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

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 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
Volume27
Issue number2
DOIs
Publication statusPublished - Feb 1996
Externally publishedYes

Fingerprint

Nitric Oxide
Oxygen
Calcium
Neurons
Glucose
Nitric Oxide Synthase
Fura-2
Argon
Wounds and Injuries
Cell Shape
Propidium
Fluorescence Microscopy
Cell Death
Gases
Cell Membrane

Keywords

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

ASJC Scopus subject areas

  • Neuroscience(all)
  • Clinical Neurology
  • Molecular Biology

Cite this

Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation. / Mei, Jian Ming; Chi, Wei Ming; Trump, Benjamin F.; Eccles, Christine U.

In: Molecular and Chemical Neuropathology, Vol. 27, No. 2, 02.1996, p. 155-166.

Research output: Contribution to journalArticle

@article{d5cfde25bc1b42ccbee92dbe59a1b35d,
title = "Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation",
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 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.",
keywords = "cerebellar granule cells, combined glucose-oxygen deprivation (CGOD), cytosolic calcium, fura-2 AM/fura-2, deregulation, Nitric oxide (NO), NO synthase inhibitor",
author = "Mei, {Jian Ming} and Chi, {Wei Ming} and Trump, {Benjamin F.} and Eccles, {Christine U.}",
year = "1996",
month = "2",
doi = "10.1007/BF02815091",
language = "English",
volume = "27",
pages = "155--166",
journal = "Journal of Molecular Neuroscience",
issn = "0895-8696",
publisher = "Humana Press",
number = "2",

}

TY - JOUR

T1 - Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation

AU - Mei, Jian Ming

AU - Chi, Wei Ming

AU - Trump, Benjamin F.

AU - Eccles, Christine U.

PY - 1996/2

Y1 - 1996/2

N2 - 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.

AB - 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.

KW - cerebellar granule cells

KW - combined glucose-oxygen deprivation (CGOD)

KW - cytosolic calcium

KW - fura-2 AM/fura-2, deregulation

KW - Nitric oxide (NO)

KW - NO synthase inhibitor

UR - http://www.scopus.com/inward/record.url?scp=0029863198&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029863198&partnerID=8YFLogxK

U2 - 10.1007/BF02815091

DO - 10.1007/BF02815091

M3 - Article

VL - 27

SP - 155

EP - 166

JO - Journal of Molecular Neuroscience

JF - Journal of Molecular Neuroscience

SN - 0895-8696

IS - 2

ER -