Glutamine transport at the blood-brain and blood-cerebrospinal fluid barriers

Glutamine has multiple physiological and pathophysiological roles in the brain. Because of their position at the interface between blood and brain, the cerebral capillaries and the choroid plexuses that form the blood-brain barriers (BBB) and blood-cerebrospinal fluid (CSF) barriers, have the potential to influence brain glutamine concentrations. Despite this, there has been a paucity of data on the mechanisms and polarity of glutamine transport at these barrier tissues. In situ brain perfusion in the rat, indicates that blood to brain L-[14C]glutamine transport at the blood-brain barrier is primarily mediated by a pH-dependent, Na(+)-dependent, System N transporter, but that blood to choroid plexus transport is primarily via a pH-independent System N transporter and a Na(+)-independent carrier that is not System L. Transport studies in isolated rat choroid plexuses and primary cultures of choroid plexus epithelial cells indicate that epithelial L-[14C]glutamine transport is polarized (apical uptake>basolateral) and that uptake at the apical membrane is mediated by pH dependent System N transporters (identified as SN1 and SN2 by polymerase chain reaction) and the Na(+)-independent System L. Blood-brain barrier System N transport is markedly effected by cerebral ischemia and may be a good marker of endothelial cell dysfunction. The multiple glutamine transporters at the blood-brain and blood-CSF barriers may have role in meeting the metabolic needs of the brain and the barrier tissues themselves. However, it is likely that the main role of these transporters is removing glutamine, and thus nitrogen, from the brain.     

Effects of cell-type specific leptin receptor mutation on leptin transport across the BBB

The functions of leptin receptors (LRs) are cell-type specific. At the blood-brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood-brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted.     

Drug metabolizing enzymes in cerebrovascular endothelial cells afford a metabolic protection to the brain.

The brain is partially protected from chemical insults by a physical barrier mainly formed by the cerebral microvasculature, which prevents penetration of hydrophilic molecules in the cerebral extracellular space. This results from the presence of tight junctions joining endothelial cells, and from a low transcytotic activity in endothelial cells, inducing selective permeability properties of cerebral microvessels that characterize the blood-brain barrier. The endothelial cells provide also, as a result of their drug-metabolizing enzymes activities, a metabolic barrier against potentially penetrating lipophilic substances. It has been established that in cerebrovascular endothelial cells, several families of enzymes metabolize potentially toxic lipophilic substrates from both endogenous and exogenous origin to polar metabolites, which may not be able to penetrate further across the blood-brain barrier. Enzymes of drug metabolism present at brain interfaces devoid of blood-brain barrier, like circumventricular organs, pineal gland, and hypophysis, that are potential sites of entry for xenobiotics, display higher activities than in cerebrovascular endothelial cells, and conjugation activities are very high in the choroid plexus. Finally, xenobiotic metabolism normally results in detoxication, but also in some cases in the formation of pharmacologically active or neurotoxic products, possibly altering some blood-brain barrier properties.     

Fatty acid transport into the brain: of fatty acid fables and lipid tails

The blood-brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Brain capillaries are a continuous layer of endothelial cells with highly developed tight junctional complexes and a lack of fenestrations. The presence of these tight junctions in the cerebral microvessel endothelial cells aids in the restriction of movement of molecules and solutes into the brain. Fatty acids are important components of biological membranes, are precursors for the biosynthesis of phospholipids and sphingolipids and are utilized for mitochondrial β-oxidation. The brain is capable of synthesizing only a few fatty acids. Hence, most fatty acids must enter into the brain from the blood. Here we review current mechanisms of transport of free fatty acids into cells and describe how free fatty acids move from the blood into the brain. We discuss both diffusional as well as protein-mediated movement of fatty acids across biological membranes.     

Saturable Retention of Vasopressin by Hippocampus Vessels In Vivo, Associated with Inhibition of Blood-Brain Transfer of Large Neutral Amino Acids

Vasopressin receptors have been reported in the endothelium of brain capillaries. The function of these receptors is not known. To test the prediction that vasopressin receptors in brain capillary endothelium affect amino acid transport across the blood-brain barrier and to assess the role of vasopressin transport across the cerebral vascular endothelium, we measured (a) the endothelial permeability to the large neutral amino acid leucine in the absence and presence of arginine vasopressin (AVP) and (b) the permeability of the blood-brain barrier to AVP relative to manitol. In brain regions protected by the blood-brain barrier, after circulation for 20 s, coinjection of leucine and AVP intravenously led to a decrease of leucine transport unrelated to changes of blood flow. The decrease was most pronounced in hippocampus (42%) and least pronounced in olfactory bulb and colliculi (17 and 19%, respectively). In the latter regions, the endothelial permeability to AVP did not significantly exceed that of mannitol. In hippocampus and in regions with no blood-brain barrier (pituitary and pineal glands), AVP retention in excess of mannitol retention was blocked by unlabeled AVP. The findings do not contradict the hypothesis of a role for AVP in the regulation of large neutral amino acid transfer into brain tissue.     

Transport of ions across the blood-brain barrier

Capillaries in the brain are formed by a uniquely specialized endothelial cell that regulates the movement of substances between blood and brain. Although they provide an impermeable barrier to some solutes, brain capillary endothelial cells facilitate the transcapillary exchange of others. In addition, they contain specific enzymes that contribute to a metabolic blood-brain barrier by limiting the movement of compounds such as neurotransmitters across the capillary wall. Studies of sodium and potassium transport by brain capillaries indicate that the endothelial cell contains distinct types of ion transport systems on the two sides of the capillary wall, i.e., the luminal and antiluminal membranes of the endothelial cell. As a result, specific solutes can be pumped across the capillary against an electrochemical gradient. These transport systems are likely to play a role in the active secretion of fluid from blood to brain and in maintaining a constant concentration of ions in the brain's interstitial fluid. In this way, the brain capillary endothelium is structurally and functionally related to an epithelium.     

The complementary membranes forming the blood-brain barrier

Brain capillary endothelial cells form the blood-brain barrier. They are connected by extensive tight junctions, and are polarized into luminal (blood-facing) and abluminal (brain-facing) plasma membrane domains. The polar distribution of transport proteins allows for active regulation of brain extracellular fluid. Experiments on isolated membrane vesicles from capillary endothelial cells of bovine brain demonstrated the polar arrangement of amino acid and glucose transporters, and the utility of such arrangements have been proposed. For instance, passive carriers for glutamine and glutamate have been found only in the luminal membrane of blood-brain barrier cells, while Na-dependent secondary active transporters are at the abluminal membrane. This organization could promote the net removal of nitrogen-rich amino acids from brain, and account for the low level of glutamate penetration into the central nervous system. Furthermore, the presence of a gamma-glutamyl cycle at the luminal membrane and Na-dependent amino acid transporters at the abluminal membrane may serve to modulate movement of amino acids from blood-to-brain. Passive carriers facilitate amino acid transport into brain. However, activation of the gamma-glutamyl cycle by increased plasma amino acids is expected to generate oxoproline within the blood-brain barrier. Oxoproline stimulates secondary active amino acid transporters (Systems A and B(o)+) at the abluminal membrane, thereby reducing net influx of amino acids to brain. Finally, passive glucose transporters are present in both the luminal and abluminal membranes of the blood-brain barrier. Interestingly, a high affinity Na-dependent glucose carrier has been described only in the abluminal membrane. This raises the question whether glucose entry may be regulated to some extent. Immunoblotting studies suggest more than one type of passive glucose transporter exist in the blood-brain barrier, each with an asymmetrical distribution. In conclusion, it is now clear that the blood-brain barrier participates in the active regulation of brain extracellular fluid, and that the diverse functions of each plasma membrane domain contributes to these regulatory functions.     

Cationization, a process for the delivery of antibodies to the central nervous system. Problems encountered in its application for immunotherapy strategies such as those for clostridial poisoning

The central nervous system is separated from the rest of the body by the blood-brain barrier. This barrier prevents many substances, such as the antibodies, to penetrate into the brain making it difficult to use them for the treatment of brain diseases, such as tetanus and botulism. These two diseases are caused by the development of bacilli of the genus Clostridium which release neurotropic toxins. Specific antibodies can neutralize toxin activity when the toxin is in the blood but are ineffective when it is transported into nerve cells. Various invasive strategies have been used to deliver antibodies to the brain. However, they can induce seizures and transient neurologic deficits and may be applicable only for diseases restricted to the brain surface. Physiologically based strategies utilizing transport systems naturally present at the blood-brain barrier appear to be a more promising approach to brain delivery of antibodies. Cationization is a chemical treatment that causes the conversion of superficial carboxyl groups on a protein into extended primary amino groups. This is used to increase interactions of this protein with the negative charges at the luminal plasma membrane of the brain endothelial cells. The cationized protein can then undergo adsorptive mediated transcytosis through the blood-brain barrier. There are many problems yet to be solved in successfully carrying out in vivo applications of cationized antibodies. One of these problems is that cationization can cause damage to an antibody molecule and, thus, can compromise its binding affinity. Depending on the radiolabelling of the cationized antibodies, a serum inhibition phenomenon can possibly alter the pharmacokinetics and the organ distribution of these molecules. The antibodies can be cationized using various, synthetic (hexamethylenediamine) or naturally occuring (e.g., putrescine) polyamines. Hexamethylenediamine-induced and putrescine-induced brain uptakes of various antibodies and proteins have been shown, but the results obtained suggest that cationization with putrescine may be a more efficient approach to blood-brain barrier delivery. The development of animal or cellular models to check for therapeutic efficacy of cationized antibodies is necessary. In spite of the difficulties, the studies described in this paper indicate that cationization can be a realistic delivery strategy for carrying antibodies across the blood-brain barrier. The advances made in antibody technologies help generate more appropriate immunological structures for brain transfer with better effector functions and decreased immunogenicity or toxicity. Taken together, these two aspects can lead to further developments in treatment of intoxications caused by the clostridial neurotoxins.     

Vascular Perfusion and Blood-Brain Glucose Transport in Acute and Chronic Hyperglycemia

We studied the effects of acute and streptozotocin-induced chronic hyperglycemia on regional brain blood flow and perfusion characteristics, and on the regional transport of glucose across the blood-brain barrier in awake rats. We found (1) a generalized decrease in regional brain blood flow in both acute and chronic hyperglycemia; (2) that chronic, but not acute, hyperglycemia is associated with a marked and diffuse decrease in brain L-glucose space; and (3) that chronic hyperglycemia does not alter blood-to-brain glucose transport. Taken together, these results suggest that in streptozotocin-induced chronic hyperglycemia, there is a reduction in the proportion of perfused brain capillaries and/or an alteration in brain endothelial membrane properties resulting in decreased noncarrier diffusion of glucose.     

An Easier, Reproducible, and Mass-Production Method to Study the Blood–Brain Barrier In Vitro

To provide an "in vitro" system for studying brain capillary function, we have developed a process of coculture that closely mimics the "in vivo" situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier, including tight junctions and gamma-glutamyl transpeptidase activity. The average electric resistance for the monolayers was 661 omega cm2. The system is impermeable to inulin and sucrose but allows the transport of leucine. Arabinose treatment increases transcellular transport flux by 70%. The relative ease with which such monolayers can be produced in large quantities would facilitate the "in vitro" study of brain capillary functions.     

Blood-brain barrier and new approaches to brain drug delivery.

Morbidity caused by brain dysfunction affects more than 50 million persons in the United States. Although new neuropharmaceuticals have the potential for treating specific brain diseases, they may not effectively enter brain from blood. Safe strategies are needed for drug delivery through the brain capillary wall, which makes up the blood-brain barrier in vivo. Two of these strategies are reviewed, as are related new developments in the molecular and cell biology of the brain capillary endothelium. The production of chimeric peptides represents a physiologic-based strategy for drug delivery. It entails the covalent coupling of the neuropharmaceutical to a brain transport vector, allowing transportation through the blood-brain barrier. Another strategy is biochemical opening of the blood-brain barrier: intracarotid leukotriene infusion is a method for selectively increasing blood-brain barrier permeability in brain tumors without affecting barrier permeability in normal brain tissue.     

Blood-Brain Barrier Effects of the Fusarium Mycotoxins Deoxynivalenol, 3 Acetyldeoxynivalenol,and Moniliformin and Their Transfer to the Brain

Background

Secondary metabolites produced by Fusarium fungi frequently contaminate food and feed and have adverse effects on human and animal health. Fusarium mycotoxins exhibit a wide structural and biosynthetic diversity leading to different toxicokinetics and toxicodynamics. Several studies investigated the toxicity of mycotoxins, focusing on very specific targets, like the brain. However, it still remains unclear how fast mycotoxins reach the brain and if they impair the integrity of the blood-brain barrier. This study investigated and compared the effects of the Fusarium mycotoxins deoxynivalenol, 3-acetyldeoxynivalenol and moniliformin on the blood-brain barrier. Furthermore, the transfer properties to the brain were analyzed, which are required for risk assessment, including potential neurotoxic effects.

Methods

Primary porcine brain capillary endothelial cells were cultivated to study the effects of the examined mycotoxins on the blood-brain barrier in vitro. The barrier integrity was monitored by cellular impedance spectroscopy and ¹⁴C radiolabeled sucrose permeability measurements. The distribution of the applied toxins between blood and brain compartments of the cell monolayer was analyzed by high performance liquid chromatography-mass spectrometry to calculate transfer rates and permeability coefficients.

Results

Deoxynivalenol reduced the barrier integrity and caused cytotoxic effects at 10 μM concentrations. Slight alterations of the barrier integrity were also detected for 3-acetyldeoxynivalenol. The latter was transferred very quickly across the barrier and additionally cleaved to deoxynivalenol. The transfer of deoxynivalenol and moniliformin was slower, but clearly exceeded the permeability of the negative control. None of the compounds was enriched in one of the compartments, indicating that no efflux transport protein is involved in their transport.     

Adipokines and the blood-brain barrier

Just as the blood-brain barrier (BBB) is not a static barrier, the adipocytes are not inert storage depots. Adipokines are peptides or polypeptides produced by white adipose tissue; they play important roles in normal physiology as well as in the metabolic syndrome. Adipokines secreted into the circulation can interact with the BBB and exert potent CNS effects. The specific transport systems for two important adipokines, leptin and tumor necrosis factor alpha, have been characterized during the past decade. By contrast, transforming growth factor beta-1 and adiponectin do not show specific permeation across the BBB, but modulate endothelial functions. Still others, like interleukin-6, may reach the brain but are rapidly degraded. This review summarizes current knowledge and recent findings of the rapidly growing family of adipokines and their interactions with the BBB.     

Vascular growth factors in cerebral ischemia

During the past decade, there has been a surge of interest in growth factors (GFs) that act selectively on vascular endothelium and perivascular cells. Studies employing mutant mice or the administration of recombinant proteins have suggested that these factors not only mediate the proliferation of endothelial cells, but also regulate vascular differentiation, regression, and permeability. During and after cerebral ischemia, brain vasculature becomes leaky and unstable, and the normally impermeable blood-brain barrier breaks down. Several days after the ischemic insult, endothelial cells begin to proliferate, and angiogenesis occurs. Expression studies have shown that key vascular GFs are regulated, during these processes, in a complex and coordinated manner. The distinct pattern of regulation exhibited by each vascular GF suggests a unique role for each factor during the initial vascular destabilization and subsequent angiogenesis that occurs after cerebral ischemia. Data from studies in other biological systems support these suggested roles. Thus, manipulation of vascular GFs may prove to be an effective means of stabilizing or enriching brain vasculature after ischemia, and ameliorating the detrimental effects of blood-brain barrier breakdown and vessel regression after stroke.     

Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier

Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Functional expression of the serotonin transporter in immortalized rat brain microvessel endothelial cells

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5-hydroxytryptamine (5-HT)]. This neurotransmitter is expected to be involved in the regulation of the blood-brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor-mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5-HT molecules. To realize this, a similar mechanism of 5-HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT-PCR that 5-HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5-HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5-HT by an active saturable process with a Km value of 397 +/- 64 nmol/L and a transport capacity of 51.7 +/- 3.5 pmol x g(-1) x min(-1). The 5-HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5-HT uptake was sensitive to specific 5-HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5-HT, which supports the concept of direct serotoninergic regulation of the BBB function.     

Ouabain-Sensitive Choline Transport System in Capillaries Isolated from Bovine Brain

In physiological conditions, there is a net transport of choline from brain to blood, despite the fact that the choline concentration is higher in plasma than in CSF. Because of the blood-brain barrier characteristics, such passage against the concentration gradient takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, [3H]choline uptake properties have been analyzed in capillaries isolated from bovine brain. [3H]Choline uptake was linear with time for up to 1 h. Nonlinear regression analysis of the uptake rates at different substrate concentrations gave the best fit to a system of two components, one of which was saturable (Km = 17.8 +/- 4.8 microM; Vmax = 11.3 +/- 3.4 pmol/min/mg of protein) and the other of which was nonsaturable at concentrations up to 200 microM. The [3H]choline transport was significantly reduced in the absence of sodium and after incubation with 10(-4) M ouabain for 30 min. Ouabain also inhibited choline uptake in purified cerebral endothelial cells, but not in the endothelium isolated from bovine aorta. Accordingly, cerebral endothelial cells were able to concentrate [3H]choline, with this effect being abolished by ouabain, whereas in aortic endothelial cells the [3H]choline intracellular concentration was never higher than that of the incubation medium. These results suggest that the blood-brain barrier endothelium is specifically provided with an energy-dependent choline transport system, which may explain the choline efflux from the brain and the maintenance of a low choline concentration in the cerebral extracellular space.     

Hypoxanthine transport through the blood-brain barrier

The unidirectional influx of hypoxanthine across cerebral capillaries, the anatomical locus of the blood=brain barrier, was measured with an in situ rat brain perfusion technique employing [³H]hypoxanthine. Hypoxanthine was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 0.4 mM. The permeability-surface area product was 3×10^–4 sec^–1 with a hypoxanthine concentration of 0.02 M in the perfusate. Adenine (4 mM) and uracil and theophylline (both 10 mM), but not inosine (10 mM) or leucine (1 mM), inhibited hypoxanthine transfer through the blood-brain barrier. Thus, hypoxanthine is transported through the blood-brain barrier by a high-capacity, saturable transport system with a half-saturation concentration about 100 times the plasma hypoxanthine concentration. Although involved in the transport hypoxanthine from blood into brain, this system is not powerful enough to transfer important quantities of hypoxanthine from blood into brain.