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

Abstract

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 ET A and ET B receptors, respectively, produced concentration-dependent rightward displacements of the ET-1(1-31) and ET-1 concentrationresponse 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 ET A receptors to induce contraction in the rabbit aorta.

The interaction between endothelin-1 and nitric oxide in the vasculature: new perspectives

“…Binding of ET-1 to ET A and ET B receptors in VSMC results in vasoconstriction, whereas the predominant effect of ET-1 binding to ET B receptors in the endothelium is increased NO and prostacyclin synthesis (79,99). Whereas ET-1 binds both ET A and ET B receptors, evidence suggests ET-1 1-31 selectively binds ET A receptors (61,81,95), although evidence that ET1 1-31 binds ET B receptors and mediates NO release also exists (68). Whether ET-1 1-32 preferentially binds one endothelin receptor over the other is not yet known.…”

“…Owing to its cleavage of the amino side of hydrophobic amino acids, NEP is also capable of cleaving bET-1 to yield ET-1 . Interestingly, unlike ECE, which requires the COOH terminus of bET-1 (105), NEP also cleaves ET-1 to generate ET-1 (40), albeit ET-1 1-31 is also functional without this cleavage step (95). As both bET-1 and ET-1 (48), whereas the BQ-123-insensitive ET A2 receptor has been localized in human and rabbit saphenous veins (67,91).…”

Prenatal Hypoxia Causes Long-Term Alterations in Vascular Endothelin-1 Function in Aged Male, but Not Female, Offspring

“…7 However, alternative pathways have been identified, including those mediated by matrix-metalloproteinase, 11 chymase, 12 and neutral endopeptidase. 13 To gain insights into the source of bET-1 conversion to active ET-1, we performed concentration-response curves to bET-1 in the presence of inhibitors of these enzymes, as well as L-NAME. The rationale for using L-NAME was 2-fold: (1) in control male offspring, vascular responses to bET-1 with intact NO synthase (ie, without L-NAME treatment) were relatively low, and almost completely absent in some cases; consequently, removal of inhibitory NO was believed to be necessary to accurately assess the relative contributions of bET-1 cleaving enzymes; (2) we sought to remove NO as a confounding factor in the differences in bET-1 conversion between control and IUGR offspring because we observed that NO had different modulatory effects on bET-1 in control and IUGR offspring ( …”

Mechanisms Underlying the Vascular Actions of Endothelin 1, Angiotensin II and Bradykinin in the Rat Carotid

“…Interestingly, the onset of contraction and the increase in tone are slow upon application of big ET-1 when compared to ET-1. This observation suggests the involvement of an enzymemediated step in the contractile action of exogenously applied big ET-1 as previously observed in other vascular tissues such as the rabbit aorta [16] , the human umbilical vein [17] , the human placental artery [18] and the rat basilar artery [15] . Additionally, our results show that phosphoramidon, a non-selective inhibitor of ECE and neutral endopeptidase, produces rightward displacements of the big ET-1 concentration-response curves with a reduction in the maximal response, further indicating that this peptide must be converted to exert its biological activity.…”

“…The approach to verify vessel wall ECE activity by measuring the vasomotor effects induced by big ET-1 using functional assays has been extensively employed [14][15][16] . In the rat carotid, we recently observed that big ET-1 induces concentration-related contraction in both endothelium-intact and -denuded rings [4] .…”