Inhibition of the sodium pump in squid axons by cardiac glycosides: dependence on extracellular ions and metabolism

Abstract

1. The rate of inhibition of the Na pump by ouabain was examined both by direct measurement of the rate of decline of the Na efflux and by the binding of [(3)H]ouabain.2. The onset of inhibition of the Na efflux was concentration-dependent; but did not follow simple first order kinetics. The time course of inhibition was roughly exponential although in about 30% of the axons inhibition was preceded by a transient stimulation of the Na efflux.3. Inhibition of the Na efflux by both ouabain and strophanthidin was apparently irreversible.4. The onset of inhibition was slowed markedly at low temperatures.5. Replacement of external Na by choline, dextrose or potassium slowed the rate of inhibition. Li behaved like Na and inhibition was faster in K-ASW than in choline-ASW.6. The rate of inhibition of Na-Na exchange was similar to that of Na-K exchange, but ouabain failed to bind securely to fully poisoned axons.7. Two components of [(3)H]ouabain-binding could be distinguished. A linear component which probably reflects uptake into the cells and a saturable component which seems to reflect binding to Na-pumping sites.8. The saturable component of binding followed a similar time course to the inhibition of the Na efflux and the rate of binding was reduced in choline-ASW and in fully poisoned axons.9. Measurements of [(3)H]ouabain-binding indicate that the number of Na pumping sites in the axon membrane is probably between 10(3) and 10(4)/mu(2).