NMDA receptor regulation of nNOS phosphorylation and induction of neuron death.
Stimulation of NMDA receptors activates neuronal nitric oxide synthase (nNOS) and the production of nitric oxide (NO). Dephosphorylation of nNOS increases nNOS enzymatic activity. We have examined the regulation of nNOS phosphorylation in rat cortical neurons following NMDA receptor activation. We show that nNOS is constitutively phosphorylated and that NMDA receptor activation decreases the level of nNOS phosphorylation by a mechanism that is blocked specifically by NMDA receptor antagonists and inhibitors of the Ca2+-regulated phosphatases calcineurin and PP1/PP2A. Using quantitative digital microscopy, we show that NMDA receptor activation induces the accumulation of nitrotyrosine, a measure of nNOS activity, and TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) positivity, a measure of cell death. A calcineurin inhibitor blocked the increase in both TUNEL and nitrotyrosine positivity. Notably, TUNEL was increased in those neurons that were most strongly positive for nitrotyrosine. We conclude that NMDA receptor activation induces death of neurons by a cell autonomous pathway involving nNOS dephosphorylation by a calcineurin-dependent mechanism.
Rameau
Gerald A
GA
Department of Biochemistry, Howard Hughes Medical Institute, NYU School of Medicine, New York, NY 10016, USA.
Chiu
Ling-Yu
LY
Ziff
Edward B
EB
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D009410:Nerve Degeneration / Q000201:enzymology* / Q000503:physiopathology; D019636:Neurodegenerative Diseases / Q000201:enzymology / Q000503:physiopathology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D016194:Receptors, N-Methyl-D-Aspartate; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases