The Influence of Oxygen-Glucose Deprivation on Nitric Oxide and Intracellular Ca²⁺ in Cultured Hippocampal Neurons

ZHANG Mu, NING Gang-Min, HONG Di-Hui, YANG Yong, KUTOR John, ZHENG Xiao-Xiang*

( Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China )

Abstract 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 Ca²⁺ was also detected as its closely relationship with NO. The generation of NO and changes in intracellular Ca²⁺ were evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2 DA), an NO probe, and Fluo-3, a Ca²⁺ 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 Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺ 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 Ca²⁺ change and Ca²⁺-CaM pathway.

Key words nitric oxide; calcium; glutamate; calmodulin; hippocampal neuron; oxygen-glucose deprivation

*Corresponding author: Tel, 86-571-87951091; Fax, 86-571-87951676; e-mail, [email protected]