Endothelin (ET) is a potent vasoconstrictor peptide of 21 amino- acids (aa), which was originally isolated from the supernatant of cultured endothelial cells. In addition to its known vasoconstricting effects, ET exerts a wide range of biological actions, with preferential effects on renal tubules and blood vessels. ET is derived from a 38 aa precursor peptide, Big ET (BET). This cleavage is performed by an unidentified protease, endothelin converting enzyme (ECE). We sought to determine: 1) if neutral endopeptidase EC.3.4.24.11 converts BET to ET, using recombinant neutral endopeptidase (rNEP), and 2) if endothelium is required for the conversion of BET to ET, by aortic rings. Incubation of 125-I-ET-1 with 1microg/ml rNEP resulted in degradation of the peptide within minutes, whereas its incubation with 10 microg/ml of the enzyme resulted in its total cleavage within seconds. Both phosphoramidon (10 microM) and SQ-28,603 (100microM) fully protected ET-1 from degradation by rNEP. The degradation of 125-I-BET-1 was much slower than that of ET-1. Even after incubation with 10 microg/ml of the enzyme, most of radioactive counts in the sample eluted as intact peptide. Identical to their effect on ET, phosphoramidon and SQ-28,603 protected the degradation of BET by rNEP. At no time point during the incubation of BET with rNEP, was ET found in the reaction mixture. In isolated rat aortic rings with and without the endothelium, both ET-1 and BET-1 caused dose- dependent contractions. There was no significant shift of the dose-response curve in the presence or absence of the endothelium. These findings indicate that: 1) neutral endopeptidase EC.3.4.24.11 is not an endothelin converting enzyme, 2) endothelium is not required for the vascular effects of ET-1 and BET-1, and 3) ECE exists in vascular smooth muscle.
