title: A live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity.
abstract: Endothelin-1 (ET-1) is formed from its precursor preproET-1 via the cleavage of the intermediate bigET-1 by endothelin-converting enzyme (ECE-1). However, the subcellular site at which this step occurs is not clear: It could occur intravesicularly along the secretory pathway or bigET-1 might be released and processed extracellularly. To address this point, we have developed an integrated autocrine system that uses a recombinant Chinese hamster ovary (CHO) luciferase reporter cell line that permanently expresses the human ET(A) receptor. Into these cells we transiently transfected human ECE-1a cDNA, either together with the human preproET-1 cDNA (as an endogenous source of bigET-1), or alone (in which case exogenous bigET-1 was added). Phosphoramidon inhibited the conversion of exogenous bigET-1 (IC50 = 5 to 30 micromol/L) much better than that of endogenous bigET-1 (IC50 > 1 mmol/L). Both conversions showed similar high yields (20% to 100%) that depended on the amount of ECE-1a expressed. Thus, ECE-1a has two equally relevant activities in this recombinant system for CHO cells: (1) an intracellular, probably intravesicular activity, corresponding to the ECE-1a-mediated step of ET-1 biosynthesis and (2) an extracellular activity at the plasma membrane. If this is also the case for endothelial cells, ECE-1a inhibitors would have to cross the plasma and vesicle membranes to be effective. The present system could be useful for screening such inhibitors.
authors.affiliation: Roussel-Uclaf, Romainville, France.
authors.forename: C
authors.initials: C
authors.lastname: Parnot
authors.forename: J M
authors.initials: JM
authors.lastname: Le Moullec
authors.forename: M A
authors.initials: MA
authors.lastname: Cousin
authors.forename: D
authors.initials: D
authors.lastname: Guédin
authors.forename: P
authors.initials: P
authors.lastname: Corvol
authors.forename: F
authors.initials: F
authors.lastname: Pinet
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D016466:CHO Cells / Q000378:metabolism / Q000502:physiology; D006224:Cricetinae; D003584:Cytological Techniques; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology*; D008666:Metalloendopeptidases; D011498:Protein Precursors / Q000378:metabolism; D017466:Receptors, Endothelin / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D017466:Receptors, Endothelin; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes