Yonsei Med J.  2016 Nov;57(6):1329-1338. 10.3349/ymj.2016.57.6.1329.

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation

• Nguyen MC
• Park JT
• Jeon YG
• Jeon BH
• Hoe KL
• Kim YM
• Lim HK
• Ryoo S

• KMID: 2427149
• DOI: http://doi.org/10.3349/ymj.2016.57.6.1329

Abstract

PURPOSE
Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism.
MATERIALS AND METHODS
Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay.
RESULTS
SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition.
CONCLUSION
These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.

Keyword

Peroxynitrite; arginase inhibition; endothelial nitric oxide synthase phosphorylation; endothelial dysfunction; nitric oxide; L-arginine

MeSH Terms

Animals
Arginase/*antagonists & inhibitors/metabolism
Arginine/analogs & derivatives/*metabolism
Endothelium, Vascular
Human Umbilical Vein Endothelial Cells/*drug effects/metabolism
Humans
Nitric Oxide/*metabolism
Nitric Oxide Synthase Type III/drug effects/*metabolism
Peroxynitrous Acid
Phosphorylation/*drug effects
Reactive Oxygen Species/metabolism
Vascular Diseases
Reactive Oxygen Species
Peroxynitrous Acid
Nitric Oxide
Arginine
Nitric Oxide Synthase Type III
Arginase

Figure

• 

  Fig. 1 Peroxynitrite generated by SIN-1 increased arginase activity and expression. (A) HUVECs were incubated with SIN-1 (50 µM) at different time points, and then arginase activity was measured (*p<0.05 vs. untreated). (B) HUVECs were incubated with increasing doses of SIN-1 (5–100 µM) for 18 h, after which cellular arginase activity was measured (*p<0.01 vs. untreated). (C) HUVECs were incubated with SIN-1 (50 µM, 18 h) in the presence or absence of the peroxynitrite scavenger, FeTPPS (5 µM), and arginase activity was measured (*p<0.01 vs. untreated, #p<0.01 vs. SIN-1 alone). ONOO-(50 µM, 18 h) was used as the control (*p<0.01 vs. untreated). (D) HUVECs were incubated with SIN-1 at different time points, and cellular NOx was measured. SIN-1 resulted in a significant decrease in endothelial-cell NOx production. Preincubation of cells with FeTPPS (5 µM) prevented the SIN-1-induced decrease in NOx production [*p<0.01 vs. untreated, #p<0.01 vs. SIN-1 (18 h)]. HUVECs, human umbilical vein endothelial cells; NOx, nitrite/nitrate. (E) Protein abundance was analyzed in HUVECs treated with SIN-1 (50 µM, 18 h). Densitometry analysis was performed [F, G, and H, *p<0.05 vs. untreated; PC, positive control for iNOS (RAW264.7 macrophage stimulated with lipopolysaccharide)]. HUVECs, human umbilical vein endothelial cells; NOx, nitrite/nitrate; iNOS, inducible nitric oxide synthase.

• 

  Fig. 2 Effects of peroxynitrite on eNOS phosphorylation and dimerization were prevented by arginase inhibition and L-arginine supplement. (A) Western blot and (B) quantitative analysis of eNOS phosphorylation at Ser1177 and Thr495. SIN-1 treatment reduced p-eNOS Ser1177 and increased p-eNOS Thr495 (*p<0.05 vs. untreated), which was reversed by arginase inhibitor, ABH (10 µM for 2 h) and L-arginine supplement (10 mM for 2 h; #p<0.05 vs. SIN-1 alone, ##p<0.01 vs. SIN-1 alone). (C) Native low-temperature SDS-PAGE and Western blot analysis were performed after SIN-1 treatment (*p<0.05 vs. untreated). (D) The reduced ratio of eNOS dimer/monomer was significantly restored by preincubation with ABH (10 µM, *p<0.01 vs. SIN-1 alone) and L-arginine supplement (10 mM, #p<0.01 vs. SIN-1 alone). L-Arg, L-arginine; eNOS, endothelial nitric oxide synthase.

• 

  Fig. 3 Incubation with arginase inhibitor and L-arginine altered NO production and ROS generation in SIN-1-stimulated mouse aortas. (A) Isolated aortic rings were incubated with SIN-1 (50 µM, 18 h), and endothelial NO production (endothelial side up) was measured using DAF-FM (5 µM). SIN-1 incubation resulted in a decreased slope of DAF-FM (*p<0.01 vs. untreated) that was restored to untreated control levels by treatment with the arginase inhibitor, ABH (10 µM), and L-arginine (10 mM; #p<0.01 vs. SIN-1 alone). L-NAME was used as a control. (B) ROS generation from the aortic endothelium was measured after preloading with DHE (5 µM; *p<0.01 vs. untreated; #p<0.01 vs. SIN-1 alone), whereas L-NAME (10 µM) treatment prevented the ROS generation induced by SIN-1 stimulation (NS, non-significant among the groups). Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) was used as a superoxide scavenger. NO, nitric oxide; ROS, reactive oxygen species; DHE, dihydroethidine; L-NAME, NG-Nitro-L-arginine methyl ester.

• 

  Fig. 4 Effect of arginase inhibition on intracellular L-arginine concentration. Intracellular L-arginine concentration was measured using HPLC (see Materials and Methods section). Treatment of HUVECs with SIN-1 resulted in decreased intracellular L-arginine that was recovered by pretreatment with the arginase inhibitor, ABH (*p<0.01 vs. untreated, #p<0.01 vs. SIN-1 alone; n=6). HPLC, high-performance liquid chromatography; HUVECs, human umbilical vein endothelial cells.

• 

  Fig. 5 Arginase inhibition restored impaired vascular function induced by SIN-1 in mouse aortas. (A) Constriction was induced by high potassium in SIN-1-treated and -untreated descending aortas. Constrictions induced by a high-potassium solution did not differ between groups (NS, non-significant). (B) Contractile responses to the agonist, U46619, were significantly enhanced in SIN-1-incubated aortic rings, and incubation with the arginase inhibitor (ABH, 10 µM) attenuated vasoconstrictor responses to U46619 (*p<0.01 vs. untreated; #p<0.01 vs. SIN-1 alone). Vessels were preconstricted, and cumulative dose responses to Ach (C) and SNP (D) were performed. The vasorelaxant EC50 to Ach was attenuated in aortic rings incubated with SIN-1 (*p<0.05 vs. untreated). Arginase inhibition enhanced maximal vasorelaxation in SIN-1-treated vessels (#p<0.01 vs. SIN-1 alone). De-endothelialized vessels did not respond to Ach. Vasorelaxant responses to SNP did not differ among groups. Ach, acetylcholine; SNP, sodium nitroprusside.

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