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Journal of the Korean Child Neurology Society 2003;11(2):238-248.
Published online November 30, 2003.
The Role of ATP on Reactive Oxygen Species Generation and Cellular Proliferation in BV-2 Cells.
Young Hoon Kim, Jeung Ki Cho, Pil Woo Huh, Young Lim, Hyoung Kyun Rha, Kyung Tae Whang
1Department of Pediatrics, Catholic Neuroscience Center, College of Medicine, The Catholic University of Korea, Seoul, Korea. hkrha@catholic.ac.kr
2Department of Occupational and Environmental Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Abstract
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-kB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-kB 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.
Key Words: Microglia, Adenosine triphosphate, Intracellular free calcium, Reactive oxygen species, Cell proliferation, NF-kB
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