Human Endothelin Receptors Characterized Using Reverse Transcriptase-Polymerase Chain Reaction, In Situ Hybridization, and Subtype-Selective Ligands BQ123 and BQ3020: Evidence for Expression of ETB Receptors in Human Vascular Smooth Muscle

Summary: Our aim was to characterize and determine the function of endothelin (ET) receptor subtypes in human vascular tissue. Reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers detected the presence of mRNA encoding both ETA and ETB receptors in the media from aorta and pulmonary and coronary arteries. In situ hybridization confirmed the presence of mRNA for both subtypes in the media of coronary arteries. Saturation binding assays using 125I-ET-1 found a single population of high-affinity ET receptors (n = three patients, ± SEM) in aorta (Kd = 0.507 ± 0.020 nM; Bmax = 9 ± 4 fmol/mg protein) and pulmonary (Kd = 0.845 ± 0.245 nM; Bmax = 15 ± 10 fmol/mg protein) and coronary arteries (Kd = 0.141 ± 0.020 nM; Bmax = 71 ± 21 fmol/mg protein). Using media from coronary arteries, the ETA-selective ligand BQ123 (cycloD-Asp-L-Pro-D-Val-L-Leu-D-Trp) and the ETB-selective ligand BQ3020 (Ala11.15-Ac-ET-16-21) both produced biphasic competition binding curves against 125I-ET-1, confirming the presence of highand low-affinity sites corresponding to the two subtypes: BQ123 (KdETA = 0.85 ± 0.03 nM; KdETB = 7.58 ± 2.27 μM; ETA/ETB, 87%: 13%) and BQ3020 (KdETA = 0.22 ± 0.04 μM; KdETB = 0.77 ± 0.34 nM; ETA/ETB, 62%: 38%). BQ123 (0.1 μM) caused a significant parallel rightward shift of ET-1-induced vasoconstriction of coronary arteries in vitro, but BQ3020 and Ala1,3,11,15-ET-1 failed to show any agonist activity when tested at concentrations of ≤3 μM in three vessels. The results show that BQ123 is highly selective (9,000-fold) for the human vascular ETA subtype and functions as an antagonist. ETB receptors and mRNA are expressed by the media in human vessels. Although BQ3020 is selective for the human ETB receptor in binding assays and is a potent constrictor of some animal vessels in vivo, this compound has no detectable agonist activity in any of the human vessels tested. The responsiveness of ETB subtypes could vary in different vascular beds, or vasoconstrictor activity may be lost in pathophysiologic tissue. A third intriguing possibility is that human ETB receptors differ from those in animal tissues and may represent a previously uncharacterized subtype.