Identification of ET(B) receptor subtypes using linear and truncated analogs of ET

Using canine spleen and lung membranes as model systems, we have shown the presence of subtypes of ET, receptors. This classification was done based on the binding profiles of various analogs of ET-1 that have been identified as ET(B)-selective. Saturation binding experiments performed with [I-125] ET-3 and [I-125] IRL-1260 (ET(B)-selective ligands) indicated that [I-125] IRL-1620 labeled 80-90% of [I-125] ET-3 binding sites in canine lung, whereas in canine spleen, the binding of [I-125] IRL-1620 was 10-20% Of [I-125] ET-3 binding. In addition, competition binding experiments using ET(B)-selective agonists [Ala(1,3,11,15)]-ET-1 (also known as 4-Ala ET-1), N-acetyl-[Ala(11,15)] ET-1 (6-21) also known as BQ3020 and Suc-[Glu(9), Ala(11,15)] ET-1 (8-21) also known as IRL-1620 indicated that all three ligands displaced [I-125] ET-3 from canine lung membranes with similar to 500-1000 fold greater affinity than canine spleen membranes. On the other hand, ET-1, ET-3, S6a, S6b, and S6d displayed very similar IC50 values in both preparations, except S6c which was similar to 20 fold less potent in canine spleen compared to lung. These data indicate that ET(B) receptors present in canine spleen are different from those present in lung and that ET(B)-selective linear as well as truncated analogs of ET-1 are good tools to identify these subtypes of ET(B) receptors.