Nitric oxide inhibits the production of soluble endothelin converting enzyme-1

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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 microM), NOS substrate L-Arg (200-1,000 microM), a L-Arg transport inhibitor (L-Lys; 10 microM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 microM). The effect of L-Arg (1,000 microM) was also tested in the presence of L-Lys (10 microM), L-Gln (100 microM) and L-NAME (10-100 microM). Ultracentrifugation (100,000xg, 4 degrees 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 microM) and L-Arg (60.97 +/- 9.22; 1,000 microM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 microM; 99.19 +/- 0.58) and L-Gln (100 microM; 104.41 +/- 0.65). In addition, the presence of L-Lys (10 microM) significantly blocked the L-Arg (1,000 microM)-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.
Original languageEnglish
Pages (from-to)49 - 54
Number of pages6
JournalMolecular and Cellular Biochemistry
Volume396
Issue number1-2
DOIs
Publication statusPublished - 2014

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