presence of extracellular K+; (2) saturability with increasing concentration of ouabain, and (3) sensitivity to a reduction in ATP levels. Binding to Na+-K+ ATPase sites gradually reached an equilibrium during 60 min of labelling at 10–7M. At equilibrium, specific binding was 25% of the total tissue content. Half-maximal binding occurred at 5.3 X 10–8M ouabain, and binding capacity was 381 x 10–15 mol/mg dry weight under K+-free conditions. A Na+-K+ ATPase site density of approximately 74 sites/μm2 of surface membrane was calculated, based on a volume to surface area of 0.6 μm. The parameters of 3H-ouabain binding in arteries are similar to previously reported values for binding to Na+-K+ sites in intestinal smooth muscle.