The Role of ATP on Reactive Oxygen Species Generation and Cellular Proliferation in BV-2 Cells

목 적 : Microglia에서 ATP는 세포증식을 포함한 여러 세포반응을 유발한다. 그러나 micorglia의 반응성 산소종(ROS) 생성에 있어서 purinoceptor의 역할은 분명하지 않다. 저자들은 micorglia에서 purinoceptor의 기능을 알기 위하여 흰쥐 BV-2 micorglia에서 ATP가 산소유리기 생성 및 세포증식에 미치는 영향을 조사하였다. 또한 여러 가지 억제제를 사용하여 신호전달기전을 규명하였다. 방 법 : 세포내 유리 칼슘은 칼슘에 민감한 지시자인 Fura-2/AM을 이용하였고, 반응성 산소종의 측정은 2,7-dichlorofluorescein(DCF) probe와 confocal laser scanning microscope을 이용하여 측정하였으며, 세포증식은 염색을 통해 세포수를 실측정하였다. 결 과 : ATP는 micorglia의 세포내 유리 칼슘을 농도-의존성 방식(concentration-dependent manner)을 통해 증가시켰다. ATP에 의한 세포내 유리 칼슘의 증가는 EGTA 전처지를 통한 Ca²+-free condition과 protein kinase C(PKC)의 억제제인 U-73122의 전처치에 의해서 감소하였다. ATP(0.1 mM)는 microglia의 산소 유리기의 생성을 유발하였으며, uridine triphosphate(UTP)와 adenosine은 산소 유리기의 생성을 유발하지 않았다. 산소 유리기의 생성은 EGTA, U-73122, P2 recrptor antagonist(suramin), P2X7 receptor blocker(KN67), protein kinase C(PKC) inhibitor(sphingosine) 그리고 NADPHoxidase inhibitor(DPI)에 의해서 억제되었다. ATP(0.1 mM)는 microglia의 유의한 세포증식을 유발하였다. NF-κB 활성은 0.3 mM에서 최고치가 되었다. 결 론 : 이상과 같은 결과는 ATP가 microglia에서 반응성 산소종의 생성과 세포증식의 효과를 나타내며 이러한 반응에는 calcium, CaM kinase Ⅱ, PLC, protein kinase C, phospholipase A₂ 및 NADPH oxidase가 관여함을 알 수 있었다.

Purpose : Adenosine triphosphate (ATP) evokes several cellular responses in microglia including propagation. However, the role of the purinoceptor on ROS generation in microglia is unclear. In order to determine the action of the purinoceptor in microglia, the effects of ATP on ROS generation and cellular proliferation in BV-2 murine microglial cells were evaluated. An additional aim of this study was to investigate signal transduction pathways using several inhibitors. Methods : The [Ca²+] was measured using Ca²+ sensitive indicator, Fura-2/AM. ROS production was observed by fluorescence-confocal microscope and cell proliferation was evaluated by counting cell number. Results : ATP increased the intracellular calcium levels ([Ca²+]i) in BV-2 cells in a dose-dependent manner. This increase was attenuated by pretreatment with a calcium chelator (EGTA) and a phospholipase C (PLC) inhibitor (U-73122) while the protein tyrosine kinase (PTK) inhibitor (genistein) had no inhibitory effects. To identify the effects of the nucleotides, ROS generation was observed in the nucleotide-stimulated BV-2 cells. The treatment with 100 M ATP induced ROS generation, but 100 M adenosine and 100 M UTP did not. To investigate the signal transduction pathway in ATP-induced ROS generation, several inhibitors were pretreated before adding ATP. ATP- induced ROS production was blocked by pretreatment with either 0.5 mM EGTA or 10 M U73122 while 40 M genistein had an inhibitory effect on ATP action. Correspondingly, 40 M KN62 (CaM kinase II inhibitor), 1 M sphingosine (protein kinase C inhibitor), 1 nM DPI (NADPH oxidase inhibitor) and 50 M mepacrine (phospholipase A₂ inhibitor) could suppress ATP-induced ROS generation. The effects of ATP on cell proliferation was observed 3 days after ATP treatment and its peak velocity after 4 days. NF-κB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-κB activation was obtained with 0.3 mM ATP while higher concentrations were less effective. Conclusion : Overall, we conclude that ATP in BV-2 cells induces ROS generation and cell propagation. The signal transduction pathways including calcium, CaM kinase II, PLC, protein kinase C, phospholipase A₂ and NADPH oxidase are involved in ATP-induced ROS generation.