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Cardiovascular Research 1999 42(3):814-822; doi:10.1016/S0008-6363(99)00009-7
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Endothelin converting enzyme is located on {alpha}-actin filaments in smooth muscle cells

Kay Barnes and Anthony J Turner*

School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

a.j.turner{at}leeds.ac.uk

* Corresponding author. Tel.: +44-113-233-3131; fax: +44-0113-242-3187

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. Conclusion: 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.

KEYWORDS CHO-K1, Chinese hamster ovary cells; ECE, endothelin converting enzyme; ECL, enhanced chemiluminescence; ET, endothelin; FITC, fluorescein isothiocyanate; HPLC, high performance liquid chromatography; TRITC, tetramethylrhodamine isothiocyanate; TRLEC-03, transformed rat lung endothelial cells; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; SMC, smooth muscle cells


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