Received for publication February 28, 2006.
Revised June 13, 2006.
Accepted for publication June 23, 2006.

Mitochondrial arginase II modulates nitric oxide synthesis through non-freely exchangeable L-arginine pools in human endothelial cells

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 N-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.

Key words: Arginase, Arginase inhibitor, Endothelial nitric oxide synthase, Human endothelial cells, L-arginine pools, L-arginine uptake

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