Objectives:

To elaborate on the unique ligand-receptor complex of endothelin-1 (ET-1) with its ETA-receptor, we characterized i) functional properties of ET-1 fragments, ii) steric hindrance by irreversibly ETA-bound ET-1 of bulky competitive antagonists and iii) constrictor effects of the low-affinity ETA-agonist ET-3.

Methods:

Concentration-response curves (CRC) for commercially available endothelinergic peptides, antagonists, locally synthesized peptidergic fragments and fluorescently-labeled antagonists were recorded in isolated 2nd order rat mesenteric resistance arteries by wire- myography in conditions minimizing endothelial and sensory-nervous effects.

Results:

0.25–16nM ET-1 (ET-1(1–21)) caused contractions that were neither mimicked nor antagonized by 1mM ET(16–21) or ET-1(1–15). The ETA-antagonists PD156707 and BQ123 caused a rightward shift of the CRC for ET-1 and could only partly and transiently inhibit ET-1- induced contractions. Cy5.5-PD156707 and FITC-BQ123 displayed antagonist potencies that did not differ from the unlabeled antagonists, but were less effective in reversing ET-1-induced contractions. In presence and absence of 1mM of the ETB-antagonist BQ788, ET-3 caused contractions with similar efficacy but 30-times lower potency than ET-1. These were less persistent (T1/2 >4min) than ET-1-initiated contractions (T1/2>20min). The ET-3-induced contractions were fully prevented by 1mM BQ123, but occurred after removal of free agonist and free antagonist. Also, BQ123 could terminate contractions initiated by ET-3 while only transiently reducing those initiated by ET-1.

Conclusions:

ETA-receptors have two binding sites; one for the N-terminus and one for the C- terminus of agonists and for small antagonists. The full ET-1 peptide is required for irreversible binding and activation of ETA-receptors and displays charnière-behavior. ET-3 might mediate contractions via receptor-dimerization.

This study was performed within the framework of TI Pharma project T2-301.