Novel receptors for ouabain: Studies in adrenocortical cells and membranes

Abstract

Sodium-potassium pumps (Na pumps) are the only known plasma membrane receptors for cardiac glycosides (CG). Adrenocortical cells secrete CGs including an endogenous ouabain (EO) via an unknown mechanism that is subject to feedback inhibition via the cell surface. In addition, recent studies show that the ability of ouabain analogs to induce sustained hypertension in rats may be independent of their ability to inhibit the Na pumps. We used bovine adrenocortical cells and membranes to search for novel ouabain receptors (NORs) that may mediate these effects. In high extracellular K+ solutions, binding of ouabain to Na pumps was suppressed, yet residual specific binding o f 3H-ouabain was observed. Under these conditions, Scatchard analyses revealed two classes o f binding sites with high (1 pM, 4.5 x 106 sites/cell, Na pump) apparent affinities. Dissociation kinetics of 3H-ouabain from intact cells revealed distinct components with half times of 6.5 min, 3.3 hr and 33 hr corresponding to NOR, Na pumps, and 3H-ouabain recycling via lysosomes, respectively. Studies with isolated membranes showed time and concentration-dependent specific binding under conditions where participation of Na pumps was minimized. NOR showed high affinity (Kd ~ 15 nM) for ouabain and an apparent Bmax of 0.23 pmoles/mg (c.f., Na pumps = 10.2 pmoles/mg). KC1 had a biphasic effect on binding of ouabain to NOR while NaCl and related ions stimulated ouabain binding. Binding of ouabain to NOR was inhibited by sub-micromolar concentrations of CGs but not affected by 30 pM aldosterone or cortisol. Photoaffinity studies followed by SDS-PAGE showed ouabainprotectable labeling o f polypepties unrelated to Na pumps. NOR was seen in bovine kidney, brain and heart membranes but not in skeletal muscle or liver membranes. NOR was more labile in storage and more sensitive to SDS than Na pumps. These results provide the first demonstration o f specific, high affinity, and tissue-specific binding sites for ouabain on the cell surface that are distinct from Na pumps. These new receptors may mediate the transmembrane transport and/or feedback regulation of EO secretion and underlie the ability o f ouabain to induce hypertension.