title: Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase.
abstract: The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na(+) ([Na(+)](e)), a condition that by reducing intracellular Ca(2+) ([Ca(2+)](I)) prevents Ca(2+)-dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as N(omega)-L-allyl-L-arginine (L-ALA), N(omega)-propyl-L-arginine (NPLA), and L-nitro-arginine-methyl-ester (L-NAME), respectively. The results demonstrated that the removal of [Na(+)](e) hampered the [Ca(2+)](i) increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na(+)](e). Furthermore, the absence of [Na(+)](e) in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L-ALA (200 nM), and L-NAME (500 microM) was able to effectively reduce NO(*) production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases.
authors.affiliation: Division of Pharmacology, Department of Neuroscience, School of Medicine, University of Naples Federico II, Naples, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Scorziello
authors.forename: C
authors.initials: C
authors.lastname: Pellegrini
authors.forename: A
authors.initials: A
authors.lastname: Secondo
authors.forename: R
authors.initials: R
authors.lastname: Sirabella
authors.forename: L
authors.initials: L
authors.lastname: Formisano
authors.forename: L
authors.initials: L
authors.lastname: Sibaud
authors.forename: S
authors.initials: S
authors.lastname: Amoroso
authors.forename: L M T
authors.initials: LM
authors.lastname: Canzoniero
authors.forename: L
authors.initials: L
authors.lastname: Annunziato
authors.forename: G F
authors.initials: GF
authors.lastname: Di Renzo
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000201:enzymology* / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology* / Q000503:physiopathology; D004789:Enzyme Activation; D005947:Glucose / Q000172:deficiency* / Q000378:metabolism; D000860:Hypoxia / Q000201:enzymology / Q000503:physiopathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000201:enzymology* / Q000473:pathology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D018384:Oxidative Stress / Q000502:physiology; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D015427:Reperfusion Injury / Q000201:enzymology* / Q000503:physiopathology; D012964:Sodium / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D013778:Tetrazolium Salts; D013844:Thiazoles; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C022616:thiazolyl blue; D005947:Glucose; D010100:Oxygen; D002118:Calcium