Expression of the endothelin-converting enzyme-1 isoforms in endothelial cells

The transformed human endothelial cell line EA.hy926 is commonly used for s tudying in vitro different aspects of endothelial cell biology such as sign al transduction, expression or angiogenesis. These cells have the ability t o process big endothelin (big-ET) into endothelin (ET), and express the end othelin-converting enzyme ECE-1. Several isoforms of ECE-1 which differ onl y in their N-terminal part (i.e. the end of the cytosolic domain) have now been identified. We could detect the co-expression of all four isoforms. Re cent works have shown that the variable cytosolic domain is responsible for the differential intracellular localization of ECE-1 isoforms. Using antib odies directed against ECE-1a and ECE-1b/c/d, we have characterized the int racellular distribution of these isoforms in EA.hy926 cells by immunofluore scence. Electron microscopy allowed us to identify further the intracellula r compartment that contains ECE-1 as multivesicular bodies, a compartment i nvolved in the endocytic pathway. In addition, using an antibody directed a gainst the: catalytic domain, we could demonstrate that no monomeric ECE-1 is present at the plasma membrane. Indeed, detection of ECE-1 immunoreactiv ity at the cell surface of living cells required a dithiothreitol (DTT) tre atment. Altogether, these results demonstrate that the EA.hy926 cell Line i s a helpful model for studying the regulation of the production of endothel in by ECE.