F-19 NMR-STUDY OF THE UPTAKE OF 2'-FLUORO-5-METHYL-BETA-L-ARABINOFURANOSYLURACIL IN ERYTHROCYTES - EVIDENCE OF TRANSPORT BY FACILITATED ANDNONFACILITATED PATHWAYS

The F-19 NMR resonances of intra- and extracellular 2'-fluoro-5-methyl -beta-L-arabinofuranosyluracil (L-FMAU) in suspensions of human erythr ocytes are well resolved. This phenomenon allows its transport behavio r to be monitored in a F-19 NMR time-course experiment. The rate of L- FMAU uptake at 25 degrees in a suspension containing L-FMAU at an init ial extracellular concentration of 4 mM was 7.6 +/- 1.0 X 10(-7) pmol cell(-1) sec(-1) (N = 5). Concentration-dependent uptake studies of L- FMAU indicate the existence of both saturable and nonsaturable transpo rt mechanisms, with a K-m for the saturable uptake of approximately 1 mM. Although the transport of L-FMAU at 25 degrees was inhibited signi ficantly (54-65%) by nitrobenzylthioinosine (NBTI) and dipyridamole, c onsistent with the participation of the nucleoside transporter, these inhibitors did not achieve complete blockage of L-FMAU uptake. The par ticipation of the nucleobase transporter in L-FMAU uptake was ruled ou t by the absence of competition with uracil uptake, and by the lack of inhibition by papaverine. In addition, the NBTI-insensitive uptake of L-FMAU was not affected by pretreatment of the cells with the sulfhyd ryl reagent, p-chloromercuriphenylsulfonic acid (pCMBS). However, the NBTI- and dipyridamole-insensitive transport of L-FMAU was found to in crease upon treatment of the erythrocytes with butanol, an agent that affects membrane fluidity. The partition coefficient of L-FMAU in octa nol/phosphate-buffered saline determined by absorption spectrophotomet ry was 0.31. These data indicate that under the conditions of the stud ies, L-FMAU uptake by erythrocytes proceeds by both the nucleoside tra nsporter and nonfacilitated membrane diffusion. (C) 1998 Elsevier Scie nce Inc.