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.     

Phorbol ester stimulates hexose uptake by brain microvessel endothelial cells

Glucose uptake into cultured endothelial cells (EC) derived from brain microvessels was determined in the absence and presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), EGTA, the calcium ionophore A23187, and insulin. EC were obtained from dog and human (autopsy) brain microvessels and maintained in culture for up to four passages. Monolayers of EC were treated with TPA and other compounds immediately prior to harvesting for hexose uptake measurements using 3-O-[3H]methyl-D-glucose, 2-[3H]deoxy-D-glucose, or D-[3H]glucose. Typically, treatment with TPA (0.1-100 ng/ml) resulted in hexose uptake levels 2 to 3 times those of controls, although occasionally levels 5 to 10 times those of controls were observed. Similar stimulation was observed with all radiolabeled hexoses. Stimulation by TPA was greatest in primary or first passage cells and was greatly diminished in older cells. Neither chelation of extracellular calcium with EGTA nor the presence of both EGTA and A23187 in the culture medium prevented the stimulatory effect of TPA. Insulin (1200 ng/ml) failed to stimulate hexose uptake. Treatment with 100 ng/ml TPA did not alter the appearance of actin filaments in canine EC as visualized with rhodamine phalloidin. These results, in combination with other recent studies, suggest that blood-brain glucose transport may be regulated by phorbol ester-activated protein kinase C.     

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.     

Uptake of glucose analogues into cultured cerebral microvessel endothelium

Tritiated glucose analogues 3-O-methylglucose (3-OMG) and 2-deoxyglucose (2-DG) were used to study glucose uptake properties in established lines of cultured mouse cerebral microvessel endothelium. Uptake of both analogues was similar in terms of rate and absolute amount for the first two minutes. Thereafter, intracellular accumulation of 2-DG continued at a more rapid rate because of intracellular phosphorylation of this substrate. The uptake of 3-OMG uptake was temperature-dependent, independent of Na+, and not inhibited by ouabain or 2,4-dinitrophenol. Phloretin and cytochalasin B both significantly inhibited 3-OMG uptake. Other hexoses in high concentration acted as competitive inhibitors at the endothelial cell membrane. Pre-incubation of cells with 50 mM D-glucose resulted in higher levels of 3-OMG accumulation than in control cells (counter-transport phenomenon). In contrast to findings at the blood-brain barrier in vivo, insulin was found to stimulate 3-OMG uptake. Maximal stimulation of approximately 3-fold was found at ambient insulin concentrations of 1,000 ng/ml or higher. The findings provide support at the cellular level for some components of the model of carrier-mediated glucose transport across the blood-brain barrier which has been postulated to exist in vivo. The effect of insulin is discussed in the light of new data that show stimulation of glucose analogue transport into isolated cerebral capillaries.     

Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide

The importance of cell adhesion molecules in maintaining the cellular integrity of the endothelial layer is well recognized, yet their exact participation in regulating the blood–brain barrier (BBB) is poorly understood. Both Ca²⁺-dependent and Ca²⁺-independent cell adhesion molecules are found in endothelial cells. In this study, we used immunofluorescence, ELISA, Western blot and cell adhesion assay to identify a Ca²⁺-dependent cell adhesion molecule, E-cadherin, in bovine brain microvessel endothelial cells (BBMECs). Monoclonal anti-E-cadherin antibody specifically interacted with cultured BBMECs and decorated the cellular junctions with a series of punctate fluorescence spots as seen by indirect immunofluorescence using a confocal microscope. The intensity of these fluorescence spots increased after brief treatment with hIFN-γ or CPT-cAMP. In the cellular extract of BBMECs, a 120 kDa protein was immunoprecipitated with anti-E-cadherin antibody. BBMECs did not react with anti-N-cadherin antibody, but recognized the FITC-labeled LRAHAVDVNG-NH₂, a decapeptide generated from the EC-1 domain of N-cadherin, which decorated the lateral margins of the cells with fluorescence spots. A concentration-dependent binding of this decapeptide was also observed in the flow cytometry assay. BBMECs dissociated with trypsin plus Ca²⁺ were able to reaggregate only in the presence of Ca²⁺. However, such cell-cell aggregations of BBMECs were prevented by the presence of either anti-E-cadherin antibody or the decapeptide in the assay medium. These results confirm that BBMECs possess a distinct Ca²⁺-dependent cell adhesion mechanism that can be modulated by the decapeptide. This modulation of cell-cell adhesion in BBMECs by the decapeptide is thought-provoking for creating channels for paracellular drug delivery across the BBB.     

Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood-brain barrier

The uptake of biotin and the closely related biocytin was characterized in primary cultures of calf brain microvessel endothelial (CBME) cells. Biotin uptake was found to be Na(+)-gradient dependent and independent of changes in the membrane potential. Concentration dependence revealed a single saturation mechanism with a K(m) of 47 microM and a V(max) of 101 pmol/min/mg. Inhibition studies demonstrated dependence on metabolic energy and the necessity for a free carboxyl group for transport activity. The anticonvulsants primidone and carbamazepine had no inhibitory effect. Biotin uptake into CBME cells is a secondary active, electroneutral, saturable and specific process. Biocytin which accumulates in biotinidase deficiency, a human congenital disorder, did not inhibit biotin uptake and was not transported into these cells. The presence of human serum with normal biotinidase activity significantly reduced biotin uptake by about 50%. Further, added biocytin was hydrolyzed to biotin, which accumulated intracellularly but to a lesser extent than added free biotin. Biotin uptake after addition of plasma of biotinidase-deficient patients was not different from that in the presence of normal serum. These results indicate that the absence of biotinidase activity in serum does not reduce blood-brain barrier transport of biotin.     

Lipid synthesis by rat brain microvessel endothelial cells in tissue culture

Lipid synthesis and its regulation by serum lipoproteins at the microvascular blood-brain barrier were studied using primary cultures of microvascular endothelial cells from rat brain. These cells are capable of synthesizing all their lipids (neutral lipids, phospholipids, glycolipids) from the water-soluble compounds, glucose, acetate, acetoacetate and beta-hydroxybutyrate. The ketone bodies, especially acetoacetate, are the preferred substrates for lipid synthesis. The incorporation patterns of acetate, acetoacetate and beta-hydroxybutyrate are very similar, indicating that these precursors contribute to lipid synthesis via the same metabolic route. However, the metabolic pathway is different for glucose, which is preferentially incorporated into phospholipids. The existence of an inverse relationship between lipid synthesis and the serum lipoprotein concentration suggests that cultured cerebral endothelial cells are capable of taking up lipids, principally cholesterol, contained in the serum lipoproteins. Cellular lipids would thus be supplied both by intracellular lipid synthesis and by serum lipoproteins. The difference between cholesterol synthesis rates in cultured cerebral endothelial cells and in isolated brain microvessel cells could be partly explained by the fact that the lipoprotein concentration is much lower in the culture medium than in rat serum.     

Rhesus macaque brain microvessel endothelial cells behave in a manner phenotypically distinct from umbilical vein endothelial cells

Activation of endothelium is a critical step in leukocyte recruitment to the CNS and in development of neurological diseases, such as HIV-associated dementia. Due to limited availability of early disease course data, it is important to develop in vitro models of the blood-brain barrier (BBB) that can be used to address these early events. No such model of the BBB has been established for the macaque. Here, we characterize rhesus microvascular brain endothelial cells (MBEC), comparing them with rhesus umbilical vein endothelial cells (RUVEC), and discuss their suitability for future use in developing in vitro models of simian immunodeficiency virus (SIV) neuropathogenesis. We conclude that MBEC are distinct from RUVEC with respect to growth characteristics, culture requirements, morphology and expression of surface molecules important for leukocyte adhesion and immune activation.     

Mercury-stimulated histamine uptake and binding in cultured astroglial and cerebral endothelial cells

The effects of mercuric compounds on histamine uptake and binding to uptake carrier in cultured rat astroglial and cerebral endothelial cells were investigated. Experimental results showed that mercuric compounds produced strong stimulation of glial and cerebroendothelial histamine uptake over a concentration range of 25–500 μM. The stimulated histamine uptake showed characteristics similar to those described for basal uptake in terms of sensitivity to inhibitory agents (e.g., impromidine) and the requirement of external Na⁺. Mercury-induced stimulation of histamine uptake could be abolished by sulfhydryl agents, dithiotreitol and cysteamine, indicating a complete reversal of, and not simply a protection from, the action of mercury. Basal and stimulated uptake of histamine represent bindings to uptake carrier with high and closely equal affinities but markedly higher capacities for stimulated uptake. In controls, the mean value of apparent K_D (derived from saturation kinetics at equilibrium) was obtained as 26.7 ± 3.9 nM for astroglial cells; and 100 μM mercuric chloride did not modify it significantly. In contrast, the apparent B_max values differed markedly; found as 0.63 ± 0.10 pmol/mg protein and 3.32 ± 0.47 pmol/mg protein in the absence and the presence of 100 μM mercuric chloride respectively. For the cerebral endothelial cell line, RBE4, the apparent K_D was calculated as 22.5 ± 3.2 nM and was comparable to that obtained for astroglial cells in control and mercury-stimulated conditions. The apparent B_max values were less, but markedly different in these conditions, obtained as 0.18 ± 0.03 pmol/mg protein and 1.2 ± 0.36 pmol/mg protein in the absence and the presence of mercuric ion respectively. In both cells, impromidine, the potent inhibitor of basal and stimulated histamine uptake, decreased the enhanced capacities of histamine binding (B_max) (without affecting the dissociation constant, K_b) in micromolar range, comparable to its inhibiting potency. Results confirmed that mercuric ion might enhance the binding capacity of histamine carrier and protein sulftydryls might play a role in this eject. The observed stimulations by mercuric compounds suggest close similarities in the mechanism of histamine uptake and the structure of histamine carrier in astroglial and cerebral endothelial cells. J. Neurosci. Res. 48:71–81, 1997. © 1997 Wiley-Liss, Inc.     

Stimulation of calcium uptake in cultured astrocytes by hypoosmotic stress — effect of cyclic AMP

To investigate the role of Ca²⁺ in astrocyte volume regulation, we determined Ca²⁺ fluxes following hypoosmotic stress and how these fluxes were modified by cyclic AMP. In isoosmotic conditions treatment with dibutyryl cyclic AMP (dBcAMP) caused almost a twofold increase in ⁴⁵Ca²⁺ uptake. Efflux studies of ⁴⁵Ca²⁺ in dBcAMP-treated cells showed three Ca²⁺ compartments while only two Ca²⁺ compartments were identified in non-dBcAMP-treated cells. Following hypoosmotic stress a twofold stimulation of ⁴⁵Ca²⁺ uptake occurred in both non-dBcAMP-treated and dBcAMP-treated astrocytes. Stimulation of Ca²⁺ uptake begins at 270 mOsm and is half-maximally stimulated at 100 mOsm. This uptake is partly mediated through L-type ‘slow’ inactivating Ca²⁺ channels. Hypoosmotic stress also induces the release of Ca²⁺ from intracellular stores. The influx of extracellular Ca²⁺ and efflux of intracellular Ca²⁺ appear to be important factors in volume regulation after hypoosmotic stress. Cyclic AMP plays an important role in modulating hypoosmotically stimulated Ca²⁺ uptake.     

Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood–brain barrier

The uptake of biotin and the closely related biocytin was characterized in primary cultures of calf brain microvessel endothelial (CBME) cells. Biotin uptake was found to be Na⁺-gradient dependent and independent of changes in the membrane potential. Concentration dependence revealed a single saturation mechanism with a K_m of 47 μM and a V_max of 101 pmol/min/mg. Inhibition studies demonstrated dependence on metabolic energy and the necessity for a free carboxyl group for transport activity. The anticonvulsants primidone and carbamazepine had no inhibitory effect. Biotin uptake into CBME cells is a secondary active, electroneutral, saturable and specific process. Biocytin which accumulates in biotinidase deficiency, a human congenital disorder, did not inhibit biotin uptake and was not transported into these cells. The presence of human serum with normal biotinidase activity significantly reduced biotin uptake by about 50%. Further, added biocytin was hydrolyzed to biotin, which accumulated intracellularly but to a lesser extent than added free biotin. Biotin uptake after addition of plasma of biotinidase-deficient patients was not different from that in the presence of normal serum. These results indicate that the absence of biotinidase activity in serum does not reduce blood–brain barrier transport of biotin.     

Plasminogen activator activity of isolated cardiac muscle microvessel endothelial cells

Isolated capillary and other microvessel (<50 μm) endothelial cells obtained from rat cardiac muscle show plasminogen activator activity that is similar to observations reported for cultured rabbit and bovine aortic and venous intimal endothelium. The extent of plasminogen activator activity is observed to be a function of time and endothelial lysate protein concentration. Microvessel endothelium does not appear to secrete an acid-labile fibrinolytic inhibitor similar to that reported for cultured intimal cells, which when inactivated at pH 2.7 enhanced plasminogen activator activity.     

Effects of energy deprivation induced by fluorocitrate in immortalised rat brain microvessel endothelial cells

The effects of the mitochondrial aconitase inhibitor, fluorocitrate on the immortalised rat brain endothelial cell line (RBE4) were investigated. Treatment with different concentrations of fluorocitrate (0–1 mM) for 24 h induced a significant, concentration-dependent decrease in the MTT reduction (an index of mitochondrial function), intracellular ATP content, glucose consumption and lactate production by RBE4 cell monolayers but did not alter the glucose to lactate ratio at concentrations lower than 0.5 mM. At all concentrations, fluorocitrate induced a significant decrease in the protein content per well. Fluorocitrate treatment of confluent RBE4 cells induced a marked redistribution of the F-actin cytoskeleton from a characteristic marginal band to a more diffuse cytosolic pattern. This redistribution of the cytoskeleton coincided with a reduction in the total cellular F-actin content of the RBE4 cells at fluorocitrate concentrations greater than 0.5 mM. Treatment of confluent RBE4 cells with fluorocitrate had no significant effect on RBE4 cell monolayer permeability measured by FITC-dextran or [¹⁴C]sucrose. These results show that whilst energy deprivation following fluorocitrate treatment induces significant changes in the RBE4 cell F-actin cytoskeleton and cellular metabolism, it does not have any significant effect on endothelial cell monolayer permeability. These results demonstrate that profound toxic effects on endothelial cell structure and metabolism are not necessarily accompanied by changes in endothelial cell monolayer permeability.     

Ethanol inhibits G-protein-mediated glucose uptake by C6 glioma cells

The mechanism of ethanol inhibition of glucose uptake was investigated using C6 glioma cells. Basal [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was inhibited by ethanol in a concentration-dependent manner. Fifty, 75 and 100 mM ethanol significantly inhibited basal 2DG uptake by 12, 20 and 23%, respectively (p < 0.05). Carbachol (an agonist acting via G protein-coupled receptors) stimulated the uptake by 26% (p < 0.05). In the presence of 100 mM ethanol, the ability of carbachol to stimulate 2DG uptake was abolished. In contrast, ethanol did not inhibit the ability of insulin to stimulate 2DG uptake. These results suggest that ethanol inhibits 2DG uptake by selectively interfering with G protein-mediated signal transduction pathway.     

Hypoxia: modulation of endothelial cell proliferation by soluble factors released by retinal cells.

A devastating complication of ischemic retinopathies is retinal neovascularization. We studied the impact on retinal endothelial cell proliferation of soluble factors released from cultured retinal glial (Müller) cells and from retinal explant cultures. Hypoxia strongly stimulated VEGF release by all types of cultures but endothelial cell growth was not further increased by the corresponding conditioned media if compared to supernatants obtained under normoxia. When the final concentration of the hypoxia-conditioned media was adjusted to the VEGF level of normoxia-conditioned media, they even inhibited endothelial cell proliferation. Inhibition may be exerted by TGF-beta 2 but TGF-beta 2 mRNA and protein expression in Müller cells were found to be down-regulated under hypoxia. We conclude that retinal endothelial cell proliferation is controlled by the balance of the amount and/or efficacy of several stimulatory and inhibitory factors.