Enzymatic pathways involved in the generation of endothelin‐1(1–31) from exogenous big endothelin‐1 in the rabbit aorta

1 We investigated whether blood vessels contribute to the production of ET‐1(1–31) from exogenous big endothelin‐1 (BigET‐1) in the rabbit and assessed which enzymes are involved in this process. 2 Vascular reactivity experiments, using standard muscle bath procedures, showed that BigET‐1 induces contraction in endothelium‐intact rabbit aortic rings. Preincubation of the rings with phosphoramidon, CGS35066 or thiorphan reduced BigET‐1‐induced contraction. Conversely, chymostatin did not affect BigET‐1‐induced contraction. 3 Thiorphan and phosphoramidon, but not CGS35066 or chymostatin, reduced ET‐1(1–31)‐induced contraction. None of the enzymatic inhibitors affected the contraction afforded by ET‐1. 4 BQ123‐, but not BQ788‐, selective antagonists for ETA and ETB receptors, respectively, produced concentration‐dependent rightward displacements of the ET‐1(1–31) and ET‐1 concentration–response curves. 5 By the use of enzymatic assays, we found that the aorta, as well as the heart, lung, kidney and liver, possess a chymase‐like activity. 6 Enzyme immunoassays detected significant levels of Ir‐ET‐1(1–31) in bathing medium of aortas after the addition of BigET‐1 (30 nM). Neither thiorphan nor chymostatin altered the levels of Ir‐ET‐1(1–31). Conversely, the levels of Ir‐ET‐1(1–31) were increased in the presence of phosphoramidon. This marked increase of the 31‐amino‐acid peptide was abolished when phosphoramidon and chymostatin were added simultaneously. 7 The major new finding of the present work is that the rabbit aorta generates ET‐1(1–31) from exogenously administered BigET‐1. Additionally, by measuring the production of ET‐1(1–31), we showed that a chymase‐like enzyme is involved in this process when ECE and NEP are inhibited by phosphoramidon. Our results also suggest that ET‐1(1–31) is an alternate intermediate in the production of ET‐1 following BigET‐1 administration. Finally, we showed that NEP is the predominant enzymatic pathway involved in the cleavage of ET‐1(1–31) to a bioactive metabolite that will act on ETA receptors to induce contraction in the rabbit aorta. British Journal of Pharmacology (2006) 148, 527–535. doi:10.1038/sj.bjp.0706735