Does biased agonism explain the discrepant pharmacology of ET-1 and S6b?

Janet Maguire, Rhoda Kuc, Lowell Ling, Anthony Davenport. University of Cambridge, Clinical Pharmacology Unit, CB20QQ, United Kingdom

We have previously reported discrepancies in the pharmacology of ET-1 and the snake toxin sarafotoxin 6b (S6b) that also binds to and activates ETA and ETB receptors (Maguire et al., 1996). Although vasoconstrictor responses to the two agonists were similar in human isolated saphenous vein (that predominantly express ETA receptors), cross-desensitisation experiments suggested S6b had lower efficacy than ET-1. Additionally, the ETA selective antagonist BQ123 was a more effective blocker of S6b responses than ET-1, with the Schild-derived affinity of BQ123 dependent on the agonist used. Finally using saturation analysis we discovered that [125I]ET-1 identified a significantly larger population of ET receptors in vein homogenates than [125I]S6b. Based on these data we suggested that ET-1 may bind to an additional population of ETA receptors that was insensitive to both BQ123 and S6b.

More recently, agonists and antagonists for some G-protein coupled receptors have been shown to demonstrate functional selectivity with respect to activation of signalling pathways. We considered whether this could explain the anomalies in the human ETA pharmacology of S6b and BQ123. As an initial step we have compared the ability of ET and sarafotoxin peptides to recruit β-arrestin and determined whether this can be antagonised by BQ123 using the DiscoverX Pathhunter Express EDNRA β-arrestin GPCR assay.

CHO-K1 cells expressing human ETA were incubated with BQ123 (1μM) or vehicle for 60 min at 37ºC. ET peptides/sarafotoxins were added and incubated for a further 90 minutes after which the detection reagent was added and cells incubated for 2 hours at room temperature. The resulting chemiluminescent signal was measured and concentration-response curves to the agonists in the absence and presence of BQ123 expressed as relative light units (RLU).

Table 1. Potency (EC50) and maximum response (EMAX) of ET and sarafotoxin peptides in the absence (control) and presence of BQ123. Data are derived from 4-parameter logistic curves fitted to the mean of triplicate observations.

These data suggest that for recruitment of β-arrestin the order of agonist potency was ET-1≥ET-2 = S6b>ET-3 as expected for an ETA response and confirmed by the lack of response to the ETB selective agonist S6c. However, the maximum response to S6b, and ET-3, was approximately half that of ET-1 and ET-2. This may be consistent with S6b activating only a subpopulation of ETA receptors, as suggested by our saturation binding experiments. Or these data may support the hypothesis that S6b is a biased agonist that couples preferentially to G-protein mediated vasoconstriction but is less likely to cause ETA receptor desensitisation via β-arrestin recruitment.

Maguire JJ, Kuc RE, Rous BA, Davenport AP (1996). Br J Pharmacol. 118:335-342.