Acta Neurologica Belgica

, Volume 113, Issue 4, pp 507–513 | Cite as

Intermittent hypoxia protects cerebral mitochondrial function from calcium overload

  • Jian Chen
  • Weigong Liao
  • Wenxiang Gao
  • Jian Huang
  • Yuqi GaoEmail author
Original Article


Hypoxia leads to Ca2+ overload and results in mitochondrial uncoupling, decreased ATP synthesis, and neuronal death. Inhibition of mitochondrial Ca2+ overload protects mitochondrial function after hypoxia. The present study was aimed to investigate the effect of intermittent hypoxia on mitochondrial function and mitochondrial tolerance to Ca2+ overload. Wistar rats were divided into control and intermittent hypoxia (IH) groups. The IH group was subject to hypoxia for 4 h daily in a hypobaric cabin (5,000 m) for 7 days. Brain mitochondria were isolated on day 7 following hypoxia. The baseline mitochondrial functions, such as ST3, ST4, and respiratory control ratio (RCR = ST3/ST4), were measured using a Clark-type oxygen electrode. Mitochondrial adenine nucleotide concentrations were measured by HPLC. Mitochondrial membrane potential was determined by measuring rhodamine 123 (Rh-123) fluorescence in the absence and presence of high Ca2+ concentration (0.1 M), which simulates Ca2+ overload. Our results revealed that IH did not affect mitochondrial respiratory functions, but led to a reduction in AMP and an increase in ADP concentrations in mitochondria. Both control and IH groups demonstrated decreased mitochondrial membrane potential in the presence of high Ca2+ (0.1 M), while the IH group showed a relative higher mitochondrial membrane potential. These results indicated that the neuroprotective effect of intermittent hypoxia was resulted partly from preserving mitochondrial membrane potential, and increasing mitochondrial tolerance to high calcium levels. The increased ADP and decreased AMP in mitochondria following intermittent hypoxia may be a mechanism underlying this protection.


Intermittent hypoxia Mitochondria Calcium Mitochondrial membrane potential Respiratory control rate 



This work was supported by grants from the National Natural Science Foundation of China (grant number 81055828) and the Major State Basic Research Development Program of China (Grant number 2012CB518201).


  1. 1.
    Wang J, Green PS, Simpkins JW (2001) Estradiol protects against ATP depletion, mitochondrial membrane potential decline and the generation of reactive oxygen species induced by 3-nitroproprionic acid in SK-N-SH human neuroblastoma cells. J Neurochem 77:804–811PubMedCrossRefGoogle Scholar
  2. 2.
    Kim JS, He L, Lemasters JJ (2003) Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304:463–470PubMedCrossRefGoogle Scholar
  3. 3.
    Petrosillo G, Di Venosa N, Moro N, Colantuono G, Paradies V et al (2011) In vivo hyperoxic preconditioning protects against rat-heart ischemia/reperfusion injury by inhibiting mitochondrial permeability transition pore opening and cytochrome c release. Free Radic Biol Med 50:477–483PubMedCrossRefGoogle Scholar
  4. 4.
    Rousset CI, Baburamani AA, Thornton C, Hagberg H (2012) Mitochondria and perinatal brain injury. J Matern Fetal Neonatal Med 25(Suppl 1):35–38PubMedCrossRefGoogle Scholar
  5. 5.
    Follstad BD, Wang DI, Stephanopoulos G (2000) Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures. Eur J Biochem 267:6534–6540PubMedCrossRefGoogle Scholar
  6. 6.
    Korge P, Honda HM, Weiss JN (2001) Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation. Am J Physiol Cell Physiol 280:C517–C526PubMedGoogle Scholar
  7. 7.
    Chen L, Lu XY, Li J, Fu JD, Zhou ZN et al (2006) Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms. Am J Physiol Cell Physiol 290:C1221–C1229PubMedCrossRefGoogle Scholar
  8. 8.
    Dong JW, Zhu HF, Zhu WZ, Ding HL, Ma TM et al (2003) Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res 13:385–391PubMedCrossRefGoogle Scholar
  9. 9.
    Manukhina EB, Downey HF, Mallet RT (2006) Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia. Exp Biol Med (Maywood) 231:343–365Google Scholar
  10. 10.
    Prass K, Scharff A, Ruscher K, Lowl D, Muselmann C et al (2003) Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke 34:1981–1986PubMedCrossRefGoogle Scholar
  11. 11.
    Sharp FR, Ran R, Lu A, Tang Y, Strauss KI et al (2004) Hypoxic preconditioning protects against ischemic brain injury. NeuroRx 1:26–35PubMedCrossRefGoogle Scholar
  12. 12.
    Sun K, Liu ZS, Sun Q (2004) Role of mitochondria in cell apoptosis during hepatic ischemia-reperfusion injury and protective effect of ischemic postconditioning. World J Gastroenterol 10:1934–1938PubMedGoogle Scholar
  13. 13.
    Dirnagl U, Meisel A (2008) Endogenous neuroprotection: mitochondria as gateways to cerebral preconditioning? Neuropharmacology 55:334–344PubMedCrossRefGoogle Scholar
  14. 14.
    Clark JB, Nicklas WJ (1970) The metabolism of rat brain mitochondria. Preparation and characterization. J Biol Chem 245:4724–4731PubMedGoogle Scholar
  15. 15.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  16. 16.
    Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17:65–134PubMedGoogle Scholar
  17. 17.
    Kwast KE, Hand SC (1996) Oxygen and pH regulation of protein synthesis in mitochondria from Artemia franciscana embryos. Biochem J 313(Pt 1):207–213PubMedGoogle Scholar
  18. 18.
    Brustovetsky N, Brustovetsky T, Jemmerson R, Dubinsky JM (2002) Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane. J Neurochem 80:207–218PubMedCrossRefGoogle Scholar
  19. 19.
    Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469–477PubMedCrossRefGoogle Scholar
  20. 20.
    Gorgias N, Maidatsi P, Tsolaki M, Alvanou A, Kiriazis G et al (1996) Hypoxic pretreatment protects against neuronal damage of the rat hippocampus induced by severe hypoxia. Brain Res 714:215–225PubMedCrossRefGoogle Scholar
  21. 21.
    Liu XQ, Sheng R, Qin ZH (2009) The neuroprotective mechanism of brain ischemic preconditioning. Acta Pharmacol Sin 30:1071–1080PubMedCrossRefGoogle Scholar
  22. 22.
    Ran R, Xu H, Lu A, Bernaudin M, Sharp FR (2005) Hypoxia preconditioning in the brain. Dev Neurosci 27:87–92PubMedCrossRefGoogle Scholar
  23. 23.
    Stadler B, Phillips J, Toyoda Y, Federman M, Levitsky S et al (2001) Adenosine-enhanced ischemic preconditioning modulates necrosis and apoptosis: effects of stunning and ischemia-reperfusion. Ann Thorac Surg 72:555–563 (Discussion 563–554)PubMedCrossRefGoogle Scholar
  24. 24.
    da Silva MM, Sartori A, Belisle E, Kowaltowski AJ (2003) Ischemic preconditioning inhibits mitochondrial respiration, increases H2O2 release, and enhances K+ transport. Am J Physiol Heart Circ Physiol 285:H154–H162PubMedGoogle Scholar
  25. 25.
    Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341(Pt 2):233–249PubMedCrossRefGoogle Scholar
  26. 26.
    Ishikawa Y, Yamamoto Y, Kume M, Yamagami K, Yamamoto H et al (1999) Heat shock preconditioning on mitochondria during warm ischemia in rat livers. J Surg Res 87:178–184PubMedCrossRefGoogle Scholar
  27. 27.
    Peralta C, Bartrons R, Riera L, Manzano A, Xaus C et al (2000) Hepatic preconditioning preserves energy metabolism during sustained ischemia. Am J Physiol Gastrointest Liver Physiol 279:G163–G171PubMedGoogle Scholar
  28. 28.
    Frassetto SS, Schetinger MR, Schierholt R, Webber A, Bonan CD et al (2000) Brain ischemia alters platelet ATP diphosphohydrolase and 5′-nucleotidase activities in naive and preconditioned rats. Braz J Med Biol Res 33:1369–1377PubMedCrossRefGoogle Scholar
  29. 29.
    Zhivotovsky B, Galluzzi L, Kepp O, Kroemer G (2009) Adenine nucleotide translocase: a component of the phylogenetically conserved cell death machinery. Cell Death Differ 16:1419–1425PubMedCrossRefGoogle Scholar
  30. 30.
    Ryu SY, Peixoto PM, Teijido O, Dejean LM, Kinnally KW (2010) Role of mitochondrial ion channels in cell death. Biofactors 36:255–263PubMedCrossRefGoogle Scholar
  31. 31.
    Delcamp TJ, Dales C, Ralenkotter L, Cole PS, Hadley RW (1998) Intramitochondrial [Ca2+] and membrane potential in ventricular myocytes exposed to anoxia-reoxygenation. Am J Physiol 275:H484–H494PubMedGoogle Scholar
  32. 32.
    Lee WT, Yin HS, Shen YZ (2002) The mechanisms of neuronal death produced by mitochondrial toxin 3-nitropropionic acid: the roles of N-methyl-D-aspartate glutamate receptors and mitochondrial calcium overload. Neuroscience 112:707–716PubMedCrossRefGoogle Scholar
  33. 33.
    Weinberg JM, Venkatachalam MA, Roeser NF, Nissim I (2000) Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates. Proc Natl Acad Sci USA 97:2826–2831PubMedCrossRefGoogle Scholar
  34. 34.
    Broekemeier KM, Dempsey ME, Pfeiffer DR (1989) Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J Biol Chem 264:7826–7830PubMedGoogle Scholar

Copyright information

© Belgian Neurological Society 2013

Authors and Affiliations

  • Jian Chen
    • 1
    • 2
    • 3
  • Weigong Liao
    • 1
    • 2
    • 3
  • Wenxiang Gao
    • 1
    • 2
    • 3
  • Jian Huang
    • 1
    • 2
    • 3
  • Yuqi Gao
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Pathophysiology and High Altitude Physiology, College of High Altitude Military MedicineThird Military Medical UniversityChongqingChina
  2. 2.The Key Laboratory of High Altitude Medicine, Ministry of EducationThird Military Medical UniversityChongqingChina
  3. 3.The Key Laboratory of High Altitude Physiology and High Altitude Disease, PLAThird Military Medical UniversityChongqingChina

Personalised recommendations