Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells.
Endothelin is a potent vasoconstrictive peptide that is produced by vascular endothelial cells; it is formed from its precursor, big endothelin, by endothelin-converting enzyme (ECE). In this work, ECE was studied using specific monoclonal antibodies. In immunoblotting, ECE was estimated to be a 300 kDa protein on SDS/PAGE under non-reducing conditions, and 130 kDa under reducing conditions. Cross-linking experiments revealed that ECE is composed of two disulphide-linked subunits. Localization of ECE was studied at the cellular and subcellular levels in various rat tissues and cells. High-level expression of ECE was observed in membrane fractions of simian virus 40-transformed rat endothelial cells by immunoblotting, but the immunoreactive band was absent form aortic smooth muscle cells and cytosolic fractions of endothelial cells. In immunohistochemical analysis, ECE was found to be localized in the endothelial cells of the aorta, lung, kidney, liver and heart. Confocal immunofluorescent microscopy showed that most of the ECE in endothelial cells and cells transfected with ECE cDNA was clustered along the plasma membrane. Intact COS or CHO cells transfected with ECE cDNA rapidly and efficiently cleaved big endothelin-1 added to the culture medium. Thus endothelial cells express ECE on the plasma membrane and the active site of the enzyme faces outside the cells, i.e. it is an ectoenzyme. Other than endothelial cells, ECE was also present in some secretory cells. The enzyme was abundant in the adrenal gland, and localized in chromaffin cells. ECE was also highly condensed in pancreatic islet beta cells. It is concluded that ECE and endothelin may be involved in the regulated secretion of hormones.
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D000911:Antibodies, Monoclonal; D001012:Aorta, Abdominal / Q000201:enzymology; D016282:Aspartic Acid Endopeptidases / Q000032:analysis*; D016466:CHO Cells / Q000201:enzymology; D002461:Cell Line, Transformed / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008666:Metalloendopeptidases / Q000032:analysis; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D051381:Rats; D013347:Subcellular Fractions
D000911:Antibodies, Monoclonal; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes