Search

Ribavirin, an antiretroviral agent targeting the hepatitis C virus, causes male reproductive toxicity. This study investigated the mechanism of ribavirin transport at the blood-testis barrier (BTB). In vivo mouse integration plot analysis after intravenous administration revealed that the net influx clearance of [ 3 H]ribavirin in the testis was 3.6-fold greater than that of [ 14 C]D-mannitol, a paracellular transport marker, implying transcellular transport of ribavirin across the BTB. Moreover, [ 3 H]ribavirin uptake by TM4 cells, mouse-derived Sertoli cells, was time- and concentration-dependent, with a K m value of 2.49 mM. S-[(4-nitrophenyl)methyl]-6-thioinosine, an inhibitor of Na + -independent equilibrative nucleoside transporters (ENTs), strongly inhibited the [ 3 H]ribavirin uptake by TM4 cells at 100 µM. Compared to the uptake of [ 3 H]adenosine, a typical endogenous nucleoside, [ 3 H]ribavirin uptake was relatively similar to ENT2 transport. [ 3 H]Ribavirin uptake was also observed in mouse ENT2-expressing Xenopus laevis oocytes, and gene silencing via the transfection of ENT2 small interfering RNA significantly reduced the [ 3 H]ribavirin transport into TM4 cells by 13%. Taken together, these results suggest that ENT2 partially contributes to ribavirin transport at the BTB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cerebral creatine deficiency syndromes (CCDS) are neurodevelopmental disorders caused by a decrease in creatine levels in the central nervous system (CNS) due to functional mutations in creatine synthetic enzymes or creatine transporter (CRT/SLC6A8). Although SLC6A8 mutations have been reported to be the most frequent cause of CCDS, sufficient treatment for patients with CCDS harboring SLC6A8 mutations has not yet been achieved. This study aimed to elucidate the molecular mechanism of SLC6A8 dysfunction caused by the c. 1699T > C missense mutation, which is thought to induce dysfunction through an unidentified mechanism. A study on SLC6A8-expressing oocytes showed that the c.1699T > C mutation decreased creatine uptake compared to that in wild-type (WT) oocytes. In addition, a kinetics study of creatine uptake revealed that the c.1699T > C mutation reduced the maximum uptake rate but not Michaelis-Menten constant. In contrast, the c.1699T > C mutation did not attenuate SLC6A8 protein levels or alter its cellular localization. Based on the SLC6A8 structure in the AlphaFold protein structure database, it is possible that the c.1699T > C mutation alters the interaction between the S567 and Y143 residues of SLC6A8, leading to decreased creatine transport function. These findings contribute to the understanding of the pathology of CCDS and to the development of strategies for CCDS treatment.

Lysosomal trapping, a physicochemical process in which lipophilic cationic compounds are sequestered in lysosomes, can affect drug disposition and cytotoxicity. To better understand lysosomal trapping at the outer blood-retinal barrier (BRB), we investigated the distribution of LysoTracker Red (LTR), a probe compound for lysosomal trapping, in conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. LTR uptake by RPE-J cells was dependent on temperature and attenuated by ammonium chloride and protonophore, which decreased the pH gradient between the lysosome and cytoplasm, suggesting lysosomal trapping of LTR in RPE-J cells. The involvement of lysosomal trapping in response to cationic drugs, including neuroprotectants such as desipramine and memantine, was also suggested by an inhibition study of LTR uptake. Chloroquine, which is known to show ocular toxicity, induced cytoplasmic vacuolization in RPE-J cells with a half-maximal effective concentration of 1.35 μM. This value was 59 times lower than the median lethal concentration (= 79.1 μM) of chloroquine, suggesting that vacuolization was not a direct trigger of cell death. These results are helpful for understanding the lysosomal trapping of cationic drugs, which is associated with drug disposition and cytotoxicity in the outer BRB., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Japanese Society for the Study of Xenobiotics. All rights reserved.)

Putrescine is a bioactive polyamine. Its retinal concentration is strictly controlled to maintain a healthy sense of vision. The present study investigated putrescine transport at the blood-retinal barrier (BRB) to gain a better understanding of the mechanisms of putrescine regulation in the retina. Our microdialysis study showed that the elimination rate constant during the terminal phase was significantly greater (1.90-fold) than that of [ 14 C]D-mannitol, which is a bulk flow marker. The difference in the apparent elimination rate constants of [ 3 H]putrescine and [ 14 C]D-mannitol was significantly decreased by unlabeled putrescine and spermine, suggesting active putrescine transport from the retina to the blood across the BRB. Our study using model cell lines of the inner and outer BRB showed that [ 3 H]putrescine transport was time-, temperature-, and concentration-dependent, suggesting the involvement of carrier-mediated processes in putrescine transport at the inner and outer BRB. [ 3 H]Putrescine transport was significantly reduced under Na + -free, Cl - -free, and K + -replacement conditions, and attenuated by polyamines or organic cations such as choline, a choline transporter-like protein (CTL) substrate. Rat CTL1 cRNA-injected oocytes exhibited marked alterations in [ 3 H]putrescine uptake, and CTL1 knockdown significantly reduced [ 3 H]putrescine uptake in model cell lines, suggesting the possible participation of CTL1 in putrescine transport at the BRB.

Blood-to-retina transport across the inner blood-retinal barrier (BRB) is a key determinant of retinal drug concentration and pharmacological effect. Recently, we reported on the amantadine-sensitive drug transport system, which is different from well-characterized transporters, at the inner BRB. Since amantadine and its derivatives exhibit neuroprotective effects, it is expected that a detailed understanding of this transport system would lead to the efficient retinal delivery of these potential neuroprotective agents for the treatment of retinal diseases. The objective of this study was to characterize the structural features of compounds for the amantadine-sensitive transport system. Inhibition analysis conducted on a rat inner BRB model cell line indicated that the transport system strongly interacts with lipophilic amines, especially primary amines. In addition, lipophilic primary amines that have polar groups, such as hydroxy and carboxy groups, did not inhibit the amantadine transport system. Furthermore, certain types of primary amines with an adamantane skeleton or linear alkyl chain exhibited a competitive inhibition of amantadine uptake, suggesting that these compounds are potential substrates for the amantadine-sensitive drug transport system at the inner BRB. These results are helpful for producing the appropriate drug design to improve the blood-to-retina delivery of neuroprotective drugs.

At the inner blood-retinal barrier (BRB), P-glycoprotein (P-gp) contributes to maintaining the homeostasis of substance concentration in the retina by transporting drugs and exogenous toxins from the retina to the circulating blood. Under inflammatory conditions, P-gp activities have been reported to be altered in various tissues. The purpose of this study was to clarify the alterations in P-gp activity at the inner BRB due to lipopolysaccharide (LPS), an inflammatory agent, and the molecular mechanisms of the alterations induced by LPS. Ex vivo P-gp activity was evaluated as luminal accumulation of 7-nitro-2,1,3-benzoxadiazole-cyclosporin A (NBD-CSA), a fluorescent P-gp substrate, in freshly prepared rat retinal capillaries. The luminal NBD-CSA accumulation was significantly decreased in the presence of LPS, indicating that P-gp activity at the inner BRB is reduced by LPS. This LPS-induced attenuation of the luminal NBD-CSA accumulation was abolished by inhibiting toll-like receptor 4 (TLR4), a receptor for LPS. Furthermore, an inhibitor/antagonist of tumor necrosis factor receptor 1, endothelin B receptor, nitric oxide synthase, or protein kinase C (PKC) significantly restored the LPS-induced decrease in the luminal NBD-CSA accumulation. Consequently, it is suggested that the TLR4/PKC pathway is involved in the reduction in P-gp function in the inner BRB by LPS.

Conjugation of angiopep-2 (Ang2) with drugs/compounds is known to increase plasma membrane permeability across endothelial barriers. The inner blood-retinal barrier (BRB) regulates retinal drug distribution and is formed by retinal capillary endothelial cells, supported by Müller cells and retinal pericytes. To elucidate the potential of Ang2 conjugation in promoting retinal drug distribution after peripheral administration across the inner BRB, an in vivo administration study and in vitro transport experiments using newly developed multicellular inner BRB spheroids were performed. After intravenous administration of Ang2-linked green fluorescence protein (GFP-Ang2) in mice, GFP-derived signals were observed in the neural retina. In contrast, GFP-derived signals were not observed after intravenous GFP administration, suggesting the promotion of the retinal distribution of substances by Ang2 conjugation. To overcome the limitations of in vitro studies using cells cultured on dishes, inner BRB spheroids were established using conditionally immortalized rat retinal capillary endothelial cells, Müller cells, and retinal pericytes. Immunocytochemistry of marker molecules suggests that the central part of the spheroids is occupied by Müller cells, and encapsulated by retinal pericytes and capillary endothelial cells. Studies on the expression and functions of tight junctions suggest that tight junctions are formed on the surface of the inner BRB spheroids by retinal capillary endothelial cells. The functional expression of drug transporters, such as P-glycoprotein, was observed in the spheroids, implying that the inner side of the spheroids reflects the retinal side of the inner BRB. In the inner BRB spheroids, energy-dependent accumulation of GFP-Ang2 and Ang2-linked 5(6)-carboxyfluorescein (FAM-Ang2) was observed. Moreover, an endocytic inhibition study revealed that clathrin-dependent endocytosis/transcytosis was involved in the transcellular transport of Ang2-conjugated drugs/compounds across the inner BRB. Consequently, it is suggested that the Ang2 linkage is useful for promoting retinal drug distribution via clathrin-dependent transcytosis at the inner BRB., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

3'-Azido-3'-deoxythymidine (AZT), an antiretroviral drug, is often adopted in the therapy for human immunodeficiency virus (HIV) infection, and the characteristics of AZT transport at the blood-testis barrier (BTB) were investigated in this study. In the integration plot analysis that evaluates the transport activity in vivo, the apparent influx clearance of [ 3 H]AZT was significantly greater than that of [ 14 C]D-mannitol, a non-permeable paracellular transport marker. In the uptake study in vitro with TM4 cells derived from mouse Sertoli cells, [ 3 H]AZT uptake exhibited a time- and concentration-dependent manner, of which K m and V max values being 20.3 µM and 102 pmol/(min·mg protein), respectively. In the inhibition analysis, [ 3 H]AZT uptake was not affected by extracellular inorganics and some substrates of transporters putatively involved in AZT transport. In the further inhibition analyses to elucidate the characteristics of AZT transport, [ 3 H]AZT uptake was strongly reduced in the presence of several nucleosides, that are categorized as 2'-deoxynucleosides with pyrimidine, whereas little effect on [ 3 H]AZT uptake was exhibited in the presence of other nucleosides, nucleobases, and antiretrovirals. These results suggest the influx transport of AZT from the circulating blood to the testis, and the involvement of carrier-mediated process at the BTB, which selectively recognizes 2'-deoxynucleosides with a pyrimidine base., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Guanidinoacetate (GAA) induces epileptogenesis and neurotoxicity in the brain. As epileptic animal models have been reported to show elevated cerebral GAA levels, the processing mechanism of GAA in the brain is important for maintaining brain homeostasis. We have revealed that GAA in the cerebrospinal fluid (CSF) is removed by incorporation into the choroid plexus epithelial cells (CPxEpic), which form the blood-CSF barrier (BCSFB). However, the processing mechanism of GAA incorporated into CPxEpic remains unknown. We have reported that monocarboxylate transporter 12 (MCT12) functions as an efflux transporter of GAA and creatine, a metabolite of GAA, in the kidneys and liver. Therefore, we aimed to clarify the role of MCT12 in GAA dynamics in CPxEpic., Methods: Protein expression and localization in CPxEpic were evaluated using immunohistochemistry. Metabolic analysis was performed using high-performance liquid chromatography (HPLC) 24 h after the addition of [ 14 C]GAA to TR-CSFB3 cells, which are conditionally immortalized rat CPxEpic. The efflux transport of [ 14 C]creatine was evaluated in TR-CSFB3 cells after transfection with MCT12 small interfering RNA (siRNA). The CSF-to-brain parenchyma transfer of creatine was measured after intracerebroventricular injection in rats., Results: Immunohistochemical staining revealed that MCT12-derived signals merged with those of the marker protein at the apical membrane of CPxEpic, suggesting that MCT12 is localized on the apical membrane of CPxEpic. The expression levels of guanidinoacetate N-methyltransferase (GAMT), which catalyzes the conversion of GAA to creatine, in TR-CSFB3 cells was also indicated, and GAA was considered to be metabolized to creatine after influx transport into CPxEpic, after which creatine was released into the CSF. Creatine release from TR-CSFB3 cells decreased following MCT12 knockdown. The contribution ratio of MCT12 to the release of creatine was more than 50%. The clearance of CSF-to-brain parenchyma transfer of creatine was 4.65 µL/(min·g brain), suggesting that biosynthesized creatine in CPxEpic is released into the CSF and supplied to the brain parenchyma., Conclusions: In CPxEpic, GAA is metabolized to creatine via GAMT. Biosynthesized creatine is then released into the CSF via MCT12 and supplied to the brain parenchyma., (© 2022. The Author(s).)

Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3',5'-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Creatine (Cr) is needed to maintain high energy levels in cells. Since Cr plays reportedly a critical role in neurodevelopment and the immune system, Cr dynamics should be strictly regulated to control these physiological events. This review focuses on the role of transporters that recognize Cr and/or Cr precursors. Our previous studies revealed physiological roles of SLC6A and SLC16A family transporters in Cr dynamics. Creatine transporter (CRT/SLC6A8) contributes to the influx transport of Cr in Cr distribution. γ-Aminobutyric acid transporter 2 (GAT2/SLC6A13) mediates incorporation of guanidinoacetate (GAA), a Cr precursor, in the process of Cr biosynthesis. Monocarboxylate transporter 12 (MCT12/SLC16A12) functions as an efflux transporter for Cr and GAA, and contributes to the process of Cr biosynthesis. Accordingly, the SLC6A and SLC16A family of transporters play important roles in the process of Cr biosynthesis and distribution via permeation of Cr and Cr precursors across the plasma membrane., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: The present study aimed to elucidate the transport properties of imipramine and paroxetine, which are the antidepressants, across the blood-brain barrier (BBB) in rats., Methods: In vivo influx and efflux transport of imipramine and paroxetine across the BBB were tested using integration plot analysis and a combination of brain efflux index and brain slice uptake studies, respectively. Conditionally immortalized rat brain capillary endothelial cells, TR-BBB13 cells, were utilized to characterize imipramine and paroxetine transport at the BBB in vitro., Results: The in vivo influx clearance of [ 3 H]imipramine and [ 3 H]paroxetine in rats was determined to be 0.322 mL/(min·g brain) and 0.313 mL/(min·g brain), respectively. The efflux clearance of [ 3 H]imipramine and [ 3 H]paroxetine was 0.380 mL/(min·g brain) and 0.126 mL/(min·g brain), respectively. These results suggest that the net flux of paroxetine, but not imipramine, at the BBB in vivo was dominated by transport to the brain from the circulating blood. The uptake of imipramine and paroxetine by TR-BBB13 cells exhibited time- and temperature-dependence and one-saturable kinetics with a K m of 37.6 μM and 89.2 μM, respectively. In vitro uptake analyses of extracellular ion dependency and the effect of substrates/inhibitors for organic cation transporters and transport systems revealed minor contributions to known transporters and transport systems and the difference in transport properties in the BBB between imipramine and paroxetine., Conclusions: Our study showed the comprehensive outcomes of imipramine and paroxetine transport at the BBB, implying that molecular mechanism(s) distinct from previously reported transporters and transport systems are involved in the transport., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Taurine transport was investigated at the blood-testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [ 3 H]taurine (27.7 μL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [ 3 H]taurine uptake with a K m of 13.5 μM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [ 3 H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as β-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [ 3 H]taurine uptake revealed an IC 50 of 378 μM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [ 3 H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.

The total synthesis of two decahydroquinoline poison frog alkaloids ent- cis - 195A and cis - 211A were achieved in 16 steps (38% overall yield) and 19 steps (31% overall yield), respectively, starting from known compound 1 . Both alkaloids were synthesized from the common key intermediate 11 in a divergent fashion, and the absolute stereochemistry of natural cis - 211A was determined to be 2 R , 4a R , 5 R , 6 S , and 8a S . Interestingly, the absolute configuration of the parent decahydroquinoline nuclei of cis - 211A was the mirror image of that of cis - 195A , although both alkaloids were isolated from the same poison frog species, Oophaga ( Dendrobates ) pumilio , from Panama.

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 [ 3 H]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 ( K m in TR-iBRB2 cells, 79.4 µM; K m in RPE-J cells, 90.5 and 9830 µM). The inhibition of [ 3 H]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.

Creatine (Cr)/phosphocreatine has the ability to buffer the high-energy phosphate, thereby contributing to intracellular energy homeostasis. As Cr biosynthetic enzyme deficiency is reported to increase susceptibility to colitis under conditions of inflammatory stress, Cr is critical for maintaining intestinal homeostasis under inflammatory stress. Cr is mainly produced in the hepatocytes and then distributed to other organs of the body by the circulatory system. Since monocarboxylate transporter 9 (MCT9) and monocarboxylate transporter 12 (MCT12) have been reported to accept Cr as a substrate, these transporters are proposed as candidates for Cr efflux transporter in the liver. The aim of this study was to elucidate the transport mechanism on Cr supply from the hepatocytes. Immunohistochemical staining of the rat liver sections revealed that both MCT9 and MCT12 were localized on the sinusoidal membrane of the hepatocytes. In the transport studies using Xenopus laevis oocyte expression system, [ 14 C]Cr efflux from MCT9- or MCT12-expressing oocytes was significantly greater than that from water-injected oocytes. [ 14 C]Cr efflux from primary cultured hepatocytes was significantly decreased following MCT12 mRNA knockdown, whereas this efflux was not decreased after mRNA knockdown of MCT9. Based on the extent of MCT12 protein downregulation and Cr efflux after knockdown of MCT12 in primary cultured rat hepatocytes, the contribution ratio of MCT12 in Cr efflux was calculated as 76.4%. Our study suggests that MCT12 substantially contributes to the efflux of Cr at the sinusoidal membrane of the hepatocytes. NEW & NOTEWORTHY Our study is the first to identify the role of monocarboxylate transporter 12 (MCT12) as a transporter of creatine (Cr) in the liver. MCT12 was found to significantly contribute to the efflux of Cr on the sinusoidal membrane of the hepatocytes. Since hepatocytes are known to be involved in creatine biosynthesis, the present findings can be beneficial for the regulation of Cr biosynthesis and supply.

Purpose: In this study, we investigated in detail the transport of phenytoin across the blood-brain barrier (BBB) to identify the transporter(s) involved in BBB-mediated phenytoin efflux from the brain., Methods: We evaluated the brain-to-blood efflux transport of phenytoin in vivo by determining the brain efflux index (BEI) and uptake in brain slices. We additionally conducted brain perfusion experiments and BEI studies in P-glycoprotein (P-gp)-deficient mice. In addition, we determined the mRNA expression of monocarboxylate transporter (MCT) in isolated brain capillaries and performed phenytoin uptake studies in MCT-expressing Xenopus oocytes., Results: [ 14 C]Phenytoin brain efflux was time-dependent with a half-life of 17 min in rats and 31 min in mice. Intracerebral pre-administration of unlabeled phenytoin attenuated BBB-mediated phenytoin efflux transport, suggesting carrier-mediated phenytoin efflux transport across the BBB. Pre-administration of P-gp substrates in rats and genetic P-gp deficiency in mice did not affect BBB-mediated phenytoin efflux transport. In contrast, pre-administration of MCT8 inhibitors attenuated phenytoin efflux. Moreover, rat MCT8-expressing Xenopus oocytes exhibited [ 14 C]phenytoin uptake, which was inhibited by unlabeled phenytoin., Conclusion: Our data suggest that MCT8 at the BBB participates in phenytoin efflux transport from the brain to the blood.

Since the retina continuously receives light to enable vision, reactive oxygen species (ROS) are easily generated in neural retina. The oxidative stress induced by ROS may be involved in the onset and progression of blinding aging diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma. Although supply of antioxidants to the retina is important to maintain the redox homeostasis in neural retina, the blood-retinal barrier (BRB) is created by complex tight-junctions of retinal capillary endothelial cells and retinal pigment epithelial cells to prevent the free diffusion of substances. The BRB is equipped with several membrane transporters to supply nutrients and essential molecules including antioxidants and drugs which exhibit antiaging effect to the retina from the circulating blood. In this review, the transporter-mediated retinal distribution of key endogenous compounds and drugs, such as vitamin C, l-cystine and gabapentin, is introduced for antiaging of the retina.

Guanidinoacetate (GAA), which is a precursor of creatine, is mainly biosynthesized in the renal proximal tubular epithelial cells (RPTECs). Plasma concentration of GAA has been reported to be reduced in patients with monocarboxylate transporter 12 (MCT12) mutation (p.Q215X). However, the mechanism underlying GAA release from the RPTECs remains unclear. Therefore, to elucidate the role of MCT12 in renal GAA release, MCT12-mediated GAA transport was evaluated using the human and rat MCT12-expressing Xenopus laevis oocytes and primary-cultured rat RPTECs. [ 14 C]GAA uptake by the human and rat MCT12-expressing oocytes was significantly higher than that by the water-injected oocytes. Rat MCT12-mediated uptake of [ 14 C]GAA by the oocytes was found to be sodium ion (Na + )-independent and exhibited saturable kinetics with a Michaelis-Menten constant of 3.38 mM. Transport activities of rat MCT12 tend to increase along with increasing of extracellular pH. In addition, the efflux transport of [ 14 C]GAA from the human and rat MCT12-expressing oocytes was significantly higher than that from the water-injected oocytes. These results suggest that both the influx and efflux transport of GAA is mediated by MCT12. In the primary-cultured rat RPTECs, [ 14 C]GAA efflux transport was significantly reduced by the transfection of MCT12-specific siRNAs, suggesting that MCT12 participates in GAA efflux transport in rat RPTECs. Therefore, it suggests that MCT12 is involved in GAA release from RPTECs to the circulating blood, since MCT12 is known to be localized on the basal membrane of RPTECs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Disclosure The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The retinal pericytes contribute to the supply of collagen to the basement membrane, and thus, form the structural support of the blood-retinal barrier. Since l-proline (L-Pro) is a major component of collagen, the uptake of L-Pro is an important process for the synthesis of collagen. This study was aimed to elucidate L-Pro transport mechanism(s) in the retinal pericytes. The transport of [ 3 H]L-Pro was evaluated in the conditionally immortalized rat retinal pericyte cell line, TR-rPCT1 cells. The expression of the candidate transporter was examined by qualitative/quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunostaining. The [ 3 H]L-Pro uptake by TR-rPCT1 cells showed Na + -dependence, Cl - -independence, and concentration-dependence with a K m of 810 μM. The substrates for system A, such as 2-(methylamino)isobutyric acid (MeAIB), significantly inhibited the L-Pro uptake, suggesting the involvement of system A in the uptake of L-Pro. Among the subtypes of system A, the mRNA expression levels of ATA2 were the highest in TR-rPCT1 cells. Immunostaining analysis of the isolated rat retinal capillaries containing pericytes indicated the protein expression of ATA2 in retinal pericytes. In conclusion, it is suggested that ATA2, at least in part, is involved in the transport of L-Pro in the retinal pericytes., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Lysosomal trapping at the blood-retinal barrier (BRB) was investigated through quinacrine and fluorescence-labeled verapamil (EFV) uptake. Quinacrine uptake by conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) cells suggested saturable and non-saturable transport processes in the inner BRB. The reduction of quinacrine uptake by bafilomycin A1 suggested quinacrine distribution to the acidic intracellular compartments of the inner BRB, and this notion was also supported in confocal microscopy. In the study using the lysosome-enriched fraction of TR-iBRB2 cells, quinacrine uptake was inhibited by bafilomycin A1, suggesting the lysosomal trapping of quinacrine in the inner BRB. Pyrilamine, clonidine, and nicotine had no effect on quinacrine uptake, suggesting the minor role of lysosomal trapping in their transport across the inner BRB. Bafilomycin A1 had no effect on EFV uptake, and lysosomal trapping driven by the acidic interior pH was suggested as a minor mechanism for EFV transport in the inner BRB. The minor contribution of lysosomal trapping was supported by the difference in inhibitory profiles between EFV and quinacrine uptakes. Similar findings were observed in the outer BRB study with the fraction of conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. These results suggest the usefulness of lysosome-enriched fractions in studying lysosomal trapping at the BRB.

We evaluated the contribution of organic anion transporting polypeptide 2A1 (OATP2A1/SLCO2A1), a high-affinity carrier for prostaglandins (PGs), to the parturition process. At gestational day (GD) 15.5, OATP2A1 is co-localized with 15-hydroxy-PG dehydrogenase in the mouse placental junctional zone and facilitates PG degradation by delivering PGs to the cytoplasm. Slco2a1 (+/-) females mated with Slco2a1 (-/-) males frequently showed elevated circulating progesterone at GD18.5 and delayed parturition. Progesterone receptor inhibition by RU486 treatment at GD18.5 blocked the delay of parturition. In the junctional zone, PGE 2 stimulated placental lactogen II (PL-II) production, resulting in higher expression of PL-II in Slco2a1 (-/-) placenta at GD18.5. Indomethacin treatment at GD15.5 suppressed the PL-II overproduction at GD18.5 in Slco2a1 (-/-) embryo-bearing dams, which promoted progesterone withdrawal and corrected the delayed parturition. These results suggest that extracellular PGE 2 reduction by OATP2A1 at mid-pregnancy would be associated with progesterone withdrawal by suppressing PL-II production, triggering parturition onset., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pannexin (Px) and connexin (Cx) hemichannels mediate bidirectional membrane transport in response to various stimuli and are involved in drug efficacy and toxicity. The purpose of the present study was to clarify in detail the transport characteristics of Px1 and Cx32 hemichannels by establishing transport assay systems using human Px1- and P2RX7 receptor-overexpressing HEK293 cells (Px1/P2RX7/HEK293) and Cx32-overexpressing HEK293 cells (Cx32/HEK293), in which P2RX7 and an extracellular Ca 2+ -depleted condition serve as the opening trigger, respectively. Uptake of the cationic fluorescent dye propidium iodide (PI) was significantly increased in Px1/P2RX7/HEK293 cells compared to that in mock cells, whereas there was no significant uptake of the anionic fluorescent dye sulforhodamine 101 (SR101). Uptake of [ 3 H]cholesterol by Px1/P2RX7/HEK293 cells was significantly decreased, whereas that of [ 3 H]taurine was not, compared to mock cells. On the other hand, uptakes of PI and SR-101 by Cx32/HEK293 cells were both significantly increased compared to mock cells. The PI uptake by Cx32/HEK293 cells was significantly inhibited by thioacetamide, acetaminophen, and N-acetyl-p-benzoquinoneimine. Cellular uptake of [ 3 H]cholesterol was significantly increased in Cx32/HEK293 cells and that of [ 3 H]taurine was significantly decreased. These results support the idea that Px1 and Cx32 hemichannels have distinct substrate recognition specificities and transport directions., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) transport systems regulate the supply of nutrients, amino acids, vitamins, and hormones to the developing brain, as well as blocking the entry of xenobiotics and drugs. The purpose of this study was to clarify the developmental changes in the absolute protein expression levels of BBB transport-related proteins in developing rat brain capillaries, using quantitative targeted absolute proteomics (QTAP). The changing patterns of ATP-binding cassette (ABC) and solute carrier (SLC) transporters, receptors, and tight junction/adherence junction-related proteins were classified into 4 types: uphill (continuously increasing expression from postnatal day (P) 1 to P56), bell-shape/inverted bell-shape (increased/decreased expression from P1 to P14 followed by decreased/increased expression from P21 to P56), downhill (continuously decreasing expression from P1 to P56), and constant (no significant difference from P1 to P56). Proteins showing uphill-type expression included P-glycoprotein/Mdr1a/Abcb1, Mrp4/Abcc4, Bcrp/Abcg2, Glut1/Slc2a1, Oatp1c1/Slco1c1, FcRn, 4F2hc/Slc3a2, claudin-5, caveolin-1, Cd29/integrin β1. Those showing bell-shape/inverted bell-shape expression included Mct1/Slc16a1, Oat3/Slc22a8, Tfr1, Lrp1, and CD147. On the other hand, Cat1/Slc7a1 and Cd54/Icam-1 showed downhill expression, and Insr showed constant expression. These results suggest that the protein expression levels of transporters and receptors at the BBB change in various ways to meet the changing requirements of the developing brain., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Prostaglandin (PG) D 2 is a lipid mediator, and in the brain, overproduction of PGD 2 is reportedly involved in the progression and exacerbation of neuroinflammation. The objective of this study was to elucidate PGD 2 efflux transport, under normal and inflammatory conditions, across the blood-brain barrier (BBB), which is formed by brain capillaries. Elimination of [ 3 H]PGD 2 across the BBB of normal and lipopolysaccharide (LPS)-induced inflammatory rats was examined by the intracerebral microinjection technique. After intracerebral injection, the percentage of [ 3 H]PGD 2 remaining in the ipsilateral cerebrum decreased with time, with a half-life of 13 min. This [ 3 H]PGD 2 elimination across the BBB was significantly inhibited by the co-administration of unlabeled PGD 2 , which suggests carrier-mediated PGD 2 efflux transport at the BBB. In isolated rat brain capillaries, mRNA expression of organic anion transporter (Oat) 3, organic anion-transporting polypeptide (Oatp) 1a4, and multidrug resistance-associated protein (Mrp) 4 was observed. In addition, co-administration of substrates/inhibitors for Oat3, Oatp1a4, and/or Mrp4, such as benzylpenicillin and cefmetazole, reduced [ 3 H]PGD 2 elimination across the BBB. Data suggest that Oat3 and Mrp4, but not Oatp1a4 are involved in PGD 2 elimination across the BBB, as Oatp1a4-expressing Xenopus (X.) oocytes did not show the significant [ 3 H]PGD 2 uptake compared with water-injected X. oocytes. In LPS-treated rats, [ 3 H]PGD 2 elimination across the BBB and mRNA expression levels of Oat3 and Mrp4 were significantly decreased. Our data suggest that Oat3- and Mrp4-mediated PGD 2 elimination across the BBB is attenuated under inflammatory conditions.

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E 2 (PGE 2 ), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE 2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.

Dysregulation of blood-brain barrier (BBB) transport exacerbates brain damage in acute ischemic stroke. Here, we aimed to investigate the mechanism of this BBB transport dysregulation by studying the localization and function of pannexin (Px) and connexin (Cx) hemichannels in blood-brain barrier endothelial cells of rat (TR-BBB13 cells) and human (hCMEC/D3 cells) under acute ischemic stroke-mimicking oxygen/glucose deprivation (OGD) and extracellular Ca 2+ ([Ca 2+ ] e )-free conditions. TR-BBB13 cells showed increased uptake of hemichannel-permeable sulforhodamine 101, and this increase was markedly inhibited by carbenoxolone, a hemichannel inhibitor. Transcripts of Px1 and Cx43 were detected in TR-BBB13 cells and freshly isolated brain microvascular endothelial cells. The basal compartment-to-cell uptake of hemichannel-permeable propidium iodide was selectively enhanced in hCMEC/D3 cells under [Ca 2+ ] e -free conditions in the basal Transwell chamber. Immunohistochemical analysis revealed the predominant localization of Cx43 on the lateral membranes of hCMEC/D3 cells. [ 3 H]Taurine uptake by hCMEC/D3 cells was significantly reduced in the absence of [Ca 2+ ] e . Functional knock-down of Px1 and Cx43 with mimetic peptides significantly inhibited the increase of ATP release from hCMEC/D3 cells under [Ca 2+ ] e -free conditions. These results suggest that polarized Px1/Cx43 hemichannel opening in brain capillary endothelial cells under acute ischemic stroke-mimicking conditions contributes to dysregulation of BBB transport function, resulting in release of intracellular taurine and ATP., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Retinal pigment epithelial (RPE) cells form the outer blood-retinal barrier (BRB) and regulate drug/compound exchange between the neural retina and blood in the fenestrated blood vessels of retinal choroid via membrane transporters. Recent studies have elucidated that RPE cells express hemichannels, which are opened by extracellular Ca 2+ depletion and accept several drugs/compounds as a transporting substrate. The objective of this study was to elucidate the hemichannel-mediated compound transport properties of the outer BRB. In human RPE cells, namely ARPE-19 cells, time-dependent uptake of fluorescent hemichannel substrates, such as Lucifer Yellow, sulforhodamine-101 (SR-101), and propidium iodide (PI) was promoted under Ca 2+ -depleted conditions. The uptake of these substrates under Ca 2+ -depleted conditions exhibited saturable kinetics with a Michaelis-Menten constant (K m ) of 87-109 µM. In addition, SR-101 and PI uptake by ARPE-19 cells was dependent of extracellular Ca 2+ concentration, and that under Ca 2+ -depleted conditions was significantly decreased by typical substrates and/or inhibitors for hemichannels. Moreover, Ca 2+ -depleted conditions promoted the efflux transport of calcein from ARPE-19 cells, and the promoted calcein efflux transport was significantly inhibited by a typical hemichannel inhibitor. These results suggested that hemichannels at the outer BRB were involved in the influx and efflux transport of drugs/compounds.

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B 2 ) 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 [ 3 H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [ 3 H]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, [ 3 H]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 [ 3 H]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)., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The cause of antiseizure drug (ASD) resistance in epilepsy is poorly understood. Here, we focus on the transporter P-glycoprotein (P-gp) that is partly responsible for limited ASD brain uptake, which is thought to contribute to ASD resistance. We previously demonstrated that cyclooxygenase-2 (COX-2) and the prostaglandin E receptor, prostanoid E receptor subtype 1, are involved in seizure-mediated P-gp up-regulation. Thus, we hypothesized that inhibiting microsomal prostaglandin E 2 (PGE2) synthase-1 (mPGES-1), the enzyme generating PGE2, prevents blood-brain barrier P-gp up-regulation after status epilepticus (SE). To test our hypothesis, we exposed isolated brain capillaries to glutamate ex vivo and used a combined in vivo - ex vivo approach by isolating brain capillaries from humanized mPGES-1 mice to study P-gp levels. We demonstrate that glutamate signaling through the NMDA receptor, cytosolic phospholipase A2, COX-2, and mPGES-1 increases P-gp protein expression and transport activity levels. We show that mPGES-1 is expressed in human, rat, and mouse brain capillaries. We show that BI1029539, an mPGES-1 inhibitor, prevented up-regulation of P-gp expression and transport activity in capillaries exposed to glutamate and in capillaries from humanized mPGES-1 mice after SE. Our data provide key signaling steps underlying seizure-induced P-gp up-regulation and suggest that mPGES-1 inhibitors could potentially prevent P-gp up-regulation in epilepsy.-Soldner, E. L. B., Hartz, A. M. S., Akanuma, S.-I., Pekcec, A., Doods, H., Kryscio, R. J., Hosoya, K.-I., Bauer, B. Inhibition of human microsomal PGE2 synthase-1 reduces seizure-induced increases of P-glycoprotein expression and activity at the blood-brain barrier.

It has been known that organic anion-transporting polypeptides (Oatps) involve hepatic transports several organic anionic compounds and drugs. This study aimed to investigate sulforhodamine-101 (SR-101) distribution in the rat liver, determine the molecules responsible for the distribution, and delineate the manner of distribution. After intravenous SR-101 administration, its distribution in frozen rat hepatic sections was examined. SR-101-derived signals were detected in regions around the hepatic central vein (CV), where immunohistochemistry (IHC) indicated high Oatp1a4 expression. The signals decreased with treatment by digoxin, a specific substrate for Oatp1a4. In vitro studies using isolated rat hepatocytes and rat Oatp1a4-expressing Xenopus laevis oocytes have suggested that SR-101 is an Oatp1a4 substrate and is taken up into rat hepatocytes mainly via Oatp1a4. Therefore, results suggested SR-101 zonation because of Oatp1a4 involvement and that Oatp1a4 function is dominant in the region around the hepatic CV in rat hepatic lobules., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The blood-to-retina supply of cyanocobalamin (vitamin B 12 ) across the blood-retinal barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin (Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the retinal pigment epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine, and chloroquine and was also significantly reduced under Ca 2+ -free conditions. Confocal microscopy of the TR-iBRB2 cells showed fluorescence signals of Cy5-cyanocobalamin and GFP-TCII protein, and these signals merged with each other. The RT-PCR, Western blot, and immunohistochemistry clearly suggested the expression of TCII receptor (TCII-R) in the inner and outer BRB. These results suggested the involvement of receptor-mediated endocytosis in the blood-to-retina transport of cyanocobalamin at the inner BRB with implying its possible involvement at the outer BRB.

Gabapentin is an antiseizure drug that is known to also have beneficial effects on the retinal cells. To use gabapentin in retinal pharmacotherapy, it is critical to understand gabapentin distribution in the retina. The purpose of this study was to clarify the kinetics of gabapentin influx transport across the inner and outer blood-retinal barrier (BRB), which regulates the exchange of compounds/drugs between the circulating blood and the retina. In vivo blood-to-retina gabapentin transfer was evaluated by the rat carotid artery injection technique. In addition, gabapentin transport was examined using in vitro models of the inner (TR-iBRB2 cells) and outer BRB (RPE-J cells). The in vivo [ 3 H]gabapentin transfer to the rat retina across the BRB was significantly reduced in the presence of unlabeled gabapentin, suggesting transporter-mediated blood-to-retina distribution of gabapentin. Substrates of the Na + -independent l-type amino acid transporter 1 (LAT1), such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), also significantly inhibited the in vivo [ 3 H]gabapentin transfer. [ 3 H]Gabapentin uptake in TR-iBRB2 and RPE-J cells exhibited Na + -independent and saturable kinetics with a K m of 735 and 507 μM, respectively. Regarding the effect of various transporter substrates/inhibitors on gabapentin transport in these cells, LAT1 substrates significantly inhibited [ 3 H]gabapentin uptake in TR-iBRB2 and RPE-J cells. In addition, preloaded [ 3 H]gabapentin release from TR-iBRB2 and RPE-J cells was trans-stimulated by LAT1 substrates through the obligatory exchange mechanism as LAT1. Immunoblot analysis indicates the protein expression of LAT1 in TR-iBRB2 and RPE-J cells. These results imply that LAT1 at the inner and outer BRB takes part in gabapentin transport between the circulating blood and retina. Moreover, treatment of LAT1-targeted small interfering RNA to TR-iBRB2 cells significantly reduced both the level of LAT1 protein expression and [ 3 H]gabapentin uptake activities in TR-iBRB2 cells. In conclusion, data from the present study indicate that LAT1 at the inner BRB is involved in retinal gabapentin transfer, and also suggest that LAT1 mediates gabapentin transport in the RPE cells.

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.

l-Arginine is required for regulating synapse formation/patterning and angiogenesis in the developing brain. We hypothesized that this requirement would be met by increased transporter-mediated supply across the blood-brain barrier (BBB). Thus, the purpose of this work was to test the idea that elevation of blood-to-brain l-arginine transport across the BBB in the postnatal period coincides with up-regulation of cationic acid transporter 1 (CAT1) expression in developing brain capillaries. We found that the apparent brain-to-plasma concentration ratio (Kp, app) of l-arginine after intravenous administration during the first and second postnatal weeks was 2-fold greater than that at the adult stage. Kp, app of l-serine was also increased at the first postnatal week. In contrast, Kp, app of d-mannitol, a passively BBB-permeable molecule, did not change, indicating that increased transport of l-arginine and l-serine is not due to BBB immaturity. Double immunohistochemical staining of CAT1 and a marker protein, glucose transporter 1, revealed that CAT1 was localized on both luminal and abluminal membranes of brain capillary endothelial cells during the developmental and adult stages. A dramatic increase in CAT1 expression in the brain was seen at postnatal day 7 (P7) and day 14 (P14) and the expression subsequently decreased as the brain matured. In accordance with this, intense immunostaining of CAT1 was observed in brain capillaries at P7 and P14. These findings strongly support our hypothesis and suggest that the supply of blood-born l-arginine to the brain via CAT1 at the BBB plays a key role in meeting the elevated demand for l-arginine in postnatal brain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 NH 4 Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH 4 Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH 4 Cl 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 IC 50 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.

The changes in the transport function of the outer blood-retinal barrier (BRB), formed by retinal pigment epithelial (RPE) cells, under pathological conditions need to be understood to normalize the retinal homeostasis in retinal diseases. Connexin 43 (Cx43) is known to be one of the basic units of gap junctions and hemichannels, which are opened by changes in extracellular conditions. The purpose of this study was to clarify the expression of Cx43 in RPE cells of the retina and Cx43 contribution to compound transport functions in RPE cells. Immunohistochemistry using guinea pig-derived polyclonal anti-Cx43 antibodies indicated that Cx43 is localized at the apical and intercellular membrane of mouse RPE cells. In addition, the immunoprecipitation study using the anti-Cx43 antibodies suggested that Cx43 at the intercellular membrane is associated with gap and adherent junctions in mouse RPE cells. The intercellular transfer after scrape loading of Lucifer Yellow (457 g/mol) among a human RPE cell line, ARPE-19 cells, was greater than that of fluorescein isothiocyanate-dextran (∼3000 g/mol). This Lucifer Yellow transfer was significantly inhibited by carbenoxolone, a connexin inhibitor, suggesting that connexins take part in compound transfer via gap junctions. In addition, Lucifer Yellow uptake by ARPE-19 cells in the absence of extracellular Ca 2+ , which is a condition of hemichannel opening, was increased compared with that under normal conditions. This uptake of Lucifer Yellow in the absence of extracellular Ca 2+ was significantly reduced in the presence of hemichannel inhibitors and Cx43-gene silencing conditions. This study suggests the involvement of Cx43 in dye transfer via gap junctions among RPE cells and hemichannel-mediated compound transport between the neural retina and RPE cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Guanidinoacetate (GAA) is a biosynthetic precursor of creatine, which plays a critical role in homeostasis of high-energy phosphates in the brain, but cerebral accumulation of GAA leads to neurological complications, such as epilepsy and seizures. The purpose of the present study was to clarify the contribution of the γ-aminobutyric acid (GABA) transport systems to GAA transport in astrocytes by means of uptake studies in rat brain slices, primary astrocyte cultures and Chinese hamster ovary (CHO) cells expressing human GABA transporters (GATs). GAA uptake by rat brain slices was Na + - and Cl - -dependent, and GABA-sensitive. The inhibitory effect of GABA, a common substrate of GATs, on GAA uptake by the brain slices was similar to that of β-alanine, a selective substrate of GAT2/Slc6a13, GAT3/Slc6a11, and taurine transporter (TauT)/Slc6a6. Taurine, a high-affinity substrate of TauT/Slc6a6, exhibited a lesser inhibitory effect. In contrast, betaine, a substrate of betaine-GABA transporter 1 (BGT1)/Slc6a12, and creatine, a substrate of creatine transporter (CRT)/Slc6a8, had little inhibitory effect. A similar inhibition profile was observed in primary-cultured astrocytes. CHO cells expressing human GAT2/SLC6A13, GAT3/SLC6A11 and BGT1/SLC6A12 exhibited GAA transport, whereas CHO cells expressing GAT1/SLC6A1 did not. The Michaelis-Menten values in CHO cells expressing GAT2/SLC6A13 and GAT3/SLC6A11 were similar to those in primary-cultured astrocytes. Overall, our results suggest that astrocytic GAT2/Slc6a13 and GAT3/Slc6a11 play major roles in GAA uptake as regulatory mechanisms of GAA in rat brain, while TauT/Slc6a6, BGT1/Slc6a12, and CRT/Slc6a8 make relatively small contributions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: para-Tyramine (p-TA) is a biogenic amine which is involved in multiple neuronal signal transductions. Since the concentration of p-TA in dog cerebrospinal fluid (CSF) has been reported to be greater than that in plasma, it is proposed that clearance of cerebral p-TA is important for normal function. The purpose of this study was to examine the role of the blood-brain barrier and blood-cerebrospinal fluid barrier (BCSFB) in p-TA clearance from the brain., Methods: In vivo [ 3 H]p-TA elimination from rat cerebral cortex and from CSF was examined after intracerebral and intracerebroventricular administration, respectively. To evaluate BCSFB-mediated p-TA transport, [ 3 H]p-TA uptake by isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells, TR-CSFB3 cells, was performed., Results: The half-life of [ 3 H]p-TA elimination from rat CSF was found to be 2.9 min, which is 62-fold faster than that from rat cerebral cortex. In addition, this [ 3 H]p-TA elimination from the CSF was significantly inhibited by co-injection of excess unlabeled p-TA. Thus, carrier-mediated p-TA transport process(es) are assumed to take part in p-TA elimination from the CSF. Since it is known that transporters at the BCSFB participate in compound elimination from the CSF, [ 3 H]p-TA transport in ex vivo and in vitro models of rat BCSFB was examined. The [ 3 H]p-TA uptake by isolated rat choroid plexus and TR-CSFB3 cells was time-dependent and was inhibited by unlabeled p-TA, indicating carrier-mediated p-TA transport at the BCSFB. The p-TA uptake by isolated choroid plexus and TR-CSFB3 cells was not reduced in the absence of extracellular Na + and Cl - , and in the presence of substrates of typical organic cation transporters. However, this p-TA uptake was significantly inhibited by cationic drugs such as propranolol, imipramine, amantadine, verapamil, and pyrilamine. Moreover, p-TA uptake by TR-CSFB3 cells took place in an oppositely-directed H + gradient manner. Therefore, this suggested that p-TA transport at the BCSFB involves cationic drug-sensitive transport systems which are distinct from typical plasma membrane organic cation transporters., Conclusion: Our study indicates that p-TA elimination from the CSF is greater than that from the cerebral cortex. Moreover, it is suggested that cationic drug-sensitive transport systems in the BCSFB participate in this p-TA elimination from the CSF.

Docosahexaenoic acid (DHA, 22 : 6) is an essential omega-3 long-chain polyunsaturated fatty acid that plays a pivotal role in vision. The purpose of this study was to clarify the cellular uptake and binding processes of free and protein-bound unesterified DHA in retinal pigment epithelial cell (RPE) line ARPE-19 as a model of the human outer blood-retinal barrier and isolated porcine RPE cell fractions. Uptake of free [ 14 C]DHA by ARPE-19 cells was saturable with a Michaelis-Menten constant of 283 µM, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid. Further, the uptakes of [ 14 C]DHA associated with retinol-binding protein ([ 14 C]DHA-RBP), [ 14 C]DHA associated with low-density lipoprotein ([ 14 C]DHA-LDL) and [ 14 C]DHA associated with bovine serum albumin ([ 14 C]DHA-BSA) in ARPE-19 cells increased time-dependently at 37°C, and were significantly reduced at 4°C, suggesting the involvement of energy-dependent transport processes. [ 14 C]DHA-LDL uptake by ARPE-19 cells was significantly inhibited by excess unlabeled LDL, but not by an inhibitor of scavenger receptor B type I. Fatty acid transport protein (FATP) 2 and 4 mRNAs were expressed in ARPE-19 cells, and [ 14 C]DHA uptake was observed in FATP2- and FATP4-expressing Xenopus oocytes. Photo-reactive crosslinking and mass spectrometry analyses identified 65-kDa retinal pigment epithelium-specific protein (RPE65) as a DHA-binding protein in porcine RPE cell membrane fractions. Thus, RPE cells possess multiple cellular transport/binding processes for unesterified DHA, involving at least partly FATP2, FATP4, LDL, RBP, and RPE65.

Regulating γ-aminobutyric acid (GABA) uptake transport on the plasma membranes is required for its efficient clearance from the brain interstitial fluid. The purpose of this study was to clarify the assembly of taurine transporter (TauT/Slc6a6) and PSD-95/Disc-large/Zo-1 (PDZ) domain of Na + -H + exchanger regulatory factor 1 (NHERF1) as a regulatory mechanism of TauT-mediated GABA transport activity. In vitro glutathione S-transferase (GST)-pull down assay and immunoblotting with anti-NHERF1 antibody revealed that NHERF1 protein was present in rat brain lysates as the binding protein of the GST-fusion TauT C-terminal protein with the PDZ-binding ETMM motif but not its corresponding deletion mutant lacking the motif. [ 3 H]GABA uptake by TauT-NHERF1-coexpressing oocytes and TauT-singly expressing oocytes exhibited saturable kinetics with Michaelis-Menten constant values of 0.835±0.288 and 0.982±0.569 mM and a maximal transport velocity of 206±37 and 283±28 pmol/(h·oocyte), respectively. These results suggest that the assembly of TauT PDZ-binding motif and NHERF1 increases the maximal transport velocity of GABA rather than changes the affinity.

Nicotine, an addictive substance, is absorbed from the lungs following inhalation of tobacco smoke, and distributed to various tissues such as liver, brain, and retina. Recent in vivo and in vitro studies suggest the involvement of a carrier-mediated transport process in nicotine transport in the lung, liver, and inner blood-retinal barrier. In addition, in vivo studies of influx and efflux transport of nicotine across the blood-brain barrier (BBB) revealed that blood-to-brain influx transport of nicotine is more dominant than brain-to-blood efflux transport of nicotine. Uptake studies in TR-BBB13 cells, which are an in vitro model cell line of the BBB, suggest the involvement of H + /organic cation antiporter, which is distinct from typical organic cation transporters, in nicotine transport at the BBB. Moreover, inhibition studies in TR-BBB13 cells showed that nicotine uptake was significantly reduced by central nervous system (CNS) drugs, such as antidepressants, anti-Alzheimer's disease drugs, and anti-Parkinson's disease drugs, suggesting that the nicotine transport system can recognize these molecules. The cumulative evidence would be helpful to improve our understanding of smoking-CNS drug interaction for providing appropriate medication.

The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood-retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood-brain barrier (BBB), and blood-cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/μg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/μg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/μg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/μg protein), and sodium-potassium adenosine triphosphatase (Na + /K + -ATPase; 53.7 fmol/μg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB (R 2 = 0.8906), followed by outer BRB (R 2 = 0.7988) and BCSFB (R 2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6) and multidrug resistance-associated protein 1 (MRP1, ABCC1) were expressed at the outer BRB (0.378 and 1.03 fmol/μg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.

1-Methyl-4-phenylpyridinium (MPP + ) transport at the blood-retinal barrier (BRB) was investigated. The retinal uptake index estimated for [ 3 H]MPP + was similar to that of [ 3 H]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 [ 3 H]MPP + in the vitreous humor decreased in a biexponential manner, and the rate constant for [ 3 H]MPP + elimination during the terminal phase was greater than that of [ 14 C]d-mannitol. The inhibition study of [ 3 H]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 [ 3 H]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)., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The placenta is an organ that secretes prostaglandin (PG) E 2 into the fetal-placental circulation to regulate both vascular tone and remodeling of the fetal ductus arteriosus. Placental PGE 2 synthesis might be mediated by microsomal PGE synthase-1 (mPGES-1), in addition to cyclooxygenase (COX) isoforms. Thus, the purpose of this study is to clarify the temporal and spatial expression patterns of mPGES-1, together with COX-1 and COX-2, in murine placenta. We found that mPGES-1 and COX-1 protein levels continuously increased in the placental labyrinth from gestational day (GD) 13.5 to GD19.5, becoming higher than in the decidua or the junctional zone by GD17.5. The PGE 2 level at GD17.5 was also highest in the labyrinth. Immunofluorescence stainings for mPGES-1 and COX-1 in the labyrinth at GD17.5 overlapped and were located on the fetal side of the signals for connexin 26, which forms gap junctions between maternal-facing (SynT-I) and fetal-facing (SynT-II) syncytiotrophoblast layers, and on the maternal side of the signals for glucose transporter 1 on the basal plasma membrane of SynT-II. On the other hand, the signals for COX-2 did not overlap with those for mPGES-1. These results indicate that COX-1 and mPGES-1 are co-localized in murine placental SynT-II, facing the fetal-placental circulation. Therefore, SynT-II could contribute to placental synthesis of PGE 2 for release into the fetal-placental circulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Riboflavin (vitamin B 2 ) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [ 3 H]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 [ 3 H]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 [ 3 H]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. [ 3 H]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. [ 3 H]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., (Copyright © 2016. Published by Elsevier Ltd.)

Spermine is the end-product in the polyamine biosynthetic pathway, and its excess accumulation induces neuroexcitatory responses and neurotoxicity. The purpose of this study was to elucidate the involvement of transport systems at the brain barriers in the clearance of spermine. In vivo rat spermine elimination from brain parenchyma across the blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCSFB) was assessed by intracerebral and intracerebroventricular administration techniques, respectively. To characterize spermine transport at the BCSFB, a transport study using rat choroid plexus was performed. After the intracerebral microinjection of [ 3 H]spermine, no time-dependent decrease in [ 3 H]spermine in the ipsilateral cerebrum was observed, suggesting the low contribution of the BBB to spermine clearance from the brain. In contrast, the [ 3 H]spermine concentration in the CSF after intracerebroventricular administration was time-dependently decreased with an elimination rate constant of 0.352 min -1 , and the elimination clearance of [ 3 H]spermine was 6.6-fold greater than that of [ 14 C]D-mannitol, reflecting bulk flow of the CSF. This [ 3 H]spermine elimination was attenuated by co-administration of unlabeled excess spermine, indicating carrier-mediated elimination of spermine from the CSF. [ 3 H]Spermine transport into the choroid plexus was strongly inhibited by unlabeled spermine, other polyamines (spermidine and putrescine), and organic cation transporter substrates such as corticosterone and 1-methyl-4-phenylpyridinium. However, other substrates/inhibitors for organic cation transporters (decynium-22 and tetraethylammonium) had little effect. Consequently, our study indicates that transporting molecules at the BCSFB, distinct from typical organic cation transporters, are involved in spermine clearance from the CSF.

The retina is a tissue essential for vision, and the blood-retina barrier (BRB) helps to maintain an optimal microenvironment for the neural system in the retina. Recent findings concerning the BRB showed the involvement of transporters at the inner and outer BRB in drug and nutrient transport, suggesting their utility in the development of novel drug delivery systems to the retina. An in vitro-in vivo relationship study of permeability suggested the influx transport of verapamil, a cationic drug, across the BRB, and further in vivo and in vitro studies of cationic drugs, such as verapamil, propranolol and clonidine, revealed the involvement of carrier-mediated process in their influx transport at the BRB. Studies on substrate specificity in TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests the involvement of novel organic cation transporter in the influx transport of cationic drugs at the inner BRB. Considering the neuroprotective effect previously reported for several cationic drugs, such as propranolol and clonidine, the study of cation transport at the BRB is widely expected to improve the treatment of retinal diseases, such as diabetic retinopathy and age-related macular degeneration.

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., (© 2016 International Society for Neurochemistry.)

Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 μM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and β2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with β2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)