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Acetylcholine inhibits long-term hypoxia-induced apoptosis by suppressing the oxidative stress-mediated MAPKs activation as well as regulation of Bcl-2, c-IAPs, and caspase-3 in mouse embryonic stem cells

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Abstract

This study examined the effect of acetylcholine (ACh) on the hypoxia-induced apoptosis of mouse embryonic stem (ES) cells. Hypoxia (60 h) decreased both the cell viability and level of [3H] thymidine incorporation, which were prevented by a pretreatment with ACh. However, the atropine (ACh receptor [AChR] inhibitor) treatment blocked the protective effect of ACh. Hypoxia (90 min) increased the intracellular level of reactive oxygen species (ROS). On the other hand, ACh inhibited the hypoxia-induced increase in ROS, which was blocked by an atropine treatment. Subsequently, the hypoxia-induced ROS increased the level of p38 mitogen activated protein kinase (MAPK) and Jun-N-terminal kinase (JNK) phosphorylation, which were inhibited by the ACh pretreatment. Moreover, hypoxic exposure (90 min) increased the level of nuclear factor-κB (NF-κB) phosphorylation, which was blocked by a pretreatment with SB 203580 (p38 MAPK inhibitor) or SP 600125 (JNK inhibitor). However, hypoxia (60 h) decreased the protein levels of Bcl-2 and c-IAPs (cellular inhibitor of apoptosis proteins) but increased the level of caspase-3 activation. All these effects were inhibited by a pretreatment with ACh. In conclusion, ACh prevented the hypoxia-induced apoptosis of mouse ES cells by inhibiting the ROS-mediated p38 MAPK and JNK activation as well as the regulation of Bcl-2, c-IAPs, and caspase-3.

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References

  1. Zhu LL, Wu LY, Yew DT et al (2005) Effects of hypoxia on the proliferation and differentiation of NSCs. Mol Neurobiol 31:231–242

    Article  PubMed  CAS  Google Scholar 

  2. Bunn HF, Poyton RO (1996) Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 76:839–885

    PubMed  CAS  Google Scholar 

  3. Duranteau J, Chandel NS, Kulisz A et al (1998) Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem 273:11619–11624

    Article  PubMed  CAS  Google Scholar 

  4. Vexler ZS, Ferriero DM (2001) Molecular and biochemical mechanisms of perinatal brain injury. Semin Neonatol 6:99–108

    Article  PubMed  CAS  Google Scholar 

  5. Millar TM, Phan V, Tibbles LA (2007) ROS generation in endothelial hypoxia and reoxygenation stimulates MAPkinase signaling and kinase-dependent neutrophil recruitment. Free Radic Biol Med 42:1165–1177

    Article  PubMed  CAS  Google Scholar 

  6. Grow J, Barks JD (2002) Pathogenesis of hypoxic–ischemic cerebral injury in the term infant: current concepts. Clin Perinatol 29:585–602

    Article  PubMed  Google Scholar 

  7. Johnston MV, Nakajima W, Hagberg H (2002) Mechanisms of hypoxic neurodegeneration in the developing brain. Neuroscientist 8:212–220

    PubMed  CAS  Google Scholar 

  8. Santos SC, Vala I, Miguel C et al (2007) Expression and subcellular localization of a novel nuclear acetylcholinesterase protein. J Biol Chem 282:25597–25603

    Article  PubMed  CAS  Google Scholar 

  9. Malinger G, Zakut H, Soreq H (1989) Cholinoceptive properties of human primordial, preantral, and antral oocytes: in situ hybridization and biochemical evidence for expression of cholinesterase genes. J Mol Neurosci 1:77–84

    PubMed  CAS  Google Scholar 

  10. Paraoanu LE, Steinert G, Klaczinski J et al (2006) On functions of cholinesterases during embryonic development. J Mol Neurosci 30:201–204

    Article  PubMed  CAS  Google Scholar 

  11. Wessler I, Kirkpatrick CJ, Racke K (1999) The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans. Clin Exp Pharmacol Physiol 26:198–205

    Article  PubMed  CAS  Google Scholar 

  12. Paraoanu LE, Steinert G, Koehler A et al (2007) Expression and possible functions of the cholinergic system in a murine embryonic stem cell line. Life Sci 80:2375–2379

    Article  PubMed  CAS  Google Scholar 

  13. Wessler I, Kirkpatrick CJ, Racke K (1998) Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans. Pharmacologist 77:59–79

    CAS  Google Scholar 

  14. Kawashima K, Fujii T (2004) Expression of non-neuronal acetylcholine in lymphocytes and its contribution to the regulation of immunefunction. Front Biosci 9:2063–2085

    Article  PubMed  CAS  Google Scholar 

  15. Dajas-Bailador FA, Lima PA, Wonnacott S (2000) The α7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca2+ dependent mechanism. Neuropharmacology 39:2799–2807

    Article  PubMed  CAS  Google Scholar 

  16. Costa LG, Guizzetti M (1999) Muscarinic cholinergic receptor signal transduction as a potential target for the developmental neurotoxicity of ethanol. Biochem Pharmacol 57:721–726

    Article  PubMed  CAS  Google Scholar 

  17. Bhuiyan MB, Murad F, Fant ME (2006) The placental cholinergic system: localization to the cytotrophoblast and modulation of nitric oxide. Cell Commun Signal 4:4

    Article  PubMed  Google Scholar 

  18. Heo JS, Han HJ (2006) ATP stimulates mouse embryonic stem cell proliferation via protein kinase C, phosphatidylinositol 3-kinease/Akt, and mitogen-activated protein kinase signaling pathways. Stem Cells 24:2637–2648

    Article  CAS  Google Scholar 

  19. Han HJ, Heo JS, Lee YJ (2006) Estradiol-17β stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDK as well as protooncogenes. Am J Physiol Cell Physiol 290:C1067–C1075

    Article  PubMed  CAS  Google Scholar 

  20. Lee SH, Heo JS, Han HJ (2007) Effect of hypoxia on 2-deoxyglucose uptake and cell cycle regulatory protein expression of mouse embryonic stem cells: involvement of Ca2+/PKC, MAPKs and HIF-1α. Cell Physiol Biochem 19:269–282

    Article  PubMed  CAS  Google Scholar 

  21. Tohgi H, Utsugisawa K, Nagane Y (2000) Hypoxia-induced expression of C1q, a subcomponent of the complement system, in cultured rat PC12 cells. Neurosci Lett 291:151–154

    Article  PubMed  CAS  Google Scholar 

  22. Utsugisawa K, Nagane Y, Obara D, Tohgi H (2002) Over-expression of α7 nicotinic acetylcholine receptor prevents G1-arrest and DNA fragmentation in PC12 cells after hypoxia. J Neurochem 81:497–505

    Article  PubMed  CAS  Google Scholar 

  23. Chen CH, Ho ML, Chang JK et al (2005) Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line. Osteoporos Int 16:2039–2045

    Article  PubMed  CAS  Google Scholar 

  24. Zhang E, Li X, Zhang S et al (2005) Cell cycle synchronization of embryonic stem cells: effect of serum deprivation on the differentiation of embryonic bodies in vitro. Biochem Biophys Res Commun 333:1171–1177

    Article  PubMed  CAS  Google Scholar 

  25. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  26. Fujii T, Yamada S, Yamaguchi N et al (1995) Species differences in the concentration of acetylcholine, a neruotransmitter, in whole blood and plasma. Neurosci Lett 201:207–210

    Article  PubMed  CAS  Google Scholar 

  27. Sastry BVR (1997) Human placentai cholinergic system. Biochem Pharmacol 53:1577–1586

    Article  PubMed  CAS  Google Scholar 

  28. Liu H, Bradley C, Zhu X et al (2001) Role of nitric oxide and protein kinase C in Ach-induced cardioprotection. Am J Physiol Heart Circ Physiol 281:191–197

    Google Scholar 

  29. Zhang Y, Kakinuma Y, Ando M et al (2006) Acetylcholine inhibits the hypoxia-induced reduction of connexin43 protein in rat cardiomyocytes. J Pharmacol Sci 101:214–222

    Article  PubMed  CAS  Google Scholar 

  30. Kakinuma Y, Ando M, Kuwabara M et al (2005) Acetylcholine from vagal stimulation protects cardiomyocytes against ischemia and hypoxia involving additive non-hypoxic induction of HIF-1α. FEBS Lett 579:2111–2118

    Article  PubMed  CAS  Google Scholar 

  31. De Sarno P, Shestopal SA, King TD et al (2003) Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition. J Biol Chem 278:11086–11093

    Article  PubMed  Google Scholar 

  32. Joseph JA, Fisher DR, Carey A et al (2004) The M3 muscarinic receptor i3 domain confers oxidative stress protection on calcium regulation in transfected COS-7 cells. Aging Cell 3:263–271

    Article  PubMed  CAS  Google Scholar 

  33. McPherson BC, Zhu X, Liu H et al (2002). Acetylcholine attenuates cardiomyocyte oxidant stress during simulated ischemia and reoxygenation. Pharmacology 64:49–56

    Article  PubMed  CAS  Google Scholar 

  34. Sun HY, Wang NP, Halkos M et al (2006) Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways. Apoptosis 11:1583–1593

    Article  PubMed  CAS  Google Scholar 

  35. Wang T, Zhang X, Li JJ (2002) The role of NF-κB in the regulation of cell stress responses. Int Immunopharmcol 2:1509–1520

    Article  CAS  Google Scholar 

  36. Zhao ZQ, Vinten-Johansen J (2002) Myocardial apoptosis and ischemic preconditioning. Cardiovasc Res 55:438–455

    Article  PubMed  CAS  Google Scholar 

  37. Shinozaki Y, Koizumi S, Ishida S et al (2005) Cytoprotection against oxidative stress-induced damage of astrocytes by extr acellular ATP via P2Y1 receptors. Glia 49:288–300

    Article  PubMed  Google Scholar 

  38. Echeverria V, Clerman A, Dore S (2005) Stimulation of PGE2 receptors EP2 and EP4 protects cultured neurons against oxidative stress and cell death following β-amyloid exposure. Eur J Neurosci 22:2199–2206

    Article  PubMed  Google Scholar 

  39. Yang B, Lin H, Xu C et al (2005) Choline produces cytoprotective effects against ischemic myocardial injuries: Evidence for the role of cardiac M3 subtype muscarinic acetylcholine receptors. Cell Physiol Biochem 16:163–174

    Article  PubMed  CAS  Google Scholar 

  40. Moon DO, Park SY, Heo MS et al (2006) Key regulators in bee venom-induced apoptosis are Bcl-2 and caspase-3 in human leukemic U937 cells through downregulation of ERK and Akt. Int Immunopharmacol 6:1796–1807

    Article  PubMed  CAS  Google Scholar 

  41. Caulfield MP (1993) Muscarnic receptors—characterization, coupling, and function. Pharmacol Ther 58:319–379

    Article  PubMed  CAS  Google Scholar 

  42. Cui QL, Fogle E, Almazan G (2006) Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src-like tyrosine kinases and PI3K/Akt pathways. Neurochem Int 48:383–393

    Article  PubMed  CAS  Google Scholar 

  43. Sato-Bigbee C, Pal S, Chu AK (1999) Different neuroligands and signal transduction pathways stimulate CREB phosphorylation at specific developmental stages along oligodendrocyte differentiation. J Neurochem 72:139–147

    Article  PubMed  CAS  Google Scholar 

  44. Pugazhenthi S, Nesterova A, Sableet C et al (2000) Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem 275:10761–10766

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by Grant SC 2210 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology. We acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project, Republic of Korea.

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Authors and Affiliations

  1. Department of Rehabilitation Science, Graduate school of Daegu University, Daegu, 705-714, Korea

    Min Hee Kim

  2. Department of Veterinary Physiology, Biotherapy Human Resources Center (BK 21), College of Veterinary Medicine, Chonnam National University, Gwangju, 500-757, Korea

    Mi Ok Kim, Jung Sun Heo & Ho Jae Han

  3. Department of Physical Therapy, College of Rehabilitation Science, Daegu University, Daegu, 705-714, Korea

    Jin Sang Kim

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  1. Min Hee Kim
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Correspondence to Ho Jae Han.

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Kim, M.H., Kim, M.O., Heo, J.S. et al. Acetylcholine inhibits long-term hypoxia-induced apoptosis by suppressing the oxidative stress-mediated MAPKs activation as well as regulation of Bcl-2, c-IAPs, and caspase-3 in mouse embryonic stem cells. Apoptosis 13, 295–304 (2008). https://doi.org/10.1007/s10495-007-0160-y

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