title: Regulation of endothelin-converting enzyme-1 (ECE-1) by the calcimimetic R-568.
abstract: Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and Western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP.
authors.affiliation: Research Unit, Biomedical Research Foundation, Prince of Asturias University Hospital, Alcalá de Henares, Madrid, Spain; Nephrology Section, Biomedical Research Foundation, Prince of Asturias University Hospital, Alcalá de Henares, Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Martínez-Miguel
authors.forename: Diana
authors.initials: D
authors.lastname: Medrano-Andrés
authors.forename: Vanessa
authors.initials: V
authors.lastname: Lopes-Martín
authors.forename: Ignacio
authors.initials: I
authors.lastname: Arribas-Gómez
authors.forename: Manuel
authors.initials: M
authors.lastname: Rodríguez-Puyol
authors.forename: Diego
authors.initials: D
authors.lastname: Rodríguez-Puyol
authors.forename: Susana
authors.initials: S
authors.lastname: López-Ongil
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis / Q000378:metabolism*; D001794:Blood Pressure / Q000187:drug effects; D002118:Calcium / Q000819:agonists*; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D019332:Endothelin-1 / Q000097:blood / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D006801:Humans; D008297:Male; D008666:Metalloendopeptidases / Q000032:analysis / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000378:metabolism; D010627:Phenethylamines; D011437:Propylamines; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000814:Aniline Compounds; D019332:Endothelin-1; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D002118:Calcium
keywords: Blood pressure; CaR; Calcimimetic; DMEM; Dulbecco's Modified Eagle Media; EA; EA.hy926 is a human endothelial cell line; ECE-1; ET-1; Endothelin-1; Endothelin-converting enzyme-1; NO; Nitric oxide; PTH; R-568; SBP; TTBS; Tween Tris buffered saline; calcimimetic R-568; calcium sensing receptor; eNOS; endothelial nitric oxide synthase; endothelin-1; endothelin-converting enzyme-1; nitric oxide; parathyroid hormone; systolic blood pressure