Endogenous Nos Inhibitor Adma Effects And Mechanism Of Anti-glutamic Acid Damage In Pc12 Cells

【BACKGROUD】Glutamate excitotoxicity plays an important role in hypoxic-ischemic brain injury and chronic neurodegenerative diseases. It is very crucial to restrain the glutamate excitotoxicity for treating and preventing these diseases. Compelling evidence showed that excessive activation of nitric oxide synthase (NOS), leading to overproduction of nitric oxide (NO), was involved in glutamate excitotoxicity.Asymmetric dimethylarginine (ADMA), known as an endogenous competitive NOS inhibitor, can inhibit NOS activity and reduce the amount of NO product. On the other hand, it also uncouples NOS to generate reactive oxygen species (ROS) and cause oxidative stress.It remains unclear whether ADMA alleviates the glutamate excitotoxictiy by inhibiting NOS activity and NO product or aggravates the excitotoxicity through NOS uncoupling and ROS accumulation.【OBJECTIVE】In the current study, we used PC12 cells treated with various concentrations of glutamate as an in vitro excitotoxic trauma model. Our objective is to investigate the effect of ADMA on glutamate-induced PC12 cell damage and explore the underlying mechanisms.【METHODS】1 ) Cell viability was measured by 3-(4,5-Dimethylthiazol-2yl)-2,5- Diphenyl Tetrazolium Bromide (MTT) assay;2)Glutamate cytotoxicity was evaluated by Lactate dehydrogenase (LDH) release assay;3)Mitochondrial membrane potential (MMP) was detected by rhodamine 123 (Rh123) staining, followed by photo fluorography and flow cytometric (FCM) analysis ; 4)Intracellular ROS was analyzed by photo fluorography and FCM assay, following dihydrorhodamin123 (DHR) staining;5)NOS activity in PC12 cells was evaluated using NOS kit;6)The contents of NO in cell culture media was detected using NO kit.【RESULTS】1. Excitotoxic effect of glutamate on PC12 cells and underlying mechanism1) PC12 cells were treated with 0.5 mM, 1 mM, 2 mM, 4 mM or 6 mM glutamate for 24 hours and MTT assay was used to evaluate cell viability. The results showed that glutamate, from 1mM to 6mM, decreased cell survival rate in a dose-dependent manner;2)LDH release was significantly increased after PC12 cells were treated with 1 mM, 2 mM or 4 mM glutamate for 24 hours;3)Treatment with 2 mM glutamate for 4 hours resulted in remarkable decrease of MMP;4) Exposure of PC12 cells to 2 mM glutamate for 4 hours led to increase of the accumulation of intra-cellular ROS;5) Treatment with glutamate for 4 hours raised the NOS activity of PC12 cells. NOS activity was increased by 0.6 or 1-fold after 1 mM or 2 mM glutamate treatment, respectively. Compared with 2mM glutamate treatment, 4 mM glutamate resulted in no further increase of the NOS activity;6)The production of NO was significantly increased after PC12 cells were exposed to glutamate for 4 hours. The production of NO treated by 1mM or 2mM glutamate was increased by 0.7 or 2-fold, respectively. Compared with 2mM glutamate treatment, 4mM glutamate led to no further increase in the production of NO.2. Effect of ADMA on glutamate-induced excitotoxicity and underlying mechanism1)Application of ADMA (20μM to 320μM ) didn't significantly change the viability of PC12 cells;2)Pretreatment with 20μM and 40μM ADMA for 30 min attenuated the cytotoxicity led by glutamate (1 mM~6 mM) in PC12 cells;3)Pretreatment with 20μM ADMA for 30 min inhibited LDH release aroused by 1 mM, 2 mM or 4 mM glutamate, respectively;4)Pretreatment with 20μM ADMA ameliorated the loss of MMP caused by 2 mM glutamate;5)Pretreatment with 20μM ADMA decreased ROS accumulation elicited by 2 mM glutamate;6)Pretreatment with 20μM or 40μM ADMA counteracted excessive activation of NOS induced by glutamate;7 ) Pretreatment with 20μM or 40μM ADMA prevented the overproduction of NO created by glutamate.【CONCLUSION】ADMA protects PC12 cells against excitotoxicity induced by glutamate. It probably plays the role by inhibiting NOS activity and attenuating the glutamate-induced overproduction of NO, which results in the decrease of intracellular ROS and preservation of MMP.