| Research Abstract |
The blood-retinal barrier (BRB), which forms complex tight junctions of retinal capillary endothelial cells (inner BRB) and retinal pigment epithelial cells (outer BRB), restricts nonspecific transport between the neural retina and the circulating blood and maintains a constant milieu in the neural retina. Although understanding the transport mechanisms of organic anions at the inner BRB will provide pharmacologically important information about the effective delivery of many anionic drugs to the retina, currently very little is known about the efflux transport process for organic anions at the BRB and the roles of organic anion transporters at the inner BRB remain to be elucidated. Moreover the transport processes for organic anions at the inner BRB are physiologically important since hormones and neurotransmitters are mostly metabolized in the form of organic anions in the retina and need to undergo efflux transport from the retina to the circulating blood. The purpose of this study
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was to determine efflux transport of organic anions across the BRB by the use of microdialysis and to quantify gene expression levels of the organic anion transporters in rat retinal vascular endothelial cells (RVEC) using a combination of a magnetic isolation method for rat RVEC and real time quantitative PCR analysis. [^3H]Estradiol 17-β glucutonide ([^3H]E17βG) or [^3H]p-aminohippuric acid ([^3H]PAH) and [^14C]D-mannitol, which were used as a model compound for organic anion and a bulk flow marker from the vitreous humor to the Schlemm's canal, respectively, were injected into the vitreous humor of rat eye, and a microdialysis probe was placed into the vitreous humor [^3H]E17βG and [^H]PAH were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [^3H]E17βG and [^3H]PAH during the terminal phase was about 2-fold greater than that of [^14C]D-mannitol and reduced the level of [^14C]D-mannitol in the retinal presence of respective cold compound, suggesting that [^3H]E17βG and [^3H]PAH is transported via a carrier-mediated efflux transport process across the BRB. The efflux transport of [^3H]E17βG was significantly inhibited by probenecid, sulfobromophthalein (BSP), digoxin, and dehydroepiandrosterone sulfate, whereas it was not inhibited by PAR On the other hand, the efflux transport of [^3H]PAH was significantly inhibited by probenecid, BSP, benzylpenicillin, whereas it was not inhibited by digoxin. These results suggest that organic anion transporting polypeptide (oatp) and organic anion transporter (OAT) are involved in the efflux transport of E17βG and PAH, respectively. The transcript levels of oatps, such as oatp1a1(Slc21a1), oatp1a4(Slc21a5), oatp1a5(Slc21a7), and oatp1c1(Slc21a14) and OATs, such as OAT1(S1c22a6), OAT2(Slc22a7) and OAT3(Slc22a8), were examined in the RVEC. Oatp1a4, oatp1c1 and OAT3 were detected in the RVEC fraction, whereas oatp1a1, oatp1a5, OAT1, and OAT2 were not. These results support that oatp1a4 and/or oatp1c1 and OAT3 are involved in the efflux transports of [^3H]E17βG and [^3H]PAH across the rat BRB has been evaluated by applications of a microdialysis probe to the vitreous humor. A functional in vivo inhibition and mRNA expression studies suggest that oatp1a4 and OAT3 are involved in efflux transport of E17βG and PAH, respectively, at the inner BRB. Less
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