Document Detail

NMDA induces NOS 1 translocation to the cell membrane in NGF-differentiated PC 12 cells.
MedLine Citation:
PMID:  12763249     Owner:  NLM     Status:  MEDLINE    
Glutamatergic-mediated nitric oxide (NO) production occurs via the N-methyl-D-aspartic acid (NMDA) postsynaptic density protein 95 (PSD95)-neuronal nitric oxide synthase (NOS1) ternary complex. To determine whether NOS1 is targeted to the membrane subsequent to NMDA receptor activation, we examined the effect of NMDA on NOS1 subcellular localization in nerve growth factor (NGF) differentiated PC12 cells. No effect on cell viability was observed using a range of NMDA concentrations from 500 to 1000 microM. Within 3 min of stimulation with 750 microM NMDA, increased cytoplasmic NOS1 immunostaining was observed with rapid membrane staining thereafter. This was inhibited by NMDAR inhibition with MK801. This observation was confirmed using subcellular fractionation and immunoblotting. Using 4, 5-diaminofluorescein diacetate (DAF2-DA) staining and a diazotization assay, concurrent NO production was observed. When PC 12 cells were co-treated with either NMDA and N(6)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK-801), nitric oxide (NO) generation was inhibited. Stimulation in a calcium-free medium did not increase NO levels. Although no evidence of cytotoxicity was observed utilizing either the MTT assay or measures of apoptosis within the maximal interval of NOS1 translocation, cell viability was reduced following 10 h of continuous NMDA exposure. While it has been shown that NMDA triggers NOS1 activation, these results indicate that NMDAR activation also mediates NOS1 targeting to the membrane. Our data validate that NGF-differentiated PC12 cells may be employed as a useful in vitro model to further study the regulation of NOS1 subsequent to NMDAR activation.
Mark Arundine; Teresa Sanelli; Bei Ping He; Michael J Strong
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Brain research     Volume:  976     ISSN:  0006-8993     ISO Abbreviation:  Brain Res.     Publication Date:  2003 Jun 
Date Detail:
Created Date:  2003-05-23     Completed Date:  2003-07-28     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0045503     Medline TA:  Brain Res     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  149-58     Citation Subset:  IM    
The Department of Pathology, The University of Western Ontario, Rm. 7 OF 10, UC-LHSC, 339 Windermere Road, London, N6A 5C1, Ontario, Canada.
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MeSH Terms
Cell Differentiation
Cell Membrane / enzymology
Cell Survival / drug effects
Excitatory Amino Acid Agonists / pharmacology*
N-Methylaspartate / pharmacology*
Neurons / cytology,  drug effects,  enzymology*
Nitric Oxide / metabolism
Nitric Oxide Synthase / metabolism*
Nitric Oxide Synthase Type I
PC12 Cells
Subcellular Fractions / enzymology
Reg. No./Substance:
0/Excitatory Amino Acid Agonists; 10102-43-9/Nitric Oxide; 6384-92-5/N-Methylaspartate; EC Oxide Synthase; EC Oxide Synthase Type I; EC protein, rat

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