Asymmetric dimethylarginine induces apoptosis via p38 MAPK/caspase-3-dependent signaling pathway in endothelial cells.
Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributor for endothelial dysfunction and its effects on endothelium are not yet completely defined. The aim of this study was to investigate ADMA-induced apoptosis and its mechanisms in human umbilical vein endothelial cells (HUVECs). Apoptosis was evaluated by in situ terminal uridine nick end labeling (TUNEL) assay and DNA fragmentation analysis. Caspase-3 activity was measured using a colorimetric protease assay kit. Activations of mitogen-activated protein kinases (MAPKs) were characterized by Western blot and immunofluorescence. Intracellular oxidant production was measured using H(2)DCF-DA, an oxidant-sensitive fluorescent indicator. ADMA (3-30 microM) induced apoptosis of HUVECs in a dose- and time-dependent manner. Caspase-3 was activated during apoptosis and its specific inhibitor DEVD-CHO significantly attenuated ADMA-induced apoptosis. Phosphorylation of p38 MAPK was induced by ADMA, and p38 MAPK specific inhibitor SB203580 concentration-dependently prevented ADMA-induced caspase-3 activation and cell apoptosis. ADMA increased intracellular oxidant production, which was significantly suppressed by intracellular antioxidant PDTC, l-arginine or antisense endothelial NOS mRNA. They also markedly prevented ADMA-induced phosphorylation of p38 MAPK and cell apoptosis. In conclusion, our present results demonstrate that ADMA induces apoptosis of endothelial cell via elevation of intracellular oxidant production, which involves p38 MAPK/caspase-3-dependent signaling pathway.
Jiang
De-Jian
DJ
Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Xiang-Ya Road (#)110, Changsha 410078, China.
Jia
Su-Jie
SJ
Dai
Zhong
Z
Li
Yuan-Jian
YJ
D017209:Apoptosis / Q000187:drug effects*; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; C018524:N,N-dimethylarginine; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D048051:p38 Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C093642:SB 203580