BACKGROUND AND PURPOSE NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available l‐arginine, reducing NO and increasing superoxide (O₂^.−) production via NOS uncoupling. Elevated O₂^.− combines with NO to form peroxynitrite (ONOO^‐), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide‐induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC‐activated RhoA/Rho kinase (ROCK) pathway.EXPERIMENTAL APPROACH Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO^‐ generator (SIN‐1) or H₂O₂. Pretreatment with inhibitors of Rho kinase (Y‐27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation.KEY RESULTS Exposure to SIN‐1 (25 µM, 24 h) or H₂O₂ (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y‐27632, PKC inhibitor, Gö6976 or siRNA to p115‐Rho GEF. There was an early activation of p115‐Rho GEF (SIN‐1, 2 h; H₂O₂, 1 h) and Rho A (SIN‐1, 4 h; H₂O₂, 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN‐1 and H₂O₂ also reduced NOS activity, which was blocked by pretreatment with p115‐RhoGEF siRNA.CONCLUSIONS AND IMPLICATIONS Our data indicate that the oxidative species ONOO^‐ and H₂O₂ increase arginase activity/expression through PKC‐mediated activation of RhoA/Rho kinase pathway.

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT₁receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg⁻¹·day⁻¹) or arginase (ABH, 8 mg·kg⁻¹·day⁻¹) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT₁receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.

Type 2 diabetes mellitus (T2DM) is becoming a major contributor to cardiovascular disease. One of the early signs of T2DM associated cardiovascular events is the development of vascular dysfunction. This dysfunction has been implicated in increasing the morbidity and mortality of T2DM patients. One of the important characteristics of vascular dysfunction is the impaired ability of endothelial cells to produce nitric oxide (NO). Additionally, decreases in the availability of NO is also a major contributor of this pathology. NO is produced by the activity of endothelial NO synthase (eNOS) on its substrate, L-arginine. Reduced availability of L-arginine to eNOS has been implicated in vascular dysfunction in diabetes. Arginase, which metabolizes L-arginine to urea and ornithine, competes directly with NOS for L-arginine. Hence, increases in arginase activity can decrease arginine levels, reducing its availability to eNOS and decreasing NO production. Diabetes has been linked to elevated arginase and associated vascular endothelial dysfunction. We aimed to determine levels of plasma NO and arginase activity in (T2DM) patients and the effects of L-citrulline supplementation, a natural arginase inhibitor, on inhibiting arginase activity in these patients. Levels of arginase correlated with HbA1c levels in diabetic patients. Twenty-five patients received L-citrulline supplements (2000 mg/day) for 1 month. Arginase activity decreased by 21% in T2DM patients after taking L-citrulline supplements. Additionally, plasma NO levels increased by 38%. There was a modest improvement on H1Ac levels in these patients, though not statistically significant. The effect of L-citrulline on arginase activity was also studied in bovine aortic endothelial cells (BAECs) grown in high glucose (HG) conditions. HG (25 mM, 72 h) caused a 2-fold increase in arginase activity in BAECs and decreased NO production by 30%. L-citrulline (2.5 mM) completely prevented the increase in arginase activity and restored NO production levels. These data indicate that L-citrulline can have therapeutic benefits in diabetic patients through increasing NO levels and thus maintaining vascular function possibly through an arginase inhibition related pathway.