Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation

Jian Ming Mei, Wei Ming Chi, Benjamin F. Trump, Christine U. Eccles

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22 Citations (Scopus)


Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca2+]i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca2+]i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca2+]i, which returned to near basal levels on restoration of O2-containing medium. Prolonged hypoxic conditions (>60 min) caused irreversible elevation of [Ca2+]i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake ("point of no return"). Pretreatment with NG-nitro-l-arginine methyl ester (l-NAME, 100 μM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca2+]i. The hypoxia-induced elevation of [Ca2+]i was also greatly attenuated if l-NAME (100 μM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca2+]i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the "point of no return", as characterized above, greatly potentiated [Ca2+]i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca2+]i and subsequent cell death, was not blocked by NOS inhibitor, l-NAME.

Original languageEnglish
Pages (from-to)155-166
Number of pages12
JournalMolecular and Chemical Neuropathology
Issue number2
Publication statusPublished - Feb 1996
Externally publishedYes


  • NO synthase inhibitor
  • Nitric oxide (NO)
  • cerebellar granule cells
  • combined glucose-oxygen deprivation (CGOD)
  • cytosolic calcium
  • fura-2 AM/fura-2, deregulation

ASJC Scopus subject areas

  • Clinical Neurology
  • Molecular Biology
  • General Neuroscience


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