Neuroprotective Effect Of Asymmetric Dimethylarginine Against 1-Methyl-4-Phenylpyridinium Ion-induced Damage In PC12 Cells

【Background and Objective】1-Methy-4-phenylpyridiniumion (MPP+), the active metabolite of MPTP, is selectively accumulated in the mitochondria of dopaminergic neurons and confers toxicity and neuronal death through inhibiting complex I of the mitochondrial electron transport chain. Inhibition of complex I by MPP+ caused the disintegration of oxidative phosphorylation and Ca2+ influx,resulting in nNOS excess activation and NO overproduction. In addition, mitochondria also produce large amounts of peroxides and other free radicals. NO produced cytotoxicity not only by itself, but also by reacting with superoxide and forming peroxynitrite, an important cell death mediators, which mediate one or two electron oxidation and then damage lipids, proteins and DNA, ultimately resulting in dopaminergic cell death. NOS excess activation and NO overproduction involve in the neurotoxicity of MPP+.Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, is synthesized during the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and is released during proteolysis. ADMA can inhibit competitively all three NOS isoforms (iNOS, nNOS, and eNOS) and may decrease the production of NO. It is not known whether or not ADMA, as a NOS inhibitor, has the ability to protect against MPP+-induced neurotoxicity through inhibiting NOS activity. Although ADMA is a novel risk marker in cardiovascular medicine and chronic kidney disease, we speculated that in some states associated with excess of NO, such as MPP+-induced neuronal injury, ADMA may be protective by limiting toxic effect of high concentrations of NO.In the current study, we used PC12 cells treated with various concentrations of MPP+ as the Parkinson's disease model in vitro. Our objective is to investigate the effect of ADMA on MPP+-induced PC12 cell damage and explore the underlying mechanisms.【Methods】1. The viability of PC12 cells was observed by the trypan blue dye exclusion assay;2. The morphological change of nuclear in apoptotic cell was observed by Hoechst 33258 staining under fluorescence microscope;3. Mitochondrial membrane potential (MMP) was measured using rhodamine 123 (Rh123) probe and the Rh123 fluorescence is visualized under a fluorescence microscope;4. Intracellular reactive oxide species (ROS) was measured using DCFH-DA staining and the DCF fluorescence is analyzed by fluorescence microscope and flow cytometry (FCM) assay;5. The activity of NOS in PC12 cells was evaluated using NOS kit;6. The contents of NO in cell culture media was detected using NO kit;7. The production of H2S and the activity of cystathionine-beta-synthase (CBS) in the PC12 cells were detected by methylene blue spectrophotometric method;8. The expression of cytochrome C (Cyt-c), Bcl-2, paraoxonase-1 (PON1), and 【Results】1. ADMA protects PC12 cells against MPP+-induced cytotoxicityTreatment of PC12 cells with 2 mmol/L MPP+ significantly decreased cell viability. This damage was reversed by ADMA at 40~320μmol/L in a concentration-dependent manner. ADMA at 320μmol/L inhibited MPP+ (2 mmol/L)-induced morphological defect and apoptosis in PC12 cells. These data suggest that ADMA protects PC12 cells against MPP+-induced cytotoxicity.2. The mechanisms of ADMA protection from MPP+-induced injury in PC12 cells.2.1 ADMA prevents MPP+-induced excess of NOS activation and NO generationExposure of PC12 cells to 2 mmol/L MPP+ for 9 h induced a marked increase in NOS activation and NO generation, however, co-treatment with 0.32 mmol/L ADMA significantly attenuated MPP+-induced excess of NOS activation and NO generation.2.2 ADMA protects against MPP+-induced cytotxicity by up-regulation of endgenous H2S generationThe protein expression and activity of CBS in PC12 cell was down-regulated by 2 mmol/L MPP+, but the effect of MPP+ on CBS was inhibited by pre-treatment with 320μmol/L ADMA. The content of H2S in PC12 cells culture medium was decreased by 2 mmol/L MPP+, but the effect of MPP+ on the down-regulation of H2S was inhibited by pre-treatment with 320μmol/L ADMA. Furthermore, amino-oxyacetate (300μmol/L, AOAA), a potent inhibitor of CBS, aggravated the MPP+-induced PC12 cells damage. Those results indicated that ADMA protects PC12 cells by up-regulation of endogenous H2S generation.2.3 ADMA protects PC12 cells may be associated with upregulation of PON1 expression.MPP+ (2 mmol/L)-induced inhibition of PON1 expression was inhibited by pre-treatment with 320μmol/L ADMA. The protective effect of ADMA on cytotoxicity induced by MPP+ in PC12 cells could be significantly attenuated by pre-treatment with 2-HQ (100,200 and 400μmol/L), which inhibited PON1 activation. The data demonstrated that the cytotoxicity of MPP+ could be blocked by ADMA via inhibiting the down-regulation of PON1 caused by MPP+.2.4 ADMA attenuates MPP+-induced increases in intracellular level of reactive oxygen species (ROS)Exposure of PC12 cells to 2 mmol/L MPP+ increased markedly the levels of intracellular reactive oxygen species (ROS). However, after co-treatment with 320μmol/L ADMA, the levels of intracellular ROS in PC12 cells were attenuated significantly, compared with that of MPP+-treated alone group.2.5 ADMA prevents MPP+-induced mitochondrial apoptotic pathwayWhen PC12 cells were exposed to 2 mmol/L MPP+ for 9 h, the mitochondrial membrane potential (MMP) was obviously reduced, as shown by the decrease in the Rh123 fluorescence intensity compared with non-treated control cell. Pretreatment with 320μmol/L ADMA ameliorated the loss of MMP caused by 2 mmol/L MPP+.The expression of Bcl-2 in PC12 cell was down-regulated by 2 mmol/L MPP+, but the down-regulatory effect of MPP+ on Bcl-2 expression was inhibited by pre-treatment with 320μmol/L ADMA, indicated that the cytotoxicity of MPP+ could be inhibited by ADMA via the up-regulation of Bcl-2.The expression of Cyt-c in PC12 cell was up-regulated by 2 mmol/L MPP+, but the up-regulatory effect of MPP+on Cyt-c expression was inhibited by pre-treatment with 320μmol/L ADMA, indicated that the cytotoxicity of MPP+ could be inhibited by ADMA via the down -regulation of Cyt-c.【Conclusion】ADMA protects PC12 cells against MPP+-induced neurotoxicity by up-regulating Bcl-2 expression and inhibiting MPP+-induced mitochondrial apoptotic pathway via attenuating ROS production, as the results of the decreases in MPP+-induced excess activation of NOS and the overproduction of NO, the increase in endogenous H2S generation, and the upregulation of PON1 expression.