Amiloride transport in rabbit renal brush-border membrane vesicles

Rabbit renal brush-border membrane vesicles (BBMV) were used to study amiloride transport across the luminal membrane of proximal tubular cells. An outwardly directed H⁺ gradient (pH(i) 6.0; pH(o) 7.5) stimulated 8 μM [¹⁴C]-amiloride uptake into BBMV and supported a transient 'active' accumulation of substrate consistent with the presence of an amiloride-H⁺ exchange process. Uptake was inhibited, in the presence or absence of a pH gradient, by 1 mM unlabeled amiloride or 20 mM tetraethylammonium (TEA). Amiloride transport was not directly affected by the presence of 100 mM Na⁺ in the extravesicular medium, suggesting that Na-H exchange did not mediate amiloride flux. Amiloride transport was a saturable process with a maximal flux (under pH gradient conditions) of 3 nmol·mg^-1·min^-1 and an apparent K(t) of 8 μM. TEA acted as a competitive inhibitor of this process with an apparent K(i) of ~80 μM, similar to the K(t) of TEA transport via the TEA-H⁺ exchanger. Likewise, amiloride acted as a competitive inhibitor of TEA uptake with an apparent K(i) of ~11 μM. Preloading BBMV with 1-2 mM TEA stimulated the rate of amiloride uptake and supported a transient active accumulation of amiloride. We conclude that amiloride and TEA are transported by a common pathway in BBMV, which involves a carrier-mediated exchange with H⁺ and which may play a role in the tubular secretion of these compounds.