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 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 microm 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 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm 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 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.
Ishii
Hirotaka
H
Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Tokyo, Japan.
Shibuya
Keisuke
K
Ohta
Yoshihiro
Y
Mukai
Hideo
H
Uchino
Shigeo
S
Takata
Norio
N
Rose
John A
JA
Kawato
Suguru
S
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002135:Calcium-Binding Proteins; D006224:Cricetinae; D000076122:Disks Large Homolog 4 Protein; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005818:Genetic Engineering; D006131:Growth Inhibitors / Q000494:pharmacology; D051528:Guanylate Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D046911:Macromolecular Substances / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D002135:Calcium-Binding Proteins; D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006131:Growth Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; C495639:yellow cameleon; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D051528:Guanylate Kinases; D002118:Calcium