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(Hypertension. 2002;39:536.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Novel Receptors for Ouabain: Studies in Adrenocortical Cells and Membranes

Stephen C. Ward; Bruce P. Hamilton; John M. Hamlyn

From the Departments of Physiology and Medicine, School of Medicine, University of Maryland, and The Veterans Administration Medical Center, Baltimore, Md.

Correspondence to John M. Hamlyn, PhD, Department of Physiology, School of Medicine, University of Maryland, 655 West Baltimore St, Baltimore, MD 21201. E-mail jhamlyn{at}umaryland.edu

Sodium-potassium pumps (Na pumps) are the only known plasma membrane receptors for cardiac glycosides. However, adrenocortical cells secrete an endogenous ouabain via an unknown mechanism that is subject to feedback inhibition via the cell surface. In addition, recent studies suggest that the induction of sustained hypertension by ouabain analogs in rats may be independent of Na pump inhibition. Accordingly, we used bovine adrenocortical cells and membranes to search for novel binding sites for ouabain. In high extracellular potassium solutions, the binding of ouabain to the Na pumps of cultured cells was suppressed, yet residual specific binding of ³H-ouabain was observed. In high extracellular potassium, Scatchard analyses revealed a novel class of ouabain binding sites with high affinity (<50 nmol/L, <2.5x10⁵ sites/cell) that was distinct from the low-affinity Na pump sites (>1 µmol/L, 4.5x10⁶ sites/cell). Analysis of the kinetics for the dissociation of ³H-ouabain from intact cells revealed components whose t_0.5 values were 6.5 minutes, 3.3 hours, and 33 hours and associated with novel sites, Na pumps, and lysosomal recycling, respectively. Studies with isolated membranes under ligand conditions where the participation of Na pumps was minimized revealed specific ouabain binding to novel sites that was saturable, time-dependent, of high affinity (K_d15 nmol/L), and of low density (apparent B_max=0.23 pmol/mg, c.f., Na pumps=10.2 pmol/mg). Ouabain binding to the novel sites was stimulated by high concentrations of KCl but was not affected by aldosterone or cortisol up to 30 µmol/L. Novel sites were not detected in skeletal muscle or liver membranes. Photoaffinity studies followed by SDS-PAGE showed ouabain-protectable labeling of membrane polypeptides with apparent molecular weights of 143, 113, and 65 kDa. We conclude that adrenocortical cells express ouabain receptors that are distinct from Na pumps. These novel receptors may be involved in the regulation and/or secretion of endogenous ouabain.

Key Words: digitalis • ouabain • adrenal gland • sodium pump • glycosides

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