Comparison of the cardiovascular and neural activity of endothelin‐1, −2, −3 and respective proendothelins: effects of phosphoramidon and thiorphan
Authors: Mattera Giovan G. , Eglezos Anthony , Renzetti Anna Rita , Mizrahi Jacques 
Published: July 2012

In the anaesthetized, ganglion‐blocked rat, intravenous boluses of endothelin‐1, endothelin‐2 and endothelin‐3 induced a transient hypotensive effect followed by a potent long lasting pressor response (ED50 mmHg: 0.72 ± 0.05, 1.8 ± 0.2 and 2.7 ± 0.3 nmol kg−1, respectively). The maximal effect for the three peptides was of a similar order of magnitude (ΔMAP: 84 to 89 mmHg). Neither of these effects was influenced by phosphoramidon or thiorphan (10 mg kg−1, i.v.).
Intravenously administered big‐endothelin‐1 and −2 induced a transient (1–2 min) hypotension followed by a potent long lasting (> 25 min) vasopressor effect (ED50 mmHg: 1.8 ± 0.2 and 6.7 ± 0.4 nmol kg−1, respectively), with a similar maximal activity (Δ MAP: 85 ± 4 and 81 ± 2.4 mmHg, respectively). The onset of the big‐endothelin‐1 vasopressor effect was more rapid (5–6 min) than that of big‐endothelin‐2 (10–13 min). Big‐endothelin‐3 was found to induce only a potent, long lasting (> 35 min) hypertension, with a maximal effect of 75 ± 4.6 mmHg at 10 nmol kg−1 and an ED50 mmHg of 6.5 ± 0.4 nmol kg−1. The onset of this effect was much slower (20–25 min) than that of the other proendothelins. Pressor responses induced by big‐endothelin‐1, −2 and −3 (3, 15 and 10 nmol kg−1, respectively) were markedly reduced (60, 80 and 100%) in the presence of phosphoramidon (10 mg kg−1, i.v.). Thiorphan (10 mg kg−1, i.v.) did not inhibit the effects of big‐endothelin‐1, −2 and −3.
In the electrically stimulated rat vas deferens, endothelin‐1 and −2 were found to be equipotent enhancers of the twitch response (EC100%: 4.0 ± 0.4 nm and 7.9 ± 4.8 nm, respectively), both about 3–4 fold as active as endothelin‐3 (EC100%: 19 ± 2.5 nm). Endothelin‐1 and −3 showed a comparable maximal stimulatory effect (Emax: 296 ± 30 and 262 ± 24%) while endothelin‐2 was less active (Emax: 194 ± 30%).
Big‐endothelin‐1 and −2 were potent enhancers of the twitch reponse too (EC100%: 10.0 ± 2.6 nm and 21.6 ± 3.2 nm, respectively), with a comparable maximal stimulatory effect (Emax: 254 ± 22 and 264 ± 24%). Big‐endothelin‐3 was found to be less potent (EC100%: 275 ± 21 nm), but retained a marked potentiating effect (Emax: 200 ± 38%). Phosphoramidon, but not thiorphan, concentration‐dependently (10 and 100 μm) reduced big‐endothelin‐1 (58 and 86% respectively) and big‐endothelin‐2 (21 and 56%)‐mediated responses. Conversely, the big‐endothelin‐3 effect was reduced by phosphoramidon only at 100 μm (−70%), while thiorphan acts concentration‐dependently (31 and 71% at 10 and 100 μm respectively); thus, in the rat vas deferens, big‐endothelin‐1 and −2 were as potent as their corresponding endothelins, while big‐endothelin‐3 was about 20 times less potent than endothelin‐3.
The increasing effect of endothelin‐2 (194 ± 30% over baseline) was significantly enhanced by either 10 μm phosphoramidon (277 ± 42%) or thiorphan (318 ± 15%). The endothelin‐1 and endothelin‐3‐mediated twitch enhancement was not affected by the two protease inhibitors (10 μm).
These results suggest that in vivo big‐endothelin‐1, −2 and −3, are processed through a similar phosphoramidon‐sensitive enzymatic pathway although with different apparent affinity. This enzymatic process is probably attributable to a neutral endoprotease, distinct from neutral‐endopeptidase 24.11 (NEP). On the other hand, a NEP‐like enzymatic activity may be involved, in the rat vas deferens, in the activation of big‐endothelin‐3 to endothelin‐3 and in the metabolism of endothelin‐2, but not of endothelin‐1 or endothelin‐3.