Induction of γ-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes

γ-Glutamyl transpeptidase (γGTP) is an enzyme found in cerebral capillary endothelial cells, the presumed site of the blood-brain barrier, but not in endothelial cells lining blood vessels in other parts of the body. Using a line of mouse cerebral microvessel endothelial cells (ME-ly cells) and a sensitive colorimetric assay to measure γGTP levels we demonstrated that primary cultures of mouse astrocytes and a line of rat C₆ glioma cells released a soluble product(s) that induced the production of γGTP in cultured endothelial cells by 34% and 39%, respectively, over control levels. Cerebrovascular smooth muscle cells had no significant effect on γGTP levels in ME-ly cells, and the astrocyte product(s) had no effect on rabbit aortic endothelial cells. The induction of γGTP levels in ME-ly cells was apparent after one day of exposure to the astrocyte product(s) and increased in magnitude with increasing time of exposure of the ME-ly cells to the product(s). Removal of the product(s) from the ME-ly cells resulted in a return to control levels of γGTP in the ME-ly cells within 2 days. The presence of a protein synthesis inhibitor during incubation with the productd(s) blocked the induction of γGTP in ME-ly cells, and treatment of the product(s) with 200 U/ml TPCK-trypsin destroyed its inductive properties. These results show that glial cells release a protein into their medium which induces de novo protein synthesis of γGTP in cerebral microvessel endothelial cells, and indicate that the glial cells may induce the cerebral capillary endothelial cells to express differentiated properties which allow the endothelium to function as the blood-brain barrier.     

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) expression by human brain microvessel endothelial cells in primary culture

PECAM-1 expression was investigated in primary cultures of human brain microvessel endothelial cells (HBMEC). HBMEC constitutively express PECAM-1 along their apical cell surface, advancing processes and on the basal surface at points of contact with the extracellular matrix. Surface expression is not altered by cytokine or lipopolysaccharide treatment. This distribution may mediate cell-cell contact and migration during angiogenesis and HBMEC-leukocyte interactions in CNS inflammation.     

Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells

The mechanisms of carrier-mediated transport of γ-aminobutyric acid (GABA) at the blood–brain barrier (BBB) were examined by investigating [ ]GABA uptake by isolated bovine brain capillaries, monolayers of primary cultured brain capillary endothelial cells (BCECs) attached to plates or suspended BCECs. The uptake of [ ]GABA was concentration-dependent and saturable. Nonlinear regression analysis of the original data indicated the existence of two distinct high and low-affinity GABA transporters on isolated brain capillaries or suspended BCECs, with K_m1, K_m2, V_m1 and V_m2 equal to 25.3 μM, 485.2 μM, 3.6 and 8.4 nmol/5 min/mg protein, respectively, for the capillaries, and 21.3 μM, 322.0 μM, 6.1 and 15.7 nmol/5 min/mg protein, respectively, for the suspended BCECs. In contrast, a single low-affinity transporter was found for monolayers of BCECs attached to plates with K_m and V_m equal to 338.7 μM and 18.8 nmol/5 min/mg protein, respectively. Subcellular location of the two distinct transporters on BCECs is discussed, suggesting that the low-affinity GABA transporter is probably localized to the luminal membrane of BCECs, and the high-affinity GABA transporter is probably localized to the antiluminal membrane. Low temperature (4°C) and metabolic inhibitors markedly diminished both high and low-affinity uptakes of [ ]GABA by isolated brain capillaries. The substitution of Na⁺ with choline⁺, K⁺ or Li⁺ with the counter anion Cl⁻ almost completely abolished both uptakes. Substitution of Cl⁻ with Br⁻, I⁻, F⁻ or NO₃⁻ in the presence of Na⁺ significantly reduced both uptakes to different extents. Alanine, leucine, phenylalanine, arginine, glutamate and pyruvate had no obvious effect on either uptake. Probenecid, amino-oxyacetic acid, β-alanine, taurine, betaine, and nipecotic acid significantly reduced both uptakes. These data suggested that both the GABA transporters at the BBB were temperature, metabolic energy, Na⁺ and Cl⁻-dependent, and may be specific and different from the known monocarboxylic acid, GABA and other amino acid transporters, which may play a role in the disposition of GABA in the brain.     

-DOPA transport properties in an immortalised cell line of rat capillary cerebral endothelial cells, RBE 4

The present study aimed to determine the kinetics of -3,4-dihydroxyphenylalanine ( -DOPA) uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4 and RBE 4B), to define the type of inhibition produced by -5-hydroxytryptophan ( -5-HTP), 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC) and N-(methylamino)-isobutyric acid (MeAlB) and its sodium dependence. Non-linear analysis of the saturation curves for -DOPA and -5-HTP revealed in RBE 4 cells K_m values (in μM) of 72 and 102 and in RBE 4B cells K_m values (in μM) of 60 and 118, respectively. IC₅₀ values for -5-HTP (RBE 4, 1026 μM; RBE 4B, 831 μM) obtained in the presence of a nearly saturating (250 μM) concentration of -DOPA were almost 5-fold those obtained when non-saturating (25 μM) concentrations of -DOPA were used. IC₅₀ values for BHC obtained in the presence of a nearly saturating (250 μM) concentration of -DOPA were also 6- to 5-fold those obtained when non-saturating (25 μM) concentrations of -DOPA were used. MeAlB (up to 2.5 mM) was found not to interfere with the uptake of -DOPA. In RBE 4 cells, V_max values for -DOPA uptake were identical in the absence and the presence of 150 μM -5-HTP or 150 μM BHC, but K_m values (μM) were significantly greater (P<0.05) when -DOPA uptake was studied in the presence of -5-HTP or BHC. Similar findings were observed when RBE 4B cells were used. Uptake of (250 μM) -DOPA in the absence of sodium in the incubation medium was similar to that observed in the presence of increasing concentrations of sodium (20 to 140 mM). It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which -DOPA and -5-HTP can be taken up, and suggested that this immortalised cell line of rat capillary cerebral endothelium might constitute an interesting in vitro model for the study of BBB mechanisms, namely those concerning solute and nutrient transfer across the brain capillary endothelium.     

Urocortin trafficking in cerebral microvessel endothelial cells

Urocortin, a potent peptide inhibitor of feeding behavior, can enter the brain from blood by leptin-facilitated permeation across the blood-brain barrier. Here, we show in cultured RBE4 cerebral microvessel endothelial cells that urocortin endocytosis is increased by leptin in a time- and dose-dependent manner. Fluorescently labeled urocortin (Alexa488-urocortin) shows vesicular trafficking localized in early endosomes at 1 min and the Golgi complex at 20 min. The endocytosis at 20 min was increased by 10 μg/mL, but not 2 μg/mL, of leptin. The facilitating effect of leptin at the dose of 10 μg/mL was seen at 20 and 30 min but not at 10 min. This increase could be abolished by excess unlabeled urocortin in radio-tracer uptake studies, indicating selective rather than nonsaturable entry. The specificity of the effect was further supported by the lack of changes in γ-glutamyl transpeptidase activity and endothelial nitric oxide synthase upon stimulation by high doses of leptin and urocortin. Leptin did not affect the level of expression of the urocortin corticotropin-releasing hormone receptor (CRHR) after 30 min of treatment but appeared to slow the turnover of CRHRs induced by urocortin. In MDCK cells overexpressing CRHR2, leptin facilitated urocortin uptake, whereas ObRa coexpression did not exert an additional effect. Thus, urocortin endocytosis is a saturable process leading to vesicular intracellular transport that can be enhanced by cell-surface leptin.     

原代大鼠脑毛细血管内皮细胞培养与鉴定简

目的介绍一种从大鼠脑组织中分离培养原代大鼠脑血管内皮细胞（BCEC）的方法。方法采用酶消化、滤网机械分离结合右旋糖苷密度离心的方法分离脑毛细血管段,接种在涂有明胶的培养皿培养BCEC。结果通过环境扫描电镜观察细胞形态,量子点超敏细胞免疫荧光鉴定,我们培养出来了纯度较高的原代BCEC。结论将分离的鼠脑毛细血管段置于涂有明胶的培养皿上培养原代BCEC的新方法比传统的培养法可以得到纯度更高的BCEC,为后期基于BCEC为载体的动物实验奠定了细胞基础。     

Serum-derived factors weaken the barrier properties of cultured porcine brain capillary endothelial cells in vitro

Cultured cerebral capillary endothelial cells are often used as a functional in vitro model of the blood-brain barrier (BBB) to determine drug uptake or to study barrier properties. Usually serum is supplemented to these cultures for cell proliferation. Here, we demonstrate the effect of serum and the serum-derived factors lysophosphatidic acid (LPA) and vascular endothelial growth factor (VEGF) on the barrier properties of cultured porcine brain capillary endothelial cells (PBCEC). Serum prevents tight junction formation of confluent PBCEC monolayers and moreover, opens already established tight junctions shown by decreasing transendothelial electrical resistances (TER). These effects are highly polarised with serum almost exclusively acting from the basolateral side of the cell culture. Immunocytochemistry of PBCEC revealed a delocalisation of the cell border lining tight junction proteins ZO-1, occludin and claudin-5 when serum was added. A serum fraction of 67 kDa was isolated by size-exclusion chromatography, identified as albumin and found to cause a serum-like decrease of the TER. However, fatty acid-free serum albumin does not develop this barrier weakening effect, indicating that small protein-bound factors might be responsible. For instance, serum-bound LPA demonstrated a TER-decreasing effect as well, but in contrast to serum mainly when added to the apical side of PBCEC. Addition of VEGF caused a serum-like decrease of the TER with the same polar effect; however, VEGF will be denatured by heat and could thus not be the heat-sensitive factor. Thus, we hypothesise that serum contains a variety of factors which weaken the tightness of a PBCEC monolayer from the apical side as expected but also from the basolateral side. Although the structure of the 67 kDa factor could not be analysed, this finding is of importance for in vitro models not only of the blood-brain barrier mostly using serum-containing media.     

Exocytotic release of alanine from cultured cerebellar neurons

Many neurons release a variety of amino acids in response to depolarizing stimuli. Although some of these amino acids, namely, glutamate, aspartate, and gamma-aminobutyric acid (GABA), have been qualified as neurotransmitters, functional roles of the other amino acids including alanine remain obscure. We investigated the mechanism and the origin of alanine release from cultured rat cerebellar cells. High-K(+)-induced depolarization produced a considerable amount (139+/-8 pmol/2 min/dish) of alanine release, comparable to that of glutamate (103+/-7 pmol/2 min/dish). Other depolarizing agents including veratridine or 4-aminopyridine also induced alanine release, suggesting that the major source is excitable neurons, rather than non-excitable glial cells. Depolarization-evoked alanine release was suppressed in the absence of extracellular Ca(2+), and was almost abolished by treating the cells with botulinum type B neurotoxin (BoNT/B), indicating that alanine is released by Ca(2+)-dependent exocytosis of vesicle-associated membrane protein-2 (VAMP-2)-containing vesicles. The properties of alanine release were different from those of glutamate and GABA in several aspects: (a) Depolarization-dependent alanine release appeared as early as 7 days in vitro, much earlier than that of GABA. (b) Fifty microM kainate, which causes selective cell death of GABAergic neurons in the culture, only partially reduced alanine release, whereas it had no effect on glutamate release. (c) Alanine release was not affected by phorbol ester, which enhanced glutamate and GABA release in a kinase-dependent manner. We therefore conclude that alanine release occurs via exocytosis of a pool of synaptic vesicles distinct from those containing glutamate or GABA.     

大鼠脑微血管内皮细胞的体外培养

目的探讨大鼠脑微血管内皮细胞的培养方法.方法取Wistar大鼠乳鼠脑组织,采用筛网过滤、胶原酶消化、离心等技术获取脑微血管内皮细胞,并进行培养.通过形态学、免疫细胞化学方法进一步鉴定,采用MTT方法测定生长曲线.结果经形态学、免疫细胞化学方法鉴定所培养的细胞为脑微血管内皮细胞,观察到原代脑微血管内皮细胞有3种表型,细胞呈单层生长,并可传代培养.结论建立大鼠脑微血管内皮细胞的培养方法可为体外研究脑血管病提供有益帮助.     

Vascular lipoxygenase activity: synthesis of 15-hydroxyeicosatetraenoic acid from arachidonic acid by blood vessels and cultured vascular endothelial cells

Although indirect pharmacologic evidence has suggested the presence of a lipoxygenase pathway of arachidonic acid (AA) metabolism in blood vessels, direct biochemical evidence has been difficult to demonstrate. We have investigated lipoxygenase metabolism in both fresh vessel preparations and cultured vascular cells from various sources and species. Lipoxygenase-derived [³H]HETE (composed of 12-HETE, 15-HETE and 5-HETE), which was abolished by ETYA but not by aspirin, was formed when [³H]AA was incubated with fresh sections of rat aorta. Lipoxygenase activity was lost following deendothelialization. A single peak of [³H]15-HETE was produced by cultured bovine aortic and human umbilical vein endothelial cells (EC) in response to exogenous [³H]AA or from [³H]AA released by ionophore A23187 from endogenous EC membrane phospholipid pools. Cultured bovine, rabbit or rat aorta smooth muscle cells had no detectable 15-lipoxygenase activity. ¹⁴C]Linoleic acid was converted by EC to its 15-lipoxygenase metabolite, [¹⁴C]13-hydroxyoctadecadienoic acid. These results indicate that blood vessels from different sources and species have a 15-lipoxygenase system, and this activity resides predominantly in the endothelial cells.     

血管内注射可溶性β-淀粉样蛋白1-40对毛细血管内皮细胞和胶质细胞毒性的研究

目的研究血液内较高浓度的可溶性β-淀粉样蛋白1-40(Aβ1-40)对毛细血管内皮细胞和胶质细胞的毒性,以探讨其在阿尔茨海默病发病中的作用.方法成年雄性SD大鼠90只,随机分为注射Aβ1-40组、注射生理盐水组以及正常大鼠(非手术)组,每组30只.采用右侧颈外静脉血管插管并留置的方法,通过导管向血管内中每日2次注射10μg的可溶性的Aβ1-40,连续14天后,采用免疫组化和免疫印迹(Western blot)方法观察星形胶质细胞、小胶质细胞的激活状态以及转糖蛋白-1、转糖蛋白-3表达的改变.结果注射Aβ1-40以后,毛细血管周围星形胶质细胞的胶质纤维酸性蛋白表达明显增多;小胶质细胞呈明显的激活状态;Western blot法检测到脑实质内转糖蛋白-1以及转糖蛋白-3表达明显减少.结论血管内连续注射Aβ1-40以后,对血-脑屏障的内皮细胞有明显的损害,对胶质细胞有明显的激活作用,可能与AD发病有关.     

Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations,micronuclei, and apoptosis

There is evidence accumulating that brain microvasculature is involved critically in oxidative stress-mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system.     

Phorbol ester stimulates acetylcholine synthesis in cultured endothelial cells isolated from porcine cerebral microvessels

Acetylcholine (ACh) is one of the factor which induces vasodilation through the release of endothelium-derived relaxing factor. The aim of this study was to clarify whether endothelial cells can synthesize ACh and the types of substance which regulate the synthesis of ACh in endothelial cells. We determined the ACh content of endothelial cells isolated from porcine cerebral microvessels and of the culture medium. ACh was detected in the medium after 12 h incubation in the presence of diisopropylfluorophosphate, a non-specific cholinesterase inhibitor, and increased linearly up to 24 h. Phorbol 12-myristate 13-acetate (PMA, 10⁻⁷ M) increased the ACh content of the medium in a dose-dependent manner. The effect of PMA was most apparent between 12 and 24 h after treatment, and was inhibited by cycloheximide. Calphostin C, a specific inhibitor of protein kinase C (PKC), did not inhibit the effect of PMA. Dioctanoyl glycerol, a specific activator of PKC, did not increase the intracellular ACh content or the amount released into the culture medium. ACh synthesis was not inhibited by bromoacetylcholine, a specific inhibitor of choline acetyltransferase (ChAT). PMA treatment did not affect the specific activity of ACh synthesis in endothelial cells. These data show that endothelial cells are able to synthesize ACh, and that ACh synthesis is up-regulated by PMA through the PKC independent mechanism via protein induction. The enzyme which synthesizes ACh in endothelial cells is not ChAT. The increase in ACh synthesis induced by PMA may not be due to induction of the ACh synthetic enzyme.     

Reverse pinocytosis induced in cerebral endothelial cells by injection of histamine into the cerebral ventricle

Summary Histamine dihydrochloride (10 g of 500 g/ml) was infused during 1 min into the lateral cerebral ventricle of rats, which resulted in a significant stimulation of pinocytosis in the endothelial cells. Systemic injections of mepyramine or metiamide could not prevent this activation. In contrast, ranitidine, injected with histamine was able to inhibit the stimulation of pinocytosis. Albumin exudation from the blood was not found. There was also no change in water and electrolyte contents of the brain tissue. The results suggest that histamine reaching the abluminal membrane can activate the pinocytosis in the cerebral endothelial cells in the reverse direction, i.e., from brain to blood, without opening the blood-brain barrier.     

An ultrastructural study of cultured human meningioma cells

Summary Ultrastructural alterations in human meningioma cells grown in vitro are reported. In early passages the cells retain some of the characteristic features of the original tumors. These include interdigitations associated with intercellular junctional devices (e.g., desmosomes, gap junctions). However, with repeated subculture these features tend to be less frequent. Typical whorl formations are observed only in primary cultures. The nunber of cytoplasmic filaments, lipid inclusions and other dense bodies increases with time in culture. Cytoplasmic invaginations into nuclei and the appearance of very large cells become more frequent in repeatedly transferred cultures.     

Flow-through cytometry of meningiomas and cultured meningioma cells

Summary Flow cytometric techniques were used to compare the DNA content, size and viability of meningioma cells obtained directly from surgical specimens with the same cells after a period of culture. Cells isolated from the original meningiomas and cells in primary culture were similar with regard to size and DNA content, regardless of the histologic subclassification of tumor. The cell populations were essentially diploid with a small proportion of tetraploid cells. Viable cells were smaller and more uniform in size than the nonviable cells. An increase in the number of cells having an elevated DNA content was seen with cultures repeatedly transferred. The latter results suggest that any transfer of information from long-term cultured meningioma cells to the in vivo situation must be done with caution.     

Indirect ultrastructural evidence for lithium uptake by cultured rat cerebral cells through the sodium channel

Cerebral cortices, prepared from 18-day-old rat embryos, were grown in explant cultures for 18 days. They were then incubated for 25 min in media of 3 types of cationic composition with and without tetrodotoxin and/or veratridine. Qualitative and quantitative ultrastructural observations revealed a considerable swelling of neuronal elements following veratridine exposure in both Na⁺ and Li⁺ media; this swelling was prevented by tetrodotoxin. By contrast, veratridine failed to induce swelling of neuronal profiles in a choline⁺ medium. The results indirectly indicate that Li⁺ ions enter cultured rat cerebral cells through the Na⁺ channels.