Intercellular adhesion molecule 1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelial cells.

Inflammatory diseases of the central nervous system, such as multiple sclerosis or experimental autoimmune encephalomyelitis, are characterized by adhesion of lymphocytes on cerebral microvascular endothelium, followed by transendothelial migration into the brain parenchyma. T lymphocyte adhesion to vascular endothelial cells is mediated by several types of adhesion molecules, including the integrin leukocyte function-associated molecule 1 and its endothelial counter receptor intercellular adhesion molecule 1 (ICAM-1), of the immunoglobulin superfamily. In order to understand the molecular mechanisms that support lymphocyte extravasation, we intended to investigate a putative role of ICAM-1 in signal transduction in brain microvessel endothelial cells. Here we describe, using a well differentiated rat brain endothelial cell line (RBE4 cells), that ICAM-1 activation by a specific monoclonal antibody, or by syngeneic encephalitogenic T cells, induces tyrosine phosphorylation of several proteins together with stimulation of the tyrosine kinase p60src activity. One of the major tyrosine-phosphorylated proteins, of 85 kDa, has been identified by immunoprecipitation and immunoblotting, as the recently described actin-binding protein, p60src substrate, cortactin. These findings demonstrate that ICAM-1 activation transduces signals in brain endothelial cells, which may lead to cytoskeleton changes and transendothelial migration of lymphocytes into the brain.

Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells.

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.

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 IFN gamma and TNF alpha, (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.

Catecholamines stimulate MHC class I, class II, and invariant chain gene expression in brain endothelium through different mechanisms.

Bovine brain microvessel endothelial cells in primary culture retain a differentiated phenotype reminiscent of the in vivo blood-brain barrier endothelium. The IFN-gamma-induced surface expression of MHC class II molecules on those cells is stimulated by catecholamines through a cAMP-independent mechanism. We report that both the induction of MHC class II molecule expression by IFN-gamma and its potentiation by isoproterenol, a catecholamine analog, are preceded by increases of steady-state levels of the corresponding mRNA. Similar results were obtained for the regulation of invariant chain expression. In addition, isoproterenol alone is able to increase class I mRNA levels, but, in contrast to what is observed for class II expression, this effect appears to be mainly mediated by cAMP. These studies demonstrate that, in bovine brain microvessel endothelial cells, catecholamines up-regulate MHC class I and class II Ag, as well as invariant chain with different kinetics and through different mechanisms and suggest that these regulatory effects occur at the transcriptional level.

Catecholamines stimulate the IFN-gamma-induced class II MHC expression on bovine brain capillary endothelial cells.

The brain has been considered for a long time as an immunologically privileged site because of the lack of a true lymphatic system and the existence of several barriers that isolate it from the periphery. In the last few years, it became evident that cells in the central nervous system (astrocytes, microglial cells, and brain capillary endothelial cells) can be induced to express class II MHC and present Ag to T lymphocytes. The brain capillary endothelial cells, which are strategically located at the interface between blood and brain, could be involved in the initiation of immune responses within the brain parenchyma. We have previously characterized bovine brain capillary endothelial cells in culture and shown that they maintain in vitro a fully differentiated phenotype associated with the blood-brain barrier endothelium. In order to assess the role of these cells in the development of immune responses in the brain, we initiated the present study on the regulation of their class II MHC surface expression. Our data indicate that this expression on bovine brain capillary endothelial cells is inducible by IFN-gamma and further stimulated by catecholamines through activation of beta-adrenergic receptors. However, this latter effect is not mimicked by forskolin, theophylline, or dibutyryl-cAMP, suggesting the involvement of a cAMP-independent mechanism.

Immortalization of brain capillary endothelial cells with maintenance of structural characteristics of the blood-brain barrier endothelium.

Early passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation, and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme and Griffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover, SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functional beta 1- and beta 2-adrenergic receptors, as observed on isolated bovine brain capillaries.

cAMP-dependent down-regulation of endothelin-1 receptors on rat astrocytoma C6 cells.

The density of endothelin-1 (ET-1) receptors on rat astrocytoma C6 cells is down-regulated by activation of protein kinase C (PKC). We have investigated whether intracellular accumulation of cyclic adenosine monophosphate (cAMP) may also modulate surface ET-1 receptor number. The density of ET-1 receptors was measured by binding of [125I]ET-1 on rat astrocytoma C6 intact cells exposed to catecholamines, dibutyryl-cAMP or forskolin. Prolonged exposure of the cells to the beta-adrenergic agonists, isoproterenol or noradrenaline, results in a time- and dose-dependent decrease in cell surface ET-1 receptor number. This decrease proceeds slowly: maximal down-regulation is obtained by 6-7 h and sustained for up to 24 h in the presence of 10 microM isoproterenol. Since this down-regulation is mimicked by dibutyryl-cAMP (4 microM) and by forskolin (10 microM), we conclude that ET-1 receptors are susceptible to down-regulation through a cAMP-dependent pathway.

Are several G proteins involved in the different effects of endothelin-1 in mouse striatal astrocytes?

High-affinity specific receptors of endothelin (ET-1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I-ET-1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET-1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide-binding proteins.

Functional endothelin-1 receptors in rat astrocytoma C6.

Rat astrocytoma C6 cells have been recently identified as target cells for ET-1, which stimulates inositol lipid turnover in these cells. It is shown here that binding of ET-1 to high-affinity receptors on C6 cells leads to 40-45% inhibition of isoproterenol-induced intracellular cyclic AMP accumulation, as well as to stimulation of inositol lipid turnover, both effects characterized by an absolute requirement of extracellular calcium. Moreover, ET-1, which has been generally reported to have a mitogenic effect on a variety of target cells including primary rat astrocytes, is shown here to stimulate or, alternatively, inhibit DNA synthesis in C6 cells, depending on the subclone considered.

Coexpression of beta 1- and beta 2-adrenergic receptors on bovine brain capillary endothelial cells in culture.

The pharmacological study of the blood-brain barrier has often been hampered by the unavailability of a large number of pure and fully differentiated brain capillary endothelial cells. Here we describe a homogeneous culture of brain capillary endothelial cells isolated from bovine brain (BBECs), which retain at least some phenotypic characteristics of the functional blood-brain barrier: intracellular tight junctions and monoamine oxidase activity. These cells were subcultured in vitro, in the absence of any neuronal or glial influences, for greater than 100 doublings without any sign of senescence. The present study is focused on the expression of beta-adrenergic receptors on BBECs. By Northern blot hybridization, subtype-specific ligand binding, and cyclic AMP accumulation experiments, we demonstrate that beta 1- and beta 2-adrenergic receptors are coexpressed (in the respective proportions of 42 and 58%) on BBEC membranes and are functionally coupled to adenylate cyclase. This is the first report documenting a significant number of beta 1-adrenergic receptors on brain capillary endothelial cells. The results are discussed in light of the known noradrenergic innervation of brain capillaries.

Comparison of binding characteristics of endothelin receptors on subpopulations of astrocytes.

Astrocytes in primary culture originating from different brain areas of the mouse embryo (striatum, cerebral cortex and mesencephalon) were compared for their [125I]-Endothelin-1 (ET-1) binding characteristics, in terms of affinity, binding capacity and specificity. Our results indicate that astrocytes from mesencephalon express about twice as many receptors as astrocytes from striatum or cortex (149,000 +/- 9700 vs 63,700 +/- 5600 and 81,900 +/- 5300, respectively), with similar affinities. Specificity patterns for the various peptides of the endothelins/sarafotoxins family (ET-1, -2, -3; SRTXa, b, c) are comparable in the three subpopulations of astrocytes: ET-1, -2 and SRTXb exhibit higher affinities than SRTXa and SRTXc. In addition, ET-3 and SRTXc seem to discriminate between different subsets of [125I]-ET-1 binding sites in the three subpopulations.

Modulation of muscarinic-receptor expression in human embryonic lung fibroblasts by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is known to have regulatory control of a large number of cellular components, including various receptors. We show that muscarinic acetylcholine receptors of the m2 subtype on CCL 137 human fibroblasts in culture are affected by PDGF treatment. A time-dependent down-regulation is observed in steady-state RNA levels, followed by a decrease in ligand-binding capacity. Minimum RNA levels are attained at 11 h; minimum binding capacity is observed after 24 h of treatment. To our knowledge, this is the first example of negative gene control by PDGF.

A monoclonal antibody directed against the beta 1-adrenergic receptor from turkey erythrocyte membranes.

A monoclonal antibody was raised against the affinity purified beta 1-adrenergic receptor from turkey erythrocytes. This antibody, B120, of the IgG1 isotype, precipitates the photoaffinity-labeled purified receptor and the corresponding binding activity. The antibody recognizes the 42 kDa component of the receptor of erythrocyte membranes, after electrotransfer on nitrocellulose. B120 does not inhibit binding of dihydroalprenolol on membranes and has no effect on the activity of adenylate cyclase. Since specific immunofluorescence was detected only after ethanol treatment of the erythrocytes, the epitope recognized by B120 appears not to be accessible at the cell surface.

The beta 2-adrenergic receptors of human epidermoid carcinoma cells bear two different types of oligosaccharides which influence expression on the cell surface.

The beta 2-adrenergic receptors of the human epidermoid carcinoma A431 cells reside on two polypeptide chains revealed by photoaffinity labelling with [125I]iodocyanopindolol-diazirine. These proteins correspond to two distinct populations of N-asparagine-linked glycoproteins: the 55-52 kDa molecules are associated with complex carbohydrate chain(s), the 65-63 kDa component with polymannosidic carbohydrate chain(s). Both types of receptors are present in preconfluent cells, but only the polymannosidic type is found in the postconfluent cells. Moreover, complex chains appear to be associated with the receptors with the highest affinity for (-)-isoproterenol and polymannosidic chains with the receptors with the lowest affinity for this agonist. the carbohydrate moiety of the beta-adrenergic receptor is involved in the expression and function of the beta 2-adrenergic receptors at the surface of the A431 cells, since tunicamycin and monensin, complete and partial inhibitors of glycosylation respectively, diminish the number of binding sites at the cell surface and increase the total number of sites in the cell. In these conditions a diminution of cyclic AMP accumulation is also observed.

Structure of the gene for human beta 2-adrenergic receptor: expression and promoter characterization.

The genomic gene coding for the human beta 2-adrenergic receptor (beta 2AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with beta 2AR properties. Southern blot analyses with beta 2AR-specific probes show that a single beta 2AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two "TATA boxes," a "CAAT box," and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the beta 2AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

Biochemical and immunochemical analysis of avian beta 1 and mammalian beta 2-adrenergic receptors.

We have studied the molecular properties of avian beta 1-adrenergic receptor and human beta 2-adrenergic receptor. The turkey erythrocytes beta 1-receptor has been solubilized in active form by digitonin and has been purified to homogeneity by affinity chromatography followed by electroelution from polyacrylamide gel. The photoactivable ligand, iodocyanopindololdiazirine, labels specifically a major 45 kDa and minor 55 kDa polypeptide in turkey erythrocytes, whereas in A431, it labels two polypeptides of molecular weights 65 kDa and 55 kDa. Both types of receptors are N- and possibly O-glycosylated but the turkey beta 1 receptor has only complex carbohydrates whereas the human beta 2 receptor has in addition oligo mannosidic polysaccharidic moiety. Polyclonal and monoclonal antibodies were raised against the beta 1- and beta 2-adrenergic receptors. Polyclonal antibodies were found to mimic beta-adrenergic agonists by stimulating adenylate cyclase upon binding to the receptors. The monoclonal antibodies precipitated both intact and affinity labeled receptors which they also revealed on immunoblots.

The oligosaccharide moiety of the beta 1-adrenergic receptor from turkey erythrocytes has a biantennary, N-acetyllactosamine-containing structure.

The turkey erythrocyte beta 1-adrenergic receptor can be purified by affinity chromatography on alprenolol-Sepharose and characterized by photoaffinity labeling with N-(p-azido-m-[125I]iodobenzyl)-carazolol. Through the use of the specific glycosidases neuraminidase and endo-beta-N-acetylglucosaminidase H and affinity chromatography on lectin-Sepharose gels, we show here that the receptor is an N-glycosyl protein that contains complex carbohydrate chains. No high-mannose carbohydrate chains appear to be present. The binding of the radiolabeled antagonist dihydroalprenolol to the receptor is affected neither by the enzymic treatments nor by the presence of lectins, suggesting that the carbohydrate moiety is not involved in the catecholamine binding site.

Altered binding properties of beta-adrenergic receptors and lack of coupling to adenylate cyclase in P815 mastocytoma cells.

P815, a murine mastocytoma cell line, possesses beta-adrenergic binding sites as assessed by using [3H]dihydroalprenolol (antagonist) and [3H]hydroxybenzylisoproterenol (agonist). The number of binding sites per cell was 29 000 for the agonist and 75 000 for the antagonist, as determined by direct binding assays and inhibition experiments on intact cells. On membrane preparations from the same cells, binding of alprenolol was only displaceable by antagonists, while stereospecific binding of hydroxybenzylisoproterenol was only displaceable by agonists. The P815 membranes also possessed an adenylate cyclase stimulated by Gpp(NH)p and NaF but not by 1-isoproterenol. The intracellular cAMP level of intact cells was not modulated by 1-isoproterenol or by 1-epinephrine, but was increased by forskolin. These results suggest that the beta-adrenergic receptor of P815 mastocytoma cells is non-functional. This may explain the failure of agonists to stimulate adenylate cyclase activity in these cells.