| pA2 online © Copyright 2004 The British Pharmacological Society |
022P
University of Newcastle Winter Meeting December 2004 |
Clearance of circulating endothelin-1 is mediated by the endothelial cell endothelin B receptor
NF Kelland1, AJ Bagnall1, FH Gulliver-Sloan1, RE Kuc2, JJ Maguire2, AP Davenport2, GA Gray1, YV Kotelevtsev1 and DJ Webb1. 1Centre for Cardiovascular Science, University of Edinburgh. 2Clinical Pharmacology Unit, University of Cambridge. |
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Print abstractInactivation of endothelin type B receptors (ETB), through selective pharmacological antagonism (Fukuroda et al., 1994) or genetic mutation (Gariepy et al., 2000 ; Berthiaume et al., 2000) increases the circulating concentration of endothelin-1 (ET-1), suggesting that the ETB plays an important role in the clearance of this peptide. By crossing floxed ETB mice (FF/NoCre) with Tie2-Cre mice (WW/Tie2-Cre), we have generated an endothelial cell (EC) specific ETB KO mouse (FF/Tie2-Cre) to investigate the relative contribution of the EC-ETB to the clearance of ET-1, without the confounding effects of receptor deletion in all other tissues.
Studies were carried out under Home Office approval, on male mice (8-16 weeks old; 25-35g). Endothelin type A receptor (ETA) and ETB expression was assessed by quantitative autoradiography using the radiolabelled ETA specific ligand [125I]-PD151242 and the ETB specific ligand [125I]-BQ-3020. Plasma ET-1 and big ET-1 concentrations were measured by radioimmunoassay. Clearance of ET-1 was measured by injecting a bolus of [125I]-ET-1 (0.37 pmol/mouse; 28 kBq/mouse) intravenously under isoflurane anaesthesia. Arterial blood was sampled over the subsequent 2 minutes and the radioactivity in each sample was measured (cpm). All results were compared using one-way ANOVA with Bonferroni post-test analysis.
Autoradiography revealed significant downregulation of ETB in EC-rich tissues such as lung of FF/Tie2-Cre animals (8 ± 3 amol.mm-2) compared to WW/-- controls (80 ± 21 amol.mm-2 (n=4; p<0.05). Levels of ETA expression were preserved despite four-fold higher concentrations of plasma ET-1 in the FF/Tie2-Cre samples (12.4 ± 3.0 pg.ml-1) compared to WW/-- controls (3.0 ± 0.8 pg.ml-1) (n=6; p<0.001). Plasma big ET-1 concentrations were only marginally increased in FF/Tie2-Cre animals (200 ± 8 pg.ml -1) relative to control animals (137 ± 13 pg.ml-1) (pooled samples; n=4; p<0.05). The area under the curve (AUC) for the cpm:time graph was significantly increased in FF/Tie2-Cre mice [235056 ± 21333 cpm.sec] compared to the WW/-- [86726 ± 10968 cpm.sec] (n=5; p<0.01). Pharmacological blockade of the ETB in WW/-- mice resulted in an AUC unchanged from that of untreated FF/Tie2-Cre animals (n=4).
We have shown tissue specific KO of the EC-ETB results in much higher plasma concentrations of ET-1 than its precursor big ET-1, most likely due to impaired ET-1 clearance. Our data are consistent with EC-ETB being the site of ET-1 scavenging.
Berthiaume , N., et al . (2000). Hypertension , 36, 1002-7.
F ukuroda , T., et al . (1994). Biochem Biophys Res Commun, 199, 1461-5.
G ariepy , C.E., et al . (2000). J Clin Invest, 105, 925-33.