[en] [³H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system. This paper extends the analysis of the properties of interaction of [³H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [³H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [³H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [³H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers

[en] The interaction of amiloride and amiloride derivatives with the Na+ channels of pig kidney membranes was studied from 22Na+ uptake experiments. The order of potency of the different molecules tested is: phenamil greater than benzamil greater than amiloride, ethylisopropylamiloride. [3H]labelled phenamil was prepared and used to titrate Na+ channels in pig kidney membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]phenamil and unlabelled phenamil indicate that phenamil recognizes a single family of binding sites with a Kd value of 20 nM and a maximum binding capacity of 11.5 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]phenamil competition experiments is identical to that found for the inhibition of 22Na+ uptake by apical Na+ channels

[en] Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the [³H]phenamil binding sites associated with the epithelial Na⁺ channel from pig kidney is 90 +/- 10 kDa. The [³H]phenamil receptor was solubilized by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na⁺ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na⁺ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO₄/polyacrylamide gel electrophoresis of the purified Na⁺ channel under nonreducing conditions showed the presence of a single major polypeptide chains of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na⁺ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na⁺ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges