Kinetics of the effect of amiloride on the permeability of the apical membrane of rabbit descending colon to sodium

The effects of the addition of graded concentrations of amiloride, (A)_m, to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (P_Na^m) were determined when the Na activity in the mucosal bathing solution, (Na)_m, was 18, 32 or 100 mm. P_Na^m was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz. S.G. 1983. J. Membrane Biol.76:299-309). The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983), P_Na^m decreases with increasing (Na)_m. (2)P_Na^m also decreases hyperbolically with increasing (A)_m. Kinetic analyses of the effect of amiloride on P_Na^m are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease in P_Na^m is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A)_m needed to halve P_Na^m at a fixed (Na)_m is 0.6-1.0 μm; and, (iv) this value is independent of (Na)_m over the fivefold range studied. These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane are kinetically distinct from the sites with which (Na)_m interacts to bring about closure (i.e., "self-inhibition"). In short, the effects of (Na)_m and (A)_m on P_Na^m in this tissue appear to be independent and additive.