[Identification and characterization of neutral endopeptidase in endothelial cells of arterial or venous origin].
Neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is a cell membrane associated zinc metalloprotease, which cleaves peptides like atrial natriuretic peptide (ANP) on the amino-side of hydrophobic amino acids. Although NEP is mainly located in reabsorptive epithelia (kidney proximal tubule), it is also present in non-epithelial cells like neuronal cells. As the renal NEP cannot account for the entire ANP metabolism, other locations were postulated. The present experiments show its expression in endothelial cells (EC) from arterial (bovine pulmonary, porcine and human aorta) and venous (human umbilical, rabbit ear marginal) origins. Three different methods were used to demonstrate the presence of the protein and its mRNA: 1) NEP enzymatic activity was estimated using both a synthetic ([D-Ala2, Leu5] enkephalin) and a natural substrate (bradykinin). Using the synthetic substrate, the enzymatic activity in EC was completely blocked by thiorphan, a specific NEP inhibitor with an IC50 value in the nM range. In contrast, captopril, bestatin, GEMSA, inhibitors of angiotensin-converting enzyme, aminopeptidases and carboxypeptidases, respectively, were 10,000 times less active, revealing an inhibition profile similar to that of the purified enzyme. Bradykinin, a natural substrate of NEP, was in part metabolized by NEP, in presence of captopril, since 50% of the formation of the major metabolite bradykinin 1-7 was inhibited by thiorphan. 2) Immunoreactive NEP was detected on the plasma membrane of rabbit EC using a monoclonal antibody directed against the homologous renal enzyme. 3) NEP mRNA was detected by Northern blot analysis on rabbit EC as a major transcript of 3.9 kb. Reverse transcriptase PCR amplification showed the presence of a specific transcript in all EC tested. Therefore, endothelial NEP could play an important role in the inactivation of ANP, bradykinin and endothelins by its localization facing the circulating vasoactive peptides.
Llorens-Cortes
C
C
INSERM U36, Collège de France, Paris.
Huang
H
H
Gasc
J M
JM
Corvol
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P
D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015260:Neprilysin / Q000378:metabolism*; D011817:Rabbits; D013552:Swine
D015260:Neprilysin
Identification et caractérisation de l'endopeptidase neutre dans les cellules endothéliales d'origine artérielle ou veineuse.