Direct nitric oxide imaging in cultured hippocampal neurons with diaminoanthraquinone and confocal microscopy.
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 increased the intercellular level of glutamate (Glu, 5 min after stimulation) and the intracellular NO production (30 min throughout stimulation). The increase of NO production could be reversed by N(w)-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.
Chen
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Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, The People's Republic of China.
Sheng
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Zheng
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D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000818:Animals; D000880:Anthraquinones / Q000737:chemistry*; D002478:Cells, Cultured; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006624:Hippocampus / Q000737:chemistry*; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods*; D009474:Neurons / Q000737:chemistry / Q000187:drug effects; D009569:Nitric Oxide / Q000032:analysis*; D010400:Penicillin G / Q000494:pharmacology; D051381:Rats; D012680:Sensitivity and Specificity
D000880:Anthraquinones; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D009569:Nitric Oxide; D015763:2-Amino-5-phosphonovalerate; C118201:1,2-diaminoanthraquinone; D010400:Penicillin G