Effect of a single embryonic exposure to alcohol on glucose transporter (GLUT-1) distribution in brain vessels of aged mouse

Distribution of glucose transporter (GLUT-1) in brain microvascular endothelium, representing the anatomic site of the blood-brain barrier (BBB), was studied with electron microscopy in 24-month-old mice, which had been exposed prenatally (on 9th day of gestation) to a single teratogenic dose of ethanol. Offspring of mice that had received an equivalent volume of isocaloric dextrose served as controls. Sections of brain samples embedded at low temperature in hydrophilic resin Lowicryl K4M were exposed to anti-GLUT-1 antiserum followed by gold-labelled secondary antibodies. By using morphometry, the labelling density was recorded over luminal and abluminal plasma membranes of the endothelial cells of blood microvessels supplying four brain regions: cortex, hippocampus, cerebellum and olfactory bulb. We found that the density of immunosignals for GLUT-1, represented by colloidal gold particles, was unchanged in the olfactory bulb and slightly lowered in the abluminal plasmalemma of the vascular endothelium in the cerebral cortex of the ethanol-treated mice. In contrast, statistical analysis using Mann-Whitney U-test revealed that in the hippocampus and cerebellum, the density of immunolabelling of both plasma membranes of microvascular endothelial cells was significantly lowered in the ethanol-treated mice. These findings suggest that prenatally applied ethanol had a different influence on the vasculature supplying different brain regions. In effect, the inefficient supply of glucose to selected brain regions can be one of the factors leading to the previously observed deficit in long-term memory in a similar alcohol-treated group of mice.     

Immunogold study of effects of prenatal exposure to lipopolysaccharide and/or valproic acid on the rat blood-brain barrier vessels

The involvement of blood microvessels, representing the anatomic site of the blood-brain barrier (BBB), in brain damage induced by prenatal exposure to lipopolysaccharide (LPS) and/or valproic acid (VPA) was studied in four-week-old rats. The immunogold procedure was applied for localization at the ultrastructural level of endogenous albumin and glucose transporter (GLUT-1) in three brain regions: cerebral cortex, cerebellum and hippocampus. Four groups of rats were used: (1) untreated control, (2) prenatally VPA-treated, (3) prenatally LPS-treated, and (4) prenatally LPS- and VPA-treated. The functional state of the BBB was evaluated as follows: (a) by its tightness, i.e., permeability to blood-borne albumin, and (b) by the expression of GLUT-1 in the endothelial cells (ECs). Using morphometry, the labelling density for GLUT-1 was recorded over luminal and abluminal plasma membranes of the ECs, also providing information on their functional polarity. No extensive increase of vascular permeability and/or any considerable dysfunction of the BBB in experimental groups nos. 2 and 3 were observed, although in solitary vascular profiles, increased endocytosis or even transcytosis of albumin by ECs was noted. In experimental group no. 4, some vascular profiles showed scanty leakage (microleakage), manifested by the presence of immunosignals for albumin in the perivascular area. Although some fluctuations in the expression of GLUT-1 occurred in all experimental groups, especially in group no. 3, a most pronounced and significant diminution of the labelling density, in all three regions of the brain, was observed in group no. 4. This finding suggests the synergistic action of prenatally applied LPS and VPA that affects specific transport functions of glucose in the microvascular endothelium. The diminished or disturbed supply of glucose to selected brain regions can be one of the factors leading to previously observed behavioral disturbances in similarly treated rats.     

Immunogold study of interendothelial junction-associated and glucose transporter proteins during postnatal maturation of the mouse blood-brain barrier

The distribution of glucose transporter (GLUT-1) and of interendothelial junction—associated proteins—zonula occludens protein (ZO-1), occludin, and β-catenin—was studied using quantitative immunogold procedure. Lowicryl K4M-embedded samples of the cerebral cortex of 1-, 7-, and 14-day-, and 6-week-old (young-adult) mice were used. Ultrathin sections were exposed to specific rabbit polyclonal antibodies followed by colloidal gold-labelled secondary antibodies. We found that the density of immunosignals for GLUT-1 in both luminal and abluminal plasma membranes of the endothelial cells, and those closely related to the interendothelial junctions was low in blood microvessels from newborn mice, dropped slightly at the 7th day, and increased through the 14th day to the level of mature blood-brain barrier (BBB) observed in 6-week-old mice. The expression of ZO-1 was high in newborn mice and increased at the 7th day to the level similar to that found in 14-day- and 6-week-old mice. The expression of occludin was less intense than that of ZO-1 and increased from birth, reaching at the 14th day the level typical for mature BBB found in young-adult animals. The immunosignals for occludin were sparsely distributed inside the junctional clefts. Such a distribution indicates that the tight junctional characteristics are limited to a few short segments of the entire interendothelial cleft. The density of immunosignals for β-catenin was lowest, and it had the tendency to a gradual, although inconsiderable, drop in the time course of BBB maturation. These findings suggest that the relatively high concentration of GLUT-1 in the interendothelial junctions results from the participation of abluminal plasma membranes of adjacent endothelial cells in the formation of the junctional complexes. The interendothelial junctions of newborn mice are equipped already with the main components of the tight junctions, and the concentration of these components (ZO-1, occludin) reaches the level of the mature BBB at the 14th day of postnatal life.     

Immunogold study of regional differences in the distribution of glucose transporter (GLUT-1) in mouse brain associated with physiological and accelerated aging and scrapie infection

Distribution of glucose transporter (GLUT-1) in brain microvascular endothelia, representing the anatomic site of the blood-brain barrier (BBB), was studied in adult, physiologically aged, senescence-accelerated prone (SAMP8) and in scrapie-infected mice. Sections of tissue samples obtained from four brain regions (cerebral cortex, hippocampus, cerebellum, and olfactory bulb) and embedded in Lowicryl K4M were exposed to anti-GLUT-1 antiserum followed by gold-labeled secondary antibody. Labelling density was recorded over luminal and abluminal plasma membranes of the microvascular endothelial cells. We found that the density of immunosignals for GLUT-1 in the cerebral cortex showed a tendency toward insignificant diminution according to the following gradation-adult > SAMP8 > scrapie > aged mice-whereas in the hippocampus, this gradation was slightly different: adult > aged > scrapie > SAMP8 mice. In the cerebellum, immunolabelling was insignificantly diminished in aged mice, whereas it was significantly decreased in scrapie-infected and SAMP8 mice. The intensity of labelling of the vascular endothelium in the olfactory bulb was significantly lower than that in other brain regions, showing a slight decrease in the following sequence: adult > aged > scrapie > SAMP8 mice. These findings suggest that the process of aging as well as of related neurodegenerative disease affects unequally the distribution of GLUT-1 in the vasculature of different brain regions.     

Increased expression of β-catenin in brain microvessels of a segmentally trisomic (Ts65Dn) mouse model of Down syndrome

We examined the distribution of β-catenin and endogenous blood serum albumin at the ultrastructural level in blood microvessels (capillaries) from brains of control and trisomic Ts65Dn mice. Morphological examination revealed an increased immunolabeling for β-catenin in endothelial substructures of the capillary network, such as intercellular junctions, cytoplasm, and nuclei. These immunosignals were significantly increased in all endothelial substructures from trisomic mice. These changes, however, did not affect the blood–brain barrier function of the entire microvascular network, because the increased uptake of albumin by endothelial cells and the eventual escape of this protein (microleakage) into the perivascular neuropil were noted only in a few capillary profiles. Nevertheless, these findings suggest the involvement of some segments of the microvascular network in the brain pathology associated with DS.     

Cellular and Subcellular Aquaporin-4 Distribution in the Mouse Neurohypophysis and the Effects of Osmotic Stimulation

Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.     

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.     

Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.     

Inhibitory effect of somatostatin on neutral amino acid transport in isolated brain microvessels

In the presence of somatostatin-14 or some of its receptorial agonists, the uptake of large neutral amino acids by isolated brain microvessels was found to be inhibited up to 50%, no other transport system being affected. Although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only uptake from the abluminal side appears to be inhibited by somatostatin. The involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptor-specific somatostatin agonists, and was confirmed by the release of inhibition caused by a specific type-2 receptor antagonist. A type-2-specific mRNA was indeed shown to be present in both bovine brain microvessels ex vivo and primary cultures of endothelial cells from rat brain microvessels.     

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.     

Diet-induced ketosis increases capillary density without altered blood flow in rat brain

It is recognized that ketone bodies, such as R-beta-hydroxybutyrate (beta-HB) and acetoacetate, are energy sources for the brain. As with glucose metabolism, monocarboxylate uptake by the brain is dependent on the function and regulation of its own transporter system. We concurrently investigated ketone body influx, blood flow, and regulation of monocarboxylate transporter (MCT-1) and glucose transporter (GLUT-1) in diet-induced ketotic (KG) rat brain. Regional blood-to-brain beta-HB influx (micromol.g(-1).min(-1)) increased 40-fold with ketosis (4.8 +/- 1.8 plasmabeta-HB; mM) in all regions compared with the nonketotic groups (standard and no-fat diets); there were no changes in regional blood flow. Immunohistochemical staining revealed that GLUT-1 density (number/mm2) in the cortex was significantly elevated (40%) in the ketotic group compared with the standard and no-fat diet groups. MCT-1 was also markedly (3-fold) upregulated in the ketotic group compared with the standard diet group. In the standard diet group, 40% of the brain capillaries stained positive for MCT-1; this amount doubled with the ketotic diet. Western blot analysis of isolated microvessels from ketotic rat brain showed an eightfold increase in GLUT-1 and a threefold increase in MCT-1 compared with the standard diet group. These data suggest that diet-induced ketosis results in increased vascular density at the blood-brain barrier without changes in blood flow. The increase in extraction fraction and capillary density with increased plasma ketone bodies indicates a significant flux of substrates available for brain energy metabolism.     

Immunogold study of altered expression of some interendothelial junctional molecules in the brain blood microvessels of diabetic scrapie-infected mice

Quantitative immunogold procedure was used to study the distribution of molecular components of interendothelial junctions in blood–brain barrier (BBB) microvessels of scrapie infected SJL/J hyperglycemic mice showing obesity and reduced glucose tolerance. Samples of brain (fronto-parietal cerebral cortex and thalamo-hypothalamic region) obtained from hyperglycemic (diabetic) mice and from non- infected, normoglycemic (non-diabetic) SJL/J mice, were processed for immunocytochemical examination. The localization of the following tight junction (TJ)-associated proteins was studied: occludin as an integral membrane (transmembrane) protein, and zonula occludens one (ZO-1) as a peripheral protein. The localization of β-catenin as a representative of the cadherin/catenin complex that is typical for adherens junctions (AJs) also was studied. Morphometric analysis revealed that the density of immunosignals for occludin, represented by colloidal gold particles (GPs), was significantly lower in the brain microvessels of diabetic than in non-diabetic mice. No significant differences in the density of immunosignals for ZO-1 and β-catenin between both experimental mouse groups were observed. It indicates that abnormal glucose metabolism affects mostly occludin which is believed to play a fundamental role in the maintenance of the tightness of endothelial lining in brain microvascular network and thereby in the preservation of its barrier function. These results also support the previously expressed opinion that occludin, detected with the applied morphological method, can be considered a sensitive indicator of altered molecular architecture of the interendothelial junctions due to the action of some metabolic or pathological insults.     

Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.     

Ultrastructural localization of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium.

The distribution of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium has been disputed. Originally reported to be highly concentrated at interendothelial cell contacts, recent studies have claimed that CD31 is distributed evenly over the entire endothelial cell surface. We re-investigated this question with two different murine anti-CD31 antibodies (MEC 13.3 and M-20), using a pre-embedding immunonanogold electron microscopic procedure that allowed precise label quantitation. MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A164). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes of endothelial cells, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither antibody was reactive in CD31-null mice. We conclude that CD31 is distributed over the entire endothelial cell surface, exclusive of specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.     

Distribution of endothelial vesicles in the microvasculature of skeletal muscle and brain cortex of the rat,as demonstrated by tannic acid tracer analysis

Summary Ultrathin serial sectioning and labeling with tannic acid have demonstrated that most plasmalemmal vesicles of rat vascular endothelial cells are not free, but rather are conjoined in three dimensions to form racemose invaginations from the cell surfaces. To elucidate the distribution of vesicles in these microvascular endothelial cells, we have examined terminal arterioles, capillaries and post-capillary venules of rat skeletal muscle and brain cortex, using tannic acid labeling and stereological methods, and have determined the proportions of free vesicles and the vesicles of luminal and abluminal invaginations, as well as the numerical density of vesicles. In the case of capillaries, regional differences in distribution have also been studied. The ratio of free vesicles is 6–7% and is constant throughout the muscle microvasculature. The distribution (proportions and numerical densities) of vesicles in the brain and muscle microvascular endothelial cells shows regionally distinctive patterns. In rapid-frozen, freeze-substituted endothelial cells, there are almost as many fused vesicles as seen in chemically fixed cells. Therefore, aldehydes do not seem to induce membrane fusion, and the distribution of vesicles seems to be preserved by chemical fixation. The structure and function of plasmalemmal vesicles are discussed.     

Ultrastructural localization of integrin subunits alpha3 and alpha6 in capillarized sinusoids of the human cirrhotic liver

Normal liver sinusoids are not lined by a basement membrane (BM). In contrast, in the course of development of liver cirrhosis, a structured BM is formed de novo in the space of Disse. This BM contributes to the inhibition of the metabolic function of the liver but the pathogenic background of the formation of this perisinusoidal BM is still unclear. Integrins of the beta1-class are generally essential for BM stability and some of them (such as alpha2beta1, alpha3beta1 and alpha6beta1) appear de novo in the perisinusoidal space of the cirrhotic liver. Their cellular distribution in capillarized sinusoids as well as the correlation between their cellular distribution and the formation of the microvascular BM in the cirrhotic liver has not been shown at the ultrastructural level. In the present work we aimed to clarify this issue. We focused on integrins alpha3beta1 and alpha6beta1 and localised them ultrastructurally in human cirrhotic liver microvessels using postembedding immunogold which allows the ultrastructural localization of antigens with high resolution in the tissue. The newly formed basement membrane of capillarized sinusoids was visualized by means of fixation with addition of tannic acid, which enables the visualization of structures of the extracellular matrix with the highest resolution. Also, we carried out laminin detection using postembedding immunogold. Our results show that both alpha3beta1 and alpha6beta1 are expressed on the surface of both hepatocytes and endothelial cells, i.e. on both sides of the newly formed basement membrane. This latter shows zones of higher density both in close proximity to the endothelial and to the hepatocytic surfaces which resemble laminae densae. We propose that hepatocytes and endothelial cells may, therefore, by expressing such integrins, contribute to the formation of this pathological BM in the microvessels of the human cirrhotic liver. On stellate cells, which are major producers of BM components, both integrins alpha3beta1 and alpha6beta1 were also localized.     

Glucose transporter asymmetries in the bovine blood-brain barrier

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.     

Visualization of chemokine binding sites on human brain microvessels

The chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) aid in directing leukocytes to specific locales within the brain and spinal cord during central nervous system inflammation. However, it remains unclear how these chemokines exert their actions across a vascular barrier, raising speculation that interaction with endothelial cells might be required. Therefore, experiments were performed to determine whether binding domains for these chemokines exist along the outer surface of brain microvessels, a feature that could potentially relay chemokine signals from brain to blood. Using a biotinylated chemokine binding assay with confocal microscopy and three-dimensional image reconstruction, spatially resolved binding sites for MCP-1 and MIP-alpha around human brain microvessels were revealed for the first time. Binding of labeled MCP-1 and MIP-1alpha could be inhibited by unlabeled homologous but not heterologous chemokine, and was independent of the presence of heparan sulfate, laminin, or collagen in the subendothelial matrix. This is the first evidence of specific and separate binding domains for MCP-1 and MIP-1alpha on the parenchymal surface of microvessels, and highlights the prospect that specific interactions of chemokines with microvascular elements influence the extent and course of central nervous system inflammation.     

Cytochemical localization of alkaline phosphatase and Na+, K+-ATPase activities in the blood-brain barrier of Rana esculenta

The ultrastructural distribution of alkaline phosphatase and Na+, K+-ATPase on the brain capillaries in Rana esculenta was investigated. Alkaline phosphatase activity appears both on the luminal and abluminal walls of the endothelial capillary cells; Na+, K+-ATPase is, instead, only present on the abluminal side. This different enzymatic distribution indicates that endothelial cells of the brain capillaries are polarized and the luminal and abluminal endothelial membranes are functionally different. The role of these two enzymatic activities is discussed in relation to the blood-brain barrier.     

Isolation and Characterization of Brain Endothelial Cells: Morphology and Enzyme Activity

Microvessels were isolated from rat brain using a double collagenase treatment which removed the endothelial basement membranes. The isolate was characterized by intact luminal and abluminal membranes and an absence of pericytes and astrocyte membranes. Minimal contamination by 5′-nucleotidase, an enzyme believed exclusively localized within the plasma membranes of neuroglia, established the purity of the isolated microvessels. Enrichment of alkaline phosphatase and -γ-glutamyl transpeptidase activity in microvessel preparations supports the endothelial localization of these enzymes.