K Hosoya1, Y Horibe, K J Kim, V H Lee. 1. Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, USA.
Abstract
PURPOSE: To elucidate the mechanisms of nucleoside transport in the pigmented rabbit conjunctiva using [3H]uridine as the substrate. METHODS: Excised pigmented rabbit conjunctiva was mounted in a modified Ussing chamber for measurement of short-circuit current (Isc) and [3H]uridine transport. RESULTS: [3H]Uridine transport in the mucosal-to-serosal direction at 10 microM exhibited directionality, temperature dependency, and phlorizin sensitivity. Uridine transport appeared to be mediated via saturable Na(+)-dependent and nitrobenzylthioinosine-insensitive Na(+)-independent processes. The corresponding Michaelis-Menten constants (K(m)) were 1.9 microM and 200 microM, and the maximal uridine fluxes (Jmax) were 29.3 and 46.7 pmol/cm2 per minute. When added to the mucosal side containing 141 mM Na+, uridine increased the Isc in a dose-dependent manner from 0.005 mM to 1.0 mM at 37 degrees C. The K(m) value was 7.6 microM, and the maximal increase in Isc was 0.71 microA/cm2. Hill analysis of uridine transport at 10 microM in the presence of varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 1.1, suggesting a 1:1 coupling between Na+ and uridine. Na(+)-dependent uridine transport was inhibited by 10 microM adenosine, guanosine, and inosine, but not by thymidine, suggesting that the transport process may be mainly selective for purine nucleosides. Moreover, 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, and 5-(2-bromovinyl)-2'-deoxyuridine were potent inhibitors of Na(+)-dependent uridine transport. CONCLUSIONS: Na(+)-dependent and Na(+)-independent nucleoside transport processes appeared to be localized on the mucosal aspect of the pigmented rabbit conjunctiva. One or more Na(+)-coupled uridine cotransport processes exhibited a 1:1 stoichiometry and an apparent preference toward purine nucleosides.
PURPOSE: To elucidate the mechanisms of nucleoside transport in the pigmented rabbit conjunctiva using [3H]uridine as the substrate. METHODS: Excised pigmented rabbit conjunctiva was mounted in a modified Ussing chamber for measurement of short-circuit current (Isc) and [3H]uridine transport. RESULTS:[3H]Uridine transport in the mucosal-to-serosal direction at 10 microM exhibited directionality, temperature dependency, and phlorizin sensitivity. Uridine transport appeared to be mediated via saturable Na(+)-dependent and nitrobenzylthioinosine-insensitive Na(+)-independent processes. The corresponding Michaelis-Menten constants (K(m)) were 1.9 microM and 200 microM, and the maximal uridine fluxes (Jmax) were 29.3 and 46.7 pmol/cm2 per minute. When added to the mucosal side containing 141 mM Na+, uridine increased the Isc in a dose-dependent manner from 0.005 mM to 1.0 mM at 37 degrees C. The K(m) value was 7.6 microM, and the maximal increase in Isc was 0.71 microA/cm2. Hill analysis of uridine transport at 10 microM in the presence of varying Na+ concentrations in the mucosal bathing fluid yielded a Hill coefficient of 1.1, suggesting a 1:1 coupling between Na+ and uridine. Na(+)-dependent uridine transport was inhibited by 10 microM adenosine, guanosine, and inosine, but not by thymidine, suggesting that the transport process may be mainly selective for purinenucleosides. Moreover, 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, and 5-(2-bromovinyl)-2'-deoxyuridine were potent inhibitors of Na(+)-dependent uridine transport. CONCLUSIONS: Na(+)-dependent and Na(+)-independent nucleoside transport processes appeared to be localized on the mucosal aspect of the pigmented rabbit conjunctiva. One or more Na(+)-coupled uridine cotransport processes exhibited a 1:1 stoichiometry and an apparent preference toward purinenucleosides.