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.     

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.     

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.     

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.     

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.     

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.     

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.     

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.     

Influence of neuroglial transport on the action of gamma-aminobutyric acid on mammalian ganglion cells.

1 The effect of inhibiting the transport of gamma-aminobutyric acid (GABA) by neuroglial cells on the depolarizing action of exogenous amino acids on isolated superior cervical ganglia of the rat was studied. 1 Transport (measured by uptake of [3H]-GABA) was inhibited by (a) reducing external [na+] from 143 to 2mM and (b) administering alternative carrier-sbustrates, 3-amino-n-butyric acid (beta-amino-butyric acid, BABA) and (+/-)-nipecotic acid at a concentration of 1 mM. 3 All three procedures enhanced the depolarization produced by low concentrations of GABA (less than or equal to 10 muM) but did not alter the maximum response, nor the response to 3-aminopropanesulphonic acid (a gabamimetic with low affinity for the neuroglial carrier). 4 It is concluded that the neuroglial uptake process can limit the action of exogenous GABA upon neurones, by reducing the interstitial GABA concentration.     

Transmembrane domains I and II of the gamma-aminobutyric acid transporter GAT-4 contain molecular determinants of substrate specificity

The sodium- and chloride-dependent GABA transporters GABA transporter (GAT) 1 to 4 in the central nervous system enable efficient synaptic transmission by removing the neurotransmitter from the cleft. Taurine interacts only weakly with the GABA transporter GAT-4 (IC50 approximately 1.6 mM). Glutamate-61 is located in the conserved transmembrane domain I of GAT-4, whereas in the related taurine-transporter taurine transporter (TAUT), glycine occupies the equivalent position. [3H]GABA uptake by the GAT-4 E61G mutant becomes markedly more sensitive to inhibition by taurine (IC50 approximately 0.26 mM). Replacement of cysteine-94, located in the conserved transmembrane domain II of GAT-4, to its TAUT counterpart serine, results only in a modest increase in the ability of taurine to inhibit GABA uptake. However, introduction of glycine at this position decreases the IC50 for taurine by approximately 8-fold (IC50 approximately 0.20 mM). The inhibitory potency of taurine is inversely correlated with the volume of the side chain of the amino acid residue introduced at positions 61 and 94. It is striking that the IC50 for taurine of the E61G/C94G double mutant is decreased by approximately 35-fold (IC50 approximately 0.05 mM), and this inhibition of GABA transport is competitive. Changes in the inhibitory potency of the mutants described are also observed with beta-ala-nine and GABA, although they are much less pronounced. Our results suggest that determinants on transmembrane domains I and II can influence the specificity of the substrate binding pocket. The size of the side chain at positions 61 and 94 seems to determine the ability of substrate and substrate analogs to interact with the transporter.     

Influx and efflux transport of H1-antagonist epinastine across the blood-brain barrier.

We investigated influx and efflux transporters involved in blood-brain barrier transport of the nonsedative H1-antagonist epinastine. The basal-to-apical transport of [14C]epinastine was markedly higher than that in the opposite direction in LLC-GA5-COL150 cells stably transfected with human multidrug resistance (MDR)1 gene. The brain-to-plasma concentration ratio of [14C]epinastine in mdr1a/b(-/-) mice was 3.2 times higher than that in wild-type mice. The uptake of both [3H]mepyramine and [14C]epinastine into immortalized rat brain capillary endothelial cells (RBEC)1 showed temperature and concentration dependence. The kinetic parameters, K(m), V(max), and uptake clearance (V(max)/K(m)), of the initial uptake of [3H]mepyramine and [14C]epinastine by RBEC1 were 150 microM, 41.8 nmol/min/mg protein, and 279 microl/min/mg protein for mepyramine and 10.0 mM, 339 nmol/min/mg protein, and 33.9 microl/min/mg protein for epinastine, respectively. The uptake of [3H]mepyramine and [14C]epinastine by RBEC1 was inhibited by organic cations such as quinidine, amantadine, and verapamil, but not by other organic cations, tetraethyl ammonium, guanidine, and carnitine. Organic anions such as benzoic acid, estrone-3-sulfate, taurocholate, and neutral digoxin were not inhibitory. Furthermore, some cationic H1 antagonists (chlorpheniramine, cyproheptadine, ketotifen, and desloratadine) inhibited the [3H]mepyramine and [14C]epinastine uptake into RBEC1. In conclusion, the present study demonstrated that the combination of efficient efflux transport by P-glycoprotein and poor uptake by the influx transporter, which is identical with that responsible for the uptake of mepyramine, account for the low brain distribution of epinastine.     

Gamma-Aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers

1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.     

GABAB autoreceptors in rat cortex synaptosomes: response under different depolarizing and ionic conditions

Rat cerebral cortex synaptosomes prelabeled with [3H]gamma-aminobutyric acid [( 3H]GABA) were exposed in superfusion to various concentrations of KCl (9-50 mM). The evoked release of [3H]GABA reached a plateau at about 35 mM KCl. The K+-induced release was Ca2+-dependent, particularly at the lowest K+ concentrations. The GABAB agonist (-)-baclofen concentration dependently inhibited the release of [3H]GABA evoked by K+; this effect decreased with increasing K+ concentration and disappeared at 35 mM KCl. The GABAA agonist muscimol (1-100 microM) was totally ineffective to inhibit the release of [3H]GABA. Veratrine (1-30 microM) induced the release of [3H]GABA and the effect was tetrodotoxin-sensitive. (-)-Baclofen, but not muscimol, decreased the veratrine-induced [3H]GABA release; the GABAB agonist was particularly effective in presence of low concentrations of veratrine (1-3 microM) but the effect disappeared when 30 microM of the alkaloid was used. The inhibitory effect of (-)-baclofen on the release of [3H]GABA evoked by 15 mM KCl was dependent on the concentration of Ca2+: the effect increased as the concentration of Ca2+ was raised, reaching a plateau at 0.6 mM Ca2+. Exogenous GABA, in presence of the GABA uptake blocker SK & F 89976A, inhibited the release of [3H]GABA evoked by K+; this effect was antagonized by phaclofen. The data support the idea that terminal GABA autoreceptors in the rat cerebral cortex are of the GABAB type.     

Tolbutamide uptake via pH- and membrane-potential-dependent transport mechanism in mouse brain capillary endothelial cell line

The purpose of this study was to investigate the transport mechanism of tolbutamide across the blood-brain barrier (BBB) using MBEC4 cells as an in vitro BBB model. METHODS: The BBB transport of tolbutamide was studied by using a mouse brain capillary endothelial cell line, MBEC4, cultured on dishes with their luminal membrane facing the culture medium. RESULTS: The uptake of [14C]tolbutamide by MBEC4 cells was dependent on temperature and energy. The uptake coefficient of [14C]tolbutamide increased markedly with decreasing pH of the external medium from neutral to acidic. Valinomycin and replacement of chloride with sulfate or gluconate significantly increased the initial uptake of [14C]tolbutamide, while replacement with nitrate significantly decreased it. The uptake was significantly reduced by a proton ionophore, FCCP, and an anion-exchange inhibitor, DIDS. The initial uptake of [14C]tolbutamide was saturable with Kt of 0.61+/-0.03 mM (pH 7.4) and 1.76+/-0.19 mM (pH 6.5). At pH 6.5, the initial uptake of [14C]tolbutamide was significantly reduced by several sulfa drugs, salicylic acid, valproic acid and probenecid, and was competitively inhibited by sulfaphenazole (Ki=3.47+/-0.50 mM) and valproic acid (Ki=2.29+/-0.43 mM). CONCLUSION: These observations indicate the existence of a pH- and membrane-potential-dependent anion exchange and/or proton-cotransport system(s) for concentrative uptake of tolbutamide and sulfa drugs in MBEC4 cells.