Endothelin converting enzyme is located on alpha-actin filaments in smooth muscle cells.
OBJECTIVE
Endothelin converting enzyme is the key enzyme in the generation of endothelin-1 from big-endothelin-1. The mature endothelin-1 is a potent vasoconstrictor which also promotes mitogenesis and proliferation of smooth muscle cells. The objectives were to demonstrate in smooth muscle cells the presence of a phosphoramidon-sensitive endothelin converting enzyme activity, reveal the subcellular localization of the enzyme protein and determine the effects of the metalloproteinase inhibitor, phosphoramidon, the lysosomotrophic 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 immunofluorescence and confocal microscopy or by biotinylation of cell cultures and immunoblotting, after treatment of cell cultures with cytochalasin D, colchicine, chloroquine and phosphoramidon. Converting enzyme activity was determined by high performance liquid chromatographic assay.


RESULTS
We detected phosphoramidon-sensitive endothelin converting enzyme activity in smooth muscle cells. In addition to its plasma membrane location, for the first time we revealed a striking co-localization of endothelin converting enzyme and alpha-actin filaments in smooth muscle cells. Colchicine treatment results in a perinuclear accumulation of endothelin converting enzyme. An increased level of endothelin converting enzyme protein was shown to be present in smooth muscle cells which had been grown in the presence of phosphoramidon or chloroquine.


CONCLUSIONS
The 120 kDa endothelin converting enzyme co-localizes with alpha-actin in smooth muscle cells and resembles that found in endothelial cells in that it is present on both the plasma membrane and intracellularly.
Barnes
K
K
School of Biochemistry and Molecular Biology, University of Leeds, UK.
Turner
A J
AJ
D000199:Actins / Q000737:chemistry*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D000072396:Endothelin-Converting Enzymes; D005455:Fluorescent Antibody Technique; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D008666:Metalloendopeptidases; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D014469:Umbilical Arteries
D000199:Actins; D006020:Glycopeptides; D019384:Nucleic Acid Synthesis Inhibitors; D011480:Protease Inhibitors; D015638:Cytochalasin D; D002738:Chloroquine; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; D003078:Colchicine; C008890:phosphoramidon