[³H]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. J_MS^Na (flux of ²²Na from mucosal to serosal chamber) was 2.1 and J_SM^Na was 1.0 μmoles cm² × hr· J_SM^Na was completely abolished by 10^-3 M ouabain, placed in the serosal side. J_SM^Na 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.