1 We measured the ratio of ET(A) and ET(B) 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, [I-125]-endothelin-1 ([I-125]-ET-1) bound with high affinity to the media of each vessel (n = 3 individuals or homogenate preparations +/- s.e.mean):coronary artery, K-D = 0.14 +/- 0.02 nM, B-max = 71.0 +/- 21.0 fmol mg(-1) protein; pulmonary artery, K-D = 0.85 +/- 0.25 nM, B-max = 15.2 +/- 10.3 fmol mg(-1) protein; aorta, K-D = 0.51 +/- 0.02 nM, B-max = 9.4 +/- 4.4 fmol mg(-1) protein; internal mammary artery, K-D = 0.34 +/- 0.31 nM, B-max = 2.0 +/- 0.5 fmol mg(-1) protein and saphenous vein, K-D = 0.28 +/- 0.05 nM, B-max = 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 [I-125]-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, K(D)ET(A) = 0.85 +/- 0.03 nM, K(D)ET(B) = 7.58 +/- 2.27 mu M, ratio = 89:11%; pulmonary artery, K(D)ET(A) = 0.27 +/- 0.05 nM, K(D)ET(B) = 24.60 +/- 5.34 mu M, ratio = 92:8%; aorta, K(D)ET(A) = 0.80 +/- 0.40 nM, K(D)ET(A) = 2.67 +/- 2.60 mu M ratio = 89:11%; saphenous vein, K(D)ET(A) = 0.55 +/- 0.17 nM, K(D)ET(B) = 14.4 +/- 0.26 mu M, 85:15% (n = 3 individuals or homogenate preparations +/- s.e.mean). BQ123 showed up to 18000 fold selectivity for the ET(A) over the ET(B) sub-type. The ET(A)-selective ligand, [I-125]-PD151242 labelled 85% of the receptors detected by a fixed concentration of [I-125]-ET-1 in media of internal mammary artery, measured by quantitative autoradiography. In contrast, the density of ET(B) receptors detected with [I-125]-BQ3020 was 7.0 +/- 1.5 amol mm(-2), representing about 8% of [I-125]-ET-1. 4 A single band corresponding to the expected position for mRNA encoding the ET(A) 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 ET(B) sub-type (428 base pairs) was also always detected. 5 S-35-labelled antisense probes to ET(A) and ET(B) 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 ET(A) sub-type. These results provide further support for the hypothesis that the ET(A) 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.
