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Abstract
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 3H-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 3H-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. 3H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 μM) and apical phloridzin (100 μM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a Km of 3.4 ± 1.8 μM and the Vmax 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 3H-uridine, 3H-inosine and 3H-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.
| Abbreviations | ||
| RTEC | = | rabbit tracheal epithelial cells |
| NBMPR | = | nitro-benzyl-mercapto-purine-ribose |
| ei | = | equilibrative NBMPR-insensitive |
| es | = | equilibrative NBMPR-sensitive |
| Papp | = | apparent permeability coefficient |
| PD | = | potential difference |
| TEER | = | transepithelial electrical resistance |
| Abbreviations | ||
| RTEC | = | rabbit tracheal epithelial cells |
| NBMPR | = | nitro-benzyl-mercapto-purine-ribose |
| ei | = | equilibrative NBMPR-insensitive |
| es | = | equilibrative NBMPR-sensitive |
| Papp | = | apparent permeability coefficient |
| PD | = | potential difference |
| TEER | = | transepithelial electrical resistance |
Acknowledgements
This work was supported in part by USP Fellowship (to N.R.M), American Heart Association Grant-in-Aid 9950442N (to K.-J.K.), and National Institutes of Health Grants GM52812 (to V.H.L.L.), HL38658 (to K.-J.K.) and HL64365 (to K.-J.K. and V.H.L.L.).