[125I]‐PD151242: a selective ligand for endothelin ETA receptors in human kidney which localizes to renal vasculature

Abstract

1 The linear tetrapeptide radioligand, [¹²⁵I]-PD151242 was used to characterize ET_A receptors in human kidney which is an ET_B-rich tissue. Saturation binding assays with [¹²⁵I]-PD151242 revealed a single population of high affinity endothelin receptors: K_D = 0.75 ± 0.07 nm and B_max = 48.4 ± 1.6 fmol mg⁻¹ protein (n = 3 individuals ± s.e.mean). Hill slopes were close to unity and a one site fit was preferred to a two site model. 2 ET_A-receptor-selective ligands competed for [¹²⁵I]-PD151242 binding with sub-nanomolar affinity: BQ123 K_D = 0.43 ± 0.10 nm, B_max = 46.6 ± 7.9 fmol mg⁻¹ protein; FR139317, K_D = 0.37 ± 0.06nM, B_max = 39.5 ± 6.5 fmol mg⁻¹ protein (n = 3 individuals ± s.e.mean). In each case, monophasic inhibition curves were obtained and a one site fit was preferred to a two site model. The ET_B-selective agonist, BQ3020 at the highest concentration tested (10 μm) inhibited binding by only 50%. The non-selective RO462005 competed for the binding of [¹²⁵I]-PD151242: K_D = 1.31 ± 1.38 μm, B_max = 33.0 ± 9.7 fmol mg⁻¹ protein. Endothelin-2 and sarafotoxin S6B inhibited [¹²⁵I]-PD151242 binding to renal tissue whereas ET-3 and sarafotoxin S6C were less effective. Non-endothelin and non-sarafotoxin peptides did not compete. 3 No degradation of [¹²⁵I]-PD151242 was detected following incubation of the ligand with renal tissue under the conditions of the binding assay. 4 Polymerase chain reaction products corresponding to the expected size for mRNA encoding ET_A and ET_B receptor sub-types were detected in cortex and medulla in each of the five individuals examined. 5 Autoradiographical studies showed that ET_A receptors visualised with [¹²⁵I]-PD151242 were mainly localized to blood vessels including interlobular and arcuate arteries, arterioles and adjacent arcuate veins. ET_B receptors localized with [¹²⁵I]-BQ3020 were concentrated in the medulla and the density of binding to vessels was low. 6 These data suggest [¹²⁵I]-PD151242 is selective for ET_A receptors in human kidney and this sub-type is mainly localized to the renal vasculature. The results provide further evidence that the human vasculature mainly expresses the ET_A receptor.