Estimation of the density of sodium entry sites in frog skin epithelium from the uptake of [3H]benzamil.

Abstract

1. The inhibition of short circuit current in frog skin by benzamil (N‐benzyl‐amidino‐3,5‐diamino‐6‐chloropyrazine carboxamide) was investigated. When skins were bathed on both sides by Ringer solution (pH 7.6) the affinity was 5 x 10(7) M‐1. When the sodium concentration was reduced to 1.1 mM and the pH adjusted to 6.5 the affinity increased to 8.5 x 10(8) M‐1. 2. A method is described for measuring uptake of [3H]benzamil into the mucosal (outer) surface of pieces of isolated epithelium, 0.95 cm2 in area, under open circuit conditions. 3. The relation of [3H]benzamil uptake at the mucosal surface to its concentration was measured in solutions containing 1.1 mM‐sodium and adjusted to pH 6.5. Uptake could be resolved into a linear component (10.2 f‐mole nM‐1) and a saturable component (21.5 f‐mole cm‐2) with a half saturating concentration of 1 nM. 4. In the presence of amiloride (1 microM) or unlabelled benzamil (1 microM) uptake was linear with concentration, and was, respectively, 9.2 f‐mole nM‐1 and 8.8 f‐mole nM‐1. When the pH was reduced to 3.5 uptake was again linear but reduced to 3.3 f‐mole nM‐1. 5. The identity of the saturable component of [3H]benzamil uptake to sodium entry sites is discussed. The results suggest a sodium entry site density of around 130 micron‐2 of mucosal surface.