Design and synthesis of ETA receptor antagonists and study of ETA receptor distribution.

Many oligopeptides were designed to find ETA receptor antagonists on the hypothesis that an ETA receptor can recognize two hydrophobic parts of ET-1, i.e., Val-Tyr-Phe and Ile-Ile-Trp, over a short distance. They were synthesized from the benzyl ester of the C-terminal amino acid by stepwise chain elongation using the solution method. The binding affinity of the synthetic peptides to the endothelin receptors was examined in porcine cardiac ventricular muscle membrane for ETA receptor and in bovine whole brain membrane for ETB receptor. Hexamethyleneiminocarbonyl-Leu-trp-ala-beta ala-tyr-phe (TTA-386) was selected as an ETA receptor-selective competitive antagonist to ET-1. It competed against ET-1 at ETA receptor sites and showed one-third the binding affinity of ET-1 for ETA receptor and < 1/10,000 the affinity for ETB receptor. It inhibited the ET-1-induced increase of cytosolic free calcium concentration in A-10 cells. The 125I-labeled hexapeptide (125I-TTA-386) was prepared to distinguish the distribution of ETA receptor from ETB receptor. Scatchard plot analysis of saturation binding of 125I-TTA-386 to porcine cardiac membranes showed the same Bmax value as that of 125I-ET-1. Autoradiographic studies showed that ETA receptors are most abundant in the cardiac muscle, intestine, large bowel, spleen, and testis.