Nucleoside transport in primary cultured rabbit tracheal epithelial cells

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of H-3-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na+, substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical H-3-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na+ gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. H-3-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 mu M) and apical phloridzin (100 mu M), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K-m of 3.4 +/- 1.8 mu M and the V-max of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical H-3-uridine, H-3-inosine and H-3-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na+-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.