Endothelin-converting-enzyme–1 (ECE-1) has been shown to convert the inactive peptide big endothelin-1 (bigET-1) to the potent vasoconstrictor endothelin-1 (ET-1), however neutral endopeptidase EC24.11 (NEP) may play a similar role in this pathway (Grantham, et al. 2000). Here we describe experiments using the selective NEP and ECE-1 inhibitors candoxatrilat and PD-69185, respectively (Shepperson et al. 1991, Ahn et al. 1998) which show NEP rather than ECE-1 is responsible for the conversion of exogenous bigET-1 to ET-1 in rabbit saphenous artery in vitro.

The IC₅₀ values of candoxatrilat and PD-69185 for NEP were determined by cleavage of the fluorimetric substrate o-aminobenzoyl-d-Arg-Arg-Leu-ethylenediamine, 2,4-dinitro-phenyl by rabbit kidney NEP. Saphenous arteries were obtained from euthanised 2-3kg male New Zealand White rabbits (Harlan). 3-4mm length rings were suspended under 2g tension in Krebs-Ringer buffer at 37 °C, and gassed with 95% O₂ / 5% CO₂. To investigate effects of the inhibitors on BigET-1 evoked vasoconstriction, tissues were incubated with different concentrations of either candoxatrilat or PD-69185 or the selective ET receptor antagonist PD-156707 (Maguire et al. 1997) for 30 minutes before treatment with 30nM BigET-1. The effects of PD-69185 on ET-1 evoked vasoconstriction were also investigated. Tissues were incubated with 3, 10 or 30 μM PD-69185 for 30 minutes before constructing cumulative concentration-response curves to ET-1. The concentrations of ET-1 producing 50% of the maximal response (EC₅₀) were interpolated from these results.

PD-156707 inhibited the contractile response of rabbit saphenous artery to BigET-1, confirming the response to be dependent on BigET-1 cleavage to ET-1. Candoxatrilat (IC₅₀ against rabbit NEP = 3.6 nM ±1.2 s.e.m., n=3), inhibited bigET-1 evoked vasoconstriction with pEC₅₀ = 7.35 ±0.47 (mean ±s.e.m., n=4) The ECE inhibitor, PD-69185 (IC₅₀ against human ECE-1 = 140 nM; Ahn et al, 1997), inhibited bigET-1 evoked vasoconstriction only at high concentrations (pEC₅₀ = 5.12 ±0.1, n=4). Interestingly, 10 μM PD-69185 completely inhibited vasoconstriction to ET-1. This may indicate that at concentrations greater than 10μM, PD-69185 is an antagonist of ET-1 receptors, and this may have contributed to the observed functional antagonism of BigET-1.

These data suggest that NEP may be responsible for the cleavage of BigET-1 to ET-1 in rabbit saphenous artery as shown by the results for candoxatrilat treatment. The selective ECE-1 inhibitor PD-69185 only inhibited BigET-1 evoked vasoconstriction at a concentration of 50x IC₅₀, compared to 15x IC₅₀ for candoxatrilat, suggesting that ECE-1 does not cleave BigET-1 in this tissue. This is supported by the findings that PD-69185 is attenuating the concentration-response to ET-1 at concentrations which inhibit bigET-1 dependent responses. In conclusion, NEP, rather than ECE-1, appears to be responsible for BigET-1 cleavage in rabbit saphenous artery, and this tissue preparation may, therefore, provide an in vitro assay for assessing the functional potency of NEP inhibitors.

Ahn, K. et al. (1997) Biochem. Biophys. Res. Commun. 243, 184-190
Grantham, J.A. et al. (2000) Circulation 101, 1976-1981
Maguire, J.J. et al. (1997) J. Pharmacol. Exp. Ther. 280, 1102-1108
Shepperson, NB et al. (1991) Clin Sci (Lond) 80, 265-269