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Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production
Author(s) -
Cossenza Marcelo,
Cadilhe Daniel V.,
Coutinho Rodrigo N.,
PaesdeCarvalho Roberto
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.1471-4159.2006.03843.x
Subject(s) - nmda receptor , arginine , biochemistry , intracellular , cycloheximide , nitric oxide , extracellular , biology , protein biosynthesis , chemistry , receptor , amino acid , endocrinology
The synthesis of nitric oxide (NO) is limited by the intracellular availability of l ‐arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular l ‐arginine which supports an increase in the production of NO. Although l ‐[ 3 H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin‐layer chromatography analysis showed that free intracellular l ‐[ 3 H]arginine was six times higher in CHX‐treated than in control cultures. Extracellular l ‐[ 3 H]citrulline levels increased threefold in CHX‐treated groups, an effect blocked by N G ‐nitro‐ l ‐arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular l ‐[ 3 H]arginine in control cultures, an effect blocked by the NMDA antagonist 2‐amino 5‐phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40–50% in the incorporation of 35 [S]methionine or l ‐[ 3 H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular l ‐arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin‐dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin‐dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.