Kuruppu Sanjaya, Rajapakse Niwanthi W, Dunstan Rhys A, Smith A Ian
Department of Biochemistry and Molecular Biology, Monash University, Building 77, Wellington Rd, Clayton, VIC, 3800, Australia,
Mol Cell Biochem. 2014 Nov;396(1-2):49-54. doi: 10.1007/s11010-014-2141-0. Epub 2014 Sep 17.
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.
本研究检测了一氧化氮对可溶性ECE-1产生的影响。使用淬灭荧光底物分析法测定培养基中ECE-1的活性，并以对照的百分比表示。将内皮细胞与一氧化氮供体二乙三胺 NONOate（DETA；250 - 800 μM）、一氧化氮合酶（NOS）底物L-精氨酸（L-Arg；200 - 1,000 μM）、L-Arg转运抑制剂（L-赖氨酸；L-Lys；10 μM）和NOS抑制剂（L-谷氨酰胺和N5-[亚氨基（硝基氨基）甲基]-L-鸟氨酸甲酯，盐酸盐（L-NAME）；10 - 100 μM）一起孵育。还在存在L-Lys（10 μM）、L-谷氨酰胺（100 μM）和L-NAME（10 - 100 μM）的情况下测试了L-Arg（1,000 μM）的作用。超速离心（100,000×g，4℃，1小时）可从上清液中完全去除ECE-1活性。此外，在蔗糖密度梯度上对浓缩培养基进行分级分离表明，ECE-1活性定位于梯度的中部，因此提示外泌体在ECE-1释放中可能发挥作用。在存在DETA（75.31 ± 3.59；800 μM）和L-Arg（60.97 ± 9.22；1,000 μM）的情况下，Ea.hy926细胞产生的可溶性ECE-1受到显著抑制（P < 0.05，非配对t检验，n = 4）。使用L-NAME（100 μM；99.19 ± 0.58）和L-谷氨酰胺（100 μM；104.41 ± 0.65）抑制NOS可阻断L-Arg介导的可溶性ECE-1释放减少。此外，L-Lys（10 μM）的存在显著阻断了L-Arg（1,000 μM）诱导的可溶性ECE-1水平降低（122.38 ± 13.16）。这些处理对细胞表面ECE-1的表达没有影响。我们的数据提供了证据表明一氧化氮可抑制内皮细胞产生可溶性ECE-1。
