Characterization of a Sodium-Dependent Concentrative Nucleobase-Transport System in Guinea-Pig Kidney Cortex Brush-Border Membrane-Vesicles

The characteristics of hypoxanthine transport were examined in purified brush-border membrane Vesicles isolated from guineapig kidney. Hypoxanthine uptake in the vesicles was specifically stimulated by both Na+ and an inside-negative potential, resulting in a transient accumulation of intravesicular hypoxanthine. Nat-dependent hypoxanthine influx was saturable (apparent K-m 4.4 +/- 2.1 mu M, V-max. 128 +/- 29 pmol/min per mg of protein at 100 mM NaCl and 22 degrees C). Guanine, thymine, 5-fluorouracil and uracil inhibited hypoxanthine uptake (k(i) values 1-30 mu M), but adenine and the nucleosides inosine and thymidine were without effect. Guanine competitively inhibited Na+-dependent hypoxanthine influx, suggesting that it was a substrate for the active nucleobase transporter in guinea-pig renal membrane vesicles. A sigmoidal dependence between hypoxanthine influx and Na+ concentration was obtained (K-Na 13 +/- 2 mM; Hill coefficient, h, 2.13 +/- 0.14), suggesting that at least two Na+ ions are transported per hypoxanthine molecule. This system differs from the Na+-nucleobase carrier in cultured LLC-PK1 renal cells, which has a stoichiometric coupling ratio of 1:1. These results represent the first demonstration of an active electrogenic nucleobase carrier in renal apical membrane vesicles.