Retinal Uptake Index Research Articles

Comprehensive Evidence of Carrier-Mediated Distribution of Amantadine to the Retina across the Blood-Retinal Barrier in Rats.

Amantadine, a drug used for the blockage of NMDA receptors, is well-known to exhibit neuroprotective effects. Accordingly, assessment of amantadine transport at retinal barriers could result in the application of amantadine for retinal diseases such as glaucoma. The objective of this study was to elucidate the retinal distribution of amantadine across the inner and outer blood–retinal barrier (BRB). In vivo blood-to-retina [3H]amantadine transport was investigated by using the rat retinal uptake index method, which was significantly reduced by unlabeled amantadine. This result indicated the involvement of carrier-mediated processes in the retinal distribution of amantadine. In addition, in vitro model cells of the inner and outer BRB (TR-iBRB2 and RPE-J cells) exhibited saturable kinetics (Km in TR-iBRB2 cells, 79.4 µM; Km in RPE-J cells, 90.5 and 9830 µM). The inhibition of [3H]amantadine uptake by cationic drugs/compounds indicated a minor contribution of transport systems that accept cationic drugs (e.g., verapamil), as well as solute carrier (SLC) organic cation transporters. Collectively, these outcomes suggest that carrier-mediated transport systems, which differ from reported transporters and mechanisms, play a crucial role in the retinal distribution of amantadine across the inner/outer BRB.

Blood-to-Retina Transport of Imperatorin Involves the Carrier-Mediated Transporter System at the Inner Blood-Retinal Barrier

This study investigated the mechanism of transporting imperatorin across the inner blood-retinal barrier (iBRB). The carotid artery single injection method was used to calculate the retinal uptake index (RUI) of [3H]imperatorin in vivo, whereas the retinal capillary endothelial cell lines were used for the in vitro uptake and mRNA expression assays. RUI value of [3H]imperatorin was greater than that of the reference compound ([14C]n-butanol). [3H]Imperatorin significantly reduced the RUI in the presence of neuroprotective organic cationic drugs at 10 mM. However, tetraethylammonium and p-aminohippuric acid showed no significant effects. [3H]Imperatorin uptake by TR-iBRB2 cells was time-, pH-, energy-, and concentration-dependent with a Km value of 679 ± 130 μM. In addition, the uptake study showed insensitivity to sodium and membrane potential. Various organic cations including pyrilamine, nicotine, and clonidine significantly reduced the uptake of [3H]imperatorin, whereas organic anions and monocarboxylic acids did not. Furthermore, the mRNA expression level dropped markedly with rOCTN1, rOCTN2, rPMAT, and rMATE1 small interfering RNAs in the transfection study. Moreover, [3H]imperatorin uptake remained neutral with small interfering RNA transfections. Our results indicate that imperatorin transport across the iBRB involves carrier-mediated transporter system.

Retina-to-Blood Transport of 1-Methyl-4-Phenylpyridinium Involves Carrier-Mediated Process at the Blood-Retinal Barrier

1-Methyl-4-phenylpyridinium (MPP+) transport at the blood-retinal barrier (BRB) was investigated. The retinal uptake index estimated for [3H]MPP+ was similar to that of [3H]d-mannitol, and was insensitive to unlabeled MPP+, suggesting no positive evidence to support the involvement of carrier-mediated transport in the blood-to-retina transport of MPP+ at the BRB. A microdialysis investigation showed that the concentration of [3H]MPP+ in the vitreous humor decreased in a biexponential manner, and the rate constant for [3H]MPP+ elimination during the terminal phase was greater than that of [14C]d-mannitol. The inhibition study of [3H]MPP+ elimination showed its substrate specificity, suggesting that the retina-to-blood transport of MPP+ at the BRB involves a carrier-mediated process. The in vitro study with model cells showed the concentration-dependent transport of MPP+, supporting carrier-mediated MPP+ transport at the inner and outer BRB, and suggested membrane potential-sensitive and Na+-, Cl−-, and pH-insensitive MPP+ transport at the BRB. In the in vitro inhibition study, the transport of [3H]MPP+ was significantly inhibited by organic cations, and further reverse transcription PCR analysis and knockdown study suggested that the retina-to-blood transport of MPP+ at the BRB is carried out by an unknown transporter of which transport function is similar to plasma membrane monoamine transporter (PMAT/SLC29A4).

Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier

Riboflavin (vitamin B2) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [3H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [3H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l-ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [3H]riboflavin uptake with a Km of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [3H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na+- and Cl−-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na+- and Cl−-independent. [3H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.

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

To elucidate L-ornithine transport at the blood-retinal barrier (BRB). Integration plot and retinal uptake index (RUI) were used to investigate the in vivo [3H]L-ornithine transport across the BRB. In vitro transport studies of [3H]L-ornithine were performed with TR-iBRB2 cells and RPE-J cells, the model cells of the inner and outer BRB, respectively. Immunohistochemistry was performed on cationic amino acid transporter 1 (CAT1/SLC7A1). The apparent influx permeability clearance of [3H]L-ornithine was found to be 18. 7 μL/(min·g retina), and the RUI of [3H]L-ornithine was reduced by L-ornithine and L-arginine, suggesting the blood-to-retina transport of L-ornithine at the BRB. [3H]L-Ornithine uptake by TR-iBRB2 cells showed a time-, temperature- and concentration-dependence with a Michaelis-Menten constant (K(m)) of 33.2 μM and a nonsaturable uptake rate (Kd) of 2.18 μL/(min·mg protein). The uptake was Na+-independent, and was inhibited by L-ornithine, L-arginine, and L-lysine, suggesting the involvement of CAT1 in L-ornithine transport at the inner BRB. Immunohistochemistry revealed the luminal and abluminal localization of CAT1 at the inner BRB, and at the basal localization at the outer BRB. Retinal pigment epithelium-J cells showed that the basal-to-cell (B-to-C) uptake of [3H]L-ornithine was greater than that of the apical-to-cell (A-to-C) uptake, and the B-to-C transport was inhibited by unlabeled L-ornithine, suggesting the involvement of CAT1 in the blood-to-cell transport of L-ornithine across the basal membrane at the outer BRB. These suggest the involvement of CAT1 in L-ornithine transport at the luminal and abluminal sides of the inner BRB and the basal side of the outer BRB.

Involvement of carrier-mediated transport in the retinal uptake of clonidine at the inner blood-retinal barrier.

In the present study, the blood-to-retina transport across the inner BRB was investigated for clonidine, a compound which is expected to exhibit a neuroprotective effect for the treatment of severe retinal diseases. In the in vivo study, the integration plot analysis for [(3)H]clonidine exhibited an apparent influx permeability clearance of 457 μL/(min·g retina) in the retina. The in vivo inhibition study suggests that the blood-to-retina transport of clonidine at the BRB is organic cation-sensitive since clonidine, pyrilamine, and propranolol, at a concentration of 40 mM, significantly reduced the retinal uptake index (RUI) of [(3)H]clonidine, and an inhibitory effect on the RUI was also exhibited by verapamil, at a concentration of 3 mM. The in vitro study with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests that carrier-mediated transport is involved in the blood-to-retina transport of clonidine at the inner BRB since the results obtained demonstrated time-, temperature-, pH-, and concentration-dependent [(3)H]clonidine uptake, with a Km of 286 μM. In the in vitro inhibition study, the [(3)H]clonidine uptake was significantly reduced by several organic cations, such as clonidine, verapamil, pyrilamine, and propranolol, and was competitively inhibited by 200 μM verapamil, in spite of slight or no significant alteration being produced with organic anions. Furthermore, the typical substrates and inhibitors of well-known organic cation transporters had no significant effect on the uptake of [(3)H]clonidine to suggest the involvement of novel transporter molecules in the transport of clonidine across the inner BRB. These results suggest that the blood-to-retina transport of clonidine across the inner BRB involves a carrier-mediated transport manner, suggesting the contribution of a novel organic cation transporter expressed by the retinal capillary endothelial cells.

Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier

The elimination of spermine, the end product of cellular polyamine, from the retina to the blood across the blood-retinal barrier (BRB) was investigated. The in vivo microdialysis study revealed that the elimination of [3H]spermine from vitreous humor after vitreous bolus injection was in a biexponential manner. The rate constant for the elimination of [3H]spermine during the terminal phase was estimated to be 1.67-fold greater than that of [14C]d-mannitol, a bulk flow marker, and the difference in the terminal elimination rate constant between [3H]spermine and [14C]d-mannitol was reduced in the presence of 50 mM spermine, suggesting a retina-to-blood transport system for [3H]spermine across the BRB. The retina-to-blood transport of [3H]spermine was also supported by a study of the retinal uptake index (RUI). The in vitro transport study with TR-iBRB2 cells, a model cell line of the inner BRB, revealed time-, concentration- and temperature-dependent transport of [3H]spermine, suggesting the involvement of carrier-mediated processes in spermine transport across the inner BRB. The in vitro study also suggested that the transport of spermine at the inner BRB is pH-, membrane potential- and Cl−-sensitive and Na+-insensitive, and these functional properties of spermine transport suggest only a minor contribution of spermine transporters, such as CCC9 (SLC12A8), the expression of which was suggested at the inner BRB. In the inhibition study, [3H]spermine transport was markedly inhibited by putrescine, spermidine, spermine and agmatine while substrates of a well-characterized organic cation transporter (OCTs/SLC22A) and a cationic amino acid transporter (CATs/SLC7A) had no effect, suggesting the involvement of unknown transporters in spermine elimination from the retina.

Involvement of a Novel Organic Cation Transporter in Verapamil Transport Across the Inner Blood-Retinal Barrier

To clarify the transport and inhibition characteristics involved in verapamil transport across the inner blood-retinal barrier (inner BRB). The transport of [(3)H]verapamil across the inner BRB was investigated using retinal uptake index and integration plot analyses in rats. The detailed transport characteristics were studied using TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line that is an in vitro model of the inner BRB. The apparent influx permeability clearance of [(3)H]verapamil was 614μL/(min·g retina), which is 4.7-fold greater than that of brain. The retinal uptake of [(3)H]verapamil was slightly increased by 3mM verapamil and 10mM qunidine and inhibited by 40mM pyrilamine, supporting the carrier-mediated efflux and influx transport of verapamil across the inner BRB. TR-iBRB2 cells exhibited a concentration-dependent uptake of [(3)H]verapamil with a K (m) of 61.9μM, and the uptake was inhibited by several cations, such as pyrilamine, exhibiting a different profile from the identified transporters. These transport properties suggest that verapamil transport at the inner BRB takes place via a novel organic cation transporter. Our findings suggest that a novel organic cation transporter is involved in verapamil transport from the blood to the retina across the inner BRB.

Application of Membrane Permeability Evaluated in In Vitro Analyses to Estimate Blood–Retinal Barrier Permeability

The relationship between the in vitro membrane permeability and systemic blood-retinal barrier (BRB) permeability of drugs was investigated. To determine membrane permeability trend lines in this relationship, the apparent permeability (P(app)) and initial uptake rate (V) of 23 compounds were evaluated in a parallel artificial membrane permeability assay and the uptake study with a rat retinal endothelial cell line (TR-iBRB2 cells) for comparison with their retinal uptake index (RUI). The RUI values of compounds undergoing passive diffusion across the BRB were correlated with a log of the P(app) [RUI = 7.93 × 10 × exp (0.994 × log P(app)), r(2) = 0.660] and a log of the V [RUI = 26.5 × exp (1.55 × log V), r(2) = 0.581]. The RUI values of compounds undergoing carrier-mediated transport across the BRB were correlated with a log of the V [RUI = 26.5 × exp (0.887 × log V), r(2) = 0.559]. These results showed that the membrane permeability trend lines derived from the RUI and V values reflect the transport of drugs at the BRB, suggesting that an in vitro analysis-based estimation of the BRB permeability can be obtained using TR-iBRB2 cells and membrane permeability trend lines.

Comparison of Drug Permeabilities Across the Blood–Retinal Barrier, Blood–Aqueous Humor Barrier, and Blood–Brain Barrier

Drugs vary in their ability to permeate the blood–retinal barrier (BRB), blood–aqueous humor barrier (BAB), and blood–brain barrier (BBB) and the factors affecting the drug permeation remain unclear. In this study, the permeability of various substances across BRB, BAB, and BBB in rats was determined using the brain uptake index (BUI), retinal uptake index (RUI), and aqueous humor uptake index (AHUI) methods. Lipophilic substances showed high permeabilities across BBB and BRB. The RUI values of these substances were approximately four-fold higher than the BUI values. The AHUI versus lipophilicity curve had a parabolic shape with AHUImax values at log D7.4 ranging from −1.0 to 0.0. On the basis of the difference on the lipophilicities, verapamil, quinidine, and digoxin showed lower permeability than predicted from those across BBB and BRB, whereas only digoxin showed a lower permeability across BRB. These low permeabilities were significantly increased by P-glycoprotein inhibitors. Furthermore, anion transporter inhibition increased the absorption of digoxin to permeate into the retina and aqueous humor. In conclusion, this study suggests that efflux transport systems play an important role in the ocular absorption of drugs from the circulating blood after systemic administration. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3904–3911, 2011

Blood-to-retina transport of biotin via Na +-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of biotin transport across the inner blood-retinal barrier (inner BRB). [ 3H]Biotin transport in the retina across the inner BRB was examined using an in vivo integration plot and retinal uptake index analyses in rats. The transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro inner BRB model. The apparent influx permeability clearance ( K in) per gram retina of [ 3H]biotin was found to be 5.55 μL/(min g retina). The K in of [ 3H]biotin was 8.9-fold greater than that of [ 3H]D-mannitol, a non-permeable paracellular marker. [ 3H]Biotin uptake by the retina was found to be significantly inhibited by biotin and pantothenic acid, supporting carrier-mediated influx transport of biotin at the inner BRB. [ 3H]Biotin uptake by TR-iBRB2 cells was Na +-, temperature-, and concentration-dependent with a K m of 146 μM. These forms of transport were significantly inhibited by Na +-dependent multivitamin transporter (SMVT) substrates such as biotin, pantothenic acid, lipoic acid, and desthiobiotin. These transport properties are consistent with those of biotin transport by SMVT. SMVT mRNA was expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. Our findings suggest that SMVT is involved in the transport of biotin from the circulating blood to the retina across the inner BRB.

Involvement of OCTN2 in the Transport of Acetyl-l-Carnitine across the Inner Blood–Retinal Barrier

To elucidate the mechanisms of acetyl-L-carnitine transport across the inner blood-retinal barrier (inner BRB). In vivo integration plot and retinal uptake index (RUI) analyses were used to examine acetyl-L-[(3)H]carnitine transport in the retina across the inner BRB in rats. RUI was determined from the ratio of acetyl-L-[(3)H]carnitine and [(14)C]n-butanol, a freely diffusible internal reference, in the retina divided by the same ratio in the solution injected in the carotid artery. The transport mechanism was characterized in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner BRB model. The apparent influx permeability clearance (K(in)) per gram retina of acetyl-L-[(3)H]carnitine was found to be 2.31 microL/(minute . g retina). The K(in) of acetyl-L-[(3)H]carnitine was 3.7-fold greater than that of [(3)H]D-mannitol, a nonpermeable paracellular marker. Acetyl-L-[(3)H]carnitine uptake by the retina was found to be significantly inhibited by L-carnitine and acetyl-L-carnitine, supporting a carrier-mediated influx transport of acetyl-L-carnitine at the inner BRB. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake by TR-iBRB2 cells was Na(+)- and concentration-dependent, with a K(m) of 29 and 26 microM, respectively. These forms of transport were significantly inhibited by organic cation/carnitine transporter (OCTN) substrates and inhibitors such as L-carnitine and acetyl-L-carnitine, tetraethylammonium, quinidine, and betaine. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was predominantly expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. The findings suggest that OCTN2 is involved in the transport of acetyl-L-carnitine from the circulating blood to the retina across the inner BRB.

The uptake index method applied to studies on the blood-retinal barrier. I. A methodological study.

The validity of the uptake index method for studies on passage through the blood-retinal barrier was evaluated. Data for the brain were also obtained for comparison. A rapid intracarotid injection of 0.2 ml buffered Ringer solution in rats did not result in a measurable increase in the permeability to EDTA for either the blood-retinal or the blood-brain barriers, while a markedly hypertonic solution caused an immediate osmotic breakdown of both barriers. For determinations of the retinal uptake index (RUI) 3HOH was found to be a suitable reference substance. A more diffusible reference, such as 14C-ethanol, resulted in a lower net extraction, probably due to a more rapid wash-out by the choroidal blood flow. Clearance of both reference and test substances from the retina was considerably faster than from the brain. For this reason a 5-s interval between the intracarotid injection and the enucleation was used as routine for determinations of RUI. With this technique RUI for the transported monosaccharide 3-O-methyl-D-glucose was significantly larger than for L-glucose, 53.8 and 14.2% respectively. The RUI for L-glucose was mainly due to L-glucose confined to extravascular choroidal fluid adhering to the retinal tissue samples. By prolonging the interval between injection and enucleation, and subtracting the activity due to recirculation, this extraretinal contamination could be practically eliminated. It is concluded that the uptake index method can be a useful tool in studies on transport of metabolic substrates through the blood-retinal barrier.