Characterization of [I-125]-endothelin-1 and [I-125]-BQ3020 binding to rat cerebellar endothelin receptors

1 We performed radioligand binding experiments on rat cerebellar homogenates using [I-125]-endothelin-1 ([I-125]-ET-1) and [I-125]-BQ3020 to examine the pharmacology of endothelin receptors in rat brain. Saturation experiments demonstrated a single population of binding sites with high affinity for both radioligands ([I-125]-ET-1, pK(d)=8.94+/-0.17; [I-125]-BQ3020, pK(d)=9.18+/-0.14 nM; mean+/-s.e.mean). However, [I-125]-BQ3020 only recognised approximately one third the number of endothelin receptors measured with [I-125]-ET-1. 2 Saturation binding experiments with [I-125]-PD151242 revealed high affinity binding to a single population of ET(A) receptors in the cerebellar homogenates (pK(d)=9.95+/-0.14; B-max=30+/-15 fmol mg(-1) protein). 3 Competition experiments were performed with ligands that are either non-selective or selective for endothelin receptor subtypes. The rat cerebellar endothelin receptor displayed a high affinity for endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin-S6c (STX-6c) although the affinity for ET-3 was slightly higher than the affinity for ET-1 using both radioligands. The selective ET(A) antagonists, BQ123, EMS-182,874 and JKC-301 all displayed low affinities at the endothelin receptors. In contrast the selective ET(B) agonists, IRL1620 and [Ala(1,3,11,15])ET-1 and the selective ET(B) antagonist, BQ-788 had moderate affinities at the endothelin receptor, in the low nanomolar range. The ET(B) agonist, BQ3020, had approximately 10 fold higher affinity than IRL1620 and [Ala(1,3,11,15)]ET-1 at the rat cerebellar endothelin receptors. The non-selective antagonists, Re-46,2005, Re-47,0203 and PD-142,893 displayed moderate affinities at the cerebellar receptor. 4 Since [I-125]-BQ3020 recognises only a fraction of the [I-125]-ET-1 binding sites, the majority of the endothelin receptors in the cerebellum cannot be classed as ET(B). Although [I-125]-PD151242 was able to detect ET(A) receptors in the rat cerebellar homogenates, the small population of ET(A) receptors (2% of the total endothelin population as measured with [I-125]-ET-1) could not account for the non-ET(B) receptor population. We conclude that the rat brain cerebellar receptor has a profile similar to the ET(B1) receptor as it has a high affinity for ET-I, ET-3, STX-6c and was moderately sensitive to PD-142,893. However, as the ET(B) ligands BQ-788, IRL1620 and [Ala(1,3,11,15])ET-1 have only a moderate affinity for the rat cerebellar endothelin receptor and since ET-3 has a higher affinity as compared to ET-1, our findings suggest that the rat cerebellum contains predominately ET(c) receptors.