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(Hypertension. 1997;30:837-844.)
© 1997 American Heart Association, Inc.

Articles

A Live-Cell Assay for Studying Extracellular and Intracellular Endothelin-Converting Enzyme Activity

Charles Parnot; Jean-Michel Le Moullec; Marie-Anne Cousin; Denis Guédin; Pierre Corvol; ; Florence Pinet

From the Roussel-Uclaf, Romainville (C.P., J.-M. Le M., M.-A.C., D.G.), and INSERM U36, Collège de France, Paris (P.C., F.P.), France.

Correspondence to Florence Pinet, INSERM U36, Collège de France, 3 rue d'Ulm, 75005 Paris, France. E-mail pinet{at}infobiogen.fr

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 µmol/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.

Key Words: live-cell assay • endothelin • promoter • metalloproteases

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