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Enhancement of nitric oxide production by association of nitric oxide synthase with N ‐methyl‐ d ‐aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real‐time fluorescence imaging using nitric oxide sensitive dye
Author(s) -
Ishii Hirotaka,
Shibuya Keisuke,
Ohta Yoshihiro,
Mukai Hideo,
Uchino Shigeo,
Takata Norio,
Rose John A.,
Kawato Suguru
Publication year - 2006
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2006.03656.x
Subject(s) - nmda receptor , chinese hamster ovary cell , nitric oxide , nitric oxide synthase , receptor , biophysics , postsynaptic potential , postsynaptic density , chemistry , biology , biochemistry , endocrinology
The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N ‐methyl‐ d ‐aspartate (NMDA) receptors, via postsynaptic density 95 (PSD‐95) proteins significantly enhances nitric oxide (NO) production. Real‐time single‐cell fluorescence imaging was applied to measure both NO production and Ca 2+ influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA‐R), nNOS, and PSD‐95. We examined the relationship between the rate of NO production and Ca 2+ influx via NMDA receptors using the NO‐reactive fluorescent dye, diaminofluorescein‐FM (DAF‐FM) and the Ca 2+ ‐sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD‐95 (PSD‐95‐YC3.1). The presence of PSD‐95 enhanced the rate of NO production by 2.3‐fold upon stimulation with 100 µ m NMDA in CHO1(+) cells (expressing NMDA‐R, nNOS and PSD‐95) when compared with CHO1(–) cells (expressing NMDA‐R and nNOS lacking PSD‐95). The presence of nNOS inhibitor or NMDA‐R blocker almost completely suppressed this NMDA‐stimulated NO production. The Ca 2+ concentration beneath the NMDA‐R, [Ca 2+ ] NR , was determined to be 5.4 µ m by stimulating CHO2 cells (expressing NMDA‐R and PSD‐95‐YC3.1) with 100 μ m NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca 2+ concentration around nNOS, [Ca 2+ ] NOS , was obtained over the wide range of [Ca 2+ ] NOS . This sigmoidal curve had an EC 50 of approximately 1.2 µ m of [Ca 2+ ] NOS , implying that [Ca 2+ ] NR = 5.4 µ m can activate nNOS effectively.