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1.
Neurochem Res ; 35(7): 1003-9, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20213344

RESUMEN

The present study tests the hypothesis that hyperoxia results in increased tyrosine phosphorylation of apoptotic proteins Bcl-2, Bcl-xl, Bax & Bad in the mitochondrial fraction of the cerebral cortex of newborn piglets. Twelve newborn piglets were divided into normoxic [Nx, n = 6], exposed to a FiO(2) of 0.21 for 1 h and hyperoxic [Hyx, n = 6], exposed to FiO(2) of 1.0 for 1 h. PaO(2) in Hyx group was maintained at 400 mmHg while the Nx group was kept at 80 to 100 mmHg. The density (O.D.x mm(2)) of phosphorylated Bcl2 protein on westernblot was 19.3 +/- 3.6 in Nx and 41.5 +/- 18.3 in Hyx, (P < 0.05). The density of phosphorylated Bcl-xl protein density was 26.9 +/- 7.0 in Nx and 47.9 +/- 2.5 in Hyx, (P < 0.05). Phosphorylated Bax density was 43.5 +/- 5.0 in Nx and 43.3 +/- 5.2 in Hyx. Phosphorylated Bad density was 23.6 +/- 3.9 in Nx, 24.4 +/- 4.7 in Hyx. The data show that during hyperoxia there is a significant increase in tyrosine phosphorylation of Bcl2 and Bcl-xl, while the phosphorylation of proapototic proteins Bax & Bad was not altered. We conclude that hyperoxia leads to post translational modification of anti apoptotic proteins Bcl2 and Bcl-xl in cerebral cortical mitochondria. We propose that phosphorylation of Bcl2 will result in loss of its antiapoptotic potential by preventing its dimerization with Bax leading to activation of the caspase pathway and subsequent neuronal death in the cerebral cortex of the newborn piglets.


Asunto(s)
Corteza Cerebral/metabolismo , Hiperoxia/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tirosina/metabolismo , Animales , Animales Recién Nacidos , Fosforilación , Porcinos
2.
Neuroscience ; 141(3): 1339-49, 2006 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-16777344

RESUMEN

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.


Asunto(s)
Caspasas/metabolismo , Corteza Cerebral/patología , Fragmentación del ADN/fisiología , Expresión Génica/fisiología , Hipoxia/patología , Mitocondrias/metabolismo , Óxido Nítrico/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Western Blotting/métodos , Caspasa 9 , Activación Enzimática/fisiología , Peroxidación de Lípido/fisiología , Fosfocreatina/análogos & derivados , Fosfocreatina/metabolismo , Porcinos
3.
Neuroscience ; 140(3): 857-63, 2006 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-16581191

RESUMEN

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.


Asunto(s)
Corteza Cerebral/enzimología , Hipoxia Encefálica/enzimología , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiopatología , Infarto Cerebral/enzimología , Infarto Cerebral/fisiopatología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Hipoxia Encefálica/fisiopatología , Indazoles/farmacología , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Fosfocreatina/metabolismo , Sus scrofa , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología
4.
Neuroscience ; 123(1): 179-86, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-14667452

RESUMEN

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro-L-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic (N-nitro-L-arginine [NNLA]-Hx) 3-5 day old piglets. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance ODxmm(2). ATP levels were 4.57+/-0.45 micromoles/g brain in the Nx group, 1.29+/-0.23 micromoles/g brain in the Hx group (P<0.05 vs Nx) and 1.50+/-0.14 micromoles/g brain in the NNLA-Hx group (P<0.05 vs Nx). PCr levels were 3.77+/-0.36 micromoles/g brain in the Nx group, 0.77+/-0.13 micromoles/g brain in the hypoxic group (P<0.05) and 1.02+/-0.24 in the NNLA-Hx group (P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5+/-53.7 ODxmm(2) in the Nx group as compared with 419.6+/-63.9 ODxmm(2) in the hypoxic group (P<0.001 vs Nx) and 270.0+/-28.7 in the NNLA-Hx group (P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8+/-42.8 ODxmm(2) in the normoxic group as compared with 364.6+/-60.1 ODxmm(2) in the Hx group (P<0.002) and 254.8+/-24.8 in the NNLA-Hx group (P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration of NNLA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated


Asunto(s)
Corteza Cerebral/metabolismo , Hipoxia Encefálica/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/enzimología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Hipoxia Encefálica/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Óxido Nítrico/antagonistas & inhibidores , Porcinos
5.
Neurochem Res ; 28(9): 1351-7, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12938857

RESUMEN

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.


Asunto(s)
Caspasas/metabolismo , Corteza Cerebral , Fragmentación del ADN , Hipoxia , Neuronas , Óxido Nítrico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Caspasa 3 , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Electroforesis en Gel de Agar , Activación Enzimática , Hipoxia/enzimología , Hipoxia/metabolismo , Hipoxia/patología , Inmunohistoquímica , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/patología , Fosfocreatina/metabolismo , Porcinos
6.
Neuroscience ; 119(4): 1023-32, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12831861

RESUMEN

Previous studies have shown that poly (ADP-ribose) polymerase (PARP) and DNA polymerase beta, nuclear enzymes, are associated with cell replication and DNA repair. The present study tests the hypothesis that hypoxia results in increased PARP and DNA polymerase activity in cerebral cortical neuronal nuclei to repair the hypoxia-induced damage to genomic DNA. Studies were conducted in 13 anesthetized and ventilated newborn piglets (age 3-5 days) divided into normoxic (n=5) and hypoxic (n=8) groups. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Following isolation of the cortical neuronal nuclei, the activity of PARP and DNA polymerase beta was determined. During hypoxia, the tissue ATP level decreased by 73% from 4.12+/-0.67 micromol/g brain to 1.12+/-0.34 micromol/g brain, and PCr decreased by 78% from 4.14+/-0.68-0.90+/-0.20 micromol/g brain. In hypoxic neuronal nuclei, PARP activity significantly increased from 5.88+/-0.51 pmol NAD/mg protein/h in normoxic nuclei to 10.04+/-2.02 (P=0.001). PARP activity inversely correlated with tissue ATP (r=0.78) and PCr levels (r=0.81). Administration of N-nitro-L-arginine prior to hypoxia decreased the hypoxia-induced increase in PARP activity by 67%. Endogenous DNA polymerase beta activity increased from 0.96+/-0.13 in normoxic nuclei to 1.39+/-0.18 nmol/mg protein/h in hypoxic nuclei (P<0.005). DNA polymerase beta activity in the presence of exogenous template increased from 1.54+/-0.14 in the normoxic to 2.42+/-0.26 nmol/mg protein/h in the hypoxic group (P<0.005). DNA polymerase beta activity in the presence or absence of template inversely correlated with the tissue ATP (r=0.95 and 0.84, respectively) and PCr levels (r=0.93 and 0.77, respectively). These results demonstrate that the activity of PARP and DNA polymerase beta enzymes increase with the increase in degree of cerebral tissue hypoxia. Furthermore, the results demonstrate a direct correlation between the PARP and the DNA polymerase beta activity. We conclude that tissue hypoxia results in increased PARP and DNA polymerase beta activities indicating activation of DNA repair mechanisms that may result in potential neuronal recovery following hypoxia and the hypoxia-induced increase in PARP activity is NO-mediated.


Asunto(s)
Asfixia Neonatal/enzimología , Corteza Cerebral/enzimología , ADN Polimerasa beta/metabolismo , Reparación del ADN/fisiología , Hipoxia Encefálica/enzimología , Óxido Nítrico/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Asfixia Neonatal/genética , Núcleo Celular/enzimología , Núcleo Celular/genética , Corteza Cerebral/fisiopatología , Daño del ADN/genética , Inhibidores Enzimáticos/farmacología , Humanos , Hipoxia Encefálica/genética , Recién Nacido , NAD/metabolismo , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/etiología , Degeneración Nerviosa/genética , Neuronas/enzimología , Nitroarginina/farmacología , Fosfocreatina/metabolismo , Recuperación de la Función/genética , Sus scrofa , Regulación hacia Arriba/genética
7.
Neuroscience ; 115(3): 985-91, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12435435

RESUMEN

Previous studies have shown that hypoxia results in increased phosphorylation of CREB protein that mediates gene expression including that of the pro-apoptotic gene bax. We also have shown that hypoxia-induced expression of Bax protein is prevented by blocking nitric oxide synthase (NOS). The present study tests the hypothesis that inhibition of NOS by N-nitro-L-arginine (NNLA) will prevent the hypoxia-induced increased phosphorylation of CREB protein in neuronal nuclei of newborn piglets. To test this hypothesis, phosphorylation of CREB protein was assessed by immunoblotting neuronal nuclear proteins from five normoxic (Nx), 10 hypoxic (Hx) and five Hx-NNLA-treated 3-5-day-old piglets. NNLA (40 mg/kg) or saline was infused over 60 min prior to induction of hypoxia. Hypoxia was achieved by reducing the FiO(2) (0.15 to 0.05) for 60 min and documented biochemically by ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were separated on 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, reacted with anti-phosphorylated CREB protein antibody and conjugated with horseradish peroxidase antibody. Protein bands were detected using the enhanced chemiluminescence method and quantitated by imaging densitometry. Protein density was expressed as absorbance (OD)xmm(2). ATP levels (micromol/g brain) were 4.3+/-0.6 in the Nx group, 1.3+/-0.5 in the Hx group (P<0.001) and 1.1+/-0.2 in the Hx-NNLA group (P<0.001 vs. Nx and Hx). Similarly, PCr levels (micromol/g brain) were 3.8+/-0.6 in the Nx group, 0.7+/-0.2 in the Hx group (P<0.001) and 0.6+/-0.1 in the Hx-NNLA group (P<0.001 vs. Nx and Hx). Density of phosphorylated CREB protein (ODxmm(2)) was 134.2+/-52.4 in the Nx group compared to 746.0+/-76.8 in the Hx group (P<0.05) and 491.1+/-40.9 in the Hx-NNLA group (P<0.05 Hx). The data show that NOS inhibition attenuates the hypoxia-induced increase in CREB protein phosphorylation in the cerebral cortex of newborn piglets.


Asunto(s)
Corteza Cerebral/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hipoxia-Isquemia Encefálica/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiopatología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/fisiopatología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Fosforilación/efectos de los fármacos , Porcinos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología
8.
Neuroscience ; 112(4): 869-77, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12088746

RESUMEN

The present study tested the hypothesis that nitration is a mechanism of hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor. To test this hypothesis the effect of hypoxia on the nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor was determined. Furthermore, the effect of administration of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNLA) on the hypoxia-induced nitration of the NMDA receptor subunits as well as the NMDA receptor-mediated Ca2+ influx, an index of NMDA receptor-ion channel function, were determined in cortical synaptosomes. Studies were performed in newborn piglets divided into normoxic, hypoxic and hypoxic-NNLA groups. Hypoxia was induced by decreasing the FiO(2) to 0.07-0.09 for 60 min. Cerebral tissue hypoxia was confirmed by determining the levels of high energy phosphates ATP and phosphocreatine. Nitration of the NMDA receptor subunits was determined by immunoprecipitation using specific antibodies and western blot analysis. NMDA receptor-ion channel-mediated Ca2+ influx was determined using 45Ca2+. There was a significant increase in the nitrated NR1, NR2A and NR2B subunits following hypoxia: 104+/-11 vs. 275+/-18 optical density (OD)xmm(2) for NR1 (P<0.05), 212+/-36 vs. 421+/-16 ODxmm(2) for NR2A (P<0.05) and 246+/-44 vs. 360+/-26 ODxmm(2) for NR2B (P<0.05). This increase in nitrated NR1, NR2A and NR2B subunits of the NMDA receptor was prevented by the administration of NNLA prior to hypoxia (NR1 160+/-19, P=NS, NNLA vs. normoxic; NR2A 304+/-49, P=NS, NNLA vs. normoxic, and NR2B 274+/-19, P=NS, NNLA vs. normoxic). The increase in nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor increased as a function of decreased cerebral high-energy phosphates, ATP and phosphocreatine, during hypoxia. Furthermore, NOS blockade prior to hypoxia resulted in prevention of the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. Our results demonstrate that hypoxia results in increased nitration of the NMDA receptor subunits and that administration of an NOS inhibitor prior to hypoxia prevents the hypoxia-induced nitration of the NMDA receptor subunits as well as the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. We conclude that nitration is a mechanism of modification of the NMDA receptor function during hypoxia in the newborn piglet brain.


Asunto(s)
Corteza Cerebral/metabolismo , Inhibidores Enzimáticos/farmacología , Hipoxia/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Calcio/metabolismo , Corteza Cerebral/enzimología , Hipoxia/enzimología , Óxido Nítrico Sintasa de Tipo I , Pruebas de Precipitina , Porcinos , Sinaptosomas/metabolismo
9.
Neurochem Res ; 26(10): 1163-9, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11700960

RESUMEN

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na+, K+-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO2 of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na+, K+-ATPase activity was determined at 37 degrees C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl2, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na+, K+-ATPase activity. Following peroxynitrite exposure, the activity of Na+, K+-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na+, K+-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na+, K+-ATPase. The results demonstrate that peroxynitrite decreased activity of Na+, K+-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na+, K+-ATPase activity.


Asunto(s)
Corteza Cerebral/enzimología , Hipoxia Encefálica/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ácido Peroxinitroso/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Corteza Cerebral/embriología , Feto , Cobayas , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Valores de Referencia
10.
Pediatr Res ; 50(5): 586-9, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11641452

RESUMEN

Previous studies have shown that severe hypocapnic ventilation [arterial carbon dioxide partial pressure (PaCO(2)) 7-10 mm Hg] in newborn animals results in decreased cerebral blood flow and decreased tissue oxidative metabolism. The present study tests the hypothesis that moderate hypocapnic ventilation (PaCO(2) 20 mm Hg) will result in decreased cerebral oxidative metabolism and nuclear DNA fragmentation in the cerebral cortex of normoxemic newborn piglets. Studies were performed in 10 anesthetized newborn piglets. The animals were ventilated for 1 h to achieve a PaCO(2) of 20 mm Hg in the hypocapnic (H) group (n = 5) and a PaCO(2) of 40 mm Hg in the normocapnic, control (C) group (n = 5). Tissue oxidative metabolism, reflecting tissue oxygenation, was documented biochemically by measuring tissue ATP and phosphocreatine (PCr) levels. Cerebral cortical nuclei were purified, nuclear DNA was isolated, and DNA content was determined. DNA samples were separated, stained, and compared with a standard DNA ladder. Tissue PCr levels were significantly lower in the H group than the C group (2.32 +/- 0.66 versus 3.73 +/- 0.32 micromol/g brain, p < 0.05), but ATP levels were preserved. Unlike C samples, H samples displayed a smear pattern of small molecular weight fragments between 100 and 12,000 bp. The density of DNA fragments was eight times higher in the H group than the C group, and DNA fragmentation varied inversely with levels of PCr (r = 0.93). These data demonstrate that moderate hypocapnia of 1 h duration results in decreased oxidative metabolism that is associated with DNA fragmentation in the cerebral cortex of newborn piglets. We speculate that hypocapnia-induced hypoxia results in increased intranuclear Ca(2+) flux, which causes protease and endonuclease activation, DNA fragmentation, and periventricular leukomalacia in newborn infants.


Asunto(s)
Núcleo Celular/genética , Corteza Cerebral/metabolismo , Fragmentación del ADN , ADN/metabolismo , Hipocapnia/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/irrigación sanguínea , Electroforesis en Gel de Agar , Metabolismo Energético , Fosfocreatina/metabolismo , Flujo Sanguíneo Regional
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