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.