title: Characterization of [125I]-endothelin-1 and [125I]-BQ3020 binding to rat cerebellar endothelin receptors.
abstract: 1. We performed radioligand binding experiments on rat cerebellar homogenates using [125I]-endothelin-1 ¿[1251]-ET-1¿ and [125I]-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 ([125I]-ET-1, pKd = 8.94 +/- 0.17; [125I]-BQ3020, pKd = 9.18 +/- 0.14 nM; mean +/- s.e.mean). However, [125I]-BQ3020 only recognised approximately one third the number of endothelin receptors measured with [125I]-ET-1. 2. Saturation binding experiments with [125I]-PD151242 revealed high affinity binding to a single population of ETA receptors in the cerebellar homogenates (pKd = 9.95 +/- 0.14; Bmax = 30 +/- 15 fmol mg-1 protein). 3. Competition experiments were performed with ligands that are either non-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 ETA antagonists, BQ123, BMS-182,874 and JKC-301 all displayed low affinities at the endothelin receptors. In contrast the selective ETB agonists, IRL1620 and [Ala1,3,11,15]ET-1 and the selective ETB antagonist, BQ-788 had moderate affinities at the endothelin receptor, in the low nanomolar range. The ETB agonist, BQ3020, had approximately 10 fold higher affinity than IRL1620 and [Ala1,3,11,15]ET-1 at the rat cerebellar endothelin receptors. The non-selective antagonists, Ro-46,2005, Ro-47,0203 and PD-142,893 displayed moderate affinities at the cerebellar receptor. 4. Since [125I]-BQ3020 recognises only a fraction of the [125I]-ET-1 binding sites, the majority of the endothelin receptors in the cerebellum cannot be classed as ETB. Although [125I]-PD151242 was able to detect ETA receptors in the rat cerebellar homogenates, the small population of ETA receptors (2% of the total endothelin population as measured with [125I]-ET-1) could not account for the non-ETB receptor population. We conclude that the rat brain cerebellar receptor has a profile similar to the ETB1 receptor as it has a high affinity for ET-1, ET-3, STX-6c and was moderately sensitive to PD-142,893. However, as the ETB ligands BQ-788, IRL1620 and [Ala1,3,11,15]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 ETc receptors.
authors.affiliation: Neurotoxicology Research Group, ZENECA Central Toxicology Laboratory, Macclesfield, Cheshire.
authors.forename: P S
authors.initials: PS
authors.lastname: Widdowson
authors.forename: C N
authors.initials: CN
authors.lastname: Kirk
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002531:Cerebellum / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000494:pharmacology*; D016232:Endothelins / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D017466:Receptors, Endothelin / Q000187:drug effects*
chemical_list: D019332:Endothelin-1; D016232:Endothelins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D017466:Receptors, Endothelin; C075771:BQ 3020