Molecular Mechanism of SLC6A8 Dysfunction with c.1699T > C (p.S567P) Mutation in Cerebral Creatine Deficiency Syndromes.

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.

Carrier-Mediated Process of Putrescine Elimination at the Rat Blood-Retinal Barrier.

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 [14C]D-mannitol, which is a bulk flow marker. The difference in the apparent elimination rate constants of [3H]putrescine and [14C]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 [3H]putrescine transport was time-, temperature-, and concentration-dependent, suggesting the involvement of carrier-mediated processes in putrescine transport at the inner and outer BRB. [3H]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 [3H]putrescine uptake, and CTL1 knockdown significantly reduced [3H]putrescine uptake in model cell lines, suggesting the possible participation of CTL1 in putrescine transport at the BRB.

Construction and Functional Evaluation of a Three-Dimensional Blood-Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells.

PURPOSE: The purpose of this study was to construct and validate an in vitro three-dimensional blood-brain barrier (3DBBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs). METHODS: The 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions. RESULTS AND DISCUSSION: The expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed. CONCLUSION: The 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

Fibronectin is essential for formation of fenestrae in endothelial cells of the fenestrated capillary.

Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle-associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary.

Functional Investigation of Solute Carrier Family 35, Member F2, in Three Cellular Models of the Primate Blood-Brain Barrier.

Understanding the mechanisms of drug transport across the blood-brain barrier (BBB) is an important issue for regulating the pharmacokinetics of drugs in the central nervous system. In this study, we focused on solute carrier family 35, member F2 (SLC35F2), whose mRNA is highly expressed in the BBB. SLC35F2 protein was enriched in isolated mouse and monkey brain capillaries relative to brain homogenates and was localized exclusively on the apical membrane of MDCKII cells and brain microvascular endothelial cells (BMECs) differentiated from human induced pluripotent stem cells (hiPS-BMECs). SLC35F2 activity was assessed using its substrate, YM155, and pharmacological experiments revealed SLC35F2 inhibitors, such as famotidine (half-maximal inhibitory concentration, 160 µM). Uptake of YM155 was decreased by famotidine or SLC35F2 knockdown in immortalized human BMECs (human cerebral microvascular endothelial cell/D3 cells). Furthermore, famotidine significantly inhibited the apical (A)-to-basal (B) transport of YM155 in primary cultured monkey BMECs and hiPS-BMECs. Crucially, SLC35F2 knockout diminished the A-to-B transport and intracellular accumulation of YM155 in hiPS-BMECs. By contrast, in studies using an in situ brain perfusion technique, neither deletion of Slc35f2 nor famotidine reduced brain uptake of YM155, even though YM155 is a substrate of mouse SLC35F2. YM155 uptake was decreased significantly by losartan and naringin, inhibitors for the organic anion transporting polypeptide (OATP) 1A4. These findings suggest SLC35F2 is a functional transporter in various cellular models of the primate BBB that delivers its substrates to the brain and that its relative importance in the BBB is modified by differences in the expression of OATPs between primates and rodents. SIGNIFICANCE STATEMENT: This study demonstrated that SLC35F2 is a functional drug influx transporter in three different cellular models of the primate blood-brain barrier (i.e., human cerebral microvascular endothelial cell/D3 cells, primary cultured monkey BMECs, and human induced pluripotent stem-BMECs) but has limited roles in mouse brain. SLC35F2 facilitates apical-to-basal transport across the tight cell monolayer. These findings will contribute to the development of improved strategies for targeting drugs to the central nervous system.

Expression and Functional Characterization of Drug Transporters in Brain Microvascular Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

Brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) have been proposed as a new blood-brain barrier model, but their transport function has not been fully clarified. Therefore, in this study, we investigated the gene expression and function of transporters in hiPS-BMECs by means of quantitative reverse transcription-PCR, in vitro transcellular transport studies, and uptake experiments. mRNAs encoding ABC and SLC transporters, such as BCRP, MCT1, CAT1, and GLAST, were highly expressed in hiPS-BMECs. Transcellular transport studies showed that prazosin, [14C]l-lactate, [3H]l-arginine, and [3H]l-glutamate (substrates of BCRP, MCT1, CAT1, and GLAST, respectively) were transported asymmetrically across the hiPS-BMEC monolayer. Substrates of LAT1, OCTN2, CAT1, GLAST, MCT1, and proton-coupled organic cation (H+/OC) antiporter were taken up by hiPS-BMECs in a time-, temperature-, and concentration-dependent manner, and the uptakes were markedly decreased by inhibitors of the corresponding transporter. These results indicate that hiPS-BMECs express multiple nutrient and drug transporters.

Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line.

PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin. METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis. RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 µM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA. CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

Impact of Nicotine Transport across the Blood-Brain Barrier: Carrier-Mediated Transport of Nicotine and Interaction with Central Nervous System Drugs.

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.

Involvement of the H+/organic cation antiporter in nicotine transport in rat liver.

Nicotine is an addictive alkaloid in cigarette smoke and is responsible for tobacco dependence. It is important to consider the blood-to-liver transport of nicotine to understand the nicotine elimination from the body because most of the nicotine is converted to inactive metabolites by cytochrome P450 localized in the endoplasmic reticulum of the hepatocytes. In this study, the blood-to-liver transport of nicotine was investigated by means of an in vivo portal vein injection technique in rats, and the in vitro uptake by freshly isolated rat hepatocytes was used to clarify its mechanism. The results obtained showed that the in vivo blood-to-liver transport of [(3)H]nicotine was significantly inhibited by 50 mM nicotine and pyrilamine, suggesting involvement of a carrier-mediated transport process in the blood-to-liver transport of nicotine. The in vitro uptake study using freshly isolated rat hepatocytes showed a time- and concentration-dependent uptake of [(3)H]nicotine with a Km value of 141 µM, and the uptake was increased under alkaline extracellular conditions. In addition, intracellular acidification caused an increase in [(3)H]nicotine uptake, suggesting that the influx transport of nicotine is driven by an oppositely directed H(+) gradient in hepatocytes. Moreover, [(3)H]nicotine uptake was strongly inhibited in the presence of cationic drugs, such as pyrilamine, whereas only weak inhibitory effects were shown by substrates of typical organic cation transporters, such as tetraethylammonium, 1-methyl-4-phenylpyridinium, choline, and l-carnitine. In conclusion, a carrier-mediated system controlling the blood-to-liver transport of nicotine appears to be present on the sinusoidal membrane of hepatocytes. The pattern of inhibition and ion dependence is suggestive of an H(+)/organic cation antiporter-mediated nicotine transport system.

Blood-to-Testis Transport of Ribavirin Involves Carrier-Mediated Processes at the Blood-Testis Barrier.

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 [3H]ribavirin in the testis was 3.6-fold greater than that of [14C]D-mannitol, a paracellular transport marker, implying transcellular transport of ribavirin across the BTB. Moreover, [3H]ribavirin uptake by TM4 cells, mouse-derived Sertoli cells, was time- and concentration-dependent, with a Km value of 2.49 mM. S-[(4-nitrophenyl)methyl]-6-thioinosine, an inhibitor of Na+-independent equilibrative nucleoside transporters (ENTs), strongly inhibited the [3H]ribavirin uptake by TM4 cells at 100 µM. Compared to the uptake of [3H]adenosine, a typical endogenous nucleoside, [3H]ribavirin uptake was relatively similar to ENT2 transport. [3H]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 [3H]ribavirin transport into TM4 cells by 13%. Taken together, these results suggest that ENT2 partially contributes to ribavirin transport at the BTB.

The Structural Characteristics of Compounds Interacting with the Amantadine-Sensitive Drug Transport System at the Inner Blood-Retinal Barrier.

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.

Characterization of LysoTracker Red uptake by in vitro model cells of the outer blood-retinal barrier: Implication of lysosomal trapping with cytoplasmic vacuolation and cytotoxicity.

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.

Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood-Brain Barrier.

A proton-coupled organic cation (H+/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.

Structural Requirements for Uptake of Diphenhydramine Analogs into hCMEC/D3 Cells Via the Proton-Coupled Organic Cation Antiporter.

There is increasing evidence that a proton-coupled organic cation (H+/OC) antiporter facilitates uptake of various central nervous system-active drugs, such as the histamine H1 receptor antagonist diphenhydramine, into the brain. The purpose of this study was to clarify the structural requirements for H+/OC antiporter-mediated uptake into hCMEC/D3 cells, an established in vitro model of the human blood-brain barrier, by using a series of diphenhydramine analogs. For this purpose, we synthesized seven tertiary amine analogs and three amide analogs. Uptake of all the amines was facilitated by an outwardly directed H+ gradient and inhibited by pyrilamine, a typical substrate and a strong inhibitor of the H+/OC antiporter. Further, uptake of most of the amines was trans-stimulated by pyrilamine. Uptake of the amines was 21 times faster than that of the amides on average, even though the lipophilicity (log D7.4) of the amines is lower than that of the amides. Amines containing a pyrrolidine or piperidine ring showed the highest uptake rates. Our results suggest that an amine moiety, especially a heterocyclic amine moiety, is important for recognition and transport by the H+/OC antiporter.

Transport Characteristics of 6-Mercaptopurine in Brain Microvascular Endothelial Cells Derived From Human Induced Pluripotent Stem Cells.

The likelihood of reoccurrence of acute lymphoblastic leukemia is influenced by the cerebral concentration of the therapeutic agent 6-mercaptopurine (6-MP) during treatment. Therefore, it is important to understand the blood-brain barrier (BBB) transport mechanism of 6-MP. The purpose of this study was to characterize this mechanism using human induced pluripotent stem cell-derived microvascular endothelial cells (hiPS-BMECs). The permeability coefficient of 6-MP across hiPS-BMECs monolayer in the basal-to-apical direction (B-to-A) was significantly greater than that in the opposite direction (A-to-B). The inhibition profiles of 6-MP transport in the A-to-B direction were different from those in the B-to-A direction. Transport in the A-to-B direction was mainly inhibited by adenine (an inhibitor of equilibrative nucleobase transporter 1; ENBT1), while transport in the B-to-A direction was significantly reduced by inhibitors of multidrug resistance-associated proteins (MRPs), especially zaprinast (an MRP5 inhibitor). Immunocytochemical analyses demonstrated the expression of ENBT1 and MRP5 proteins in hiPS-BMECs. We confirmed that the cellular uptake of 6-MP is decreased by ENBT1 inhibitors in hiPS-BMECs and by knockdown of ENBT1 in hCMEC/D3 cells. These results suggest that ENBT1 and MRP5 make substantial contributions to the transport of 6-MP in hiPS-BMECs and hCMEC/D3 cells.

Design, Synthesis, and Blood-Brain Barrier Transport Study of Pyrilamine Derivatives as Histone Deacetylase Inhibitors.

We designed and synthesized a pyrilamine derivative 1 as a selective class I HDAC inhibitor that targets pyrilamine-sensitive proton-coupled organic cation antiporter (PYSOCA) at the blood-brain barrier (BBB). Introduction of pyrilamine moiety to benzamide type HDAC inhibitors kept selective class I HDAC inhibitory activity and increased BBB permeability. Our BBB transport study showed that compound 1 is a substrate of PYSOCA. Thus, our findings suggest that the hybrid method of HDAC inhibitor and substrate of PYSOCA such as pyrilamine is useful for development of HDAC inhibitors with increased BBB permeability.

Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells.

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 ß2-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 ß2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation.

Carrier-Mediated Transport of Nicotine Across the Inner Blood-Retinal Barrier: Involvement of a Novel Organic Cation Transporter Driven by an Outward H(+) Gradient.

The present study was carried out to investigate the blood-to-retina transport of nicotine across the inner blood-retinal barrier (BRB). Using the in vivo vascular injection method, the blood-to-retina influx clearance of nicotine across the BRB was determined as 131 µL/(min?g retina), which is much higher than that of a nonpermeable paracellular marker, and blood-to-retina transport of nicotine was inhibited by organic cations such as pyrilamine and verapamil. The nicotine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro model of the inner BRB, exhibited time, temperature, and concentration dependence with a Km of 492 µM. These results suggest the involvement of a carrier-mediated transport process in nicotine transport in the inner BRB. The nicotine uptake by TR-iBRB2 cells was stimulated by an outwardly directed H(+) gradient, and the uptake was significantly inhibited by bulky and hydrophobic cationic drugs, whereas inhibitors of organic cation transporters did not show inhibitory effect. These results suggest that the novel organic cation transport system driven by an outwardly directed H(+) gradient is involved in the blood-to-retina transport of nicotine across the inner BRB.

Blood-to-brain influx transport of nicotine at the rat blood-brain barrier: involvement of a pyrilamine-sensitive organic cation transport process.

Nicotine is the most potent neural pharmacological alkaloid in tobacco, and the modulation of nicotine concentration in the brain is important for smoking cessation therapy. The purpose of this study was to elucidate the net flux of nicotine transport across the blood-brain barrier (BBB) and the major contributor to nicotine transport in the BBB. The in vivo brain-to-blood clearance was determined by a combination of the rat brain efflux index method and a rat brain slice uptake study, and the blood-to-brain transport of nicotine was evaluated by in vivo vascular injection in rats and a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13 cells) as an in vitro model of the rat BBB. The blood-to-brain nicotine influx clearance was obtained by integration plot analysis as 272 µL/(ming brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 µL/(ming brain)). Thus, it is suggested that the net flux of nicotine transport across the BBB is dominated by blood-to-brain influx transport. In vivo blood-to-brain nicotine transport was inhibited by pyrilamine. [(3)H]Nicotine uptake by TR-BBB13 cells exhibited time-, temperature-, and concentration-dependence with a K(m) value of 92 µM. Pyrilamine competitively inhibited nicotine uptake by TR-BBB13 cells with a K(i) value of 15 µM, whereas substrates and inhibitors of organic cation transporters had little effect. These results suggest that pyrilamine-sensitive organic cation transport process(es) mediate blood-to-brain influx transport of nicotine at the BBB, and this is expected to play an important role in regulating nicotine-induced neural responses.