[I-125] PD151242 - A SELECTIVE LIGAND FOR ENDOTHELIN ET(A) RECEPTORS IN HUMAN KIDNEY WHICH LOCALIZES TO RENAL VASCULATURE

1 The linear tetrapeptide radioligand, [I-125]-PD151242 was used to characterize ET(A) receptors in human kidney which is an ET(B)-rich tissue. Saturation binding assays with [I-125]-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 [I-125]-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 +/- 0.06 nM, 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 mu M) inhibited binding by only 50%. The non-selective R0462005 competed for the binding of [I-125]-PD15r242: K-D = 1.31 +/- 1.38 mu M, B-max = 33.0 +/- 9.7 fmol mg(-1) protein. Endothelin-2 and sarafotoxin S6B inhibited [I-125]-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 [I-125]-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 ETA receptors visualised with [I-125]-PD151242 were mainly localized to blood vessels including interlobular and arcuate arteries, arterioles and adjacent arcuate veins. ET(B) receptors localized with [I-125]-BQ3020 were concentrated in the medulla and the density of binding to vessels was low. 6 These data suggest [I-125]-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.