Endothelin ETA and ETB mRNA and receptors expressed by smooth muscle in the human vasculature: majority of the ETA sub‐type

• Published in: British journal of pharmacology Vol. 114; no. 6; pp. 1110 - 1116
• Main Authors: Davenport, Anthony P., O'Reilly, Gillian, Kuc, Rhoda E.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.03.1995; Nature Publishing
• Subjects: Adult; aorta; Arteries; autoradiography; Biological and medical sciences; Blood vessels and receptors; BQ123; BQ3020; coronary artery; Endothelin; Endothelins - genetics; ETA mRNA; ETB mRNA; Female; Fundamental and applied biological sciences. Psychology; Humans; internal mammary artery; Male; Middle Aged; Muscle, Smooth, Vascular - metabolism; PD151242; Polymerase Chain Reaction; pulmonary artery; Radioligand Assay; Receptors, Endothelin - biosynthesis; RNA, Messenger - biosynthesis; Saphenous Vein; Vertebrates: cardiovascular system; Human; Endothelin; Saphenous vein; Blood vessel; Coronary artery; Peptidergic receptor; Smooth muscle; Aorta; Circulatory system; Internal mammary artery; In vitro; Pulmonary artery
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/j.1476-5381.1995.tb13322.x

1 We measured the ratio of ETA and ETB sub‐types in the media (containing mainly smooth muscle) of human cardiac arteries (aorta, pulmonary and coronary), internal mammary arteries and saphenous veins. 2 In saturation experiments, [125I]‐endothelin‐1 ([125I]‐ET‐1) bound with high affinity to the media of each vessel (n = 3 individuals or homogenate preparations ± s.e.mean): coronary artery, KD = 0.14 ± 0.02 nm, Bmax = 71.0 ± 21.0 fmol mg−1 protein; pulmonary artery, KD = 0.85 ± 0.25 nm, Bmax = 15.2 ± 10.3 fmol mg−1 protein; aorta, KD = 0.51 ± 0.02 nm, Bmax = 9.4 ± 4.4 fmol mg−1 protein; internal mammary artery, KD = 0.34 ± 0.31 nm, Bmax = 2.0 ± 0.5 fmol mg−1 protein and saphenous vein, KD = 0.28 ± 0.05 nm, Bmax = 52.8 ± 1.0 fmol mg−1 protein. In each vessel, over the concentration‐range tested, Hill slopes were close to unity and a one site fit was preferred to a two site model. 3 In competition binding assays, the ETA selective ligand, BQ123 inhibited the binding of 0.1 nm [125I]‐ET‐1 to the media in a biphasic manner. In each case, a two site fit was preferred to a one or three site model: coronary artery, KDETA = 0.85 ± 0.03 nm, KDETB = 7.58 ± 2.27 μm, ratio = 89:11%; pulmonary artery, KDETA = 0.27 ± 0.05 nm, KDETB = 24.60 ± 5.34 μm, ratio = 92:8%; aorta, KDETA = 0.80 ± 0.40 nm, KDETB = 2.67 ± 2.60 μm ratio = 89:11%; saphenous vein, KDETA = 0.55 ± 0.17 nm, KDETB = 14.4 ± 0.26 μm, 85:15% (n = 3 individuals or homogenate preparations ± s.e.mean). BQ123 showed up to 18000 fold selectivity for the ETA over the ETB sub‐type. The ETA‐selective ligand, [125I]‐PD151242 labelled 85% of the receptors detected by a fixed concentration of [125I]‐ET‐1 in media of internal mammary artery, measured by quantitative autoradiography. In contrast, the density of ETB receptors detected with [125I]‐BQ3020 was 7.0 ± 1.5 amol mm−2, representing about 8% of [125I]‐ET‐1. 4 A single band corresponding to the expected position for mRNA encoding the ETA receptor (299 base pairs) was found in the media in each of the five vessels (n = 3 individuals) using reverse‐transcriptase polymerase chain reaction assays. A single band corresponding to the ETB sub‐type (428 base pairs) was also always detected. 5 35S‐labelled antisense probes to ETA and ETB hybridised to the media of epicardial coronary arteries as well as intramyocardial vessels, confirming the presence of mRNA encoding both sub‐types in the vascular smooth muscle of the vessel wall. 6 Although mRNA for both receptors was detected, competition binding using BQ123 demonstrated that the majority (at least 85%) of ET receptors present in smooth muscle are the ETA sub‐type. These results provide further support for the hypothesis that the ETA sub‐type is the receptor that must be blocked in humans to produce a beneficial vasodilatation in pathophysiological conditions where there is an increase in peptide concentration or receptor density.