Evidence for vesicles that transport endothelin-1 in bovine aortic endothelial cells.
To facilitate studies of the intracellular processing of endothelin-1 (ET-1) in endothelial cells we developed an enrichment procedure for the isolation of transport or secretory vesicles containing ET-1. Cultured bovine aortic endothelial cells (BAECs) were disrupted using a tight-fitting Dounce homogenizer, and subcellular fractions were isolated by sucrose-density-gradient ultracentrifugation. ET-1 immunoreactivity (ET-IR) in the fractions was measured with a specific ET[16-21] radio-immunoassay (RIA). The major peak of ET-IR was consistently localized at the 1.0/1.2 M sucrose interface (10.96 fmol/10(6) cells) in each preparation of subcellular fractions. The mean level of ET-IR at this interface, expressed as a percentage of the total, was 47.9 +/- 3.5% (n = 6). This finding provides strong evidence for the existence of a vesicle that transports ET-1 within BAECs, which may be an important site for endogenous ET-1 processing. A number of studies have reported that the final processing step in ET-1 biosynthesis by the hypothetical endothelin-converting enzyme (ECE) is inhibited by phosphoramidon (PHOS). Therefore, levels of PHOS-sensitive ECE activity in each fraction were assessed in parallel with ET-IR levels. The ECE activity associated with the peak of ET-IR (2.67 pmol ET-1/h/10(6) cells) was insensitive to 100 microM PHOS. However, the peak of ECE-like activity (10.4 pmol ET-1/h/10(6) cells) localized in the 0.8 M sucrose band was inhibited by 79% in the presence of 100 microM PHOS.(ABSTRACT TRUNCATED AT 250 WORDS)
Harrison
V J
VJ
William Harvey Research Institute, St. Bartholomew's Hospital Medical College, London, England.
Corder
R
R
Anggård
E E
EE
Vane
J R
JR
D000818:Animals; D001011:Aorta / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011863:Radioimmunoassay; D013347:Subcellular Fractions / Q000201:enzymology
D016232:Endothelins; D006020:Glycopeptides; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon