A LIVE-CELL ASSAY FOR STUDYING EXTRACELLULAR AND INTRACELLULAR ENDOTHELIN-CONVERTING ENZYME-ACTIVITY

Endothelin-1 (ET-1) is formed from its precursor preproET-1 via the cl eavage of the intermediate bigET-1 by endothelin-converting enzyme (EC E-1). However, the subcellular site at which this step occurs is not c lear: It could occur intravesicularly along the secretory pathway or b igET-1 might be released and processed extracellularly. To address thi s 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 ETA receptor. Into these cells we transiently transfected human ECE-1a cDNA, either together with the h uman preproET-1 cDNA (as an endogenous source of bigET-1), or alone (i n which case exogenous bigET-1 was added). Phosphoramidon inhibited th e conversion of exogenous bigET-1 (IC50=5 to 30 mu mol/L) much better than that of endogenous bigET-1 (IC50 >1 mmol/L). Both conversions sho wed similar high yields (20% to 100%) that depended on the amount of E CE-1a expressed. Thus, ECE-1a has two equally relevant activities in t his recombinant system for CHO cells: (1) an intracellular, probably i ntravesicular activity, corresponding to the ECE-1a-mediated step of E T-1 biosynthesis and (2) an extracellular activity at the plasma membr ane. 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.