Characterization of endothelin-converting enzyme from endothelial cells and rat brain: detection of the formation of biologically active endothelin-1 by rapid bioassay.
Using the endothelin-1 (ET-1)-stimulated elevation in cGMP in LLC-PK1 cells as a biological detector system for the conversion of big ET-1 (bET-1) to ET-1, we detected bET-1-converting activities in subcellular fractions from bovine aortic cultured endothelial cells (BAE) and rat brain. Within the particulate fraction of BAE, we detected two activities, at pH 3.4 and pH 5.4-7.4. The latter but not the former activity was inhibited in a concentration-dependent manner by phosphoramidon (approximate IC50, 1 microM) and converted bET-1 to ET-1 at a rate of 0.6 nmol/hr/mg of protein. It could be solubilized from the particulate fraction by detergent treatment. Phosphoramidon-inhibitable converting activity was also detected in the cytosolic fraction of BAE. Within the rat brain, phosphoramidon-inhibitable conversion of bET-1 to ET-1 was detected principally in the cytoskeletal fraction, i.e., that fraction from the membrane that was not solubilized by detergent treatment. These results show the presence of at least two different endothelin-converting enzyme activities in endothelial cells and a third within the rat brain. They also demonstrate the use of LLC-PK1 cells as a rapid assay that permits the sensitive detection and measurement of the formation of biologically active ET-1 from its precursor bET-1.
Warner
T D
TD
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Mitchell
J A
JA
D'Orleans-Juste
P
P
Ishii
K
K
Förstermann
U
U
Murad
F
F
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001921:Brain / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000096:biosynthesis* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006020:Glycopeptides / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008297:Male; D008666:Metalloendopeptidases; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; D006152:Cyclic GMP; C008890:phosphoramidon