[3H]ethylpropylamiloride, a radio-labelled diuretic for the analysis of the Na+/H+ exchange system. Its use with kidney cell membranes.

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Abstract
The interaction of amiloride and several amiloride derivatives with the Na+/H+ exchange system in Madin‐Darby canine kidney cells and in rabbit renal microvillus membrane vesicles was studied from 22Na+ uptake experiments. On both types of preparation, the order of potency of the different molecules tested is: ethylisopropylamiloride greater than ethylpropylamiloride (EPA) greater than amiloride greater than benzamil. 3H‐labelled EPA was prepared and used to titrate amiloride binding sites in solubilized microvillus membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]EPA and unlabelled EPA indicate that EPA recognizes a single family of binding sites with a Kd value of 45 nM and a maximum binding capacity of 2 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]EPA competition experiments is identical to that found for the inhibition of 22Na+ uptake by the Na+/H+ exchange system, suggesting that [3H]EPA binding sites are associated with the Na+/H+ exchange system. [3H]EPA binding sites are pharmacologically distinct from those of [3H]benzamil and [3H]bumetanide in kidney membranes.