Effect of Graded Hypoxia on Cerebral Cortical Genomic DNA Fragmentation in Newborn Piglets

Akhter, Waseem; Ballesteros, Juan R.; Kubin, Joanna; Mishra, Om P.; Delivoria‐Papadopoulos, Maria

doi:10.1203/00006450-199904020-02013

Cited by 12 publications

(17 citation statements)

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“…Upon reventilation/reoxygenation, the high energy phosphate levels and the transmembrane ionic gradients are gradually restored, but the initially still increased intracellular and intramitochondrial Ca 2 + levels will activate a host of intracellular proteases and nucleases, as well as enzymatic and non-enzymatic synthesis of reactive oxygen species (ROS). These alterations will trigger mitochondrial dysfunction [17] and DNA fragmentation [22] setting the stage for further delayed and programmed neuronal cell death during the secondary energy failure stage. Based on the above delineated sequence of events, rational neuroprotective therapies aimed to mitigate neuronal injury of HIE should focus on (1) shortening the duration of asphyxia, (2) administration of cytoprotective/antioxidant agents simultaneously with the resuscitation to limit reoxygenation/reperfusion injury, and (3) to implement neuroprotective interventions before the neuronal injury related to secondary energy failure occurs.…”

Section: Discussionmentioning

confidence: 99%

Active forms of Akt and ERK are dominant in the cerebral cortex of newborn pigs that are unaffected by asphyxia

et al. 2018

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Section: Discussionmentioning

confidence: 99%

Active forms of Akt and ERK are dominant in the cerebral cortex of newborn pigs that are unaffected by asphyxia

et al. 2018

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“…Programmed cell death is also assessed by cleavage of genomic DNA and has been shown to occur in the brain following ischemia and oxygen deprivation [54,58,59]. In the newborn piglet, fragmentation of neuronal genomic DNA occurs following 1 h of hypoxia, and the degree of fragmentation correlates with the severity of cerebral hypoxia [60]. Two distinct patterns of DNA degradation during hypoxic-ischemic brain cell death have been observed [61].…”

Section: Discussionmentioning

confidence: 98%

Mechanism of Increased Tyrosine (Tyr99) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

Mishra

Ashraf

Delivoria‐Papadopoulos

2009

Neurochem Res

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The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr(99) phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr(99) and total) of calmodulin determined by Western blot using anti-phospho-(pTyr(99))-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr(99) calmodulin (ODxmm(2)) was 78.55 +/- 10.76 in Nx, 165.05 +/- 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 +/- 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 +/- 13.69 in Nx, 156.17 +/- 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 +/- 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr(99) phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr(99) phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr(99) phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.

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“…Programmed cell death is also assessed by cleavage of genomic DNA and has been shown to occur in the brain following ischemia and oxygen deprivation [57,61,62]. In the newborn piglet, fragmentation of neuronal genomic DNA occurs following 1 h of hypoxia, and the degree of fragmentation correlates with the severity of cerebral hypoxia [63]. Two distinct patterns of DNA degradation during hypoxic-ischemic brain cell death have been observed [64].…”

Section: Discussionmentioning

confidence: 98%

Mechanism of Post-Translational Modification by Tyrosine Phosphorylation of Apoptotic Proteins During Hypoxia in the Cerebral Cortex of Newborn Piglets

Delivoria‐Papadopoulos

Mishra

2009

Neurochem Res

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The present study aims to investigate the mechanism of phosphorylation of apoptotic proteins and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of apoptotic proteins Bcl-2 and Bcl-xl is Ca(2+)-influx-dependent. Piglets were divided in normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-pretreated with clonidine (Clo + Hx, n = 4) groups. Hypoxic animals were exposed to an FiO(2) of 0.06 for 1 h. Clonidine (12.5 microg/kg, IV) was administered to piglets 30 min prior to hypoxia. Hypoxia was confirmed by ATP and phosphocreatinine (PCr) levels. Cytosol was isolated and separated by 12% SDS-PAGE and probed with tyrosine phosphorylated (p) -Bax, Bad, Bcl-2 and Bcl-xl antibodies and bands were detected. The ATP levels (micromol/g brain) in the Nx, Hx, Clo + Hx were 4.3 +/- 1.0 (P < 0.05 vs. Hx, Clo-Hx), 0.9 +/- 0.8 and 1.5 +/- 0.3, respectively. The PCr levels in the Nx, Hx, Clo + Hx were 2.7 +/- 0.7 (P < 0.05 vs. Hx, Clo-Hx), 0.9 +/- 0.2 and 0.9 +/- 0.9, respectively. Ca(2+)-influx (pmoles/mg protein) was 4.96 +/- 0.94 in Nx, 11.11 +/- 2.38 in Hx, and 6.23 +/- 2.07 in Clo + Hx (P < 0.05 Nx vs. Hx and Hx vs. Clo + Hx). p-Bcl-2 density was 21.1 +/- 1.1 Nx, 58.9 +/- 9.6 Hx and 29.5 +/- 6.4 Clo + Hx (P < 0.05 vs. Hx). p-Bcl-xl density was 29.6 +/- 1.5 Nx, 50.6 +/- 7.4 Hx and 32.1 +/- 0.1 Clo + Hx (P < 0.05 vs. Hx). p-Bax density was 38.6 +/- 16.2 Nx, 46.1 +/- 5.5 Hx and 41.6 +/- 1.9 Clo + Hx groups (P = NS). p-Bad was 66.7 +/- 12.8 Nx, 71.2 +/- 6.8 Hx and 78.7 +/- 22.5 Clo + Hx groups (P = NS). Results showed that clonidine administration prior to hypoxia prevents the hypoxia-induced increased nuclear Ca(2+)-influx and increased phosphorylation of Bcl-2 and Bcl-xl while phosphorylation of Bad and Bax was not altered. We conclude that post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl during hypoxia is nuclear Ca(2+)-influx-dependent. We propose that blockade of nuclear Ca(2+)-influx that prevents phosphorylation of antiapoptotic proteins may become a neuroprotective strategy.

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Effect of Graded Hypoxia on Cerebral Cortical Genomic DNA Fragmentation in Newborn Piglets

Cited by 12 publications

References 0 publications

Active forms of Akt and ERK are dominant in the cerebral cortex of newborn pigs that are unaffected by asphyxia

Active forms of Akt and ERK are dominant in the cerebral cortex of newborn pigs that are unaffected by asphyxia

Mechanism of Increased Tyrosine (Tyr99) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

Mechanism of Post-Translational Modification by Tyrosine Phosphorylation of Apoptotic Proteins During Hypoxia in the Cerebral Cortex of Newborn Piglets

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