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


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.


CONCLUSIONS
These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.
Department of Biology, College of Natural Sciences, School of Medicine, Kangwon National University, Chuncheon, Korea.
Minh Cong
MC
Nguyen
Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea.
Jong Taek
JT
Park
Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea.
Yeong Gwan
YG
Jeon
Infectious Signaling Network Research Center, Department of Physiology, School of Medicine, Chungnam National University, Daejeon, Korea.
Byeong Hwa
BH
Jeon
Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea.
Kwang Lae
KL
Hoe
Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Korea.
Young Myeong
YM
Kim
Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea. hyunkyolim@yonsei.ac.kr.
Hyun Kyo
HK
Lim
Department of Biology, College of Natural Sciences, School of Medicine, Kangwon National University, Chuncheon, Korea. ryoosw08@kangwon.ac.kr.
Sungwoo
S
Ryoo
D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism*; D004730:Endothelium, Vascular; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000187:drug effects / Q000378:metabolism*; D030421:Peroxynitrous Acid; D010766:Phosphorylation / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014652:Vascular Diseases
D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; C026512:arginine methyl ester; D009569:Nitric Oxide; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D001119:Arginase
L-arginine; Peroxynitrite; arginase inhibition; endothelial dysfunction; endothelial nitric oxide synthase phosphorylation; nitric oxide