Apparent cooperativity of [3H]ouabain binding to myocytes obtained from guinea-pig heart.

Kinetics of [3H]ouabain binding to intact cardiac cells were examined using myocytes obtained from guinea-pig heart. In intact cells, the use of excess unlabeled ouabain results in an under-estimation of nonspecific binding, presumably due to cytotoxic effects of the unlabeled glycoside; estimation of the specific binding, as that to rapidly releasing sites yields more accurate results. Specific [3H]ouabain binding to myocytes is promoted by an increase in Na+ influx, indicating that normal intracellular Na+ concentration is insufficient to fully stimulate glycoside binding. High concentrations of [3H]ouabain seem to increase the apparent affinity of binding sites for the glycoside via increases in intracellular Na+ concentration resulting from sodium-pump inhibition; hence the binding reaction may be regarded as having a novel type of cooperativity. This cooperativity has kinetics different from those of classical positive cooperativity based on binding-site interactions, and is apparent with toxic concentrations of the glycoside that cause marked increases in intracellular Na+ concentrations.