The efficiency of glutamate uptake differs between neonatal and adult cortical microvascular endothelial cells

Glutamate transporters (excitatory amino-acid transporters (EAATs)) are essential for brain homeostasis. While previous studies indicate that the vascular endothelium contributes to glutamate efflux in the adult brain, little information is available regarding glutamate uptake in the immature brain. The present study shows a differential expression pattern of EAATs between cortical microvessels in adults and newborns. In addition, adult cortical endothelial cells take up glutamate more efficiently than neonatal cells. Our findings indicate age-specific changes in extracellular glutamate regulation by brain endothelial cells, suggesting differences in the efficiency of glutamate efflux during an excitotoxic process that, in turn, may contribute to age-specific brain vulnerability.     

Blood-brain barrier active efflux transporters: ATP-binding cassette gene family

The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from potentially harmful endogenous and exogenous substances. BBB active drug efflux transporters of the ATP-binding cassette (ABC) gene family are increasingly recognized as important determinants of drug distribution to, and elimination from, the CNS. The ABC efflux transporter P-glycoprotein (Pgp) has been demonstrated as a key element of the BBB that can actively transport a huge variety of lipophilic drugs out of the brain capillary endothelial cells that form the BBB. In addition to Pgp, other ABC efflux transporters such as members of the multidrug resistance protein (MRP) family and breast cancer resistance protein (BCRP) seem to contribute to BBB function. Consequences of ABC efflux transporters in the BBB include minimizing or avoiding neurotoxic adverse effects of drugs that otherwise would penetrate into the brain. However, ABC efflux transporters may also limit the central distribution of drugs that are beneficial to treat CNS diseases. Furthermore, neurological disorders such as epilepsy may be associated with overexpression of ABC efflux transporters at the BBB, resulting in pharmacoresistance to therapeutic medication. Therefore, modulation of ABC efflux transporters at the BBB forms a novel strategy to enhance the penetration of drugs into the brain and may yield new therapeutic options for drug-resistant CNS diseases.     

Brain-to-blood transporters for endogenous substrates and xenobiotics at the blood-brain barrier: An overview of biology and methodology

In the past decade, research into P-glycoprotein involving the blood-brain barrier (BBB) has seen a shift in the concept of the BBB as a structural barrier to that of a functional barrier for xenobiotics and changed simultaneously the strategy for the discovery and development of drugs acting in the CNS. As far as making advances in neurotherapeutics are concerned, the brain-to-blood transport function at the BBB will be one of the most important issues. Knowing the limitations of thein vivo andin vitro methods for BBB efflux research, it is essential to adopt a multidisciplinary approach in investigating the true physiological role of the BBB. Among several methods, the Brain Efflux Index method and the use of conditionally immortalized brain capillary endothelial cell lines, established from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene, are likely to be very useful tools for the BBB efflux transport research. According to our recent findings using these methods, several transporters in the brain capillary endothelial cells appear to play an important role in reducing the brain level of hydrophilic endogenous substrates produced either in the brain or peripheral organs, e.g., neurotransmitters, neuromodulators, metabolites of neurotransmitters, and uremic toxins. It has been reported also that large hydrophilic molecules, such as IgG, apo-transferrin, and amyloid-β peptide, are susceptible to brain-to-blood efflux transport. In the light of the latest findings, we have formed the hypothesis that the BBB acts as a CNS detoxifying system for both endogenous substrates and xenobiotics in the brain. A fuller understanding of the physiological role of BBB efflux transporters will provide rational insights to assist in the development of safer neurotherapeutics.     

A Model for Fatty Acid Transport into the Brain

A key function of fatty acid (FA) transport into the brain is to supply polyunsaturated fatty acids (PUFA) that are not synthesized in brain cells but are essential signaling molecules and components of the phospholipid membrane. In addition, common dietary FAs such as palmitic acid are also rapidly taken up by the brain and esterified to phospholipids or oxidized to provide cellular energy. Most evidence shows that FA crossing the blood brain barrier (BBB) is derived mainly from FA/albumin complexes and, to a lesser extent, from circulating lipoproteins. Our model proposes that FA diffuse across the lipid bilayer of the BBB without specific transporters to reach brain cells. They cross the luminal and transluminal leaflets of the endothelial cells and the plasma membrane of neural cells by reversible flip-flop. Acyl-CoA synthetases trap FA by forming acyl-CoA, which cannot diffuse out of the cell. Selection of FA is controlled largely by enzymes in the pathways of intracellular metabolism, beginning with the acyl-CoA synthetase.     

Structure and function of the blood–brain barrier

Neural signalling within the central nervous system (CNS) requires a highly controlled microenvironment. Cells at three key interfaces form barriers between the blood and the CNS: the blood–brain barrier (BBB), blood–CSF barrier and the arachnoid barrier. The BBB at the level of brain microvessel endothelium is the major site of blood–CNS exchange. The structure and function of the BBB is summarised, the physical barrier formed by the endothelial tight junctions, and the transport barrier resulting from membrane transporters and vesicular mechanisms. The roles of associated cells are outlined, especially the endfeet of astrocytic glial cells, and pericytes and microglia. The embryonic development of the BBB, and changes in pathology are described. The BBB is subject to short and long-term regulation, which may be disturbed in pathology. Any programme for drug discovery or delivery, to target or avoid the CNS, needs to consider the special features of the BBB.     

Probenecid-inhibitable efflux transport of valproic acid in the brain parenchymal cells of rabbits: a microdialysis study

Delivery of valproic acid (VPA) to the human brain is relatively inefficient as reflected by a low brain-to-unbound plasma concentration ratio (< or =0.5) at steady state. Previous pharmacokinetic studies suggested that the unfavorable brain-to-plasma gradient is maintained by coupled efflux transport processes at both the brain parenchymal cells and blood-brain barrier (BBB); one or both of the efflux transporters are inhibitable by probenecid. The present study in rabbits utilized microdialysis to measure drug concentration in the brain extracellular fluid (ECF) of the cerebral cortex during steady-state i.v. infusion with VPA alone or with VPA plus probenecid. Probenecid co-infusion elevated VPA concentration in the brain tissue surrounding the tip of the microdialysis probe to a greater extent than in the ECF (230% versus 47%). Brain intracellular compartment (ICC) concentration was estimated. In control rabbits, the ICC concentration was 2.8+/-0.28 times higher than the ECF concentration. Probenecid co-infusion elevated the ICC-to-ECF concentration ratio to 4.2+/-0.44, which confirms the existence of an efflux transport system in brain parenchymal cells. The ECF-to-unbound plasma concentration ratio was well below unity (0.029), indicating an uphill efflux transport of VPA across the BBB. Co-infusion of probenecid did not have a significant effect on VPA efflux at the BBB as evidenced by a minimal change in the ECF-to-unbound plasma concentration ratio. This study suggests the presence of distinctly different organic anion transporters for the efflux of VPA at the parenchymal cells and capillary endothelium in the brain.     

Membrane transporter proteins: a challenge for CNS drug development

Drug transporters are membrane proteins present in various tissues such as the lymphocytes, intestine, liver, kidney, testis, placenta, and central nervous system. These transporters play a significant role in drug absorption and distribution to organic systems, particularly if the organs are protected by blood-organ barriers, such as the blood-brain barrier or the maternal-fetal barrier. In contrast to neurotransmitters and receptor-coupled transporters or other modes of interneuronal transmission, drug transporters are not directly involved in specific neuronal functions, but provide global protection to the central nervous system. The lack of capillary fenestration, the low pinocytic activity and the tight junctions between brain capillary and choroid plexus endothelial cells represent further gatekeepers limiting the entrance of endogenous and exogenous compounds into the central nervous system. Drug transport is a result of the concerted action of efflux and influx pumps (transporters) located both in the basolateral and apical membranes of brain capillary and choroid plexus endothelial cells. By regulating efflux and influx of endogenous or exogenous substances, the blood-brain barrier and, to a lesser extent the blood-cerebrospinal barrier in the ventricles, represents the main interface between the central nervous system and the blood, i.e., the rest of the body. As drug distribution to organs is dependent on the affinity of a substrate for a specific transport system, membrane transporter proteins are increasingly recognized as a key determinant of drug disposition. Many drug transporters are members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily or the solute-linked carrier (SLC) class. The multidrug resistance protein MDR1 (ABCB1), also called P-glycoprotein, the multidrug resistance-associated proteins MRP1 (ABCC1) and MRP2 (ABCC2), and the breast cancer-resistance protein BCRP (ABCG2) are ATP-dependent efflux transporters expressed in the blood-brain barrier They belong to the superfamily of ABC transporters, which export drugs from the intracellular to the extracellular milieu. Members of the SLC class of solute carriers include, for example, organic ion transporting peptides, organic cation transporters, and organic ion transporters. They are ATP-independent polypeptides principally expressed at the basolateral membrane of brain capillary and choroid plexus endothelial cells that also mediate drug transport through central nervous system barriers.     

TNFalpha transport across the blood-brain barrier is abolished in receptor knockout mice

The presence of transport systems at the blood-brain barrier (BBB) enables some cytokines in blood to reach specific targets in the brain and spinal cord. The "transporters" function in a way different from conventional receptors, in that cytokines are chaperoned from blood to the CNS rather than being degraded in the specialized endothelial cells composing the BBB. Here we present the first study to determine whether the transporter for tumor necrosis factor-alpha (TNFalpha) is identical to its receptors. Three types of TNFalpha receptor knockout mice were used, and the influx of (125)I-TNFalpha from blood to brain and blood to spinal cord was measured. In either p55 or p75 receptor knockout mice, the influx of (125)I-TNFalpha was significantly, but not completely, decreased in spinal cord, whereas the decrease in brain was not statistically significant. This indicates that both receptors are partially involved in the transport of TNFalpha across the BBB but that neither receptor is the sole transporter. By contrast, in double knockout mice lacking both p55 and p75 receptors, the entry of (125)I-TNFalpha into brain and spinal cord was completely abolished. Therefore, both receptors are necessary for transporting TNFalpha across the BBB. The results clearly demonstrate that the transport of TNFalpha across the BBB is a complicated process involving additive or even synergistic activities of both receptors, thus differing from typical ligand-receptor binding and downstream signal transduction.     

Small molecular drug transfer across the blood-brain barrier via carrier-mediated transport systems

Because of the physiological nature of the blood-brain barrier (BBB), transport of chemical compounds between blood and brain has been widely believed to occur by means of passive diffusion, depending upon the lipophilicity of the compounds. However, discrepancies exist between the lipophilicity and apparent BBB permeation properties in many cases, and these discrepancies can be ascribed to the existence of multiple mechanisms of drug transport through the BBB. Molecular identification and functional analysis of influx transport proteins (from blood to brain) and efflux transport proteins (from brain to blood) have progressed rapidly. Therefore, the BBB is now considered to be a dynamic interface that controls the influx and efflux of a wide variety of substances, including endogenous nutrients and exogenous compounds such as drugs, to maintain a favorable environment for the CNS. This review focuses on the role of transport systems in the uptake of xenobiotics, including organic anionic/cationic and neutral drugs, across the BBB into the brain, as well as on strategies to increase drug delivery into the brain by blocking efflux transport protein function, or to reduce CNS side effects by modulating BBB transport processes.     

Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system that strictly controls the exchanges between the blood and brain compartments, therefore playing a key role in brain homeostasis and providing protection against many toxic compounds and pathogens. In this review, the unique properties of brain microvascular endothelial cells and intercellular junctions are examined. The specific interactions between endothelial cells and basement membrane as well as neighboring perivascular pericytes, glial cells and neurons, which altogether constitute the neurovascular unit and play an essential role in both health and function of the central nervous system, are also explored. Some relevant pathways across the endothelium, as well as mechanisms involved in the regulation of BBB permeability, and the emerging role of the BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the experimental approaches that can be used to monitor BBB properties and function in a variety of conditions and have allowed recent advances in BBB knowledge. Elucidation of the molecular anatomy and dynamics of the BBB is an essential step for the development of new strategies directed to maintain or restore BBB integrity and barrier function and ultimately preserve the delicate interstitial brain environment.     

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood–brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood–brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.     

Neuroinflammation: a common pathway in CNS diseases as mediated at the blood-brain barrier

The blood-brain barrier (BBB) is not simply a physical barrier but a regulatory interface between the central nervous system (CNS) and immune system. The BBB both affects and is affected by the immune system and connects at many levels with the CNS, including the following: (1) the BBB transports cytokines and secretes various substances with neuroinflammatory properties; (2) transporters are altered in disease states including traumatic injury, Alzheimer's disease and inflammatory processes; (3) cytokines and other immune secretions from the cells comprising the BBB are both constitutive and inducible; (4) immune cells are transported across the BBB by the highly regulated process termed diapedesis, which involves communication and interactions between the brain endothelial cells and the immune cells; (5) the neuroimmune system has various effects on the BBB, including modulation of important transport systems and in extreme pathological conditions even disruption of the BBB, and (6) the brain-to-blood efflux transporter P-glycoprotein is altered in inflammatory conditions, thus affecting drug delivery to the brain. In summary, the BBB is an interactive interface that regulates and defines many of the ways that the CNS and the immune system communicate with one another.     

Active efflux across the blood-brain barrier: Role of the solute carrier family

The brain uptake of xenobiotics is restricted by the blood-brain brain barrier formed by brain capillary endothelial cells. Active efflux transport systems in the blood-brain barrier work as a detoxification system in the brain by facilitating removal of xenobiotic compounds from the brain. Drugs, acting in the brain, have to overcome such efflux mechanisms to achieve clinically significant concentration in the brain. Multiple transporters are involved in this efflux transport in the brain capillaries. In the past few years, considerable progress has been made in the cloning of these transporters and their functional characterization after heterologous expression. Members of the solute carrier family (SLC) play an important role in the efflux transport, especially for organic anions, which include organic anion transporting polypeptides (OATP/SLCO) and organic anion transporters (OAT/SLC22A). It is believed that coordination of the members of SLC family, and ABC transporters, such as P-glycoprotein, multidrug resistance protein, and breast cancer-resistant protein (BCRP/ABCG2), allows an efficient vectorial transport across the endothelial cells to remove xenobiotics from the brain. In this review, we shall summarize our current knowledge about their localization, molecular and functional characteristics, and substrate and inhibitor specificity.     

Cellular prion protein participates in amyloid-β transcytosis across the blood-brain barrier

The blood-brain barrier (BBB) facilitates amyloid-β (Aβ) exchange between the blood and the brain. Here, we found that the cellular prion protein (PrP(c)), a putative receptor implicated in mediating Aβ neurotoxicity in Alzheimer's disease (AD), participates in Aβ transcytosis across the BBB. Using an in vitro BBB model, [(125)I]-Aβ(1-40) transcytosis was reduced by genetic knockout of PrP(c) or after addition of a competing PrP(c)-specific antibody. Furthermore, we provide evidence that PrP(c) is expressed in endothelial cells and, that monomeric Aβ(1-40) binds to PrP(c). These observations provide new mechanistic insights into the role of PrP(c) in AD.     

Atrial natriuretic peptide is eliminated from the brain by natriuretic peptide receptor-C-mediated brain-to-blood efflux transport at the blood–brain barrier

Cerebral atrial natriuretic peptide (ANP), which is generated in the brain, has functions in the regulation of brain water and electrolyte balance, blood pressure and local cerebral blood flow, as well as in neuroendocrine functions. However, cerebral ANP clearance is still poorly understood. The purpose of this study was to clarify the mechanism of blood–brain barrier (BBB) efflux transport of ANP in mouse. Western blot analysis showed expression of natriuretic peptide receptor (Npr)-A and Npr-C in mouse brain capillaries. The brain efflux index (BEI) method confirmed elimination of [¹²⁵I]human ANP (hANP) from mouse brain across the BBB. Inhibition studies suggested the involvement of Npr-C in vivo. Furthermore, rapid internalization of [¹²⁵I]hANP by TM-BBB4 cells (an in vitro BBB model) was significantly inhibited by Npr-C inhibitors and by two different Npr-C-targeted short interfering RNAs (siRNAs). Finally, treatment with 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) significantly increased Npr-C expression in TM-BBB4 cells, as determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based targeted absolute proteomics. Our results indicate that Npr-C mediates brain-to-blood efflux transport of ANP at the mouse BBB as a pathway of cerebral ANP clearance. It seems likely that levels of natriuretic peptides in the brain are modulated by 1,25(OH)₂D₃ through upregulation of Npr-C expression at the BBB.     

Brain access and anticonvulsant efficacy of carbamazepine, lamotrigine, and felbamate in ABCC2/MRP2-deficient TR- rats

Summary: Purpose: Different adenosine triphosphate (ATP)‐driven multidrug transporters have been described to be expressed in the luminal membrane of blood–brain barrier (BBB) endothelial cells. At this site, multidrug transporters have been suggested to restrict penetration of drugs into the brain. Increasing evidence suggests that overexpression of different multidrug transporters occurs in the region of the epileptic focus of pharmacoresistant epilepsy patients. Based on the assumption that antiepileptic drugs (AEDs) are substrates of these transporters, this overexpression may limit access of AEDs to epileptic neurons and may contribute to drug‐refractoriness. In a recent study, overexpression of multidrug resistance protein 2 (ABCC2; MRP2) was reported in BBB endothelial cells of epileptic focal tissue from pharmacoresistant patients. With brain microdialysis, we recently demonstrated that the AED phenytoin is subject to transport by ABCC2 at the BBB, whereas phenobarbital does not seem to be a substrate of ABCC2. Methods: We investigated whether ABCC2 is functionally involved in transport of the AEDs carbamazepine (CBZ), lamotrigine (LTG), and felbamate (FBM) across the BBB. The distribution of these AEDs into the brain of ABCC2‐deficient TR^? rats was determined. Results: AED concentrations in plasma and brain extracellular space of these mutant rats did not differ significantly from those of rats of the corresponding background strain. In the amygdala‐kindling model of epilepsy, the anticonvulsant efficacy of LTG and FBM was comparable in both groups of rats. In contrast, CBZ exhibited a higher anticonvulsant activity in kindled ABCC2‐deficient rats as compared with nonmutant rats. Conclusions: In this present study, the microdialysis results gave no evidence that ABCC2 function modulates entry of CBZ, LTG, and FBM into the CNS of naïve rats. However, ABCC2 deficiency was associated with an increased anticonvulsant response of CBZ in the kindling model. Future investigations are planned to identify the underlying mechanism for this difference, clarifying whether a pharmacokinetic difference is detectable only when brain access of CBZ is compared in kindled ABCC2‐deficient rats and kindled nonmutant rats, which may have an increased expression of ABCC2 in response to seizures. The data substantiate that ABCC2‐deficient TR^? rats are a useful tool for defining the role of ABCC2 for transport of AEDs, and give evidence that the use of kindled TR^? rats may provide important supplementary information.     

Functional characterisation of nucleoside transport in rat brain endothelial cells

Multiple nucleoside transport systems exist in the body yet the subtypes functional at the blood-brain barrier (BBB) have not been fully investigated. We have employed RBE4 immortalised rat brain endothelial cells to functionally identify the carrier subtypes involved in nucleoside transfer between blood and brain. Uptake in RBE4 cells was partially sodium dependent, indicating the presence of both equilibrative and concentrative systems. Uptake of adenosine via equilibrative transporters was sensitive to nitro-benzylmercaptopurine riboside, which showed biphasic inhibition. Uptake of [3H]-adenosine via concentrative transporters was studied using the subtype-specific inhibitors thymidine (cit), formycin-B (cif) and tubercidin (cib) and was significantly reduced by thymidine and formycin-B but not by tubercidin. This study suggests that nucleoside transport at the in situ BBB may be mediated by ei and es equilibrative transporters and by cit and cif concentrative transporters.     

P-glycoprotein-mediated efflux of phenobarbital at the blood-brain barrier evidence from transport experiments in vitro

The purpose of the present studies was to investigate characteristics of phenobarbital (PB) transport across the blood-brain barrier (BBB). Cultured rat brain microvascular endothelial cells (rBMECs) were used as an in vitro BBB model. Experiments were conducted to examine time-, concentration- and temperature-dependent elements of PB uptake, and the effect of tested agents on the steady-state uptake of PB. PB efflux from rBMECs and the polarised transport of PB were examined to evaluate whether P-glycoprotein (P-gp) was involved in the PB efflux transport across BBB. The results demonstrated that the uptake of PB by rBMECs was in a time-, concentration- and temperature-dependent manner. P-gp modulators, cyclosporin A (CsA), ketoconazole and metabolic inhibitor dinitrophenol, increased the PB steady-state uptake by more than 50% (p < 0.01), and decreased the PB efflux by more than 50% (p < 0.01). Similar results were observed in the uptake and efflux studies of rhodamine-123 by co-administration of CsA. Further results were obtained in the polarised transendothelial transport of PB in the apical to basolateral (A-B) and basolateral to apical (B-A) directions either with or without CsA. The result showed that transport of PB in the B-A direction was significantly greater than the transport in the A-B direction (p < 0.05), and the co-administration of 50microM CsA almost inhibited P-gp-involved efflux in the rBMECs. Overall, these findings suggest that P-gp may contribute to the efflux transport of PB across BBB.     

Intact lipid rafts regulate HIV-1 Tat protein-induced activation of the Rho signaling and upregulation of P-glycoprotein in brain endothelial cells

The Rho signaling has an essential function in human immunodeficiency virus (HIV)-1-mediated disruption of the integrity of the blood–brain barrier (BBB). However, it is unknown how membrane domains, such as lipid rafts, can influence HIV-1-mediated activation of the Rho pathway and how these processes can affect the expression of the efflux transporters at the BBB level. This study is focused on the function of HIV-1 protein Tat in activation of the Rho signaling and upregulation of P-glycoprotein (P-gp) in human brain endothelial cells. Treatment with Tat markedly elevated GTP-RhoA levels and the potential downstream effectors, such as myosin phosphatase target subunit 1 and myosin light chain. In addition, Tat upregulated expression and promoter activity of P-gp as well as its efflux function. Inhibition of the Rho signaling cascade effectively blocked P-gp overexpression at the level of promoter activity. Disruption of lipid rafts by depletion of membrane cholesterol by methyl-beta-cyclodextrin, but not caveolin-1 silencing, also abolished Tat-mediated RhoA activation and P-gp upregulation. The present data indicate the critical function of intact lipid rafts and the Rho signaling in HIV-1-mediated upregulation of P-gp and potential development of drug resistance in brain endothelial cells.