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 K_m 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.     

Propranolol Transport Across the Inner Blood–Retinal Barrier: Potential Involvement of a Novel Organic Cation Transporter

The influx transport of propranolol across the inner blood–retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [³H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [³H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [³H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [³H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter.     

Involvement of reduced folate carrier 1 in the inner blood-retinal barrier transport of methyltetrahydrofolate

The purpose of this study was to elucidate the mechanism of methyltetrahydrofolate (MTF) transport at the inner blood-retinal barrier (inner BRB). The characteristics and function of MTF transport at the inner BRB were examined using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model of the inner BRB. The [3H]MTF uptake by TR-iBRB2 cells increased with lowering extracellular pH and was Na+- and Cl--independent. The [3H]MTF uptake was concentration-dependent with a K(m) of 5.1 microM. This process was inhibited by reduced folate carrier 1 (RFC1) substrates, such as methotrexate and formyltetrahydrofolate, in a concentration-dependent manner with an IC50 of 8.7 and 2.8 microM, respectively, suggesting that RFC1 mediates MTF uptake in TR-iBRB2 cells. Although both RFC1 and proton-coupled folate transporter (PCFT) mRNA, which are pH-sensitive folate transporters, are expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells, the expression level of RFC1 mRNA was 83- and 49-fold greater than that of PCFT, respectively. Taken together, the above findings are consistent with the involvement of RFC1 in the inner BRB transport of MTF.     

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

Purpose

To clarify the transport and inhibition characteristics involved in verapamil transport across the inner blood-retinal barrier (inner BRB).

Methods

The transport of [³H]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.

Results

The apparent influx permeability clearance of [³H]verapamil was 614 μL/(min·g retina), which is 4.7-fold greater than that of brain. The retinal uptake of [³H]verapamil was slightly increased by 3 mM verapamil and 10 mM qunidine and inhibited by 40 mM pyrilamine, supporting the carrier-mediated efflux and influx transport of verapamil across the inner BRB. TR-iBRB2 cells exhibited a concentration-dependent uptake of [³H]verapamil with a K _m of 61.9 μM, 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.

Conclusions

Our findings suggest that a novel organic cation transporter is involved in verapamil transport from the blood to the retina across the inner BRB.     

Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier

Purpose

To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).

Methods

EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively.

Results

The signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker^® Red, a lysosomotropic agent, and was not altered by acute treatment with NH₄Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH₄Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH₄Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC₅₀ of 98.0 μM.

Conclusions

Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.

     

Advances in the cell biology of transport via the inner blood-retinal barrier: establishment of cell lines and transport functions

The retinal capillary endothelial cells are connected to each other by tight junctions that play a key role in permeability as the inner blood-retinal barrier (inner BRB). Thus, understanding the inner BRB transport mechanism is an important step towards drug targeting of the retina. Nevertheless, inner BRB transport studies have been very limited in number since it is not easy to use the retinal capillaries, which are very small in size, for in vitro transport studies. Conditionally immortalized rat retinal capillary endothelial cells (TR-iBRB), pericytes (TR-rPCT) and Müller cell lines (TR-MUL) have been established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines possess respective cell type markers and maintain certain in vivo functions. Using a combination of newly developed cell lines and in vivo studies, we have elucidated the mechanism whereby vitamin C, L-cystine, and creatine are supplied to the retina. TR-iBRB cells are also able to identify transporters and apply to study regulation of transporters under pathophysiological conditions. Furthermore, these cell lines permit the investigation of cell-to-cell interactions and the expression of inner BRB-specific genes between TR-iBRB and other cell lines.     

Riboflavin transport mediated by riboflavin transporters (RFVTs/SLC52A) at the rat outer blood-retinal barrier

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B₂) across the blood-retinal barrier (BRB). In the present study, the blood-to-retina supply of riboflavin across the outer BRB was assessed in RPE-J cells, a rat-derived in vitro cell model of the outer BRB that is formed by the retinal pigment epithelial cells. In the directional uptake analysis on collagen-coated Transwell® inserts, RPE-J cells showed higher basal-to-cell (B-to-C) uptake (22.8 μL/mg protein) of [³H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [³H]riboflavin with a K_m of 297 nM, suggesting the involvement of carrier-mediated process in the blood-to-retina transport of riboflavin across the outer BRB. In RPE-J cells, [³H]riboflavin uptake was affected under a K⁺-replacement condition while no effect was observed under a choline-replacement condition and at different pH values. Uptake of [³H]riboflavin by RPE-J cells was markedly reduced by riboflavin, flavin adenine dinucleotide (FAD), and lumichrome with no significant effect noted for other vitamins. The obtained results suggested the involvement of riboflavin transporters (SLC52A/RFVT) at the outer BRB, and this is supported by the expression and knockdown analyses of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3).     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

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.     

Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [³H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [³H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [³H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [³H]3-OMG uptake was increased at 48 h. However, [³H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [³H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [³H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.     

Recent advances in drug and nutrient transport across the blood-retinal barrier

Introduction: The blood-retinal barrier (BRB) is the barrier separating the blood and neural retina, and transport systems for low-weight molecules at the BRB are expected to be useful for developing drugs for the treatment of ocular neural disorders and maintaining a healthy retina.

Areas covered: This review discusses blood-to-retina and retina-to-blood transport of drugs and nutrients at the BRB. In particular, P-gp (ABCB1/MDR1) has low impact on the transport of cationic drugs at the BRB, suggesting a significant role of novel organic cation transporters in influx and efflux transport of lipophilic cationic drugs between blood and the retina. The transport of pravastatin at the BRB involves transporters including organic anion transporting polypeptide 1a4 (Oatp1a4). Recent studies have shown the involvement of solute carrier transporters in the blood-to-retina transport of nutrients including riboflavin, L-ornithine, β-alanine, and L-histidine, implying that dipeptide transport at the BRB is minimal.

Expert opinion: Novel organic cation transport systems and the elimination-dominant transport of pravastatin at the BRB are expected to be useful in systemic drug delivery to the neural retina without CNS side effects. The mechanism of nutrient transport at the BRB is expected to provide a new strategy for delivery of nutrient-mimetic drugs.     

Inner Blood-Retinal Barrier Transporters: Role of Retinal Drug Delivery

The inner blood-retinal barrier (inner BRB) forms complex tight junctions of retinal capillary endothelial cells to prevent the free diffusion of substances between the circulating blood and the neural retina. Thus, understanding of the inner BRB transport mechanisms could provide a basis for the development of strategies for drug delivery to the retina. Recent progress in inner BRB research has revealed that retinal endothelial cells express a variety of unique transporters which play a role in the influx transport of essential molecules and the efflux transport of xenobiotics. In this review we focus on the transport mechanism at the inner BRB in relation to its importance in influencing the inner BRB permeability of drugs.     

Evaluation of amino acid-mustard transport as L-type amino acid transporter 1 (LAT1)-mediated alkylating agents

The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na+-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [3H]L-phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [3H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards.     

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 K_m 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.     

Effects of antipsychotic drugs on neurotoxicity, expression of fos-like protein and c-fos mRNA in the retrosplenial cortex after administration of dizocilpine

To elucidate the role of P-glycoprotein in human placenta, we examined its expression in placenta, and the transcellular transport and uptake of P-glycoprotein substrates in cultured human placental choriocarcinoma epithelial cells (BeWo cells). The uptake of [(3)H]vinblastine and [(3)H]vincristine into BeWo cells was increased in the presence of a metabolic inhibitor, sodium azide. The basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was greater than the apical-to-basolateral transcellular transport. In the presence of cyclosporin A, the basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was significantly increased, and the apical-to-basolateral transcellular transport was decreased. The uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells was significantly enhanced in the presence of several inhibitors, such as verapamil or mouse monoclonal antibody anti-P-glycoprotein MX-MDR (MRK16) as well as cyclosporin A. Although progesterone significantly enhanced the uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells, the uptake of [(3)H]progesterone was not affected by these inhibitors. Immunoblot analysis revealed that P-glycoprotein with a molecular weight of 172 kDa was expressed in BeWo cells and isolated trophoblast cells. Furthermore, P-glycoprotein was detected in human placental brush-border membrane vesicles, but not in human placental basolateral membrane vesicles. In conclusion, these data suggest that P-glycoprotein is expressed on the brush-border membrane (maternal side) of human placental trophoblast cells. P-Glycoprotein is considered to regulate the transfer of several substances including vinblastine, vincristine and digoxin from mother to fetus, and to protect the fetus from toxic substances.     

Mechanisms and interaction of vinblastine and reduced glutathione transport in membrane vesicles by the rabbit multidrug resistance protein Mrp2 expressed in insect cells.

The present study examined how the multidrug resistance protein (MRP) 2, which is an ATP-dependent anionic conjugate transporter, also mediates transport of the chemotherapeutic cationic drug vinblastine (VBL). We show that ATP-dependent [(3)H]VBL (0.2 microM) uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mock-infected Sf9 cells (Sf9-mock) but could be stimulated by reduced glutathione (GSH) with a half-maximum stimulation of 1.9 +/- 0.1 mM. At 5 mM GSH, initial ATP-dependent [(3)H]VBL uptake rates were saturable with an apparent K(m) of 1.5 +/- 0.3 microM. The inhibitory effect of VBL on Mrp2-mediated ATP-dependent transport of the anionic conjugate [(3)H]leukotriene C(4) was potentiated by increasing GSH concentrations. Membrane vesicles from Sf9-Mrp2 cells exhibited a approximately 7-fold increase in initial GSH uptake rates compared with membrane vesicles from Sf9-mock cells. Uptake of [(3)H]GSH was osmotically sensitive, independent of ATP, and was trans-inhibited by GSH. The anionic conjugates estradiol-17beta-D-glucuronide and leukotriene C(4) cis-inhibited [(3)H]GSH uptake but only in the presence of ATP. Whereas ATP-dependent [(3)H]VBL uptake was stimulated by GSH, VBL did not affect [(3)H]GSH uptake. Our results show that GSH is required for Mrp2-mediated ATP-dependent VBL transport and that Mrp2 transports GSH independent of VBL.     

Contribution of an unidentified sodium-dependent nucleoside transport system to the uptake and cytotoxicity of anthracycline in mouse M5076 ovarian sarcoma cells

In the present study, we investigated whether an unidentified system for Na(+)-dependent nucleoside transport is expressed by mouse M5076 ovarian sarcoma cells, besides concentrative nucleoside transporter 2 (CNT2(M)), and is involved in the uptake and cytotoxicity of anthracyclines. In a transport assay involving CNT2(M)-transfectants, CNT2(M) was found to transport [(3)H]cytidine in a Na(+)-dependent manner, and 500 microM cytidine completely inhibited the Na(+)-dependent uptake of [(3)H]uridine via the transporter. In contrast, the Na(+)-dependent [(3)H]uridine uptake by M5076 cells decreased with 500 microM cytidine only to 70% of the control level. Furthermore, transfection of CNT2(M)-specific siRNAs into M5076 cells resulted in a reduction in the Na(+)-dependent uptake of [(3)H]uridine by only 23%, although the expression of CNT2(M) mRNA and Na(+)-dependent uptake of [(3)H]cytidine disappeared in the cells. The uptake of pirarubicin (THP), an anthracycline, by M5076 cells requiring extracellular Na(+) was significantly inhibited by 500 microM uridine, but not 500 microM cytidine. The Na(+)-dependent and cytidine-insensitive uptake of [(3)H]uridine and the that of THP by M5076 cells significantly increased on cotreatment with both cholate and taurocholate, and the enhancement of THP uptake by the bile acids was reversed by cotreatment with 500 microM uridine. Furthermore, the cytotoxicity of THP and doxorubicin, which were previously reported to be taken up via the same transporter, toward M5076 cells was enhanced by cotreatment with both the bile acids. Therefore, it was indicated that an unidentified Na(+)-dependent transport system for nucleosides is expressed by M5076 cells, and contributes to the uptake and cytotoxicity of the anthracyclines.     

Evaluation of an Immortalized Retinal Endothelial Cell Line as an In Vitro Model for Drug Transport Studies Across the Blood-Retinal Barrier

Purpose. To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies. Methods. TR-iBRB cells were grown on permeable membrane filters. The effect of coating material on cell growth was investigated. Transport of [¹⁴C]-3-O-methyl-D-glucose (3-OMG), AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol, rhodamine 123, and sotalol was measured across the cultured cell layer to determine the apparent permeability coefficients (P_app). Rhodamine 123 uptake into these cells in the presence of these test compounds was evaluated. Western blot was performed to detect the efflux transporter P-glycoprotein (P-gp). Bidirectional transport in MDR1-MDCK cell monolayers overexpressing the human P-gp was measured for AGN 197075. Results. TR-iBRB cells form confluent cell layers when grown on fibronectin-coated membrane and exhibit characteristic spindle-shaped morphology. A good correlation between P_app and cLogD (pH 7.4) of the compounds tested was observed, except for 3-OMG, AGN 197075, and rhodamine 123, which are substrates of carrier-mediated transport systems such as P-gp and a glucose transporter (GLUT1). When grown on permeable membrane, TR-iBRB cells expressed functional P-gp and GLUT1. Conclusions. TR-iBRB cells, when grown on permeable membrane, provide a useful tool for predicting permeability across the BRB. The usefulness of this model for high-throughput screening and rank ordering of drug candidates intended for the back of the eye in treatment of ocular diseases needs further characterization upon correlation with in vivo data.     

腹腔注射L-丝氨酸后大鼠梗死侧脑皮质L-丝氨酸与D-丝氨酸含量的测定

目的:制作大鼠永久性脑梗死模型,腹腔注射L-丝氨酸,测定梗死侧大脑皮质L-丝氨酸与D-丝氨酸含量的变化,为应用L-丝氨酸辅助治疗脑梗死提供方法学上的依据。方法:利用邻苯二甲醛及N-乙酰半胱氨酸柱前衍生,高效液相色谱荧光检测法测定脑组织中L-丝氨酸与D-丝氨酸含量。结果:流动相A为82%0.1 mol·L-1磷酸盐缓冲液,流动相B为18%甲醇条件下,L-丝氨酸与D-丝氨酸得到很好的分离;腹腔注射L-丝氨酸1 h后,梗死侧脑皮质L-丝氨酸含量迅速增加,2 h即达高峰[(6.85±0.30)μmol·g-1],为正常脑组织的4倍以上,12 h时仍是正常的2倍以上水平[(3.56±0.22)vs(1.52±0.04)μmol·g-1];D-丝氨酸水平升高缓慢,升高幅度低,6 h时达高峰,为正常的2倍多[(0.75±0.05)vs(0.32±0.01)μmol·g-1]。结论:腹腔注射L-丝氨酸能快速升高梗死侧大脑皮质L-丝氨酸水平,对D-丝氨酸影响相对较小。