Skip to main content
SpringerLink
Log in

Ischemia-Induced Inhibition of Mitochondrial Complex I in Rat Brain: Effect of Permeabilization Method and Electron Acceptor

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

In this study we have examined the effect of global brain ischemia/reperfusion on biochemical properties of the mitochondrial respiratory complex I (CI) in rat hippocampus and cortex. Since the inner mitochondrial membrane forms the permeability barrier for NADH, the methodology of enzymatic activity determinations employs membrane permeabilization methods. This action affects the basic character of electrostatic and hydrophobic interactions inside the membrane and might influence functional properties of membrane embedded proteins. Therefore we have performed the comparative analysis of two permeabilization methods (sonication, detergent) and their impact on CI enzymatic activities under global brain ischemic-reperfusion conditions. We have observed that ischemia led to significant decrease of CI activities using both permeabilization methods in both brain areas. However, significant differencies in enzymatic activities were registered during reperfusion intervals according to used permeabilization method. We have also tested the effect of electron acceptors (decylubiquinone, potassium ferricyanide, nitrotetrazolium blue) on CI activities during I/R. Based on our results we assume that the critical site where ischemia affects CI activities is electron transfer to electron acceptor. Further, the observed mitochondrial dysfunction was analyzed by means of one and 2-dimensional BN PAGE/SDS PAGE with the focus on 3-nitrotyrosine immunodetection as a marker of oxidative damage to proteins. Add to this, initialization of p53 mitochondrial apoptosis through p53, Bax, Bcl-XL proteins and a possible involvement of GRIM-19, the CI structural subunit, in apoptotic processes were also studied.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Saraste M (1999) Oxidative phosphorylation at the fin de siecle. Science. 283:1488–1493

    Article  PubMed  CAS  Google Scholar 

  2. Halestrap AP (2006) Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans 34:232–237

    Article  PubMed  CAS  Google Scholar 

  3. Feissner RF, Skalska J, Gaum WE et al (2009) Crosstalk signaling between mitochondrial Ca2 + and ROS. Front Biosci 14:1197–1218

    Article  PubMed  CAS  Google Scholar 

  4. Vinogradov AD (2001) Respiratory complex I: structure, redox components, and possible mechanisms of energy transduction. Biochemistry (Mosc) 66:1086–1097

    Article  CAS  Google Scholar 

  5. Brandt U (2006) Energy converting NADH:quinone oxidoreductase (complex I). Annu Rev Biochem 75:69–92

    Article  PubMed  CAS  Google Scholar 

  6. Kussmaul L, Hirst J (2006) The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci USA 103:7607–7612

    Article  PubMed  CAS  Google Scholar 

  7. Green DR, Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305:626–629

    Article  PubMed  CAS  Google Scholar 

  8. Clayton R, Clark JB, Sharpe M (2005) Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implication for apoptosis and neurodegenerative disease. J Neurochem 92:840–849

    Article  PubMed  CAS  Google Scholar 

  9. Carroll J, Fernley IM, Skehel JM et al (2006) Bovine complex I is a complex of 45 different subunits. J Biol Chem 281:32724–32727

    Article  PubMed  CAS  Google Scholar 

  10. Brant U, Kerscher S, Dröse S et al (2003) Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism? FEBS Lett 545:9–17

    Article  Google Scholar 

  11. Schägger H, de Coo R, Bauer MF et al (2004) Significance of respirasomes for the assembly/stability of human resiratory chain complex I. J Biol Chem 279:36349–36353

    Article  PubMed  Google Scholar 

  12. Vinogradov AD (1993) Kinetics, control and mechanism of ubiquinone reduction by the mammalian respiratory chain-linked NADH-ubiquinone reductase. J Bioenerg Biomembr 25:367–375

    Article  PubMed  CAS  Google Scholar 

  13. Grivennikova VG, Kapustin AN, Vinogradov AD (2001) Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria Evidence for the slow active/inactive transition. J Biol Chem 276:9038–9044

    Article  PubMed  CAS  Google Scholar 

  14. Maklashina E, Kotlyar AB, Cecchini G (2003) Active/deactive transition of respiratory complex I in bacteria, fungi and animals. Biochim Biophys Acta 1606:95–103

    Article  PubMed  CAS  Google Scholar 

  15. Moncada S, Erusalimsky JD (2002) Does nitric oxide modulate mitochondrial energy generation and apoptosis? Natl Rev Mol Cell Biol 3:214–220

    Article  CAS  Google Scholar 

  16. Angell JE, Lindner DJ, Shapiro PS et al (2000) Identification of GRIM-19, a novel cell death-regulatory gene induced by the interferon-beta and retinoic acid combination, using a genetic approach. J Biol Chem 275:33416–33426

    Article  PubMed  CAS  Google Scholar 

  17. Fearnley IM, Carroll J, Shannon RJ et al (2001) GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I). J Biol Chem 276:38345–38348

    Article  PubMed  CAS  Google Scholar 

  18. Mehrabian Z, Chandrasekaran K, Kalakonda S et al (2007) The IFN-β and retinoic acid-induced cell death regulator GRIM-19 is upregulated during focal cerebral ischemia. J Interferon Cyt Res 27:383–392

    Article  CAS  Google Scholar 

  19. Sun P, Nallar S, Kalakonda S et al (2009) GRIM-19 inhibits v-src-induced cell motility by interfering with cytoskeletal restructuring. Oncogene 28:1339–1347

    Article  PubMed  CAS  Google Scholar 

  20. Pulsinelli WA, Buchan AM (1988) The four-vessel occlusion rat model: method for complete occlusion of vertebral arteries and control of collateral circulation. Stroke 19:913–914

    Article  PubMed  CAS  Google Scholar 

  21. Polosa PL, Attardi G (1991) Distinctive pattern and translational control of mitochondrial protein synthesis in rat brain synaptic endings. J Biol Chem 266:10011–10017

    PubMed  CAS  Google Scholar 

  22. Maklashina E, Sher Y, Zhou HZ et al (2002) Effect of anoxia/reperfusion on the reversible active/de-active transition of NADH-ubiquinone oxidoreductase (complex I) in rat heart. Biochim Biophys Acta 1556:6–12

    Article  PubMed  CAS  Google Scholar 

  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  24. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  PubMed  Google Scholar 

  25. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231

    Article  PubMed  Google Scholar 

  26. Brookes PS, Pinner A, Ramachandran A et al (2002) High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2:969–977

    Article  PubMed  CAS  Google Scholar 

  27. Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6:1215–1225

    Article  PubMed  CAS  Google Scholar 

  28. Papa S, De Rasmo D, Scacco S et al (2008) Mammalian complex I: a regulable and vulnerable pacemaker in mitochondrial respiratory function. Biochim Biophys Acta 1777:719–728

    Article  PubMed  CAS  Google Scholar 

  29. Vinogradov AD, Grivennikova VG (2001) The mitochondrial complex I: progress in understanding of catalytic properties. IUBMB Life 52:129–134

    Article  PubMed  CAS  Google Scholar 

  30. Zickermann V, Kurki S, Kervinen M et al (2000) The NADH oxidation domain of complex I: do bacterial and mitochondrial enzymes catalyze ferricyanide reduction similarly? Biochim Biophys Acta 1459:61–68

    Article  PubMed  CAS  Google Scholar 

  31. Kotlyar AB, Sled VD, Vinogradov AD (1992) Effect of Ca2+ ions on the slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase. Biochim Biophys Acta 1098:144–150

    Article  PubMed  CAS  Google Scholar 

  32. Loskovich MV, Grivennikova VG, Cecchini G et al (2005) Inhibitory effect of palmitate on the mitochondrial NADH:ubiquinone oxidoreductase (complex I) as related to the active-de-active enzyme transition. Biochem J 387:677–683

    Article  PubMed  CAS  Google Scholar 

  33. Hoch FL (1992) Cardiolipins and biomembrane function. Biochim Biophys Acta 1113:71–133

    PubMed  CAS  Google Scholar 

  34. Paradies G, Petrosillo G, Pistolese M et al (2004) Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin. Circ Res 94:53–59

    Article  PubMed  CAS  Google Scholar 

  35. Pfeiffer K, Gohil V, Stuart RA (2003) Cardiolipin stabilizes respiratory chain supercomplexes. J Biol Chem 278:52873–52880

    Article  PubMed  CAS  Google Scholar 

  36. Petrosillo G, Matera M, Moro N et al (2009) Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin. Free Radic Biol Med 46:88–94

    Article  PubMed  CAS  Google Scholar 

  37. Ott M, Robertson JD, Gogvadze V et al (2002) Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci USA 99:1259–1263

    Article  PubMed  CAS  Google Scholar 

  38. Dröse S, Zwicker K, Brandt U (2002) Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids. Biochim Biophys Acta 1556:65–72

    Article  PubMed  Google Scholar 

  39. Musatov A (2006) Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase. Free Radic Biol Med 41:238–246

    Article  PubMed  CAS  Google Scholar 

  40. Racay P, Tatarkova Z, Chomova M et al (2009) Mitochondrial calcium transport and mitochondrial dysfunction after global brain ischemia in rat hippocampus. Neurochem Res 34:1469–1478

    Article  PubMed  CAS  Google Scholar 

  41. Suslick KS, Flannigan DJ (2008) Inside a collapsing bubble: sonoluminiscence and the conditions during cavitation. Annu Rev Phys Chem 59:659–683

    Article  PubMed  CAS  Google Scholar 

  42. Miller MW, Miller DL, Brayman AA (1996) A review of in vitro bioeffects of intertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 22:1131–1154

    Article  PubMed  CAS  Google Scholar 

  43. Ashokkumar M, Lee J, Kentish S, Grieser F (2007) Bubbles in an acoustic field: an overview. Ultrason Sonochem 14:470–475

    Article  PubMed  CAS  Google Scholar 

  44. le Maire M, Champeil P, Moller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111

    Article  PubMed  CAS  Google Scholar 

  45. Schägger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19:1777–1783

    Article  PubMed  Google Scholar 

  46. Acín-Pérez R, Fernández-Silva P, Peleato ML et al (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32:529–539

    Article  PubMed  Google Scholar 

  47. Marchenko N, Zaika A, Moll UM (2000) Death Signal-induced localization of p53 protein to mitochondria. J Biol Chem 275:16202–16212

    Article  PubMed  CAS  Google Scholar 

  48. Endo H, Kamada H, Nito C et al (2006) Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats. J Neurosci 26:7974–7983

    Article  PubMed  CAS  Google Scholar 

  49. Schuler M, Green DR (2001) Mechanisms of p53-dependent apoptosis. Biochem Soc Trans 29:684–688

    Article  PubMed  CAS  Google Scholar 

  50. Huang G, Lu H, Hao A et al (2004) GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I. Mol Cell Biol 24:8447–8456

    Article  PubMed  CAS  Google Scholar 

  51. Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431–1568

    PubMed  CAS  Google Scholar 

  52. Hu BR, Martone ME, Jones YZ et al (2000) Protein aggregation following transient cerebral ischemia. J Neurosci 20:3191–3199

    PubMed  CAS  Google Scholar 

  53. Racay P (2011) Ischaemia-induced protein ubiquitinylation is differentially accompanied with heat-shock protein 70 expression after naïve and preconditioned ischaemia. Cell Mol Neurobiol.[Epub ahead of print] http://www.ncbi.nlm.nih.gov/pubmed/21789629 doi: 10.1007/s10571-011-9740-z

  54. Rubbo H, Radi R (2008) Protein and lipid nitration: role in redox signaling and injury. Biochim Biophys Acta 1780:1318–1324

    Article  PubMed  CAS  Google Scholar 

  55. Bartesaghi S, Ferrer-Sueta G, Peluffo G et al (2007) Protein tyrosine nitration in hydrophilic and hydrophobic environments. Amino Acids 32:501–515

    Article  PubMed  CAS  Google Scholar 

  56. Radi R (2004) Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci USA 101:4003-4008

    Google Scholar 

  57. Ischiropoulos H (2003) Biological selectivity and functional aspects of protein tyrosine nitration. Biochem Biophys Res Commun 305:776–783

    Article  PubMed  CAS  Google Scholar 

  58. Söderling AS, Hultman L, Delbro D et al (2007) Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting. J Chromatogr B Anal Technol Biomed Life Sci 851:277–286

    Article  Google Scholar 

  59. Otto CM, Baumgardner JE (2001) Effect of culture PO2 on macrophage (RAW 264.7) nitric oxide production. Am J Physiol Cell Physiol 280:C280–C287

    PubMed  CAS  Google Scholar 

  60. Aulak KS, Koeck T, Crabb JW et al (2004) Dynamics of protein nitration in cells and mitochondria. Am J Physiol Heart Circ Physiol 286:H30–H38

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Ministry of Education of Slovak Republic, grant VEGA 1/0049/09. Authors are grateful to Zdenka Cetlova for her technical assistance.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Faculty of Medicine, Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Comenius University, Sasinkova 2, 811 08, Bratislava, Slovakia

    Maria Chomova

  2. Jessenius Faculty of Medicine, Institute of Medical Biochemistry, Comenius University, Martin, Slovakia

    Maria Chomova, Zuzana Tatarkova, Dusan Dobrota & Peter Racay

Authors
  1. Maria Chomova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zuzana Tatarkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dusan Dobrota
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter Racay
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Chomova.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chomova, M., Tatarkova, Z., Dobrota, D. et al. Ischemia-Induced Inhibition of Mitochondrial Complex I in Rat Brain: Effect of Permeabilization Method and Electron Acceptor. Neurochem Res 37, 965–976 (2012). https://doi.org/10.1007/s11064-011-0689-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-011-0689-6

Keywords

Search

Navigation