Functional and molecular characterization of adenosine transport at the rat inner blood–retinal barrier

Abstract

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.

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

“…The temperature-sensitive rat TR-iBRB2 were grown as previously described (Hosoya and Tomi, 2005;Nagase et al, 2006). TR-iBRB2 (denotes transgenic-inner blood-retinal barrier EC line 2) differentiates at 378C, but becomes conditionally immortalized at 338C.…”

Impact of Cationic Amino Acid Transporter 1 on Blood-Retinal Barrier Transport of L-Ornithine

“…1B), and K in,brain of [ 3 H]L-ornithine was 11.5 6 0.8 lL/(minÁg brain), which was 1.64-fold lower than the K in,retina and 8.27-fold greater than that of [ 3 H]D-mannitol (1.39 lL/(minÁg brain)). 16 The RUI of [ 3 H]L-ornithine was estimated as 128%, which was much higher than that of [ 3 H]Dmannitol (11.6%) ( Table 1), 39 supporting the blood-to-retina transport of L-ornithine across the BRB. The retinal uptake of [ 3 H]L-ornithine was significantly inhibited by 10 mM Lornithine and 10 mM L-arginine during no inhibition shown by 10 mM L-leucine ( Table 1).…”

“…[8][9][10][11] These pieces of evidence indicate the relevance of Lornithine to health maintenance and pathogeneses, and suggest the physiological importance of regulation of the L-ornithine concentration in the retina where the blood-retinal barrier (BRB) is formed by retinal capillary endothelial cells (inner BRB) and RPE cells (outer BRB) to separate the neural retina and the circulating blood. [12][13][14][15] At the BRB, these responsible cells form tight junctions to restrict paracellular solute transport, 12,13 and previous studies have suggested that the retinal capillary endothelial cells express various transporters, such as equilibrative nucleoside transporter 2 (ENT2/SLC29A2), taurine transporter (TAUT/SLC6A6), L (leucine-referring)-type amino acid transporter (LAT1/SLC7A5) and glucose transporter (GLUT1/SLC2A1), involved in the blood-to-retina transport of nutrients across the inner BRB nourishing two-thirds of the retina, [14][15][16][17][18][19] suggesting the involvement of transporters at the inner BRB in regulating the L-ornithine concentration in the retina.…”

The role of dinucleoside polyphosphates on the ocular surface and other eye structures

“…Furthermore, adenosine effects in retina may be regulated by equilibrative (ENT1 and ENT2) and concentrative nucleoside transporters (CNT2 and CNT3) as well as adenosine deaminase 2 (ADA2) (Akanuma et al, 2013;dos Santos-Rodrigues et al, 2011;Elsherbiny et al, 2013;Nagase et al, 2006). In fact, patients with diabetes express a high level of ADA2, which impaired the antiinflammatory activity of adenosine at retina level (Elsherbiny et al, 2013).…”