Inhibition of Na/K ATPase from rat aorta by two endogenous Na-Pump inhibitors, ouabain and marinobufagenin

Recently, we have shown that mammalian plasma cross-reacts with an antibody to a bufodienolide Na/K ATPase inhibitor, marinobufagenin (Mbg). In rat aortae, Mbg induced vasoconstriction via inhibition of the vascular smooth muscle Na/K pump, whereas ouabain had its predominant effect on pumps localized to nerve endings (Bagrov et al, Eur J Pharm, 1995, 274, 151-158). Na,K ATPase inhibitory effects of ouabain and Mbg were studied in two membrane fractions isolated from rat (Fisher 344XNB) thoracic aorta by sucrose density gradient centrifugation. One fraction contained predominantly the α-3 isoform of Na/K ATPase and represented membranes from the perivascular nerve endings (in vitro treatment of the tissue with 6ʹOHDA resulted in disappearance of α-3 isoform). The other membrane fraction, containing predominantly the α-1 isoform, was derived from the plasmalemmal membrane. The IC50 for inhibition of the Na/K ATPase by ouabain and Mbg were 1 nmol/L and 0.5 μmol/L in the neuronal membrane fraction, and 0.1 μmol/L and 10 nmol/L in plasmalemmal fraction, respectively. The concentration of Mbg-like immunoreactive material in C-18 extracted rat plasma by a fluorescent immunoassay using a rabbit polyclonal antibody to Mbg, was 0.7±0.58 nmol/L. Thus, Mbg preferentially inhibits Na/K ATPase in vascular smooth muscle membranes, and ouabain demonstrates higher affinity towards Na/K ATPase from vascular nerve endings. Rat plasma contains Mbg-like immunoreactive material in the concentrations which may provide functionally significant inhibition of vascular Na,K ATPase. These results confirm the view that differential responsiveness to the inhibition by endogenous digitalis-like factors is a functional feature of alpha-isoforms of Na/K ATPase.