Abstract
Reduced synthesis of nitric oxide (NO) contributes to the endothelial dysfunction and may be related to limited availability of l-arginine, the common substrate of constitutive nitric-oxide synthase (NOS) and cytosolic arginase I and mitochondrial arginase II. To determine whether arginases modulate the endothelial NO synthesis, we investigated the effects of the competitive arginase inhibitor Nω-hydroxy-nor-l-arginine (Nor-NOHA) on the activity of NOS, arginases, and l-arginine transporter and on NO release at surface of human umbilical vein endothelial cells (HUVECs). In unstimulated cells, Nor-NOHA dose-dependently reduced the arginase activity with maximal inhibition at 20 μM. When HUVECs were stimulated by thrombin without extracellular l-arginine, Nor-NOHA dose-dependently increased the NOS activity and the NO release with maximal effects at 20 μM. Extracellular l-arginine also dose-dependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 μM l-arginine, NOS activity and NO release were similar in untreated and Nor-NOHA-treated cells. However, despite activation of l-arginine uptake, the inhibition of arginase activity by Nor-NOHA was still significant. The depletion of freely exchangeable l-arginine pools with extracellular l-lysine did not prevent Nor-NOHA from increasing the NO release. This indicates the presence of pools, which are accessible to NOS and arginase, but not exchangeable. Interestingly, the mitochondrial arginase II was constitutively expressed, whereas the cytosolic arginase I was barely detectable in HUVECs. These data suggest that endothelial NO synthesis depends on the activity of arginase II in mitochondria and l-arginine carriers in cell membrane.
Footnotes
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J.-L.G.T. is a recipient of support from the International Society of Thrombosis and Hemostasis and the Société Française d'Athérosclérose.
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Part of this work has been presented in abstracts of the First joint French-German NO meeting, which was held in Strasbourg (France) in Oct 2-4, 2003.
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doi:10.1124/jpet.106.103747.
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ABBREVIATIONS:l-Arg, l-arginine; NO, nitric oxide; CAT, cationic amino acid transporter; NOHA, Nω-hydroxy-l-arginine; NOS, NO synthase; iNOS, inducible NOS; mtNOS, mitochondrial NOS; NOS3, endothelial NO synthase; HUVEC, human umbilical vein endothelial cell; Nor-NOHA, Nω-hydroxy-nor-l-arginine; PBS, phosphate-buffered saline.
- Received February 27, 2006.
- Accepted June 23, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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