Pharmacological characterization of the rat cerebellar endothelin B (ETB) receptor using the novel agonist radioligand [125I]BQ3020.

A novel linear peptide fragment of endothelin-1 (ET-1), N-acetyl-[Ala11,15]ET-1[6-21] (BQ3020) has been identified as a potent and ETB-selective agonist. The present studies were conducted in order to characterize the binding of [125I]BQ3020 to the ETB receptor in rat cerebellum. [125I]BQ3020 (0.1 nM) bound with high specificity (90% of total binding) and selectivity for the ETB receptor. ET-1, ET-2, and ET-3 inhibited 0.1 nM [125I]BQ3020 binding with equivalent affinity (Ki values = 55-110 pM). The sarafotoxins S6a, S6b, and S6c also potently inhibited [125I]BQ3020 binding (Ki values = 55-2000 pM). The ETA-selective antagonist, BQ123 (100 microM) did not significantly inhibit [125I]BQ3020 binding. Ligand saturation studies indicated that [125I]BQ3020 labeled a single class of recognition sites with very high affinity (Kd = 31 pM) and limited capacity (Bmax = 570 fmol/mg protein). High affinity 0.1 nM [125I]BQ3020 binding was reduced by 40-50% in the presence of 1 mM guanine nucleotides. Additional competition studies indicated that ET-1 and ET-2 produced biphasic inhibition curves in competing for 0.5 nM [125I]BQ3020. The high affinity component of the ET-1 inhibition curve was subsequently eliminated in the presence of 1 mM GTP-gamma-S The guanine nucleotide sensitivity of [125I]BQ3020 binding offers the possibility that different functional consequences of ETB receptor activation may be mediated by multiple affinity states of the receptor. The present data demonstrate that [125I]BQ3020 is a potent and selective agonist for the ETB receptor that can discriminate high and low affinity states of the ETB receptor.