ENDOTHELIN-CONVERTING ENZYME - ULTRASTRUCTURAL-LOCALIZATION AND ITS RECYCLING FROM THE CELL-SURFACE

The potent vasoconstrictor endothelin-1 (ET-1) is secreted constitutiv ely by endothelial cells and has been implicated in the pathophysiolog y of several cardiovascular diseases. It is generated from its inactiv e intermediate, big ET-1, through the action of endothelin-converting enzyme (ECE). Using several complementary techniques, we have demonstr ated that ECE is present at the cell surface and on intracellular vesi cles and that it recycles from the cell surface in endothelial cells. This is the first ultrastructural localization of ECE in lung and the first time big ET-1 and ECE have been colocalized by immunogold in a v esicular population, 50 to 100 nm in diameter. In addition, by double immunogold staining oi ultrathin cryosections, we have localized ECE t ogether with angiotensin-converting enzyme on the luminal membrane of endothelial cells. With cell. surface biotinylation oi a transformed r at endothelial cell. line and of human umbilical vein endothelial cell s, we have confirmed the presence of ECE on the plasma membrane. Alter treatment of endothelial cells with chloroquine, ECE and trans-Golgi network 38 protein were shown by immunofluorescence staining to locali ze to the same intracellular compartment.