Evidence for intracellular endothelin-converting enzyme-2 expression in cultured human vascular endothelial cells.
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 micromol/L PD159790 and by 100 micromol/L phosphoramidon but not by 0.1 micromol/L phosphoramidon. In contrast, ECE activity was unaffected by 30 micromol/L PD159790 but was inhibited markedly by 0.1 and 100 micromol/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.
Russell
F D
FD
Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge, UK.
Davenport
A P
AP
D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases; D012333:RNA, Messenger / Q000032:analysis
D007527:Isoenzymes; D012333:RNA, Messenger; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes