Fig. 6.

Time-dependent efflux of total [³H]N5-2OH metabolites (A) and [³H]N5-2OH monophosphate to the growth media (B) of CEM TK1⁺ cells. (C) Effects of transport inhibitors on the efflux of N5-2OH-MP from CEM TK1⁺ cells. CEM TK1⁺ cells were incubated 1 hour with 1 μM [³H]N5-2OH, washed with PBS, and then incubated in growth media without [³H]N5-2OH for 150 minutes. The total intracellular counts per minute (A, ■) and total counts per minute in free media (A, ●) were determined by scintillation counting at different time points after removal of the labeled nucleoside. N5-2OH (B, ●) and N5-2OH-MP (B, ■) in free media were analyzed by HPLC as described in Materials and Methods. Results shown are the average of three experiments. For the efflux inhibition experiments, cells were incubated with 1 μM labeled nucleoside solution [³H]N5-2OH for 2 hours at 37°C in growth media in the presence of 100 μM dipyridamole, indomethacin, or verapamil. [³H]N5-2OH-MP in media from cells was determined by HPLC as described in Materials and Methods. Values show the % of counts per minute of [³H]N5-2OH-MP in media with inhibitors to 100% of the control experiment without inhibitors in media.