DIRECT NITRIC OXIDE IMAGING IN CULTURED HIPPOCAMPAL NEURONS WITH DIAMINOANTHRAQUINONE AND CONFOCAL MICROSCOPY

Authors


Department of Biomedical Engineering, Zhejiang University, 20 Zheda Road, Yuquan, Hangzhou 310027, People's Republic of China. Fax: 86-571-7951676; E-mail: zxx@mail.hz.zj.cn

Abstract

A method for the direct measurement of intracellular nitric oxide (NO) production stimulated by penicillin G (PG) in cultured hippocampal neurons with diaminoanthraquinone (DAA) using laser scanning confocal microscopy (LSCM) was developed. Intracellular DAA fluorescence could specifically represent NO production based on two facts: (1) 3-morpholinosydnonimine, a NO donor, could dose-dependently increase DAA fluorescence; and (2) haemoglobin, a NO scavenger, could inhibit the increase of DAA fluorescence. The PG dose-dependently increasd the intercellular level of glutamate (Glu, 5min after stimulation) and the intracellular NO production (30min throughout stimulation). The increase of NO production could be reversed by Nw-nitro- l -arginine (a NO synthase inhibitor), and also by d (−)2-amino-5-phosphonovaleric acid, a subtype of Glu receptor antagonist. These results revealed that DAA could be used to indicate real-time and kinetic intracellular NO production of hippocampal neurons with higher sensitivity, specificity and accuracy.

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