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(Circulation Research. 1999;84:891-896.)
© 1999 American Heart Association, Inc.

Original Contribution

Evidence for Intracellular Endothelin-Converting Enzyme-2 Expression in Cultured Human Vascular Endothelial Cells

Fraser D. Russell, Anthony P. Davenport

From the Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge, UK.

Correspondence to Anthony P. Davenport, Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical Investigation, Box 110, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK. E-mail apd10{at}medschl.cam.ac.uk

Abstract—We have previously reported the intracellular localization of the endothelin-converting enzyme-1 (ECE-1) in human umbilical vein endothelial cells. In the present study, we provide the first immunocytochemical and biochemical evidence for the presence of ECE-2 in human cells. ECE activity was determined by conversion of exogenously added big endothelin-1 (big ET-1) to ET-1 in subcellular fractions obtained by sucrose density gradient centrifugation of human umbilical vein endothelial cell homogenates. ECE-1 and ECE-2 can be differentiated by pH dependence for optimal activity and by sensitivity to phosphoramidon, which shows selectivity for ECE-2 over ECE-1 and PD159790, a novel ECE-1 selective inhibitor. Optimal ECE activity was measured at pH 6.0, a value intermediate between that reported for ECE-1 (pH 6.8) and ECE-2 (pH 5.5), indicating expression of both enzymes. At pH 6.9, conversion of big ET-1 was inhibited markedly by 30 µmol/L PD159790 and by 100 µmol/L phosphoramidon but not by 0.1 µmol/L phosphoramidon. In contrast, ECE activity was unaffected by 30 µmol/L PD159790 but was inhibited markedly by 0.1 and 100 µmol/L phosphoramidon at pH 5.4 (IC₅₀ 1.5 nmol/L), consistent with ECE-2 activity. Confocal microscopy revealed a punctate pattern of ECE-2–like immunoreactive staining in the cell cytosol, suggesting localization to secretory vesicles with a possible role in processing big ET-1 while in transit to the cell surface via the constitutive secretory pathway.

Key Words: endothelin • endothelin-converting enzyme • endothelium

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