Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier.
The purpose of the present study was to characterize the adenosine transport system(s) at the inner blood-retinal barrier (inner BRB). A conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), used as an in vitro model of the inner BRB, expresses equilibrative nucleoside transporter 1 (ENT1), ENT2, concentrative nucleoside transporter 2 (CNT2), and CNT3 mRNAs. TR-iBRB2 cells exhibited an Na+-independent and concentration-dependent [3H]adenosine uptake with a Michaelis-Menten constant of 28.5 microM and a maximum uptake rate of 814 pmol/(min mg protein). [3H]Adenosine uptake by TR-iBRB2 cells was strongly inhibited by 2 mM adenosine, inosine, uridine, and thymidine. On the other hand, this process was not inhibited by 100 nM nitrobenzylmercaptopurine riboside and dipyridamole. These uptake studies suggest that ENT2 is involved in [3H]adenosine uptake by TR-iBRB2 cells. Quantitative real-time PCR revealed that the expression of ENT2 mRNA is 5.5-fold greater than that of ENT1 mRNA. An in vivo study suggested that [3H]adenosine is transported from the blood to the retina and significantly inhibited by adenosine and thymidine. The results of this study show that ENT2 most likely mediates adenosine transport at the inner BRB and is expected to play an important role in regulating the adenosine concentration in the retina.
Nagase
Katsuhiko
K
Faculty of Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama 930-0194, Japan.
Tomi
Masatoshi
M
Tachikawa
Masanori
M
Hosoya
Ken-Ichi
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism* / Q000493:pharmacokinetics; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D001813:Blood-Retinal Barrier / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D042783:Endothelial Cells / Q000502:physiology; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2 / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D066298:In Vitro Techniques; D027283:Large Neutral Amino Acid-Transporter 1 / Q000378:metabolism; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism; D033703:Nucleoside Transport Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015415:Biomarkers; D002352:Carrier Proteins; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D051272:Glucose Transporter Type 1; D027283:Large Neutral Amino Acid-Transporter 1; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; C438652:Slc29a1 protein, rat; C438655:Slc29a2 protein, rat; C080679:creatine transporter; D000241:Adenosine