J Korean Child Neurol Soc.  2003 Nov;11(2):238-248.

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

• Kim YH
• Cho JK
• Huh PW
• Lim Y
• Rha HK
• Whang KT

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.

Keyword

Microglia; Adenosine triphosphate; Intracellular free calcium; Reactive oxygen species; Cell proliferation; NF-kB

MeSH Terms

Adenosine
Adenosine Triphosphate*
Calcium
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Cell Count
Cell Proliferation*
Egtazic Acid
Genistein
Microglia
NADPH Oxidase
NF-kappa B
Nucleotides
Oxidoreductases
Phospholipases A2
Phosphotransferases
Protein Kinase C
Protein-Tyrosine Kinases
Quinacrine
Reactive Oxygen Species*
Receptors, Purinergic
Signal Transduction
Sphingosine
Type C Phospholipases
Uridine Triphosphate
Adenosine
Adenosine Triphosphate
Calcium
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Egtazic Acid
Genistein
NADPH Oxidase
NF-kappa B
Nucleotides
Oxidoreductases
Phospholipases A2
Phosphotransferases
Protein Kinase C
Protein-Tyrosine Kinases
Quinacrine
Reactive Oxygen Species
Receptors, Purinergic
Sphingosine
Type C Phospholipases
Uridine Triphosphate