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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Inner blood-retinal barrier mediates l-isomer-predominant transport of serine

D-serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the blood-to-retina transport of [(3) H]L-serine with an influx clearance of 49.9 μL/(min·g retina), which is greater than that of [(3) H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [(3) H]L-Serine and [(3) H]D-serine uptake by TR-iBRB2 cells took place in an Na(+)-dependent and a concentration-dependent manner with Michaelis constant values of 97.5 μM and 9.63 mM, respectively. The uptake process of [(3) H]L-serine and [(3) H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina.     

A peptide prodrug approach for improving bisphosphonate oral absorption

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. (14)C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[(3)H]Phe-[(14)C]pamidronate, and Pro-[(3)H]Phe-[(14)C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (F(TIBIA)) and 1.9 (F(URINE)) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.     

Carrier-Mediated Transport of H1-Antagonist at the Blood-Brain Barrier: A Common Transport System of H1-Antagonists and Lipophilic Basic Drugs

The blood-brain barrier (BBB) transport system for H₁ antagonists was studied using primary cultured bovine brain capillary endothelial cells (BCEC). The uptake of [³H]mepyramine was inhibited by various H₁-antagonists. Ketotifen competitively inhibited [³H]mepyramine uptake with an inhibition constant (K_i ) of 46.8 µM. Lipophilic basic drugs such as propranolol, lidocaine and imipramine significantly inhibited [³H]mepyramine uptake. In particular, propranolol inhibited [³H]mepyramine uptake competitively at an inhibition constant (K_i) of 51.1 µM. Moreover, in ATP-depleted BCEC, [³H]mepyramine uptake was stimulated by preloading with H₁- antagonists and lipophilic basic drugs. These results indicated that H₁-antagonists are transported across the BBB via a carrier-mediated transport system common to lipophilic basic drugs.     

Blood-brain-barrier Transport of Lipid Microspheres Containing Clinprost,a Prostaglandin I2 Analogue

Because the permeability of the blood-brain barrier to lipid microspheres (LMs) has not hitherto been demonstrated, blood-brain-barrier permeability to LM containing the prostaglandin I₂ analogue clinprost has been evaluated for an in-vitro system of primary cultured monolayers of bovine brain capillary endothelial cells (BCECs), by a capillary depletion study in rats and by an in-situ brain perfusion study in normal and 4-vessel-occluded fore brain ischaemic rats. Although energy-dependency was not observed in [³H]clinprost uptake by BCECs, in accordance with results for simple diffusional transport, uptake of [³H]clinprost contained in lipid microspheres (denoted [³H]clinprost(LM)) was significantly inhibited by the endocytosis inhibitor, dansylcadaverine. The transport of LM into BCECs by endocytosis was also confirmed by fluorescence microscopy and flow-cytometric analysis using LM labelled with a fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). The absolute uptake of DiI(LM) by BCECs, measured by HPLC, was, however, almost 1/10 that of [³H]clinprost(LM), results which suggest the superiority of simple diffusion of clinprost over endocytosis of its LM form in the uptake of clinprost(LM) by BCECs. In the capillary-depletion study with rat-brain-perfused [³H]clinprost(LM) from the internal carotid artery, the parenchyma apparent distribution volume was about 45 times larger than that of the capillary, showing that [³H]clinprost(LM) was transported through the blood-brain barrier into the brain. The permeability coefficients of [³H]clinprost and [³H]clinprost(LM) determined by in-situ brain perfusion in normal rats were considerably higher than those of the active metabolite [³H]isocarbacyclin and its LM form. In addition, the Blood-brain-barrier permeabilities to [³H]clinprost, [³H]isocarbacyclin and their LM forms in ischaemic rats were almost identical to those in normal rats. It was concluded that clinprost(LM) was transported through the blood-brain barrier by endocytosis of LM, simple diffusion of clinprost released from LM, and transport of isocarbacyclin generated by hydrolysis of clinprost. The blood-brain-barrier permeability of clinprost(LM) is not reduced in ischaemic conditions, because the simple diffusion of clinprost released from LM contributed mainly to clinprost(LM) transport.     

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).     

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.

     

Effects of pyroglutamic acid on corticostriatal glutamatergic transmission

The effects of l-pyroglutamic acid, a molecule structurally derived from l-glutamic acid (Glu), were measured on the high affinity of uptake of glutamic acid from striatal synaptosomes of the rat and on the binding of [l-³H]glutamic acid to striatal membranes. The results showed a competitive inhibition of the high affinity transport of glutamic acid by l-pyroglutamic acid in vitro with no effect on the uptake of γ-aminobutyric acid (GABA). An inhibition of the binding of [l-³H]glutamic acid to striatal membranes was also detected. Significant high affinity uptake of [l-³H]pyroglutamic acid was evident in synaptosomes from the striatum. A regional distribution study of the uptake processes for [l-³H]glutamic acid and [l-³H]pyroglutamic acid in different areas of the brain showed a similar distribution, suggesting that an uptake of [l-³H]pyroglutamic acid, although weak, occurs in glutamatergic nerve terminals. This proposal was further reinforced by measuring the effects of a large cortical lesion involving frontal and parietal areas on the uptake of [l-³H]glutamic acid and [l-³H]pyroglutamic acid in synaptosomes from the striatum. The results showed a large decrease in the uptake processes of both labelled molecules showing that the uptake of [l-³H]pyroglutamic acid, as for glutamic acid mainly occurred in corticostriatal nerve terminals, although other uptake sites are not excluded.     

Carrier-Mediated Transport of H1-Antagonist at the Blood–Brain Barrier: Mepyramine Uptake into Bovine Brain Capillary Endothelial Cells in Primary Monolayer Cultures

The transport mechanism of the H₁antagonist mepyramine at the blood-brain barrier (BBB) was studied by using primary cultured monolayers of bovine brain capillary endothelial cells (BCEC). The initial uptake of [³H]mepyramine into the BCEC showed strong temperature and concentration dependency, indicating that it involves both saturable and nonsaturable processes. Transport at the luminal membrane may be the rate-limiting process in the transcellular transport, since the values of the uptake coefficient of [³H]mepyramine at the luminal membrane (609 µl/mg protein/min) and the transcellular permeability coefficient (488 µl/mg protein/min) are very similar. The initial uptake of [³H]mepyramine was not affected by metabolic inhibitors, but was stimulated by preloading with the drug. Mepyramine appears to be transported into the BCEC by a carrier-mediated transport system which does not require metabolic energy, probably via a facilitated diffusion mechanism.     

Suppression of viability and acetyl-LDL metabolism in RAW 264 macrophage-like and smooth muscle cells by bisphosphonates in vitro

Etidronate and clodronate are bisphosphonates that inhibit the development of experimental atherosclerosis. Etidronate decreases the intimamedia thickness of carotid artery even in man. Liposome-encapsulated bisphosphonates inhibit the cellular metabolism of atherogenic, modified low-density lipoprotein (acetyl-LDL) by cultured macrophages. In the present study, the effects of new bisphosphonate tiludronate and nitrogen-containing bisphosphonate alendronate on cell viability and cellular uptake and degradation of acetyl-LDL were investigated in vitro with macrophages and arterial smooth muscle cells, which have a significant role in atherogenesis. Tiludronate and alendronate decreased the viability of RAW 264 macrophages at high concentration (1,000 microM; p < 0.05), while liposome-encapsulated drugs suppressed the viability at concentrations of 30-300 microM. At concentrations greater than or equal to 10 microM, tiludronate and alendronate inhibited the uptake and degradation of acetyl-LDL by RAW 264 cells in a concentration-dependent manner (p < 0.001). None of the bisphosphonates affected the viability of smooth muscle cells, and none but alendronate at a high concentration (1,000 microM) inhibited the uptake and degradation of acetyl-LDL by smooth muscle cells. The results show that tiludronate and alendronate inhibit the atherogenic activity of macrophages in vitro, as shown previously with etidronate and clodronate, providing further evidence for the antiatherogenic effects of bisphosphonates.