Kinetics of amiloride action in the hen coprodaeum in vitro

The kinetics of amiloride action on the isolated epithelium of the hen coprodaeum are reported. Tissues were taken from birds fed on low salt diets for 9-10 days, conditions which induce a high resting short circuit current due to sodium and sensitive to amiloride. The relation between the inhibition of amiloride sensitive short circuit current and blocker concentration obeyed simple mass laws with an apparent stoichiometry of 1:1 between amiloride and the sodium entry sites. The concentration of amiloride producing its half maximal effect (Ki) was 1.77 +/- 0.20 microM at a sodium concentration of 130 mM. There was a shallow dependence of Ki on sodium concentration, the value of Ki falling to 0.78 +/- 0.1 microM at 1.3 mM Na. The relation of Ki to Na concentration was linear indicating competitive antagonism. The sodium concentration which half saturates the amiloride site (KNa) was 80 mM. This value is very different from the concentration of sodium which half saturates SCC (Kscc = 5-7 mM) suggesting there are at least two sites at which sodium can modify the transporting characteristics. These data are compared to those for other epithelia where Kscc and KNa are rather similar. The benzyl derivative of amiloride (benzamil) was found to be 11.6 times more potent than amiloride on this tissue. The potency ration is similar to that for other sodium transporting epithelia suggesting that the structure of the ion translocation mechanism is partly conserved between species although the Ki for amiloride may vary by an order of magnitude.