Effects of ouabain at low external concentrations on 86rubidium influx and specific 3H-ouabain binding in human lymphocytes in vitro

Abstract

Changes in the sodium/potassium-adenosine triphosphatase (Na+/K+ pump) of human leukocytes may reflect changes in the Na+/K+ pump of vascular smooth muscle cells in cryptogenic ('essential') hypertension. Lymphocytes from healthy donors have significantly fewer Na+/K+ pumps than normal when incubated for 72 h with serum from patients with crytogenic hypertension. This effect might be due to Na+/K+ pump inhibition by a cardiac glycoside-like substance in the serum. We assessed the sensitivity of two techniques for measuring inhibition of cellular (Na+/K+ pumps by ouabain at low concentrations in vitro. We incubated lymphocytes from healthy subjects for 1 h and 72 h with unlabelled ouabain at different concentrations to determine the lowest concentration which inhibits specific 3H-ouabain binding (the number of Na+/K+ pumps) and ouabain sensitive 86rubidium influx (pump activity). Incubation of cells with ouabain for 1 h did not significantly affect Na+/K+ pump activity. The lowest ouabain concentrations which significantly reduced Na+/K+ pump numbers and activity during 72 h incubation were 5 x 10-9 and 1 x 10-8 mol L-1, which approximate the sensitivity of radioimmunoassay for plasma ouabain. We conclude that specific 3H-ouabain binding and ouabain sensitive 86rubidium influx are similarly sensitive at detecting lymphocyte Na+/K+ pump inhibition by ouabain. Both these measurements are reliable only if made when the binding of glycoside-like substances at low concentration is at equilibrium.