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Z-Q Han, H A Coppock, D M Smith, S Van Noorden, M W Makgoba, C G Nicholl and S Legon

ABSTRACT

An abundant, seven trans-membrane domain receptor related to the calcitonin receptor has been studied by a number of groups without identification of its ligand. A recent report claimed that the receptor was a type 1 CGRP receptor (Aiyar et al J. Biol. Chem. 271 11325-11329 (1996)). We have studied the equivalent rat sequence in transfected cells. When expressed in 293 cells the receptor interacts with CGRP and adrenomedullin with KD values of 1.2 nM for CGRP and 11 nM for adrenomedullin. Both ligands cause an elevation of intracellular cAMP with EC50 values of 4 nM and 20 nM respectively and these effects are inhibited by the antagonist CGRP8-37. The receptor is expressed at high levels in the pulmonary vascular endothelium. Both the pharmacological data and the localisation are consistent with the conclusion that the orphan receptor is a type 1 CGRP receptor. However, when expressed in COS-7 cells, no receptor activity could be demonstrated suggesting that 293 cells contain a factor necessary for functional receptor expression.

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Jéssyca Aparecida Soares Giesen, Wender do Nascimento Rouver, Eduardo Damasceno Costa, Virgínia Soares Lemos and Roger Lyrio dos Santos

& Randall 1998 , Rosano et al. 2017 ). The endothelium can release vasodilatory factors, such as nitric oxide (NO), prostacyclin (PGI 2 ), and endothelium-dependent hyperpolarizing (EDH), or vasoconstriction factors, such as thromboxane A 2 (TXA 2

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Philippe Huber, Christine Mallet, Elodie Faure, Christine Rampon, Marie-Hélène Prandini, Olivier Féraud, Stéphanie Bouillot and Isabelle Vilgrain

Introduction Vascular endothelial (VE)-cadherin (CD144) has been shown to play important roles in the establishment and maintenance of endothelium integrity ( Lampugnani et al. 1993 ). VE-cadherin is a member of the cadherin

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Eugenia Mata-Greenwood, P Naomi Jackson, William J Pearce and Lubo Zhang

We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n=15), or resistant (n=10). Real-time PCR analysis of GR 5′UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5′UTR mRNA isoforms 1C and 1D, but lower levels of the 5′UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5′UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5′UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns.

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Yanxia Tang and GuoDong Li

Introduction Endothelium plays an important role in the regulation of vascular tone and other functions via the synthesis and release of vascular modulators. Nitric oxide (NO), a gas molecule and potent vasodilator, has received the most interest in

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Agua Sobrino, Pilar J Oviedo, Susana Novella, Andrés Laguna-Fernandez, Carlos Bueno, Miguel Angel García-Pérez, Juan J Tarín, Antonio Cano and Carlos Hermenegildo

aggregation, it is important to determine the role of these enzymes in the regulation of prostanoid biosynthesis by the endothelium. Although there is evidence that PGI2 release from endothelial cells is increased by E 2 ( Mikkola et al . 1995 ), less is

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Yu-Guang Ma, Liang Liang, Yin-Bin Zhang, Bao-Feng Wang, Yun-Gang Bai, Zhi-Jun Dai, Man-Jiang Xie and Zhong-Wei Wang

administration of 100 mg/kg/day berberine for 8 weeks significantly induced the relaxation of middle cerebral artery in diabetic rats As compared with that in CON, endothelium-dependent relaxation to acetylcholine (Ach, Figs 2A and 3A ) and endothelium

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S Barker, W Marchant, M M Ho, J R Puddefoot, J P Hinson, A J L Clark and G P Vinson

ABSTRACT

We have generated hybridomas which secrete monoclonal antibodies to the AT1 subtype of the angiotensin II receptor (AT1 receptor). These were obtained after immunization of Balb C/c mice with synthetic peptides representing sequences from either the extracellular domain (residues 8-17) or the intracellular domain (residues 229-237) of the AT1 receptor.

Hybridoma populations were first screened for the production of antibodies which bound to rat liver cells. Further selection, and cloning by limiting dilution, was carried out for antibodies which bound specifically to rat adrenal glomerulosa cells. Confirmation that the antibody designated 6313/G2 interacted with the angiotensin II receptor was obtained using COS-7 cells transfected with AT1A receptor cDNA. In particular, the initial characterization of 6313/G2 showed specific immunofluorescence of vascular endothelium.

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JS Fleming, NM Hope and CJ Bolter

We have examined the expression of the ovine clusterin gene in the sheep pituitary gland, with the aim of determining its site of synthesis in this tissue. Northern blotting analysis of extracted polyadenylated RNA, using a (32)P-labelled rat clusterin cDNA probe, detected the greatest amounts of clusterin mRNA in the anterior part of dissected pituitary glands. In situ hybridisation studies showed clusterin mRNA in anterior and intermediate pituitary cells, with lower amounts in vascular endothelium and posterior pituicytes. Clusterin protein, detected by immunohistochemistry, was observed in some single secretory cells, within the capillary lumen and in cells around capillaries in the anterior and intermediate lobes, but no immunoreactivity was observed in posterior pituitary tissue. The pattern of clusterin expression in anterior and intermediate pituitary cells suggests possible roles for the protein in secretory cell turnover and/or hormone secretion or lipid uptake. Clusterin does not appear to be involved in ovine posterior pituitary hormone neurosecretion.

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S C Hodgkinson, J R Napier, G S G Spencer and J J Bass

ABSTRACT

Interactions between the IGF-binding proteins (IGFBPs) and glycosaminoglycans (GAGs) such as heparin may be involved in the regulatory control of IGF exerted by the IGFBPs at the level of the extracellular matrix and capillary endothelium, although the precise mechanisms of this remain uncertain. We have searched primary sequences of human, rat and bovine IGFBPs-1 to -6 for putative GAG-binding consensus sequences (XBBXBX and XBBBXXBX, where B represents any basic amino acid and X is undefined). At least one such sequence was identified in each IGFBP examined except human and rat IGFBP-4 and rat IGFBP-6, with IGFBP-5 containing three GAG-binding consensus sequences. Additionally, the bovine IGF type II receptor was found to contain two such sequences in the intracellular region. Affinity of the IGFBP preparations for heparin was examined experimentally by affinity chromatography using pooled fractions of fetal and adult ovine plasma obtained by size exclusion chromatography. Pooled fractions of 150 kDa (containing IGFBP-3 alone by IGF ligand blot analysis) and 40–50 kDa (containing IGFBPs-3 and -2, together with proteins of 29, 24 and 25–28 kDa which may include IGFBP-4 and IGFBPs-1, -5 and -6) were found to bind strongly to the matrix necessitating high salt concentrations for their elution; however, in contrast, a >200 kDa fraction containing the soluble form of the type II receptor failed to bind. Recombinant human non-glycosylated IGFBP-3 also bound strongly to the affinity adsorbent. No evidence of dissociation of bound IGF from binding protein complexes by association with the matrix was obtained from this experiment. This study provides a molecular basis for the interaction of IGFBPs with matrix GAGs, although precise mechanisms by which this may influence IGF bioactivity at the cellular level remain to be established.