The xanthophyll carotenoids such as lutein, zeaxanthin, and lycopene play a significant role in the maintenance of normal vision. These carotenoids are taken up into differentiated ARPE-19 cells via SR-BI (During et al. 2008), suggesting that a similar mechanism might operate in vivo in the uptake of these pigments from blood.

4.2.7.3  Cystine

Glutathione (g-Glu-Cys-Gly), a tripeptide consisting of gluatamate, cysteine, and glycine, is a major antioxidant in the retina. Since intracellular cysteine is low compared to the other two amino acids, cysteine is the rate-limiting amino acid for glutathione synthesis. Cysteine is present in plasma predominantly in the oxidized form cystine. The cystine uptake is mediated by cystine-glutamate exchanger which consists of the “transporter proper” xCT (SLC7A11) and the chaperone 4F2hc. xCT is expressed in TR-iBRB cells (Tomi et al. 2002). When the cellular levels of glutathione are depleted by treatment with diethylmaleate, the expression of xCT is up-regulated to facilitate glutathione synthesis (Hosoya et al. 2001a; Tomi et al. 2002). Functional and immunocytochemical studies have shown that RPE cells express xCT (Bridges et al. 2001; Dun et al. 2006; Gnana-Prakasam et al. 2009). The expression of the transporter is up-regulated in RPE cells in response to increased oxidative stress, indicating a protective role of xCT as an antioxidant mechanism through glutathione (Bridges et al. 2001; Gnana-Prakasam et al. 2009). Thus, xCT at the BRB may be an important factor of glutathione homeostasis in the retina.

4.2.8  Miscellaneous Protective Compounds

4.2.8.1  Creatine

Creatine plays a vital role in the storage and transmission of phosphate-bound energy in retina. The Na+- and Cl−-dependent creatine transporter (CRT, SLA6A8) mediates creatine influx into retina at the inner BRB. CRT is localized on both the luminal and abluminal membranes of rat retinal capillary endothelial cells (Nakashima et al. 2004). Creatine supplementation into retina is a potentially promising treatment for gyrate atrophy of the choroid and retina with hyperornithinemia. However, CRT at the inner BRB is almost saturated by plasma creatine (140–600 mM in mice and rats), since the Michaelis constant for creatine uptake in TR-iBRB cells (~15 mM) is much lower than these plasma concentrations (Nakashima et al. 2004). The development of drugs which increase the density of CRT on the luminal membrane and/or CRT transport activity at the inner BRB is needed for creatine therapy of the gyrate atrophy.

4.2.8.2  Taurine

Taurine, the most abundant free amino acid in retina, functions as an osmolyte to regulate cellular volume under altered osmotic conditions. High levels of taurine in the retina are maintained by the Na+- and Cl−-dependent taurine transporter (TAUT, SLC6A6) (Heller-Stilb et al. 2001). Indeed, TAUT knockout mice show markedly decreased taurine levels in the eye and loss of vision due to severe retinal degradation (Heller-Stilb et al. 2001). TAUT at the inner BRB mediates taurine transport from blood to the retina (Tomi et al. 2007b). Since the Michaelis constant for taurine uptake by TR-iBRB cells (~20 mM) is several-fold smaller than the plasma taurine concentration (100–300 mM) in rats, the blood-to-retina taurine transport appears to be more than 80% saturated by the endogenous taurine under in vivo conditions (Tomi et al. 2007b). TAUT also transports g-aminobutyric acid (an inhibitory neurotransmitter) with a lower affinity than taurine (Tomi et al. 2008). Several studies have demonstrated the functional expression and regulation of the TAUT in RPE cells (Bridges et al. 2001; El-Sherbeny et al. 2004; Leibach et al. 1993). Isolated apical membrane vesicles from bovine RPE cells demonstrate robust Na+/Cl−- coupled taurine uptake (Miyamoto et al. 1991; Sivakami et al. 1992).

4.3Efflux Transporters at the Blood-Retinal Barrier

The BRB plays an essential role in the protection of the retina from unwanted harmful effects of endobiotics and xenobiotics which are present in systemic circulation and/or produced in the retina. Two distinct mechanisms participate in this process. The endobiotics and xenobiotics including drugs in the systemic circulation might gain entry into retinal capillary endothelial cells and RPE cells either by passive diffusion or by specific influx transporters. These compounds can be effluxed out of these cells back into the circulating blood via a primary active efflux transport system. This efflux transport system consists of ATP-binding cassette (ABC) transporters which exhibit a very broad range of substrate selectivity for such toxic compounds. ABC transporters are likely to exist on the luminal membrane and basolateral membrane of RVEC and RPE cells, respectively, to carry out the efflux process (Fig. 4.1). Within the ABC transporter family, ABCA, ABCB, ABCC, and ABCG transporter subfamilies could provide a protective mechanism for the retina by restricting the entry of potentially harmful compounds into retina. The second mechanism involves transcellular transport of endobiotics and xenobiotics from subretinal space into the circulating blood via concerted actions of influx transporters in the abluminal and apical membranes and efflux transporters in the luminal and basolateral membranes of RVEC and RPE cells, respectively. Organic anion transporting polypeptides (OATPs, SLCO, SLC21A) and organic anion transporters (OATs, SLC22A) are most likely involved in the influx transport mechanism. ABC transporters play a role in the efflux transport. While it is certainly true that these transporters play a beneficial role in the protection of retina from potentially toxic

xenobiotics, the processes pose a major problem for the effective delivery of therapeutically active drugs to retina. Many of the widely used and clinically relevant drugs are substrates for the transporters and therefore such drugs are actively removed from retina across the BRB, thus preventing accumulation of these drugs in retina at therapeutically effective concentrations. This hurdle might be overcome, however, if specific inhibitors of the transporters are coadministered along with the drugs. Therefore, it is important to identify the efflux transport systems at these barriers and elucidate their substrate selectivity in terms of various drugs that are of potential use for the treatment of retinal diseases.

4.3.1  Organic Anion Transporter 3 (OAT3, SLC22A8)

The distribution of b-lactam antibiotics in the vitreous humor/retina after systemic administration is limited, resulting in reduced efficacy in the treatment of bacterial endophthalmitis (Barza et al. 1983). 6-MP is frequently used for cancer chemotherapy in patients with childhood acute lymphoblastic leukemia. Relapse of childhood acute lymphoblastic leukemia involving eye is a rare but challenging problem. This is probably due to the restricted distribution of 6-MP in the eye (Somervaille et al. 2003). One possible factor in the restricted drug distribution in the retina/eye is the retina-to-blood efflux transport of such anionic drugs across the BRB. Indeed, b-lactam antibiotic benzylpenicillin (PCG) and 6-MP are biexponentially eliminated from the vitreous humor after bolus injection into vitreous of the rat eye (Hosoya et al. 2009). The elimination rate constant of PCG and 6-MP during the terminal phase was about twofold greater than that of D-mannitol, a bulk flow marker. This efflux transport was reduced in the retina in the presence of probenecid, p-aminohippuric acid (PAH), and PCG, relatively specific substrates of organic anion transporter (OAT) 3 (SLC22A8) (Kikuchi et al. 2003). OAT3 is localized on the abluminal membrane of retinal capillary endothelial cells (Hosoya et al. 2009). OAT3 knockout mice exhibit decreased distribution and elimination of PCG (VanWert et al. 2007). Thus, OAT3 is involved in the uptake of PCG and 6-MP across the abluminal membrane of RVEC and contributes to the efflux transport of PCG and 6-MP from vitreous humor/retina into blood across the inner BRB.

4.3.2  Organic Anion Transporting Polypeptides

(OATPs, SLCO, SLC21A)

Some b-lactam antibiotics are substrates for organic anion transporting polypeptide (Oatp) 1a4 (Slco1a4; Oatp2) (Nakakariya et al. 2008). Since Oatp1a4 is expressed in RVEC (Gao et al. 2002), this transporter could also be involved in the clearance of anionic b-lactam antibiotics at the inner BRB (Katayama et al. 2006). Oatp1c1 (Slco1c1/Oatp14) mRNA is also expressed in isolated rat RVEC (Tomi and

Hosoya 2004). Oatp1c1 transports estradiol 17b-glucuronide as is the case with Oatp1a4 whereas Oatp1c1 does not have high affinity for digoxin (Sugiyama et al. 2001), a specific substrate of Oatp1a4. This suggests that Oatp1c1 and Oatp1a4 play distinct roles in the retina-to-blood efflux transport in terms of the specificity of the drugs and xenobiotics. Further studies are needed to clarify the individual contribution of Oatp1c1 and Oatp1a4 to the efflux of specific anionic drugs across the inner BRB. Oatp1a4 is expressed prominently in the apical membrane of RPE cells (Ito et al. 2002). Oatp-E (Slco4a1) is expressed in RPE cells although its exact location is not known (Ito et al. 2003).

4.3.3  P-Glycoprotein (ABCB1)

Several classes of drugs, including anticancer agents, antibiotics, steroids, and immunosuppressants are recognized as substrates for P-glycoprotein (P-gp, ABCB1). P-gp is localized on the luminal membrane of RVEC (Hosoya and Tomi 2005). TR-iBRB cells express P-gp, and the accumulation of rhodamine 123 in TR-iBRB cells is enhanced in the presence of inhibitors of P-gp (Hosoya et al. 2001b). The expression of P-gp has also been demonstrated in a number of human RPE cell lines (e.g., D407, h1RPE), but interestingly not in ARPE-19 cells (Constable et al. 2006; Kennedy and Mangini 2002; Mannermaa et al. 2009). The transporter is localized more predominantly in the RPE basolateral membrane where it can mediate active transfer of its substrates from RPE cells into blood (Kennedy and Mangini 2002). The active efflux transport function of P-gp at the BRB could lower the blood-to- retina permeability of its substrates. For example, cyclosporine A, a substrate of P-gp, was not detected in the intraocular tissues of cyclosporine A-treated rabbits, although the blood level of cyclosporine A was within the therapeutic window (BenEzra and Maftzir 1990). Daunomycin, which is used for the management of proliferative vitreoretinopathy, is a substrate for P-gp. Treatment of patients with proliferative vitreoretinopathy using daunomycin causes overexpression of P-gp, thus resulting in multidrug resistance (Esser et al. 1998). Abcb1a gene knockout mice gave evidence that penetration of central nervous system acting drugs into the brain is restricted by P-gp at the blood–brain barrier (Schinkel et al. 1996). It is therefore intriguing in future studies to investigate the contribution of P-gp to the blood-to-retina transport of drugs, possibly using Abcb1 knockout mice.

4.3.4  Multidrug Resistance-Associated Proteins (ABCCs)

Studies on multidrug resistance-associated protein 4 (MRP4) gene-disrupted mice reveal that MRP4 at the blood–brain barrier and the blood-CSF barrier restricts penetration of drugs into the brain (Kruh et al. 2007). In the retina, MRP4 functions as a BRB efflux transporter of anionic drugs. MRP4 accepts several anionic drugs