Endothelin-converting enzyme in the human vasculature: evidence for differential conversion of big endothelin-3 by endothelial and smooth-muscle cells.
Our aim was to localize endothelin-converting enzyme (ECE) in human saphenous vein grafts and to quantify enzymic activity in cultured human endothelial and smooth-muscle cells. Immunoreactive ECE localized to the endothelium and infiltrating macrophages in vein grafts, but little or no immunoreactivity was detected within the media or proliferated smooth muscle of the occlusive lesion. Cultures of human umbilical vein endothelial cells were incubated with big endothelin-1 (ET-1) (10 nM) to measure extracellular conversion. After 2 h the concentration of mature peptide in the medium was increased by 162.7 +/- 21.6 pM (n = 3 +/- SEM) above basal. Permeabilization of the cells increased conversion to 1077.9 +/- 52.8 pM, suggesting that about 85% of ECE activity was located intracellularly. In both cases, activity was inhibited by phosphoramidon but not by thiorphan. In contrast, conversion of big ET-3 (10 nM) under the same conditions was not detected in either intact or permeabilized cells after 2 h. Big ET-3 and big ET-1 were converted by a phosphoramidon-sensitive/thiorphan-insensitive enzyme on the surface of confluent cultures of human umbilical vein smooth-muscle cells, with concentrations of the corresponding mature peptides increasing by 99.5 +/- 14.5 pM and 222.2 +/- 11.6 pM, respectively. These results suggest that smooth-muscle cells could be responsible for the synthesis of ET-3 present in plasma and for additional processing of big ET-1 released by endothelial cells.
Davenport
A P
AP
Clinical Pharmacology Unit, University of Cambridge, Addenbrookes Hospital, England.
Kuc
R E
RE
Mockridge
J W
JW
D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002478:Cells, Cultured; D019332:Endothelin-1; D019334:Endothelin-3; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology*; D005110:Extracellular Space / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D011498:Protein Precursors / Q000378:metabolism*; D012501:Saphenous Vein / Q000166:cytology / Q000201:enzymology
D019332:Endothelin-1; D019334:Endothelin-3; D016232:Endothelins; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes