Characterization of the binding of endothelin ETB selective ligands in human and rat heart
Authors: Russell Fraser D. , Davenport Anthony P. 
Published: July 2012

We determined competition binding characteristics of endothelin ETB receptor selective ligands in human left ventricle and compared these values to those obtained with rat left ventricle. Sarafotoxin S6c, ET‐3, BQ788 and IRL2500 competed against [125I]‐BQ3020 (ETB selective radioligand; Molenaar et al., 1992) with high affinity and against [125I]‐PD151242 (ETA selective radioligand) with low affinity in human left ventricle, confirming the ETB selectivity of these compounds.

ET‐3 competed with moderate selectivity for ETB over ETA receptors in human left ventricle and with slightly higher selectivity in rat left ventricle (460 and 1,400 fold, respectively). There was a small difference in the affinity of ETA receptors for ET‐3 (KD ETA in human left ventricle = 0.07 ± 0.02 μm; KD ETA in rat left ventricle = 0.27 ± 0.08 μm; P = 0.05) but no difference in the affinity of ETB receptors for this ligand (KD ETB in human left ventricle = 0.15 ± 0.06 nM; KD ETB in rat left ventricle = 0.19 ± 0.03 nM).

The selectivity of sarafotoxin S6c for ETB over ETA receptors in human left ventricle was 5,900 fold compared with 59,400 fold in rat left ventricle. The affinity of ETA receptors for sarafotoxin S6c was higher in human than in rat left ventricle (KD ETA = 2.00 ± 0.20 μm and 3.50 ± 0.26 μm, respectively; P = 0.03), while the affinity of ETB receptors for this ligand was higher in rat left ventricle (KD ETB = 0.06 ± 0.02 nM) than in human left ventricle (KD ETB = 0.34 ± 0.13 nM) (P = 0.02). The affinity of ETB receptors for sarafotoxin S6c in rat left ventricle determined in the absence or presence of GTP was the same indicating that differing affinity states of ETB receptors in human and rat left ventricle do not account for the variation observed between species.

There was no difference in the affinity of ETA receptors for BQ788 (KD ETA = 1.01 ± 0.20 μm and KD ETA = 1.39 ± 0.35 μm) or for the novel ETB selective antagonist, IRL2500 (KD ETA = 30.0 ± 20.8 μm and KD ETA = 55.6 ± 9.93 μm) in human and rat left ventricle, respectively. ETB receptors had a significantly higher affinity for BQ788 (KD ETB = 9.8 ± 1.3 nM and KD ETB = 31.0 ± 5.4 nM; P = 0.02) and IRL2500 (KD ETB = 78.2 ± 9.7 nM and KD ETB = 300.0 ± 75.1 nM; P = 0.03) in human and rat left ventricle, respectively. The synthetically synthesized ETB selective antagonist RES‐701‐1 (0.1–3 μm) failed to inhibit [125I]‐ET‐1 binding in either tissue.

In conclusion, we have compared equilibrium dissociation constants for a number of ETB selective compounds in human and rat heart. The affinity of ETB receptors for sarafotoxin S6c, BQ788 and IRL2500 differed in human and rat left ventricle. No difference in affinity was detected for ET‐3 binding at ETB receptors. Sarafotoxin S6c binding was unaffected by GTP indicating that the different receptor affinities in human and rat heart cannot be explained by differing ETB receptor affinity states. This study highlights the need to consider differences in binding characteristics that may arise from the use of tissues obtained from different species.