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

Abstract

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. 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. 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.BQ123-, but not BQ788-, selective antagonists for ET(A) and ET(B) receptors, respectively, produced concentration-dependent rightward displacements of the ET-1(1-31) and ET-1 concentration-response curves. 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. 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. 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 ET(A) receptors to induce contraction in the rabbit aorta.

Figure 1

Concentration–response curves for BigET-1 obtained in endothelium-intact rabbit aortic rings. (a) Effect of phosphoramidon (0.1 mM), thiorphan (10 μM) or CGS35066 (10 μM) on BigET-1-induced contraction. (b) Effect of phosphoramidon (1 mM), thiorphan (30 μM) or CGS35066 (30 μM) on BigET-1-induced contraction. (c) Effect of chymostatin (100 μM) or the association of phosphoramidon (0.1 mM) and chymostatin (100 μM) on BigET-1-induced contraction. The rings were preincubated with the drugs for 30 min. Values are means±s.e.m. of five to eight independent preparations. ^*P<0.05 when compared to control curves of agonists+vehicle or in c) agonist+chymostatin (ANOVA followed by Bonferroni's comparison test).

Figure 2

Concentration–response curves for ET-1(1–31) (a) and ET-1 (b) obtained in endothelium-intact rabbit aortic rings. The concentration–response curves were obtained in the absence (control) or in the presence of phosphoramidon (0.1 mM), thiorphan (10 μM), CGS35066 (10 μM) or chymostatin (100 μM). The rings were preincubated with the drugs for 30 min. Values are means±s.e.m. of five to eight independent preparations. ^*P<0.05 when compared to control curves of agonists+vehicle (ANOVA followed by Bonferroni's comparison test).

Figure 3

Effect of BQ123 and BQ788 on ET-1 and ET-1(1–31)-induced contraction of endothelium-intact rabbit aortic rings. The curves for ET-1 and ET-1(1–31) were obtained in the absence (control) or presence of BQ123 (a, c) or BQ788 (b, d). Values are means±s.e.m. of five to six independent preparations.

Figure 4

Total MCA-forming activities in the rabbit aorta, heart, lung, kidney and liver. The MCA-forming activity was obtained in the absence (control) or presence of chymostatin or phosphoramidon (a) and BigET-1 (1, 10 and 100 nM) (b). ^*Compared to respective control group; ^#compared to heart, lung, kidney and liver (P<0.05; ANOVA followed by Bonferroni's comparison test). Values are means±s.e.m. of five to eight independent experiments.

Figure 5

Comparison of chymase-like activity in the rabbit aorta, heart, lung, kidney and liver. ^*Compared to heart, lung, kidney and liver (P<0.05; ANOVA followed by Bonferroni's comparison test). Values are means±s.e.m. of six to eight independent experiments.

Figure 6

Bath content of Ir-ET-1(1–31) after the administration of BigET-1 (30 nM). Ir-ET-1(1–31) was determined in the organ bath content after the administration of BigET-1 in the absence (control) or presence of phosphoramidon (0.1 mM), chymostatin (100 μM), thiorphan (10 μM) or the association of phosphoramidon (0.1 mM) and chymostatin (100 μM). ^*Compared to BigET-1 (P<0.05; ANOVA followed by Bonferroni's comparison test). ^#P<0.05. Values are means±s.e.m. of at least six independent experiments.

Figure 7

Schematic representation of the enzymatic pathways involved in the production of ET peptides. The closed arrow indicates the main pathway implicated in the production of ET-1 from BigET-1 by ECE and NEP. The opened arrows indicate the alternative pathway involved in the production of ET-1. ET-1(1–31) is an intermediate peptide in the production of ET-1 from BigET-1. The inhibition of the main pathways (i.e. ECE and NEP) with phosphoramidon unmasks the production of ET-1(1–31). ET-1, endothelin-1; BigET-1, big endothelin-1; ET-1(1–31), endothelin-1(1–31); ECE, endothelin-converting enzyme; NEP, neutral endopeptidase.