Endothelin converting enzyme is located on alpha-actin filaments in smoothmuscle cells

Objective: Endothelin converting enzyme is the key enzyme in the generation of endothelin-l from big-endothelin-l. The mature endothelin-l is a potent vasoconstrictor which also promotes mitogenesis and proliferation of smoot h muscle cells. The objectives were to demonstrate in smooth muscle cells t he presence of a phosphoramidon-sensitive endothelin converting enzyme acti vity, reveal the subcellular localization of the enzyme protein and determi ne the effects of the metalloproteinase inhibitor, phosphoramidon, the lyso somotrophic drug, chloroquine, and colchicine on the cycling pathway of the enzyme. Methods: Subcellular localization of endothelin converting enzyme on human smooth muscle cells and the rat cell line, A7r5, was by immunofluo rescence and confocal microscopy or by biotinylation of cell cultures and i mmunoblotting, after treatment of cell cultures with cytochalasin D, colchi cine, chloroquine and phosphoramidon. Converting enzyme activity was determ ined by high performance liquid chromatographic assay. Results: We detected phosphoramidon-sensitive endothelin converting enzyme activity in smooth m uscle cells. In addition to its plasma membrane location, for the first tim e we revealed a striking co-localization of endothelin converting enzyme an d a-actin filaments in smooth muscle cells. Colchicine treatment results in a perinuclear accumulation of endothelin converting enzyme. An increased l evel of endothelin converting enzyme protein was shown to be present in smo oth muscle cells which had been grown in the presence of phosphoramidon or chloroquine. Conclusion: The 120 kDa endothelin converting enzyme co-locali zes with ol-actin in smooth muscle cells and resembles that found in endoth elial cells in that it is present on both the plasma membrane and intracell ularly. (C) 1999 Elsevier Science B.V. All rights reserved.