Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Abstract

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.

• NADPH oxidase
• Rac1
• glutamate
• apoptosis
• reactive oxygen species
• SH-SY5Y cells

Abbreviations
DCFH-DA	=	2′,7′-dichlorofluorescin diacetate
DMSO	=	dimethyl sulfoxide
DPI	=	diphenylene iodonium
NAC	=	N-acetyl-l-cysteine
NDGA	=	nordihydroguaiaretic acid
NMDA	=	N-methyl-d-aspartate
PI	=	propidium iodide
ROS	=	reactive oxygen species
RT-PCR	=	reverse transcription polymerase chain reaction

Abbreviations
DCFH-DA	=	2′,7′-dichlorofluorescin diacetate
DMSO	=	dimethyl sulfoxide
DPI	=	diphenylene iodonium
NAC	=	N-acetyl-l-cysteine
NDGA	=	nordihydroguaiaretic acid
NMDA	=	N-methyl-d-aspartate
PI	=	propidium iodide
ROS	=	reactive oxygen species
RT-PCR	=	reverse transcription polymerase chain reaction