Na+-independent nucleoside transporters regulate adenosine and hypoxanthine levels in Müller cells and the inner blood-retinal barrier.
OBJECTIVE
To elucidate the mechanism(s) of hypoxanthine production in Müller cells and the elimination of hypoxanthine across the inner blood-retinal barrier (BRB).


METHODS
The hypoxanthine biosynthesis and adenosine transport in Müller cells were investigated using a conditionally immortalized rat Müller cell line, TR-MUL5 cells. The elimination of hypoxanthine across the inner BRB was assessed by an in vivo microdialysis method and an in vitro transport study using a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells.


RESULTS
[(3)H]Hypoxanthine was detected in TR-MUL5 cells and TR-MUL5 cell-cultured medium 3 hours after [(3)H]adenosine incubation, indicating that the hypoxanthine is produced in TR-MUL5 cells. [(3)H]Adenosine was taken up into TR-MUL5 cells, which express mRNAs of nucleoside transporters (ENT1-2 and CNT1-2), in an Na(+)-independent and concentration-dependent manner (Km = 20 μM). Moreover, 100 μM nitrobenzylmercaptopurine riboside (NBMPR) and azidothymidine, which are inhibitors of ENT2, inhibited [(3)H]adenosine uptake, suggesting that ENT2 is a major contributor to adenosine transport in Müller cells. [(3)H]Hypoxanthine was eliminated from the rat vitreous humor and this process was inhibited in the presence of NBMPR. [(3)H]Hypoxanthine uptake by TR-iBRB2 cells took place in an Na(+)-independent and concentration-dependent manner with Km values of 4.3 μM and 2.9 mM, and was inhibited by 100 μM NBMPR.


CONCLUSIONS
Our findings suggest that hypoxanthine is produced from adenosine in Müller cells and ENT2 plays a major role in adenosine uptake in Müller cells. Hypoxanthine in the retina is eliminated via Na(+)-independent equilibrative nucleoside transporters.
Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Shin-ichi
S
Akanuma
Tatsuya
T
Soutome
Eikichi
E
Hisada
Masanori
M
Tachikawa
Yoshiyuki
Y
Kubo
Ken-ichi
K
Hosoya
D000241:Adenosine / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001813:Blood-Retinal Barrier; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000378:metabolism; D033705:Equilibrative Nucleoside Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D019271:Hypoxanthine / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D017551:Microdialysis; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D033703:Nucleoside Transport Proteins / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D015215:Zidovudine / Q000494:pharmacology
D017931:DNA Primers; D033705:Equilibrative Nucleoside Transport Proteins; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D012333:RNA, Messenger; C444719:adenosine transporter; C065171:cif nucleoside transporter; D019271:Hypoxanthine; D013868:Thioinosine; D015215:Zidovudine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine