[H-3]OUABAIN BINDING AND DISSOCIATION IN RABBIT COLON - EFFECT OF IONS AND DRUGS

22Na fluxes (J) and [3H]ouabain binding were studied in vitro in the distal portion of the rabbit colon, mounted as a membrane, separating two perspex chambers. JMSNa (flux of 22Na from mucosal to serosal chamber) was 2.1 and JSMNa was 1.0 μmoles cm2 × hr· JSMNa was completely abolished by 10-3 M ouabain, placed in the serosal side. JSMNa was unaffected by ouabain. [3H] Ouabain binding to the serosal surface of the colon was inhibited to the same maximal effect by 'cold' ouabain (I50 = 5 × 10-7 M), by K+ placed in the serosal chamber (I50 = 3.5 mM) and by replacement of Na+ with choline on the serosal side. Increased tissue concentration of Na+ did not affect [3H]ouabain binding, suggesting that extracellular rather than intracellular sodium plays a major role in cardiac glycoside binding. Various cardiac glycosides (digoxin, digitoxin. ouabain) inhibited [3H]ouabain binding to the serosal side of the colon but other steroids (estriol, testosterone, desoxycorticosterone) had not effect. Efflux of [3H]ouabain, bound to the serosal side of the colon, was differentiated into dissociation of nonspecifically bound [3H]ouabain (95 per cent in 60 min) and dissociation of specifically bound [3H]ouabain (20 per cent in 60 min). Dissociation of specifically bound [3H]ouabain was accelerated when Na was replaced by choline (50 per cent in 60 min). The dissociation rate of nonspecifically bound ouabain was unaffected by replacement of Na+· [3H]Ouabain binding to mucosal surface was unaffected by 'cold' ouabain, by increased K+ in the medium or by replacement of Na+. It is concluded that there is no specific binding of [3H]ouabain to the mucosal surface. Omission of Ca, Mg or phosphate from the medium did not affect [3H]Ouabain binding to either mucosal or serosal surfaces of rabbit colon. © 1979.