Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes. © 1996 Wiley-Liss, Inc.     

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.     

Differential effects of sodium butyrate and dimethylsulfoxide on gamma-glutamyl transpeptidase and alkaline phosphatase activities in MCF-7 breast cancer cells

Sodium butyrate and dimethylsulfoxide (DMSO), two known chemical inducers of cell differentiation, were examined on MCF-7 breast cancer cells. Both agents reduce the proliferative capacity of MCF-7 cells, as reflected by inhibition of colony formation in semisolid agar. Sodium butyrate is shown to enhance markedly the activity of two plasma membrane-bound enzymes, alkaline phosphatase and gamma-glutamyl transpeptidase. DMSO does not enhance the activity of these enzymes, but rather induces a small decrease in gamma-glutamyl transpeptidase activity. The present results show that although both agents inhibit cell proliferation, they have a distinct effect on phenotypic expression.     

Immunohistochemical study of localization of gamma-glutamyl transpeptidase in the rat brain

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.     

Molecular characterization of a novel gamma-glutamyl transpeptidase homologue found in rat brain

A cDNA clone for a novel homologue to gamma-glutamyl transpeptidase (gamma-GTP), termed GTPH, was isolated from a rat brain expression cDNA library using antisera against total brain synaptosomal fractions. The cloned GTPH consists of 641 amino acid residues (78 kDa) and exhibits structural similarity with a conventional type of gamma-GTP that is predominantly expressed in the liver: They share significant amino acid homology (33% identity, 73% similarity) spanning over the entire sequence. RNA analyses revealed that GTPH mRNA expression is found only in the nervous system, including all brain regions, eyes and peripheral ganglia, and increases during development. Endogenous GTPH protein is a membrane-bound glycoenzyme and migrates as 90-100 kDa in polyacrylamide gels. Taken together, GTPH is a novel form of a gamma-GTP-like molecule expressed exclusively in the nervous system.     

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.     

Effect of anoxia and reoxygenation on antioxidant enzyme activities in immortalized brain endothelial cells

Summary The effects of anoxia and reoxygenation on major antioxidant enzyme activities were investigatedin vitro in immortalized rat brain endothelial cells (RBE₄ cells). A sublethal anoxic period of 12 h was assessed for RBE₄ cells using the neutral red uptake test. Anoxia markedly influenced the specific activity of catalase and superoxide dismutase, with no major effect on glutathione peroxidase or glutathione reductase. After 24 h postanoxia, the superoxide dismutase activity modulated by the presence or absence of oxygen returned to control value. Damage and recovery of RBE₄ immortalized rat brain endothelial cells in culture after exposure to free radicals and other oxygen-derived species provides a usefulin vitro model to study anoxia-reoxygenation trauma at the cellular level.     

Regulation of P-glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells

In mammalian systems, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been recognized as xenobiotic-sensors which can up-regulate the functional expression of drug transporters, such as P-glycoprotein (P-gp). In the brain, an increase in P-gp expression can further limit drug permeability across the blood-brain barrier (BBB) and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of human PXR (hPXR) and CAR (hCAR) in the regulation of P-gp expression at the human BBB is unknown. In this study, we investigate the role of hPXR and hCAR in the regulation of P-gp expression using a human cerebral microvessel endothelial cell culture system. We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P-gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand-mediated P-gp induction. Ligand-induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down-regulation of hPXR and hCAR proteins using small-interfering RNA decreased P-gp expression. Our findings provide first evidence for P-gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P-gp regulation at this site.     

Annual variations of cerebral and renal alkaline phosphatase and γ‐glutamyl transpeptidase activities in rats

Abstract

This work was undertaken to study annual variations of cerebral and renal enzymatic markers in rats. Cerebral γ‐glutamyltranspeptidase activity levels presented two peaks: in June and December (+167%, p<0,05),with minima in February and April (0%), and a mean of 89%. Cerebral alkaline phosphatase activity was highest in June (+125%, p<0,05) and lowest in February (000%) and a mean of 52%. Renal γ‐glutamyltranspeptidase activity was highest from June to August (+125%, p<0.05) and lowest in February (000%) and a mean of 79%. Whereas renal alkaline phosphatase activity peaked in October (+28%, p<0.05) with a minimum in August (000%) and a mean of 16%. These variations could be related to an endogenous oscillator, the melatonin pulsatile secretion and to different levels of 25‐hydroxyvitamin D₃ during the whole year.     

gamma-glutamyl transpeptidase and related enzyme activities inthe reproductive system of the male rat.

The γ-glutamyl transpeptidase activity of the epididymis is much higher than that of the several other organs of the reproductive system of the male rat. The epididymal caput has much more activity than the epididymal cauda. Relatively low activity was found in spermatozoa. The enzyme is present in the epididymal fluid in a particulate form suggesting that it originates from membranes of epididymal epithelial cells. The epididymal caput exhibits high γ-glutamylcysteine synthetase activity indicating an active γ-glutamyl ycle in this tissue, which plays an important role in transport phenomena.     

Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol

1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gammaglutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated.2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1–2 days.3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1–3 days.4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 μM tocotrienol at 1–3 days in culture compared to the control.     

Automated colorimetric method for the determination of serum gamma-glutamyl transpeptidase utilizing the Technicon SMAC alkaline phosphatase flow channel

An automated procedure for the determination of serum gamma-glutamyltranspeptidase using Technicon SMAC alkaline phosphatase flow channel has been developed. The flow variants are insignificant. We use L-gamma-glutamyl-3-carboxy-4-nitroanilide as a very soluble substrate. With the dyalisis procedure, hemoglobin and bilirubin do not interfere. By employing dialysis, the need of blank corrections is therefore eliminated. The procedure can been run at 130 determinations per hour with insignificant carryover and satisfactory precision.     

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.     

Nitric oxide and endothelin secretion by brain microvessel endothelial cells: Regulation by cyclic nucleotides

Endothelin (ET)-1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET-1 and NO, which, at the level of the blood-brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFNγ and TNFα, (2) they constitutively secrete ET-1, and (3) cAMP potentiates the cytokine-induced NO release and exerts a biphasic regulation on ET-1 secretion: micromolar concentrations of 8-Br-cAMP inhibit and higher doses stimulate ET-1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET-1. © 1993 Wiley-Liss, Inc.