19F NMR study of the uptake of 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil in erythrocytes: evidence of transport by facilitated and nonfacilitated pathways.
The 19F NMR resonances of intra- and extracellular 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) in suspensions of human erythrocytes are well resolved. This phenomenon allows its transport behavior to be monitored in a 19F NMR time-course experiment. The rate of L-FMAU uptake at 25 degrees in a suspension containing L-FMAU at an initial 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 transport mechanisms, with a Km for the saturable uptake of approximately 1 mM. Although the transport of L-FMAU at 25 degrees was inhibited significantly (54-65%) by nitrobenzylthioinosine (NBTI) and dipyridamole, consistent with the participation of the nucleoside transporter, these inhibitors did not achieve complete blockage of L-FMAU uptake. The participation of the nucleobase transporter in L-FMAU uptake was ruled out 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 sulfhydryl reagent, p-chloromercuriphenylsulfonic acid (pCMBS). However, the NBTI- and dipyridamole-insensitive transport of L-FMAU was found to increase upon treatment of the erythrocytes with butanol, an agent that affects membrane fluidity. The partition coefficient of L-FMAU in octanol/phosphate-buffered saline determined by absorption spectrophotometry was 0.31. These data indicate that under the conditions of the studies, L-FMAU uptake by erythrocytes proceeds by both the nucleoside transporter and nonfacilitated membrane diffusion.
Xu
A S
AS
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709-2233, USA.
Chu
C K
CK
London
R E
RE
D000998:Antiviral Agents / Q000037:antagonists & inhibitors / Q000097:blood / Q000493:pharmacokinetics*; D001086:Arabinofuranosyluracil / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000097:blood / Q000493:pharmacokinetics; D001692:Biological Transport; D004176:Dipyridamole / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D005461:Fluorine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy
D000998:Antiviral Agents; D005461:Fluorine; D001086:Arabinofuranosyluracil; D004176:Dipyridamole; C034935:clevudine