The influence of oxygen-glucose deprivation on nitric oxide and intracellular Ca(2+) in cultured hippocampal neurons.
Nitric oxide (NO) was speculated to play an important role in the pathophysiology of cerebral ischemia. In this study, the effect of oxygen-glucose deprivation (OGD) on the cellular production of NO was investigated in cultured hippocampal neurons. Intracellular Ca(2+) was also detected as its closely relationship with NO. The generation of NO and changes in intracellular Ca(2+) were evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2 DA), an NO probe, and Fluo-3, a Ca(2+) probe respectively. Extracellular glutamate level was also measured by HPLC with fluorescence detection. Results showed that OGD induced an increase in NO production and intracellular Ca(2+) concentration ([Ca(2+)](i)), the rise of DAF-2 and Fluo-3 fluorescence intensity was about 160% and 270% respectively; an increase of about 100% in glutamate level was observed after 20 min of OGD. NMDA inhibitor MK-801 significantly reduced the OGD-induced elevation of [Ca(2+)](i) and NO, DAF-2 and Fluo-3 fluorescence intensity uptake was inhibited by 69% and 74% respectively. The increase in NO production was also attenuated by extracellular Ca(2+) elimination and calmodulin (CaM) antagonist trifluoperazine dose-dependently. These results indicated that NO production increased during oxygen-glucose deprivation, and was greatly modulated by glutamate release, intracellular Ca(2+) change and Ca(2+)-CaM pathway.
Zhang
Mu
M
Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China.
Ning
Gang-Min
GM
Hong
Di-Hui
DH
Yang
Yong
Y
Kutor
John
J
Zheng
Xiao-Xiang
XX
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D015687:Cell Hypoxia; D002478:Cells, Cultured; D016291:Dizocilpine Maleate / Q000494:pharmacology; D005947:Glucose / Q000172:deficiency*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors
D002147:Calmodulin; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D016291:Dizocilpine Maleate; D005947:Glucose; D002118:Calcium