Article date: August 1996
By: P.S. Widdowson, C.N.P. Kirk, in Volume 118, Issue 8, pages 2126-2130
We performed radioligand binding experiments on rat cerebellar homogenates using [125I]‐endothelin‐1 ([125I]‐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.
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).
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 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.
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.
We performed radioligand binding experiments on rat cerebellar homogenates using [125I]‐endothelin‐1 ([125I]‐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.
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).
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 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.
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.
DOI: 10.1111/j.1476-5381.1996.tb15652.x
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