title: Nitric oxide inhibits the production of soluble endothelin converting enzyme-1.
abstract: This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 µM), NOS substrate L-Arg (200-1,000 µM), a L-Arg transport inhibitor (L-Lys; 10 µM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 µM). The effect of L-Arg (1,000 µM) was also tested in the presence of L-Lys (10 µM), L-Gln (100 µM) and L-NAME (10-100 µM). Ultracentrifugation (100,000×g, 4 °C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P < 0.05, unpaired t test, n = 4) in the presence of DETA (75.31 ± 3.59; 800 µM) and L-Arg (60.97 ± 9.22; 1,000 µM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 µM; 99.19 ± 0.58) and L-Gln (100 µM; 104.41 ± 0.65). In addition, the presence of L-Lys (10 µM) significantly blocked the L-Arg (1,000 µM)-induced reduction in soluble ECE-1 levels (122.38 ± 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Building 77, Wellington Rd, Clayton, VIC, 3800, Australia, Sanjaya.Kuruppu@monash.edu.
authors.forename: Sanjaya
authors.initials: S
authors.lastname: Kuruppu
authors.forename: Niwanthi W
authors.initials: NW
authors.lastname: Rajapakse
authors.forename: Rhys A
authors.initials: RA
authors.lastname: Dunstan
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
mesh_terms: D001120:Arginine / Q000494:pharmacology; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D000072396:Endothelin-Converting Enzymes; D006801:Humans; D008239:Lysine / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D012995:Solubility; D014461:Ultracentrifugation
chemical_list: D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D008239:Lysine; D019331:NG-Nitroarginine Methyl Ester