Abundant Expression of OCT2, MATE1, OAT1, OAT3, PEPT2, BCRP, MDR1, and xCT Transporters in Blood-Arachnoid Barrier of Pig and Polarized Localizations at CSF- and Blood-Facing Plasma Membranes.

The physiologic and pharmacologic roles of the blood-arachnoid barrier (BAB) remain unclear. Therefore, the purpose of the present study was to comprehensively evaluate and compare the absolute protein expression levels of transporters in the leptomeninges and plexus per cerebrum, and to determine the localizations of transporters at the cerebrospinal fluid (CSF)-facing and blood (dura)-facing plasma membranes of the BAB in pig. Using multidrug resistance protein 1 (MDR1) and organic anion transporter (OAT) 1 as blood (dura)-facing and CSF-facing plasma membrane marker proteins, respectively, we established that breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP) 4, organic anion-transporting polypeptide (OATP) 2B1, multidrug and toxin extrusion protein 1 (MATE1), and glucose transporter 1 (GLUT1) are localized at the blood-facing plasma membrane, and OAT3, peptide transporter (PEPT) 2, MRP3, organic cation transporter (OCT) 2, xCT, monocarboxylate transporter (MCT) 1, MCT4, and MCT8 are localized at the CSF-facing plasma membrane of the BAB. The absolute protein expression levels of OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc in the whole BAB surrounding the entire cerebrum were much larger than those in the total of the choroid plexuses forming the blood-cerebrospinal fluid barrier (BCSFB). Although MRP4, OATP2B1, MCT8, GLUT1, and MCT1 were also statistically significantly more abundant in the BAB than in the choroid plexuses per porcine cerebrum, these transporters were nevertheless almost equally distributed between the two barriers. In contrast, OATP1A2, MRP1, OATP3A1, and OCTN2 were specifically expressed in the choroid plexus. These results should be helpful in understanding the relative overall importance of transport at the BAB compared with that at the BCSFB, as well as the rank order of transport capacities among different transporters at the BAB, and the directions of transport mediated by individual transporters. SIGNIFICANCE STATEMENT: We found that BCRP, MRP4, OATP2B1, MATE1, and GLUT1 localize at the blood-facing plasma membrane of the blood-arachnoid barrier (BAB), while OAT3, PEPT2, MRP3, OCT2, xCT, MCT1, MCT4, and MCT8 localize at the CSF-facing plasma membrane. 4F2hc is expressed in both membranes. For OAT1, OAT3, MDR1, BCRP, PEPT2, xCT, MATE1, OCT2, and 4f2hc, the absolute protein expression levels in the whole BAB surrounding the entire cerebrum are much greater than the total amounts in the choroid plexuses.

Comparison of Absolute Protein Abundances of Transporters and Receptors among Blood-Brain Barriers at Different Cerebral Regions and the Blood-Spinal Cord Barrier in Humans and Rats.

This work was designed to clarify the absolute abundances of transporters and receptors at different cerebral regions of the blood-brain barriers (BBB) and blood-spinal cord barrier (BSCB) in humans and rats, using physiologically relevant units (pmol/g tissue and fmol/cm2); 39 and 29 proteins including tight-junction proteins and markers were quantified in human and rat capillary samples, respectively. Protein expression levels of almost all proteins were identical within a 2-fold range between BBB and BSCB in rats, while many proteins showed >2-fold smaller expression levels in BSCB than BBB in humans. Protein expression levels of transporters and receptors in humans were remarkably smaller than those in rats in both BBB and BSCB in units of pmol/g tissue and fmol/cm2. Protein expression levels (fmol/cm2) of MDR1 and BCRP at the BBB in humans were 9.88-fold and 5.23-fold smaller than those in rats, respectively. GLUT1 expression (pmol/g tissue) at cortical BBB in a human was 2.49- and 3.76-fold greater than that at white matter BBB and BSCB, respectively. INSR and LRP1 proteins were detected at cortical BBB, but not at white matter BBB or BSCB in humans. These findings throw light on regional differences and species differences in pharmacokinetics and physiological functions in the central nervous system.

Histamine elimination from the cerebrospinal fluid across the blood-cerebrospinal fluid barrier: involvement of plasma membrane monoamine transporter (PMAT/SLC29A4).

The elimination of histamine, an excitatory neurotransmitter, from the brain/CSF across the blood-brain barrier and blood-CSF barrier (BCSFB) was investigated using Wistar rats, which were anesthetized with pentobarbital sodium. An in vivo intracerebral microinjection study suggested that there was only partial efflux of [3 H]histamine from the rat brain across the blood-brain barrier. The [3 H]histamine elimination clearance from the rat CSF was 3.8-fold greater than that of a CSF bulk flow marker, and the elimination of [3 H]histamine was significantly inhibited by the co-administration of unlabeled histamine, suggesting the involvement of a carrier-mediated process in histamine elimination from the CSF. The uptake study of [3 H]histamine by the isolated rat choroid plexus revealed histamine elimination from the CSF across the BCSFB. The [3 H]histamine uptake by TR-CSFB3 cells, a model cell line for the BCSFB, exhibited temperature-dependent and saturable kinetics, suggesting the involvement of carrier-mediated transport of histamine at the BCSFB. In the inhibition study, [3 H]histamine uptake by TR-CSFB3 cells was significantly inhibited by substrates and/or inhibitors of plasma membrane monoamine transporter (PMAT/SLC29A4), but not affected by substrates of organic cation transporters. These results suggest the elimination of histamine from the CSF via plasma membrane monoamine transporter at the BCSFB.

Lysosomal Trapping Is Present in Retinal Capillary Endothelial Cells: Insight into Its Influence on Cationic Drug Transport at the Inner Blood-Retinal Barrier.

Lysosomal trapping was investigated in the retinal capillary endothelial cells that are responsible for the inner blood-retinal barrier (BRB) using LysoTracker(®) Red (LTR). Using confocal microscopy on TR-iBRB2 cells, an in vitro model of the inner BRB, the presence of lysosomal trapping in retinal capillary endothelial cells was suggested since TR-iBRB2 cells exhibited punctuate intracellular localization of LTR that was attenuated by NH4Cl treatment. The study confirmed that LTR uptake by retinal capillary endothelial cells took place in a time- and temperature-dependent manner, and exhibited the 1.58-fold greater uptake at pH 8.4 than that at pH 7.4 while there was no change in uptake between pH 6.4 and pH 7.4, suggesting that passive diffusion is not enough to explain LTR uptake. The inhibition study showed the possible influence of lysosomal trapping on cationic drug transport by retinal capillary endothelial cells since LTR uptake was significantly inhibited by cationic amphiphilic drugs. Inhibition profiling and the estimation of IC50 suggested the influence of lysosomal trapping on propranolol and low-affinity pyrilamine transport while lysosomal trapping had only a partial effect on verapamil, clonidine, nicotine and high-affinity pyrilamine transport in retinal capillary endothelial cells.

Excitatory Amino Acid Transporter 1-Mediated L-Glutamate Transport at the Inner Blood-Retinal Barrier: Possible Role in L-Glutamate Elimination from the Retina.

The purpose of this study was to elucidate the transport mechanism(s) of L-glutamate (L-Glu), a neuroexcitatory neurotransmitter, in the inner blood-retinal barrier (BRB). The L-Glu transport was evaluated by an in vitro uptake study with a conditionally-immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells. L-Glu uptake by TR-iBRB2 exhibited time- and concentration-dependence, and was composed of high- and low-affinity processes with Michaelis-Menten constants (Km) of 19.3 µM and 275 µM, respectively. Under Na(+)-free conditions, L-Glu uptake by TR-iBRB2 involved one-saturable kinetics with a Km of 190 µM, which is similar to that of the low-affinity process of L-Glu uptake under normal conditions. Moreover, substrates/inhibitors of system Xc(-), which is involved in blood-to-retina transport of compounds across the inner BRB, strongly inhibited the L-Glu uptake under Na(+)-free conditions, suggesting that Na(+)-independent low-affinity L-Glu transport at the inner BRB is carried out by system Xc(-). Regarding the Na(+)-dependent high affinity process of L-Glu transport at the inner BRB, L-Glu uptake by TR-iBRB2 under normal conditions was significantly inhibited by substrates/inhibitors of excitatory amino acid transporter (EAAT) 1-5, but not alanine-serine-cysteine transporters. Reverse-transcription polymerase chain reaction (RT-PCR) analysis and immunoblot analysis demonstrated that mRNA and protein of EAAT1 are expressed in TR-iBRB2 cells, whereas mRNAs and/or proteins of EAAT2-5 are not. Immunohistochemical analysis revealed that EAAT1 protein is localized on the abluminal membrane of the retinal capillaries. In conclusion, EAAT1 most likely mediates Na(+)-dependent high-affinity L-Glu transport at the inner BRB and appears to take part in L-Glu elimination from the retina across the inner BRB.

Β-alanine and l-histidine transport across the inner blood-retinal barrier: potential involvement in L-carnosine supply.

The supply of L-carnosine, a bioactive dipeptide of ß-alanine and l-histidine, to the retina across the blood-retinal barrier (BRB) was studied. The in vivo and in vitro studies revealed low uptake activities for [(3)H]Gly-Sar, a representative dipeptide, suggesting that l-carnosine transport plays only a minor role at the BRB. The in vivo study using rats showed approximately 18- and 23-fold greater retinal uptake indexes (RUI) for [(3)H]ß-alanine and [(3)H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [(3)H]ß-alanine was taurine- and γ-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [(3)H]ß-alanine uptake, suggesting that a carrier-mediated process was involved in ß-alanine transport across the inner BRB. [(3)H]ß-Alanine uptake was inhibited by taurine and ß-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of ß-alanine across the inner BRB. Regarding l-histidine, the l-leucine-sensitive RUI of [(3)H]l-histidine was identified, and the in vitro [(3)H]l-histidine uptake by TR-iBRB2 cells suggested that a carrier-mediated process was involved in l-histidine transport across the inner BRB. The inhibition profile suggested that L-type amino acid transporter (LAT1/SLC7A5) is responsible for the influx transport of l-histidine across the inner BRB. These results show that the influx transports of ß-alanine and l-histidine across the inner BRB is carried out by TAUT and LAT1, respectively, suggesting that the retinal l-carnosine is supplied by enzymatic synthesis from two kinds of amino acids transported across the inner BRB.

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).