Different pharmacology of ETA and ETB receptors revealed in β-arrestin assay

Janet Maguire, Rhoda Kuc, Anthony Davenport. University of Cambridge, Clinical Pharmacology Unit, Level 6 ACCI, Box 110 Addenbrooke's Hospital Cambridge CB20QQ, UK

G-Protein coupled receptors may couple promiscuously to multiple G-protein subclasses and may activate intracellular signalling pathways in a G-protein independent manner via the scaffolding proteins β-arrestin 1 and 2. Agonist-specific pathway activation (biased agonism), if identified, may have therapeutic potential. Our aim was to determine the pharmacology of ETA- and ETB-mediated β-arrestin recruitment and compare this to the established human pharmacology of these receptors to identify evidence for endothelin (ET) receptor biased signalling and pathway specific blockade by antagonists.

The ability of ET-1, ET-2, ET-3, sarafotoxin 6b (S6b) and sarafotoxin 6c (S6c) to activate ETA and ETB-mediated β-arrestin recruitment was investigated using DiscoveRx PathHunter® eXpress β-Arrestin GPCR assays. CHO-K1 cells expressing human ETA or ETB were incubated with antagonists or vehicle for 60 min at 37ºC. Increasing concentrations of agonists were added, incubated for 90 minutes, detection reagent was then added and cells incubated for 2 hours at room temperature. The resulting chemiluminescent signal was measured and agonist concentration-response curves expressed as relative light units. Agonist responses were normalised to the maximum response elicited by a control ET-1 curve for each assay. Affinities were obtained for ETA selective (BQ123, sitaxentan, ambrisentan), ETB selective (BQ788) and mixed (bosentan) antagonists and compared to affinities obtained in competition experiments in human heart and by Schild analysis in human saphenous vein.

Table 1. Potency (pD2) and maximum response (EMAX, % maximum control ET response) of endothelin and sarafotoxin peptides in ETA and ETB expressing cells. Data were derived from 4-parameter logistic curves and are the mean of n = 3-12 observations. *Significantly different from ET-1 control (P<0.05, Student’s 2-tailed t-test).

For β-arrestin recruitment, the relative potency of ET-1 and ET-3 was as expected for ETA (ET-1≥ET-2>>ET-3) and ETB (ET-1 = ET-3≥ET-2); for both receptors ET-2 was significantly less potent than ET-1. For ETA, S6b exhibited comparable potency to ET-2, however, unexpectedly S6b and ET-3 were partial agonists compared to ET-1. As expected S6c was inactive. In the ETB assay S6b, S6c and BQ3020 showed similar potency to ET-1 and ET-3. Antagonism by selective and mixed antagonists appeared non-competitive, particularly in the ETA assay. In this assay BQ123 (0.1-1μM) blocked responses to the ET peptides in a comparable concentration-dependent manner with similar data obtained for BQ788 in the ETB assay. However, BQ123 appeared to be more effective against the sarafotoxins and BQ3020. In competition binding experiments in human heart using [125I]ET-1, bosentan did not distinguish between the two receptor subtypes. However, bosentan (0.3-30μM) produced a greater rightward shift of the ET-1 curve in the ETA assay than the ETB assay indicating ETA selectivity for antagonism of β-arrestin recruitment. The ETA selectivity of ambrisentan was confirmed and consistent with our human pharmacology for this compound.

In conclusion these data suggest differences in the pharmacology of ETA and ETB receptors linked to G-protein- and β-arrestin mediated responses and bosentan appeared to show bias, preferentially blocking ETA mediated β-arrestin recruitment.