MBEC4 Cells Research Articles

Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells

As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS.

An apolipoprotein E modified liposomal nanoparticle: Ligand dependent efficiency as a siRNA delivery carrier for mouse-derived brain endothelial cells

A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposomal nanoparticle containing a proton-ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligand for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent manner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium.

Contribution of P-glycoprotein to efflux of ramosetron, a 5-HT3 receptor antagonist, across the blood-brain barrier.

In-situ rat and mouse brain perfusion data indicated that the brain distribution of ramosetron (R-ramosetron), a 5-hydroxytryptamine3 (5-HT3) receptor antagonist, was extremely low compared with that expected from its lipophilicity. We hypothesized the involvement of an efflux system(s) and investigated the contribution of P-glycoprotein to efflux transport of ramosetron across the blood-brain barrier by means of an in-vitro uptake study in cell lines that over-express P-glycoprotein. We examined the contributions of mdr1a, mdr1b and MDR1 P-glycoprotein by using LV500 cells, MBEC4 cells and LLC-GA5-COL300 cells, which over-express mdr1a P-glycoprotein, mdr1b P-glycoprotein and MDR1 P-glycoprotein, respectively. The uptake of [14C]ramosetron by LV500 cells and LLC-GA5-COL300 cells was significantly lower than that by the respective parental cells. Next, we studied the effects of P-glycoprotein inhibitors, verapamil and ciclosporin, on uptake of [14C]ramosetron by these cell lines. The uptake of [14C]ramosetron by LV500 cells and LLC-GA5-COL300 cells was significantly increased in the presence of verapamil or ciclosporin, while verapamil did not affect the uptake of [14C]ramosetron by MBEC4 cells. These results indicate that the efflux of [14C]ramosetron is partly mediated by mdr1a P-glycoprotein, but not by mdr1b P-glycoprotein, and that there is a difference in substrate specificity between mdr1a P-glycoprotein and mdr1b P-glycoprotein. Further, [14C]ramosetron was confirmed to be effluxed by human MDR1 P-glycoprotein. We conclude that the limited distribution of ramosetron to the brain is due, at least in part, to efflux mediated by the P-glycoprotein at the blood-brain barrier.

Tumor Necrosis Factor-α Mediates the Blood–Brain Barrier Dysfunction Induced by Activated Microglia in Mouse Brain Microvascular Endothelial Cells

The present study was designed to elucidate the involvement of tumor necrosis factor-α (TNF-α) release from activated microglia in the induction of blood–brain barrier (BBB) dysfunction in an in vitro co-culture system with mouse brain capillary endothelial cells (MBEC4) and microglia. Lipopolysaccharide (LPS)–activated microglia increased the permeability of MBEC4 cells to sodium-fluorescein, and this hyper-permeability was blocked by a neutralizing antibody against TNF-α. LPS stimulated microglia to facilitate TNF-α release. These findings suggested that TNF-α released from activated microglia is attributable to BBB dysfunction.

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase Cϵ-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, IL-17, interferon-gamma, IL-1beta, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-alpha in BCECs and consequently repairs TNF-alpha-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cepsilon (PKCepsilon) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCepsilon inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCepsilon-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.

Adverse effect of cyclosporin A on barrier functions of cerebral microvascular endothelial cells after hypoxia-reoxygenation damage in vitro.

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood-brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan's blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.

Functional Induction of P-glycoprotein in the Blood-Brain Barrier of Streptozotocin-Induced Diabetic Rats: Evidence for the Involvement of Nuclear Factor-κB, a Nitrosative Stress-Sensitive Transcription Factor, in the Regulation

The objective of this study was to investigate the transport kinetics of cyclosporin A, a well known substrate for P-glycoprotein (P-gp), across the blood-brain barrier (BBB), and the expression of the transporter in the brain of streptozotocin-induced diabetic rats. The in vivo transport clearance of cyclosporin A was significantly reduced in diabetic rats compared with that in the control. The decreased transport was associated with the increased level of mRNA and the protein for P-glycoprotein in the rat brain. The functional activity of the efflux transporter in mouse brain capillary endothelial (MBEC4) cells, an in vitro model of the BBB, was also stimulated when slow nitric oxide (NO)-releasing donors were present, whereas the stimulation was absent in the case of rapid NO-releasing donors (e.g., S-nitroso-N-acetyl-dl-penicillamine and diethylenetriamine). The stimulatory effect was highest for sodium nitroprusside (SNP) and the functional induction associated with the increased mRNA and protein level of the transporter. The pretreatment of the cell with SNP along with ascorbate, methylene blue, or superoxide dismutase attenuated the induction of function and expression for P-glycoprotein, suggesting that the reaction product between superoxide and NO is involved in the induction of function and expression. The level of nuclear translocation of nuclear factor-kappaB (NF-kappaB) and DNA binding activity of nuclear extracts to the NF-kappaB consensus oligonucleotide was increased in MBEC4 cells pretreated with SNP. Taken together, these observations suggest that nitrosative stress leads to the up-regulation of the message for the efflux transporter and, ultimately, to the enhanced function, probably via a NF-kappaB-dependent mechanism.

An inhibitory role of nitric oxide in the dynamic regulation of the blood-brain barrier function.

1. The present study aimed at elucidating the effect of nitric oxide (NO) on blood-brain barrier (BBB) function with mouse brain capillary endothelial (MBEC4) cells.2. Histamine (20-100 microM) evoked NO production (1.6-7 microM) in MBEC4 cells in a dose-dependent manner.3. The permeability coefficient of sodium fluorescein for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were increased dose-dependently by the addition of NO solutions (14 and 28 microM) every 10 min during a 30-min period.4. The present study demonstrated that NO increased the permeability and inhibited the P-glycoprotein efflux pump of brain capillary endothelial cells, suggesting that NO plays an inhibitory role in the dynamic regulation of the BBB function.

Protective Action of Indapamide, a Thiazide-Like Diuretic, on Ischemia-Induced Injury and Barrier Dysfunction in Mouse Brain Microvascular Endothelial Cells

The aim of the present study was to elucidate the effects of indapamide on ischemic damage to the blood-brain barrier (BBB) in vitro. The ischemia /reperfusion conditions employed here significantly decreased the viability of mouse brain capillary endothelial (MBEC4) cells, an effect ameliorated by indapamide. Ischemia increased the permeability of MBEC4 cells to two cellular transport markers, sodium fluorescein and Evan’s blue-albumin. Indapamide reduced the ischemia-induced hyperpermeability of cells. These results suggest that indapamide may have a protective role against ischemia-induced injury and dysfunction of the BBB.

Inhibition of Transforming Growth Factor-β Production in Brain Pericytes Contributes to Cyclosporin A-Induced Dysfunction of the Blood-Brain Barrier

: 1. The present study was designed to clarify whether brain pericytes and pericyte-derived transforming growth factor-beta1 (TGF-beta1) participate in cyclosporin A (CsA)-induced dysfunction of the blood-brain barrier (BBB).2. The presence of brain pericytes markedly aggravated CsA-increased permeability of MBEC4 cells to sodium fluorescein and accumulation of rhodamine 123 in MBEC4 cells.3. Exposure to CsA significantly decreased the levels of TGF-beta1 mRNA in brain pericytes in pericyte co-cultures. Treatment with TGF-beta1 dose-dependently inhibited CsA-induced hyperpermeability and P-glycoprotein dysfunction of MBEC4 cells in pericyte co-cultures.4. These findings suggest that an inhibition of brain pericyte-derived TGF-beta1 contributes to the occurrence of CsA-induced dysfunction of the BBB.

Brain pericytes contribute to the induction and up-regulation of blood–brain barrier functions through transforming growth factor-β production

The blood–brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-β (TGF-β) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-β1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-β1 antibody (10 μg/ml) or a TGF-β type I receptor antagonist (SB431542) (10 μM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-β1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-β production.

Functional characterization of adenosine transport across the BBB in mice

We investigated transport characteristics of adenosine across the blood–brain barrier (BBB) in mice. Uptake clearance across the BBB was measured by using an in situ mouse brain perfusion technique and cultured mouse brain capillary endothelial cell line (MBEC4 cells). Nucleoside transporter was cloned by RT-PCR and expressed on Xenopus laevis oocyte. Both in situ and in vitro studies revealed that the adenosine uptake is concentration-dependent, Na +-independent and S-( p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive. The K t values of in situ and in vitro studies were 31.7 ± 13.8 μM and 11.9 ± 2.84 μM, respectively. A good correlation was found for the inhibitory effects of nucleoside analogs to adenosine uptake between in situ and in vitro studies. RT-PCR revealed the expression of RNA of mouse equilibrative nucleoside transporter (mENT1) in mouse brain capillary and MBEC4 cells. In mENT1 expressed on X. laevis oocyte, K t value of adenosine transport was 6.9 ± 2.7 μM (and comparable to those in situ and in vitro studies). In conclusion, we characterized the adenosine transport across the BBB in mice by using in situ brain perfusion technique and MBEC4 cells and found that these transports share common characteristics with mENT1-mediated transport. Transport of adenosine across the BBB in mice may be attributable to mENT1.

Nitric oxide mediates cyclosporine-induced impairment of the blood–brain barrier in cocultures of mouse brain endothelial cells and rat astrocytes

The present study was designed to clarify the involvement of nitric oxide (NO) signaling in the adverse effect of cyclosporine on the blood–brain barrier. Cyclosporine increased the permeability of sodium-fluorescein and the cellular accumulation of rhodamine 123, a substrate of P-glycoprotein, in mouse brain endothelial (MBEC4) cells. This effect was markedly enhanced two- to threefold when MBEC4 cells were cocultured with rat astrocytes or C6 glioma cells. Direct and continuous electrochemical measurement of NO demonstrated that cyclosporine dose-dependently increased histamine- and phenylephrine-evoked NO production in MBEC4 cells and astrocytes, respectively. A NO synthase inhibitor ( N G-monomethyl- l-arginine) blocked slightly and markedly cyclosporine-induced impairment of the endothelial barrier in the monolayer and coculture system, respectively. These findings suggest that cyclosporine impairs the brain endothelial barrier function by accelerating NO production in the brain endothelial and astroglial cells. This event may be interpreted as triggering the occurrence of cyclosporine neurotoxicity.

Transforming growth factor-beta1 upregulates the tight junction and P-glycoprotein of brain microvascular endothelial cells.

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.

Uptake and efflux of quinacrine, a candidate for the treatment of prion diseases, at the blood-brain barrier.

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).

Tolbutamide Uptake Via pH- and Membrane-potential-dependent Transport Mechanism in Mouse Brain Capillary Endothelial Cell Line

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

In vivo delivery of glial cell-derived neurotrophic factor across the blood-brain barrier by gene transfer into brain capillary endothelial cells.

The gene encoding mouse glial cell-derived neurotrophic factor (mGDNF gene) was transfected into brain capillary endothelial cells (BCECs) with the aim of delivering the gene product extensively into the brain parenchyma by making use of the secretory function of BCECs. First, we transfected mGDNF gene into cultured BCECs (MBEC4; mouse brain capillary endothelial cells) in vitro. The amount of mGDNF protein secreted from the transfected cells into the medium was 1500 to 3200 pg/mg of cell protein per day, being about sevenfold higher than that accumulated intracellularly. Furthermore, the basolateral-directed secretion of mGDNF protein from the transfected MBEC4 cells was fivefold higher than the apical-directed secretion. Next, the hemagglutination virus of Japan (HVJ)-liposomes encapsulating mGDNF gene were administered to rats in vivo via the internal carotid artery. The transfected rats showed a marked increase in the brain level of GDNF as assessed by means of enzyme-linked immunosorbent assay (ELISA) and Western blotting on day 3 after the administration, and the level remained significantly elevated for at least 12 days. Furthermore, immunohistochemical staining revealed an increase in GDNF immunoreactivity throughout the transfected forebrain. These results indicate that the gene was successfully transferred in vivo from HVJ-liposomes into BCECs, where it was expressed, and the gene product was secreted into the brain. Then, using this delivery method, we evaluated the protective effect for dopamine neuron against a retrograde 6-hydroxydopamine (6-OHDA) lesion, as assessed by behavioral and neurochemical indices.

Enhancement of L-cystine transport activity and its relation to xCT gene induction at the blood-brain barrier by diethyl maleate treatment.

The purpose of the present study was to elucidate the mechanism of enhancement of L-cystine uptake at the blood-brain barrier (BBB). The uptake of [(14)C]L-cystine and [(3)H]L-glutamic acid (L-Glu) was determined using a mouse brain endothelial cell line (MBEC4) as an in vitro BBB model. The mRNA levels of L-cystine/L-Glu exchanger, system x(c)(-), which consists of xCT and 4F2hc, were determined by quantitative real-time reverse transcription-polymerase chain reaction analysis. The [(14)C]L-cystine uptake by MBEC4 cells appeared to be mediated via an Na(+)-independent saturable process. The corresponding Michaelis-Menten constant (K(m)) was 63.7 microM. In the presence of L-Glu, there was competitive inhibition with an inhibition constant (K(i)) of 83.5 microM. [(3)H]L-Glu uptake in the absence of Na(+) was saturable with a K(m) of 48.1 microM, and it exhibited competitive inhibition with a K(i) of 24.9 microM in the presence of L-cystine. The mutual inhibition between L-cystine and L-Glu and the type of inhibition suggest that system x(c)(-) operates in MBEC4 cells. The xCT and 4F2hc mRNAs were expressed in MBEC4 cells and, following diethyl maleate (DEM) treatment, the xCT mRNA level and L-cystine uptake in MBEC4 cells were enhanced in parallel with an increase in DEM concentration (up to 500 microM). Concomitantly, the glutathione concentration in MBEC4 cells was increased. In conclusion, system x(c)(-)-mediated L-cystine uptake takes place in MBEC4 cells. L-Cystine transport via system x(c)(-) at the BBB is likely to be induced under oxidative stress conditions following DEM treatment due to enhanced transcription of the xCT gene.

Transluminal Gene Transfer into Brain Capillary Endothelial Cells In Vivo with HVJ-liposomes

Bioactive proteins or peptides cannot be effectively delivered into brain capillary endothelial cells (BCECs) or brain parenchyma. In this study, we selectively transferred Escherichia coli β -galactosidase gene (β -gal) as a model gene into BCECs by using the hemagglutination virus of Japan (HVJ)-liposomes. HVJ-liposomes encapsulating a β -gal plasmid were used to transfect MBEC4 cells in vitro, and were administrated via the internal carotid artery to rat in vivo. Success of the procedure was confirmed by the detection of 116 kDa β -gal protein in transfected MBEC4 cells and in brain capillaries isolated from transfected rats, by Western blot analysis and histological staining. The enzymatic activities of β -galactosidase were 5- to 10-fold and 20-fold higher than when β -gal-containing liposomes without fusogenic activity (uncoated liposomes) or plasmid alone were employed in vitro and in vivo, respectively. Thus, HVJ-liposomes were demonstrated to be a useful vector to transfer a foreign gene into the brain capillary endothelium in vivo via the transluminal route.

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier.

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.