Potentiation of intracellular Ca2+ mobilization by hypoxia-induced NO generation in rat brain striatal slices and human astrocytoma U-373 MG cells and its involvement in tissue damage (original) (raw)
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Neuroscience Letters, 2002
Previous studies have shown that hypoxia results in the generation of nitric oxide (NO) free radicals in the cerebral cortex of newborn animals. The present study tested the hypothesis that NO increases Ca 11 -influx in neuronal nuclei as well as N-methyl-d aspartate (NMDA) receptor-mediated Ca 11 -influx in cortical synaptosomes of newborn piglets. Studies were performed in five normoxic (Nx) and 6 hypoxic (Hx) newborn piglets. Cerebral tissue hypoxia was documented by determining the levels of ATP and phosphocreatine (PCr). 45 Ca 11 -influx was determined in the presence of sodium nitroprusside (SNP, 10 mM), a NO donor, and peroxynitrite (10 mM). In the Hx group, ATP levels decreased to 1.40^0.69 vs 4.27^0.80 mmoles/g brain in the Nx group (P , 0:05). Similarly, PCr levels decreased to 0.91^0.57 vs 3.40^0.99 mmoles/g brain (P , 0:001). Nuclear 45 Ca 11 -influx increased from 3.57^1.46 pmoles/mg protein in Nx nuclei to 8.64^3.50 in Hx nuclei (P , 0:05). SNP increased neuronal nuclear Ca 11 influx in the Nx from 3.57^1.46 to 5.47^2.52 pmoles/mg protein (P , 0:05) but did not affect Ca 11 influx in the Hx group (8.64^3.50 vs. 10.17^4.00 pmoles/mg protein). The level of Ca 11 influx in the presence of SNP in Nx nuclei was similar to that seen in Hx nuclei alone. Peroxynitrite did not affect nuclear Ca 11 -influx in either Nx or Hx group. Synaptosomal Ca 11 -influx in the presence of glu 1 gly was 40^11 pmoles/mg protein in the Nx group and 80^16 pmoles/mg protein in the Hx group (P , 0:05). Both SNP and peroxynitrite increased Ca 11 influx in Nx and Hx synaptosomes. These results show that hypoxia results in increased nuclear and synaptosomal Ca 11 -influx. Further, the data demonstrate that NO increases intranuclear as well as intrasynaptosomal Ca 11 -influx and suggest that during hypoxia, the increase in intranuclear and intraynaptosomal Ca 11 is NO-mediated. We propose that NO-mediated modification (by nitrosylation/nitration) of nuclear membrane high affinity Ca 11 -ATPase and neuronal membrane NMDA receptor, resulting in increased intranuclear and intracellular Ca 11 influx, are potential NO-mediated mechanisms of Hx neuronal injury. q
Brain Research, 2000
Previous studies have shown that during hypoxia, neuronal nuclear high affinity Ca -ATPase activity is increased in the cerebral cortex of newborn piglets. The present study tests the hypothesis that pretreatment with N-nitro-L-arginine (NNLA) will prevent the 21 hypoxia-induced increase in high affinity Ca -ATPase activity in cortical neuronal nuclear membrane of newborn piglets. We also tested 21 the hypothesis that nitration is a mechanism of elevation of the high affinity Ca -ATPase activity during hypoxia. Studies were performed in five normoxic, five hypoxic, and six NNLA-pretreated (40 mg / kg) hypoxic newborn piglets. Cerebral cortical neuronal 21 nuclei were isolated and the high affinity Ca -ATPase activity was determined. Further, normoxic samples were aliquoted into two 21 sub-groups for in vitro nitration with 0.5 mM peroxynitrite and subsequent determination of the high affinity Ca -ATPase activity. The activity increased from 309640 nmol Pi / mg protein / h in the normoxic group to 5206108 nmol Pi / mg protein / h in the hypoxic group (P,0.05). In the NNLA-pretreated group, the activity was 442653 nmol Pi / mg protein / h (P,0.05), which is 25% lower than in the hypoxic group. In the nitrated group the enzyme activity increased to 554659 nmol Pi / mg protein / h (P,0.05). Thus peroxynitrite-21 induced nitration in vitro increased the high affinity Ca -ATPase activity and NNLA administration in vivo partially prevented the 21 hypoxia-induced increase in neuronal nuclear high affinity Ca -ATPase activity. We conclude that the hypoxia-induced increase in 21 nuclear membrane high affinity Ca -ATPase activity is NO-mediated and that nitration of the enzyme is a mechanism of its modification.
Hypoxia-induced expression of neuronal nitric oxide synthase in astrocytes of human corpus callosum
Brain Structure and Function, 2021
Nitric oxide (NO) is a gaseous neurotransmitter largely diffused in the brain; among other functions, it regulates the cerebral blood flow in response to hypoxia. NO can be synthetized by three different isoforms of the enzyme NO synthase: neuronal (nNOS), typical of neurons, endothelial and inducible. The aim of this study was to assess nNOS expression in human corpus callosum (CC) astrocytes, and its relationship with the hypoxia duration. Autoptic samples of CC from adult human subjects have been processed with immunohistochemistry and immunofluorescence using antibodies anti-nNOS and anti-glial fibrillary acidic protein (GFAP), the astrocyte marker. Results demonstrated for the first time the presence of nNOS-immunopositive astrocytes in the human CC. In particular, nNOS-positive astrocytes were absent in subjects deceased after a short hypoxia; their number and labeling intensity, however, increased with hypoxia prolongation. Neuronal NOS immunopositivity of CC astrocytes seems...
Biomolecules, 2021
Brain ischemia is one of the leading causes of disability and mortality worldwide. Nitric oxide (NO•), a molecule that is involved in the regulation of proper blood flow, vasodilation, neuronal and glial activity constitutes the crucial factor that contributes to the development of pathological changes after stroke. One of the early consequences of a sudden interruption in the cerebral blood flow is the massive production of reactive oxygen and nitrogen species (ROS/RNS) in neurons due to NO• synthase uncoupling, which leads to neurotoxicity. Progression of apoptotic or necrotic neuronal damage activates reactive astrocytes and attracts microglia or lymphocytes to migrate to place of inflammation. Those inflammatory cells start to produce large amounts of inflammatory proteins, including pathological, inducible form of NOS (iNOS), which generates nitrosative stress that further contributes to brain tissue damage, forming vicious circle of detrimental processes in the late stage of i...
Journal of Neurochemistry, 2002
Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2concentration ([Ca2~],)homeostasis in cultured hippocampal neurons during 20-mm exposures. In this study, we examine the relationship between late alterations of [Ca2~] homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S-nitrosocysteine (SNOC; 300 iiM) reduced survival by about one half 1 day after 20mm exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2] 1,persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca 2~] elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2d uring the 20-mm SNOC treatment failed to prevent the late [Ca2~] elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2~for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2~-activatedprotease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2~],homeostatic mechanisms, and that the resulting persistent [Ca2~] elevations contribute to the delayed neurotoxicity of NO.
Nitric oxide protects blood-brain barrier in vitro from hypoxia/reoxygenation-mediated injury
Febs Letters, 1998
A cell culture model of blood-brain barrier (BBB, coculture of rat brain endothelial cells with rat astrocytes) was used to investigate the effect of nitric oxide ( c NO) on the damage of the BBB induced by hypoxia/reoxygenation (H/R). Permeability coefficient of fluorescein across the endothelium was used as a marker of BBB tightness. The permeability coefficient increased 5.2 times after H/R indicating strong disruption of the BBB. The presence of the c NO donor S-nitroso-N-acetylpenicillamine (SNAP, 30 W WM), authentic c NO (6 W WM) or superoxide dismutase (50 units/ml) during H/R attenuated H/R-induced increase in permeability. 30 W WM SNAP or 6 W WM c NO did not influence the function of BBB during normoxia, however, severe disruption was observed using 150 W WM of SNAP and more than 24 W WM of c NO. After H/R of endothelial cells, the content of malondialdehyde (MDA) increased 2.3 times indicating radicalinduced peroxidation of membrane lipids. 30 W WM SNAP or 6 W WM authentic c NO completely prevented MDA formation. The results show that c NO may effectively scavenge reactive oxygen species formed during H/R of brain capillary endothelial cells, affording protection of BBB at the molecular and functional level. z 1998 Federation of European Biochemical Societies.
Brain Research, 2011
Cerebral vessels may regulate cerebral blood flow by responding to changes in carbon dioxide (CO 2) through nitric oxide (NO) production. To better determine the role of NO production by human adult cerebral microvascular endothelial cells and human fetal astrocytes under different CO 2 conditions, we studied endothelial cell and astrocyte production of NO under hypo-, normoand hypercapnic conditions. Human cerebral endothelial cell and fetal astrocyte cultures were exposed to hypocapnic (pCO 2 21.7±6.7 mmHg), normocapnic (pCO 2 40.1±0.9 mmHg) and hypercapnic (pCO 2 56.3±8.7 mmHg) conditions. NO production was recorded continuously over 24 hours with stable pH. N-nitro-L-arginine [NLA; a nitric oxide synthase (NOS) inhibitor] and Larginine (substrate for NO production via NOS) were used to further define the role of NOS in chemoregulation. NO levels in endothelial cells increased during hypercapnia by 36% in 8 hours and remained 25% above baseline. NO increase in astrocytes was 30% after 1 hour but returned to baseline at 8 hours. NLA blocked NO increase in endothelial cells under hypercapnia. During hypocapnia, NO levels in the endothelial cells decreased by 30% at 8 hours but were unchanged in astrocytes. L-arginine prevented NO decrease in endothelial cells under hypocapnia. NO changes in the endothelial cells correlated with changes in pCO 2 (R=0.99) and were independent of pH. This study suggests that cerebral endothelial cells and astrocytes release NO under normocapnic conditions and NO production is increased during hypercapnia and decreased during hypocapnia independent of pH. Further, this demonstrates that endothelial cells may play a pivotal role in chemoregulation by modulating NOS activity.
Activation of nitric oxide synthase gene expression by hypoxia in central and peripheral neurons
Molecular Brain Research, 1996
Ž . Ž . In the present study we examined the effects of hypobaric hypoxia on neuronal n and endothelial e nitric oxide synthase NOS Ž . gene expression in the central and peripheral nervous system. Adult rats were exposed either to normoxia room air or to hypobaric Ž . hypoxia 0.4 atm for 4, 12 or 24 h and cerebellum and nodose ganglion representing the central and peripheral neurons, respectively, were removed. Messenger RNAs encoding n-and eNOS as well as b-actin were analyzed by reverse transcriptase polymerase chain Ž . reaction RT-PCR technique. Hypoxia increased nNOS mRNA expression with maximal changes occurring after 12 h wherein mRNA levels were increased by 10.4 " 1.3 and 2 " 0.4 fold in nodose ganglion and cerebellum, respectively. Hypoxia, on the other hand, had no significant effect on eNOS and b-actin mRNA levels. Analysis of nNOS protein and enzyme activity showed near doubling of these variables in both tissues after 24 h of hypoxia, indicating that nNOS protein levels are increased and that the protein is functionally active. These observations demonstrate that 12-24 h of hypobaric hypoxia selectively activates nNOS gene expression, which is reflected in an increase in nNOS protein in central and peripheral neurons. It is suggested that up-regulation of nNOS leads to increased generation of nitric oxide, which in turn may contribute to the readjustments of cardio-respiratory systems during the early stages of chronic hypoxia.
Nitric Oxide, 2003
Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, and NO synthase (NOS) activity assays in adult rats submitted for 30 min to hypoxia, in a hypobaric chamber at a simulated altitude of 38,000 ft (11000 m) (154.9 mm Hg). The cerebral cortex was studied after different survival times, 0 and 24 h, 5, 8, 15, and 30 days of reoxygenation. This situation led to morphological alterations in the large type I interneurons, as well as immunoreactive changes in the appearance and number of the small neurons (type II), both containing neuronal NOS (nNOS). Some of these small neurons showed immunoreactive cytoplasm and short processes; others, the more numerous during all reoxygenation periods, contained the immunoreactive product mainly related to a perinuclear ring. Ultrastructurally, these small neurons exhibited changes in nuclear structures as in the shape of the nuclear membrane, in the distribution of heterochromatin, and in the nucleolar morphology. The reaction product for nitrotyrosine, as a marker of protein nitration, showed modifications in distribution of the immunoreactive product. No expression was found for inducible NOS (iNOS). All these modifications were accompained by increased nNOS and nitrotyrosine production as demonstrated by Western blotting and calcium-dependent activity, returning to control conditions after 30 days of reoxygenation, suggesting a reversible NO mechanism of action.