Journal
CELL BIOLOGY INTERNATIONAL
Volume 25, Issue 7, Pages 593-598Publisher
WILEY
DOI: 10.1006/cbir.2001.0734
Keywords
nitric oxide; confocal microscopy; diaminoanthraquinone; epilepsy; hippocampal neurons; penicillin G; glutamate
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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. (C) 2001 Academic Press.
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