Mitochondrial arginase II modulates nitric-oxide synthesis through nonfreely exchangeable L-arginine pools in human endothelial cells.
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 N(omega)-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 microM. 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 microM. Extracellular L-arginine also dose-dependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 microM 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.
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L-arginine NO Nor-NOHA L-arginine Nor-NOHA L-arginine L-arginine L-lysine Nor-NOHA NO NO l-arginine nitric-oxide nitric oxide NO NO N(omega)-hydroxy-nor-L-arginine Nor-NOHA L-arginine NO Nor-NOHA L-arginine Nor-NOHA NO L-arginine NO nitric-oxide L-arginine thrombin NOS arginase NOS arginase mitochondrial arginase II arginase I constitutive nitric-oxide synthase arginase II NOS arginase I mitochondrial arginase II arginases arginase NOS arginases L-arginine transporter arginase thrombin NOS arginase Mitochondrial arginase II