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

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 Ca²⁺ 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 Ca²⁺ influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca²⁺-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 μM 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 Ca²⁺ concentration beneath the NMDA-R, [Ca²⁺]_NR, was determined to be 5.4 μM by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 μM NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca²⁺ concentration around nNOS, [Ca²⁺]_NOS, was obtained over the wide range of [Ca²⁺]_NOS. This sigmoidal curve had an EC₅₀ of approximately 1.2 μM of [Ca²⁺]_NOS, implying that [Ca²⁺]_NR = 5.4 μM can activate nNOS effectively.