Quantitation of Na/K ATPase pump sites in the rabbit corneal endothelium

In these experiments, the binding of 3H . ouabain, a specific inhibitor of Na/K ATPase, was used to quantitate the density of Na/K ATPase pump sites in the rabbit corneal endothelium. The uptake of ouabain by the corneal endothelium shows two components: one that saturates at a ouabain concentration near 2 X 10(-7) M (specific binding), and one component that increases linearly with increasing glycoside concentration (nonspecific uptake). The nonspecific uptake can be accounted for by that ouabain equilibrating with the extracellular space, which, estimated by inulin space, amounts to 13.0 nl/mm2 of endothelium. The saturable component of endothelial ouabain uptake is displaced by K+ ions, which is consistent with this fraction being bound to Na/K ATPase. Maximal endothelial ouabain binding was measured as 20.7 fmoles/mm2 of endothelium, which corresponds to 3.0 X 10(6) pump sites per cell. The density of Na/K ATPase pump sites in the rabbit corneal endothelium is comparable to densities reported for several transporting epithelia. These data are consistent with the known function of the endothelium in corneal deturgescense and corroborate the importance of Na/K ATPase in endothelial fluid transport.