High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(E T-3) values as low as 50 nM were defined from [I-125]ET-1 binding experimen ts performed at low (5 mu g/ml) protein concentrations in the assays. Large r (up to 1 mu M) values were artefactually obtained in experiments performe d at larger protein concentrations. The three monoiodo ET-3 derivatives wer e synthetized, ([I-125]Y-14)ET-3 did not recognize ETA receptors, ([I-125]Y -6)ET-3 labelled 18% of [I-125]ET-1 binding sites with a Kd value of 320 pM , ([I-125]Y-13)ET-3 labelled 44% of [I-125]ET-1 binding sites with a Kd val ue of 130 pM. High affinity ([I-125]Y-6)ET-3 and ([I-125]Y-13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620, The three monoiodo ET-3 derivatives bound to re combinant rat ETB receptors with a pM affinity. The results suggest that ET -3, ([I-125]Y-6)ET-3 and ([I-125]Y-13)ET-3 should not be considered as ETB receptor specific ligands. (C) 1999 Academic Press.