Localization of sodium pump sites in frog urinary bladder

[³H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and authoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na⁺ transport was completely reversible in toad bladder whereas, in frog bladder, [³H] ouabain was tightly bound and Na⁺ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [³H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of cecrtain (Na⁺?K⁺)-ATPase ligands (K⁺, ATP) to levels known to reduce ouabain binding. High K⁺ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [³H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na⁺ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significatly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.