High affinity sodium-dependent nucleobase transport in cultured renal epithelial cells (LLC-PK1)

Abstract

Two distinct transporters for nucleobases have been characterized in LLC-PK1 cells. The first system accumulates hypoxanthine against a concentration gradient in the presence of sodium. The sodium-dependent uptake of hypoxanthine was saturable at 22 degrees C with a Km value of 0.79 +/- 0.43 microM, a Vmax of 15 +/- 4 pmol/mg protein/60 s, and a Na+:hypoxanthine coupling stoichiometry of 1.27 +/- 0.20. Uptake of hypoxanthine was inhibited by 5-fluorouracil, uracil, thymine, and guanine (Ki values 3-6 microM). Adenine and nucleosides were without effect. Using cell monolayers grown on a permeable filter support, Na(+)-dependent hypoxanthine uptake occurred preferentially from the apical surface. The second system exhibited no cation specificity and was saturable with a low affinity for hypoxanthine (Km of 124 +/- 22 microM) and a high Vmax of 275 +/- 38 pmol/mg protein/60 s. Adenine and guanine inhibited Na(+)-independent hypoxanthine uptake (Ki values 30 +/- 15 and 18 +/- 7 microM, respectively). Other nucleobases and nucleosides exhibited little or no inhibition of equilibrative hypoxanthine influx. Dipyridamole, dilazep, and phloridzin were effective inhibitors of Na(+)-dependent hypoxanthine uptake but had little effect on the Na(+)-independent flux. This study represents the first direct demonstration of a unique high affinity Na+ nucleobase co-transporter system in cultured animal cells.