Kazunori Katayama

Expression and regulation of L-cystine transporter, system xc−, in the newly developed rat retinal Müller cell line (TR-MUL)

The purpose of the present study was to elucidate the expression and regulation of the L‐cystine transporter, system xc−, in Müller cells. In this study, newly developed conditionally immortalized rat Müller cell lines (TR‐MUL) from transgenic rats harboring the temperature‐sensitive SV 40 large T‐antigen gene were used as an in vitro model. TR‐MUL cells express large T‐antigen and grow well at 33°C with a doubling time of 30 h, but do not grow at 39°C. TR‐MUL cells express typical Müller cell markers such as S‐100, glutamine synthetase, and EAAT1/GLAST, whereas EAAT2/GLT‐1 and EAAT5 are not detected. TR‐MUL cells also exhibit little or no expression of glial fibrillary acidic protein. We found that TR‐MUL5 cells exhibited [14C]L‐cystine uptake activity and expressed xCT and 4F2hc, which involve system xc−. The uptake of [14C]L‐cystine was significantly inhibited by L‐glutamic acid and L‐aspartic acid, whereas L‐leucine had no effect. Following diethyl maleate (DEM) treatment, the glutathione concentration in TR‐MUL5 cells was reduced in the first 24 h, then gradually recovered for more than 24 h. The L‐cystine uptake rate and the xCT expression level in TR‐MUL5 cells were enhanced by DEM treatment. In contrast, the 4F2hc expression level was unchanged. In conclusion, TR‐MUL cells have the properties of Müller cells and exhibit system xc−‐mediated L‐cystine uptake activity. The oxidative stress conditions following DEM treatment activate L‐cystine transport in TR‐MUL cells due to the enhanced transcription of the xCT gene. GLIA 9999:000–000, 2003.

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of blood‐to‐retina creatine transport across the blood–retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR‐iBRB2 cells) were examined using [14C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT‐PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [14C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [14C]Creatine uptake by TR‐iBRB2 cells was saturable, Na+‐ and Cl–‐dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT‐PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR‐iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood‐to‐retina creatine transport across the inner BRB.

Application of microdialysis to evaluate the efflux transport of estradiol 17-β glucuronide across the rat blood-retinal barrier

The purpose of the present study was to evaluate vitreous humor/retina-to-blood efflux transport in rats and determine the efflux transport of estradiol 17-beta glucuronide (E17betaG) across the blood-retinal barrier (BRB) by the use of microdialysis. [(3)H]E17betaG and [(14)C]D-mannitol, which were used as a model compound for amphipathic organic anions and a bulk flow marker, respectively, were injected into the vitreous humor of rat eye, and a microdialysis probe was placed in the vitreous humor. [(3)H]E17betaG and [(14)C]D-mannitol were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]E17betaG during the terminal phase was 1.9-fold greater than that of [(14)C]D-mannitol and reduced the level of [(14)C]D-mannitol in the retinal presence of 0.3 mM E17betaG, suggesting that [(3)H]E17betaG is transported via a carrier-mediated efflux transport process across the BRB. The efflux transport of [(3)H]E17betaG was significantly inhibited by organic anions, such as probenecid, sulfobromophthalein, digoxin, and dehydroepiandrosterone sulfate, whereas it was not inhibited by p-aminohippuric acid. In conclusion, the efflux transport of [(3)H]E17betaG across the rat BRB was evaluated by microdialysis and its inhibition by organic anions suggests organic anion transporting polypeptide 1a4-mediated E17betaG efflux transport at the BRB.