Mitochondrial Arginase II Modulates Nitric-Oxide Synthesis through Nonfreely 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 M. 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 M. Extracellular L-arginine also dosedependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 M 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.

Polarization of Macrophages toward M2 Phenotype Is Favored by Reduction in iPLA2β (Group VIA Phospholipase A2)

“…iNOS generates nitric oxide from the amino acid arginine, and Arg inhibits nitric oxide synthesis by reducing available arginine via conversion to ornithine and urea (66). However, there are two isoforms of arginase that are encoded by different genes (67): Arg1, which is cytoplasmic, and Arg2, which is mitochondrial.…”

L-Arginine Metabolism in the Lung: Reciprocal Regulation of the NOS and Arginase Pathways

“…Both arginase 1 and 2 have been implicated in regulating NOS1 activity through the depletion of intracellular L-arginine [64,65]. In cultured endothelial cells arginase 2 has been demonstrated to regulate the activity of NOS3, and this regulation has been shown to take place within non-freely exchangeable Larginine pools [66,67]. Furthermore, over expression of arginase 1 has been demonstrated to result in impaired NO production by NOS1 in cultured 293 embryonic kidney cells stably transfected with NOS1 [65].…”

Exercise and Oxidative Stress

“…Arginases and NOS compete for arginine, and -under any conditions -arginase activity exceeds NOS activity at all NOS/arginase molar ratios (Santhanam et al, 2008). Moreover, although the K M of arginases is 100-fold higher than that of NOS, the enzymes compete for arginine because the maximal catalytic rate of arginases is more than 1000 times higher than that of NOS (Wu et al, 1998;Topal et al, 2006). Therefore, increased expression and/or activity of ARG have a deep impact on NOS efficiency.…”