Neurochemical Research

, Volume 35, Issue 1, pp 76–84 | Cite as

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

  • Maria Delivoria-Papadopoulos
  • Om Prakash Mishra
Original Paper
First Online:
Received:
Accepted:

Abstract

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 Ca2+-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 FiO2 of 0.06 for 1 h. Clonidine (12.5 μg/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 (μmol/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. Ca2+-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 Ca2+-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 Ca2+-influx-dependent. We propose that blockade of nuclear Ca2+-influx that prevents phosphorylation of antiapoptotic proteins may become a neuroprotective strategy.

Keywords

Apoptotic proteins Bcl-2 Bcl-xl Tyrosine phosphorylation Hypoxia EGFR kinase 
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Notes

Acknowledgments

This study was supported by the National Institute of Health grants HD-20337 and R56 HD038079. The authors thank Mrs. Anli Zhu for her expert technical assistance.

References

  1. 1.
    Hu X, Qiu J, Grafe MR et al (2003) Bcl-2 family members make different contributions to cell death in hypoxia and/or hyperoxia in rat cerebral cortex. Int J Dev Neurosci 21:371–377CrossRefPubMedGoogle Scholar
  2. 2.
    Vexler ZS, Ferriero DM (2001) Molecular and biochemical mechanisms of perinatal brain injury. Semin Neonatol 6:99–108CrossRefPubMedGoogle Scholar
  3. 3.
    Ashraf QM, Zanelli SA, Mishra OP et al (2001) Phosphorylation of Bcl-2 and Bax proteins during hypoxia in newborn piglet. Neurochem Res 26:1–9CrossRefPubMedGoogle Scholar
  4. 4.
    Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619CrossRefPubMedGoogle Scholar
  5. 5.
    Ravishankar S, Ashraf QM, Fritz KI, Mishra OP, Delivoria-Papadopoulos M (2001) Expression of Bax and Bcl-2 proteins during hypoxia in cerebral cortical neuronal nuclei of newborn piglets: effect of administration of magnesium sulfate. Brain Res 901:23–29CrossRefPubMedGoogle Scholar
  6. 6.
    Ito T, Deng X, Carr B et al (1997) Bcl-2 phosphorylation required for anti-apoptosis function. J Biol Chem 272:11671–11673CrossRefPubMedGoogle Scholar
  7. 7.
    Haldar S, Chintapalli J, Croce CM (1996) Taxol induces Bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 56:1253–1255PubMedGoogle Scholar
  8. 8.
    Haldar S, Jena N, Croce CM (1995) Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci USA 92:4507–4511CrossRefPubMedGoogle Scholar
  9. 9.
    Mishra OP, Delivoria-Papadopoulos M (1992) NMDA receptor modification of the fetal guinea pig brain during hypoxia. Neurochem Res 17:1211–1216CrossRefPubMedGoogle Scholar
  10. 10.
    Hoffman DJ, DiGiacomo JE, Marro PJ, Mishra OP, Delivoria-Papadopoulos M (1994) Hypoxia-induced modification of the N-methyl-d-aspartate (NMDA) receptor in the brain of newborn piglets. Neurosci Lett 167:156–160CrossRefPubMedGoogle Scholar
  11. 11.
    Razdan B, Kubin JA, Mishra OP, Delivoria-Papadopoulos M (1996) Modification of the glycine (co-activator) binding site of the N-methyl-d-aspartate receptor in the guinea pig fetus brain during development following hypoxia. Brain Res 733:15–20PubMedGoogle Scholar
  12. 12.
    Zanelli S, Numagami Y, McGowan JE, Mishra OP, Delivoria-Papadopoulos M (1999) NMDA receptor mediated calcium influx in cerebral cortical synaptosomes of the hypoxic guinea pig fetus. Neurochem Res 24:437–446CrossRefPubMedGoogle Scholar
  13. 13.
    Mishra OP, Zanelli S, Ohnishi ST, Delivoria-Papadopoulos M (2000) Hypoxia-induced generation of nitric oxide free radicals in cerebral cortex of newborn guinea pigs. Neurochem Res 25:1559–1565CrossRefPubMedGoogle Scholar
  14. 14.
    Zanelli SA, Ashraf QM, Delivoria-Papadopoulos M, Mishra OP (2000) Peroxynitrite-induced modification of the NMDA receptor in the cerebral cortex of the guinea pig fetus at term. Neurosci Lett 296:5–8CrossRefPubMedGoogle Scholar
  15. 15.
    Zanelli S, Ashraf QM, Mishra OP (2002) Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia. Neurosci Lett 112:869–877Google Scholar
  16. 16.
    Mishra OP, Delivoria-Papadopoulos M (2002) Nitric oxide-mediated Ca2+-influx in neuronal nuclei and cortical synaptosomes of normoxic and hypoxic newborn piglets. Neurosci Lett 318:93–97CrossRefPubMedGoogle Scholar
  17. 17.
    Zubrow AB, Delivoria-Papadopoulos M, Ashraf QM, Fritz KI, Mishra OP (2002) Nitric oxide-mediated Ca2+/calmodulin-dependent protein kinase IV activity during hypoxia in neuronal nuclei from newborn piglets. Neurosci Lett 335:5–8CrossRefPubMedGoogle Scholar
  18. 18.
    Mishra OP, Ashraf QM, Delivoria-Papadopoulos M (2002) Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition. Neuroscience 115:985–991CrossRefPubMedGoogle Scholar
  19. 19.
    Zubrow AB, Delivoria-Papadopoulos M, Ashraf QM, Ballesteros JR, Fritz KI, Mishra OP (2002) Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets. Brain Res 954:60–67CrossRefPubMedGoogle Scholar
  20. 20.
    Mishra OP, Akhter W, Ashraf QM, Delivoria-Papadopoulos M (2003) Hypoxia-induced modification of poly(ADP-ribose)polymyrase and DNA polymerase B activity in cerebral cortical nuclei of newborn piglets: role of nitric oxice. Neuroscience 119:1023–1032CrossRefPubMedGoogle Scholar
  21. 21.
    Parikh NA, Katsetos CD, Ashraf QM, Haider SH, Legido A, Delivoria-Papadopoulos M, Mishra OP (2003) Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets. Role of nitric oxide. Neurochem Res 28:1351–1357CrossRefPubMedGoogle Scholar
  22. 22.
    Mishra OP, Delivoria-Papadopoulos M (2007) NO-mediated mechanisms of hypoxic brain injury in newborns. In: Mishra OP (ed) Mechanisms of hypoxic brain injury in the newborn and potential strategies for neuroprotection. Signpost Publishers, Trivandrum, pp 157–171Google Scholar
  23. 23.
    Delivoria-Papadopoulos M, Akhter WA, Mishra OP (2003) Hypoxia-induced Ca2+-influx in cerebral cortical neuronal nuclei of newborn piglets. Neurosci Lett 342:119–123CrossRefPubMedGoogle Scholar
  24. 24.
    Mishra OP, Delivoria-Papadopoulos M (2001) Effect of graded hypoxia on high affinity Ca2+-ATPase activity in cortical neuronal nuclei of newborn piglets. Neurochem Res 26:1335–1341CrossRefPubMedGoogle Scholar
  25. 25.
    Hornick K, Chang E, Zubrow AB, Mishra OP, Delivoria-Papadopoulos M (2007) Mechanism of Ca(2+)/calmodulin-dependent protein kinase IV activation and of cyclic AMP response element binding protein phosphorylation during hypoxia in the cerebral cortex of newborn piglets. Brain Res 1150:40–45CrossRefPubMedGoogle Scholar
  26. 26.
    Timmons SD, Geisert E, Stewart AE, Lorenzon NM, Foehring RC (2004) Alpha2-adrenergic receptor-mediated modulation of calcium current in neocortical pyramidal neurons. Brain Res 1014:184–196CrossRefPubMedGoogle Scholar
  27. 27.
    Wang WZ, Yuan WJ, Pan YX, Tang CS, Su DF (2004) Interaction between clonidine and N-methyl-d-aspartate receptors in the caudal ventrolateral medulla of rats. Exp Brain Res 158:259–264CrossRefPubMedGoogle Scholar
  28. 28.
    Gorini A, Villa RF (2001) Effect of in vivo treatment of clonidine on ATP-ase’s enzyme systems of synaptic plasma membranes from rat cerebral cortex. Neurochem Res 26:821–827CrossRefPubMedGoogle Scholar
  29. 29.
    Lowry O, Rosenbrough NJ, Farr A, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  30. 30.
    Lamprecht W, Stein P, Heinz F, Weissner H (1974) Creatine phosphate. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 4. Academic Press, New York, pp 1777–1781Google Scholar
  31. 31.
    du Plessis AJ, Volpe JJ (2002) Perinatal brain injury in the preterm and term newborn. Curr Opin Neurol 15:151–157CrossRefPubMedGoogle Scholar
  32. 32.
    Salhab WA, Perlman JM (2005) Severe fetal acidemia and subsequent neonatal encephalopathy in the larger premature infant. Pediatr Neurol 32:25–29CrossRefPubMedGoogle Scholar
  33. 33.
    Barkovich AJ, Sargent SK (1995) Profound asphyxia in the premature infant: imaging findings. Am J Neuroradiol 16:1837–1846PubMedGoogle Scholar
  34. 34.
    Gunn AJ (2000) Cerebral hypothermia for prevention of brain injury following perinatal asphyxia. Curr Opin Pediatr 12:111–115CrossRefPubMedGoogle Scholar
  35. 35.
    Bennet L, Roelfsema V, George S, Dean JM, Emerald BS, Gunn AJ (2007) The effect of cerebral hypothermia on white and grey matter injury induced by severe hypoxia in preterm fetal sheep. J Physiol 578:491–506CrossRefPubMedGoogle Scholar
  36. 36.
    Dean JM, Gunn AJ, Wassink G, George S, Bennet L (2006) Endogenous alpha(2)-adrenergic receptor-mediated neuroprotection after severe hypoxia in preterm fetal sheep. Neuroscience 142:615–628CrossRefPubMedGoogle Scholar
  37. 37.
    Anteir D, Franconi F, Sannajust F (1999) Idazoxandoes not prevent but worsens focal hypoxic–ischemic brain damage in neonatal wistar rats. J Neurosci Res 58:690–696CrossRefGoogle Scholar
  38. 38.
    Laudenbach V, Mantz J, Lagercrantz H, Desmonts JM, Evrard P, Gressens P (2002) Effects of alpha2-adrenoceptor agonists on perinatal excitotoxic brain injury: comparison of clonidine and dexmedetomidine. Anesthesiology 96:134–141CrossRefPubMedGoogle Scholar
  39. 39.
    Paris A, Mantz J, Tonner PH, Hein L, Brede M, Gressens P (2006) The effects of dexmedetomidine on perinatal excitotoxic brain injury are mediated by the alpha2A-adrenoceptor subtype. Anesth Analg 102:456–461CrossRefPubMedGoogle Scholar
  40. 40.
    Ma D, Hossain M, Rajakumaraswamy N, Arshad M, Sanders RD, Franks NP, Maze M (2004) Dexmedetomidine produces its neuroprotective effect via the alpha(2A)-adrenoceptor subtype. Eur J Pharmacol 502:87–97CrossRefPubMedGoogle Scholar
  41. 41.
    Yuan SZ, Runold M, Hagberg H, Bona E, Lagercrantz H (2001) Hypoxic–ischaemic brain damage in immature rats: effects of adrenoceptor modulation. Eur J Paediatr Neurol 5:29–35CrossRefPubMedGoogle Scholar
  42. 42.
    Dean JM, George S, Naylor AS, Mallard C, Gunn AJ, Bennet L (2008) Partial neuroprotection with low dose infusion of the α2-adrenergic receptor agonist clonidine after severe hypoxia in preterm fetal sheep. Neuropharmacology 55:166–174CrossRefPubMedGoogle Scholar
  43. 43.
    Jellish WS, Murdoch J, Kindel G, Zhang X, White FA (2005) The effect of clonidine on cell survival, glutamate, and aspartate release in normo- and hyperglycemic rats after near complete forebrain ischemia. Exp Brain Res 167:526–534CrossRefPubMedGoogle Scholar
  44. 44.
    Kamisaki Y, Hamahashi T, Hamada T, Maeda K, Itoh T (1992) Presynaptic inhibition by clonidine of neurotransmitter amino acid release in various brain regions. Eur J Pharmacol 217:57–63CrossRefPubMedGoogle Scholar
  45. 45.
    Bickler PE, Hansen BM (1996) Alpha 2-adrenergic agonists reduce glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices during hypoxia. Neuropharmacology 35:679–687CrossRefPubMedGoogle Scholar
  46. 46.
    Barret WE, Degnore JP, Keng YF, Zang ZY, Yim MB, Chock PB (1999) Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase IB. J Biol Chem 49:34543–34546CrossRefGoogle Scholar
  47. 47.
    Lee SR, Kwan KS, Kim SR, Rhu SA (1998) Reversible inactivation of protein-tyrosine phosphatase IB in A431 cells stimulated with epidermal growth factor. J Biol Chem 273:15366–15372CrossRefPubMedGoogle Scholar
  48. 48.
    Takakure K, Beckman JS, MacMillan-Cron LA, Cron JP (1999) Rapid and irreversible inactivation of protein tyrosine phosphatases PTPIB, CD45 and LAR by peroxynitrite. Arch Biochem Biophys 369:197–207CrossRefGoogle Scholar
  49. 49.
    Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent hydroxyl radical by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci 87:1620–1624CrossRefPubMedGoogle Scholar
  50. 50.
    Ischiropoulos H, Zhu L, Beckman JS (1992) Peroxynitrite formation from macrophage-derived nitric oxide. Arch Biochem Biophys 298:446–451CrossRefPubMedGoogle Scholar
  51. 51.
    Nathoo N, Goldlust S, Vogelbaum MA (2004) Epidermal growth factor receptor antagonists: novel therapy for the treatment of high-grade gliomas. Neurosurgery 54:1480–1489CrossRefPubMedGoogle Scholar
  52. 52.
    Liu B, Neufeld AH (2007) Activation of epidermal growth factor receptors in astrocytes: from development to neural injury. J Neurosci Res 85:3523–3529CrossRefPubMedGoogle Scholar
  53. 53.
    Contessa JN, Hamstra DA (2008) Revoking the privilege: targeting HER2 in the central nervous system. Mol Phamacol 73:271–273CrossRefGoogle Scholar
  54. 54.
    Cameron DA, Stein S (2008) Drug insight: intracellular inhibitors of HER2-clinical development of lapatinib in breast cancer. Nat Clin Pract Oncol 5:512–520CrossRefPubMedGoogle Scholar
  55. 55.
    Sharma PS, Sharma R, Tyagi T (2009) Receptor tyrosine kinase inhibitors as potent weapons in war against cancers. Curr Pharm Res 15:758–776CrossRefGoogle Scholar
  56. 56.
    Chen J, Zhu RL, Nakayama M, Kawaguchi K, Jin K, Stetler RA, Simon RP, Graham SH (1996) Expression of the apoptosis-effector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons following global ischemia. J Neurochem 67:64–71PubMedCrossRefGoogle Scholar
  57. 57.
    Gillardon F, Lenz C, Waschke KF, Krajewski S, Reed JC, Zimmermann M, Kuschinsky W (1996) Altered expression of Bcl-2, Bcl-X, Bax, and c-Fos colocalizes with DNA fragmentation and ischemic cell damage following middle cerebral artery occlusion in rats. Mol Brain Res 40:254–260CrossRefPubMedGoogle Scholar
  58. 58.
    Krajewski S, Mal JK, Krajewska M, Sikorska M, Mossakowski MJ (1995) Upregulation of Bax protein levels in neurons following cerebral ischemia. J Neurosci 15:6364–6376PubMedGoogle Scholar
  59. 59.
    Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B, Reed JC (1994) Tumor suppressor p53 is a regulator of bcl2 and Bax gene expression in vitro and in vivo. Oncogene 9:1799–1805PubMedGoogle Scholar
  60. 60.
    Reed JC (1996) Mechanisms of Bcl-2 family protein function and dysfunction in health and disease. Behring Inst Mitt 97:72–100PubMedGoogle Scholar
  61. 61.
    Ferrer I, Tortosa A, Macaya A, Sierra A, Moreno D, Munell F, Blanco R, Squier W (1994) Evidence of nuclear DNA fragmentation following hypoxia–ischemia in the infant rat brain, and transient forebrain ischemia in the adult gerbil. Brain Pathol 4:115–122CrossRefPubMedGoogle Scholar
  62. 62.
    Rosenbaum DM, Michaelson M, Batter DK, Doshi P, Kessler JA (1994) Evidence for hypoxia-induced, programmed cell death of cultured neurons. Ann Neurol 36:864–870CrossRefPubMedGoogle Scholar
  63. 63.
    Akhter WA, Ashraf QM, Zanelli SA, Mishra OP, Delivoria-Papadopoulos M (2001) Effects of graded hypoxia on cerebral cortical genomic DNA fragmentation in newborn piglets. Biol Neonate 79:187–193CrossRefPubMedGoogle Scholar
  64. 64.
    Tominaga T, Kure S, Yoshimoto T (1992) DNA fragmentation in focal cortical freeze injury of rats. Neurosci Lett 25:265–268CrossRefGoogle Scholar
  65. 65.
    Arends MJ, Morris RG, Wyllie AH (1990) Apoptosis. The role of the endonuclease. Am J Pathol 136:593–608PubMedGoogle Scholar
  66. 66.
    Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306CrossRefPubMedGoogle Scholar
  67. 67.
    Cohen GM, Sun XM, Snowden TT, Dinsdale D, Skilleter DN (1992) Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J 286:331–334PubMedGoogle Scholar
  68. 68.
    Fukuda K, Kojiro M, Chiu JF (1993) Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 7777 tissue: apoptosis or necrosis? Am J Pathol 142:935–946PubMedGoogle Scholar
  69. 69.
    Ishida R, Akiyoshi H, Takahashi T (1974) Isolation and purification of calcium and magnesium dependent endonuclease from rat liver nuclei. Biochem Biophys Res Commun 56:703–710CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Maria Delivoria-Papadopoulos
    • 1
  • Om Prakash Mishra
    • 1
  1. 1.Department of PediatricsDrexel University College of Medicine and St. Christopher’s Hospital for ChildrenPhiladelphiaUSA

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