Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation.
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 microM), 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 microM) 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.
Mei
J M
JM
Department of Pharmaceutical Sciences, University of Maryland at Baltimore 21201, USA.
Chi
W M
WM
Trump
B F
BF
Eccles
C U
CU
D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects*; D005947:Glucose / Q000172:deficiency*; D000860:Hypoxia / Q000139:chemically induced / Q000473:pathology* / Q000628:therapy; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects*; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D005947:Glucose; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester