Saturation kinetics of sodium efflux across isolated frog skin

Measurement of Na efflux across the frog skin epithelium from the serosal side to the outside (JNa 3 leads to 1) in a new chamber specifically designed to avoid edge damage shows that JNa 3 leads to 1 exhibits saturation kinetics with a maximal efflux (Jmax) of 31.8 nmol/cm2 per h and an apparent KNa of 4.0 mM. In contrast, JNa 3 leads to 1 measured in conventional chambers and efflux determinations in the new chamber of substances that pass the epithelium via extracellular pathways (polyethylene glycol 900, sucrose, mannitol) exhibit a linear relationship between the efflux of the substance in question and its concentration in the bath. In addition, changes in external Na concentration do not cause substantial changes in JNa 3 leads to 1. The saturation remains but both Jmax and KNa increase after application of ouabain. Amiloride, as well as dinitrophenol, eliminates the saturation and JNa 3 leads to 1 becomes a linear function of Na concentration. The separate effects of ouabain and amiloride suggest that these two inhibitors which are known to affect two distinctly different steps in the active transport pathway act also on two separate steps of JNa 3 leads to 1: the passage across the inward- (serosal) and outward-facing (apical) cell membranes of the epithelial cells, respectively. The action of dinitrophenol indicates the involvement of metabolism in JNa 3 leads to 1 probably at the latter of the two steps. The results suggest strongly that JNa 3 leads to 1 proceeds not via a paracellular but via a transcellular pathway that interacts with the active transport pathway.