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Molecular and Chemical Neuropathology

, Volume 27, Issue 2, pp 155–166 | Cite as

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
Original Articles

Abstract

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 withN G-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 ifl-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.

Index Entries

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

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Copyright information

© Humana Press Inc 1996

Authors and Affiliations

  • Jian Ming Mei
    • 1
  • Wei Ming Chi
    • 2
  • Benjamin F. Trump
    • 2
  • Christine U. Eccles
    • 1
  1. 1.Department of Pharmaceutical SciencesThe University of Maryland at BaltimoreBaltimore
  2. 2.Department of PathologyThe University of Maryland at BaltimoreBaltimore

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