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Alteration in mitochondrial thiol enhances calcium ion dependent membrane permeability transition and dysfunction in vitro: a cross-talk between mtThiol, Ca2+, and ROS

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Abstract

Mitochondrial permeability transition (MPT) and dysfunctions play a pivotal role in many patho-physiological and toxicological conditions. The interplay of mitochondrial thiol (mtThiol), MPT, Ca2+ homeostasis, and resulting dysfunctions still remains controversial despite studies by several research groups. Present study was undertaken to ascertain the correlation between Ca2+ homeostasis, mtThiol alteration and reactive oxygen species (ROS) in causing MPT leading to mitochondrial dysfunction. mtThiol depletion significantly enhanced Ca2+ dependent MPT (swelling) and depolarization of mitochondria resulting in release of pro-apoptotic proteins like Cyt c, AIF, and EndoG. mtThiol alteration and Ca2+ overload caused reduced mitochondrial electron flow, oxidation of pyridine nucleotides (NAD(P)H) and significantly enhanced ROS generation (DHE and DCFH-DA fluorescence). Studies with MPT inhibitor (Cyclosporin A), Ca2+ uniport blocker (ruthenium red) and Ca2+ chelator (BAPTA) indicated that mitochondrial dysfunction was more pronounced under dual stress of altered mtThiol and Ca2+ overload in comparison with single stress of excessive Ca2+. Transmission electron microscopy confirmed the changes in mitochondrial integrity under stress. Our findings suggest that the Ca2+ overload itself is not solely responsible for structural and functional impairment of mitochondria. A multi-factorial cross-talk between mtThiol, Ca2+ and ROS is responsible for mitochondrial dysfunction. Furthermore, minor depletion of mtThiol was found to be an important factor along with Ca2+ overload in triggering MPT in isolated mitochondria, tilting the balance towards disturbed functionality.

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References

  1. Duszynski J, Koziel R, Brutkowski W, Szczepanowska J, Zablocki K (2006) The regulatory role of mitochondria in capacitative calcium entry. Biochim Biophys Acta 1757(5-6):380–387

    Article  PubMed  CAS  Google Scholar 

  2. Romagnoli A, Aguiari P, De Stefani D, Leo S, Marchi S, Rimessi A, Zecchini E, Pinton P, Rizzuto R (2007) Endoplasmic reticulum/mitochondria calcium cross-talk. Novartis Found Symp 287:122–131

    Article  PubMed  CAS  Google Scholar 

  3. Bernardi P, Petronilli V (1996) The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J Bioenerg Biomembr 28:131–138

    Article  PubMed  CAS  Google Scholar 

  4. Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA (2006) Calcium in cell injury and death. Annu Rev Pathol 1:405–434

    Article  PubMed  CAS  Google Scholar 

  5. Kaasik A, Safiulina D, Zharkovsky A, Veksler V (2006) Regulation of mitochondrial matrix volume. Am J Physiol Cell Physiol 292(1):C157–C163

    Article  PubMed  Google Scholar 

  6. Kakkar P, Singh BK (2007) Mitochondria: a hub of redox activities and cellular distress control. Mol Cell Biochem 305:235–253

    Article  PubMed  CAS  Google Scholar 

  7. Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82:47–95

    PubMed  CAS  Google Scholar 

  8. Yuan L, Kaplowitz N (2009) Glutathione in liver disease and hepatotoxicity. Mol Aspects Med 30:29–41

    Article  PubMed  CAS  Google Scholar 

  9. Ghosh A, Sil PC (2009) Protection of acetaminophen induced mitochondrial dysfunctions and hepatic necrosis via Akt-NF-kB pathway: role of a novel plant protein. Chem Biol Interact 177:96–106

    Article  PubMed  CAS  Google Scholar 

  10. Franco R, Cidlowski JA (2009) Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ 16:1303–1314

    Article  PubMed  CAS  Google Scholar 

  11. Giacomello M, Drago I, Pizzo P, Pozzan T (2007) Mitochondrial Ca2+ as a key regulator of cell life and death. Cell Death Differ 14(7):1267–1274

    Article  PubMed  CAS  Google Scholar 

  12. Hollis BW (2005) Calcium and disease. In: Weaver CM, Heaney RP (eds) Calcium in human health. Humana Press, Totowa, NJ, pp 313–425

    Google Scholar 

  13. Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblast. Nat Protoc 2:287–292

    Article  PubMed  CAS  Google Scholar 

  14. Gusdon AM, Chen J, Votyakova TV, Mathews CE (2009) Quantification, localization, and tissue specificities of mouse mitochondrial reactive oxygen species production. Methods Enzymol 456:439–457

    Article  PubMed  CAS  Google Scholar 

  15. Nieminen AL, Ramshesh VK, Lemasters JJ (2008) Use of fluorescent reporters to measure mitochondrial membrane potential and the mitochondrial permeability transition. In: Dykens JA, Will Y (eds) Drug induced mitochondrial dysfunction. Wiley, New Jersey, pp 414–431

    Google Scholar 

  16. Kruglov AG, Teplova VV, Saris NE (2007) The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction. Biochem Pharmacol 74:545–556

    Article  PubMed  CAS  Google Scholar 

  17. Lowry OH, Rosebough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin Phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  18. Estebern-Pretel G, Lopez-Garcia MP (2006) An experimental design for the controlled modulation of intracellular GSH levels in cultured hepatocytes. Free Radic Biol Med 41:610–619

    Article  Google Scholar 

  19. Harwood DT, Kettle AJ, Brennan S, Winterbourn CC (2009) Simultaneous determination of reduced glutathione, glutathione disulphide and glutathione sulphonamide in cells and physiological fluids by isotope dilution liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 877:3393–3399

    Article  PubMed  CAS  Google Scholar 

  20. Santana DP, Faria PA, Paredes-Gamero EJ, Caires AC, Nantes IL, Rodrigues T (2009) Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis. Biochem J 417:247–256

    Article  PubMed  CAS  Google Scholar 

  21. Gonzalez D, Espino J, Bejarano I, Lopez JJ, Rodriguez AB, Pariente JA (2010) Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal. Mol Cell Biochem 333:151–157

    Article  PubMed  CAS  Google Scholar 

  22. Tripathi M, Singh BK, Mishra C, Raissudin S, Kakkar P (2010) Involvement of mitochondria mediated pathways in hepatoprotaction conferred by Fumeria parviflora Lam. extract against nimesulide induced apoptosis in vitro. Toxicol In Vitro 24:495–508

    Article  PubMed  CAS  Google Scholar 

  23. Halvey PJ, Hansen JM, Lash LH, Jones DP (2008) Compartmentation of redox signaling and control: discrimination of oxidative stress in mitochondria, cytoplasm, nuclei and endoplasmic reticulum. In: Dykens JA, Will Y (eds) Drug induced mitochondrial dysfunction. Wiley, New Jersey, pp 433–461

    Chapter  Google Scholar 

  24. Oyama TB, Oyama K, Kawanai T, Oyama TM, Hashimoto E, Satoh M, Oyama Y (2009) Tri-n-butyltin increases intracellular Zn2+ concentration by decreasing cellular thiol content in rat thymocytes. Toxicology 262:245–249

    Article  PubMed  CAS  Google Scholar 

  25. Mayevsky A, Barbiro-Michaely E (2009) Use of NADH fluorescence to determine mitochondrial function in vivo. Int J Biochem Cell Biol 41:1977–1988

    Article  PubMed  CAS  Google Scholar 

  26. Pierce RH, Campbell JS, Stephenson AB, Franklin CC, Chaisson M, Poot M, Kavanagh TJ, Rabinovitch PS, Fausto N (2000) Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line. Am J Pathol 157:221–236

    Article  PubMed  CAS  Google Scholar 

  27. Sharikabad MN, Ostbye KM, Lyberg T, Brors O (2001) Effect of extracellular Mg2+ on ROS and Ca2+ accumulation during reoxygenation of rat cardiomyocytes. Am J Physiol Heart Circ Physiol 280:344–353

    Google Scholar 

  28. Campbell RV, Yang Y, Wang T, Rachamallu A, Li Y, Watowich SJ, Weinman SA (2009) Effects of hepatitis C core protein on mitochondrial electron transport and production of reactive oxygen species. Methods Enzymol 456:363–380

    Article  PubMed  CAS  Google Scholar 

  29. Malinska D, Kudin AP, Debska-Veilhaber G, Veilhaber S, Kunz WS (2009) Quantification of superoxide production by mouse brain and skeletal muscle mitochondria. Methods Enzymol 456:419–437

    Article  PubMed  CAS  Google Scholar 

  30. Cohen G, Kesler N (1999) Monoamine oxidase and mitochondrial respiration. J Neurochem 73:2310–2315

    Article  PubMed  CAS  Google Scholar 

  31. Fu W, Luo H, Parthasarathy S, Mattson MP (1998) Catecholamines potentiates amyloid β-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction and perturbed calcium homeostasis. Neurobiol Dis 5:229–243

    Article  PubMed  CAS  Google Scholar 

  32. Kakkar P, Mehrotra S, Vishwanathan PN (1998) Influence of antioxidants on the peroxidative swelling of mitochondria in vitro. Cell Biol Toxicol 14:313–321

    Article  PubMed  CAS  Google Scholar 

  33. Cande C, Vahsen N, Garrido C, Kroemer G (2004) Apoptosis-nducing factor (AIF): caspase-independent after all. Cell Death Differ 11(6):591–595

    PubMed  CAS  Google Scholar 

  34. Kowaltowski AJ, Netto LES, Vercesi AE (1998) The thiol specific antioxidant enzyme prevents mitochondria membrane permeability transition. Evidence for the participation of reactive oxygen species in this mechanism. J Biol Chem 273:12766–12769

    Article  PubMed  CAS  Google Scholar 

  35. Kowaltowski AJ, Castilho RF, Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress. FEBS Lett 495:12–15

    Article  PubMed  CAS  Google Scholar 

  36. Maciel EN, Vercesi AE, Castilho RF (2001) Oxidative stress in Ca2+ induced membrane permeability transition in brain mitochondria. J Neurochem 79:1237–1245

    Article  PubMed  CAS  Google Scholar 

  37. Mehrotra S, Kakkar P, Vishwanathan PN (1999) Mitochondrial damage by active oxygen species in vitro. Free Radic Biol Med 10:277–285

    Article  Google Scholar 

  38. Mehrotra S, Vishwanathan PN, Kakkar P (1993) Influence of some biological response modifiers on swelling of rat liver mitochondria in vitro. Mol Cell Biochem 124:101–106

    Article  PubMed  CAS  Google Scholar 

  39. Lash LH (2008) Mitochondrial GSH transport and intestinal cell injury: a commentary on Contribution of mitochondrial GSH transport to matrix GSH status and colonic epithelial cell apoptosis. Free Radic Biol Med 44:765–767

    Article  PubMed  CAS  Google Scholar 

  40. Reed DJ, Savage MK (1995) Influence of metabolic inhibitors on mitochondrial permeability transition and glutathione status. Biochim Biophys Acta 1271:43–50

    PubMed  Google Scholar 

  41. Chernyak BV, Bernardi P (1996) The mitochondrial permeability transition pore is modulated by oxidative agents through both pyridine nucleotides and glutathione at two separate sites. Eur J Biochem 238:623–630

    Article  PubMed  CAS  Google Scholar 

  42. Costantini P, Chernyak BV, Petronilli V, Bernardi P (1996) Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two different sites. J Biol Chem 271(12):6746–6751

    Article  PubMed  CAS  Google Scholar 

  43. Halestrap AP, Woodfield KY, Connern CP (1997) Oxidative stress, thiol reagents and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J Biol Chem 272(6):3346–3354

    Article  PubMed  CAS  Google Scholar 

  44. Lu C, Armstrong JS (2007) Role of calcium and cyclophilin D in the regulation of mitochondrial permeabilization induced by glutathione depletion. Biochim Biophys Res Commun 363:572–577

    Article  CAS  Google Scholar 

  45. Starnes JW, Barnes BD, Olsen ME (2007) Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli. J Appl Physiol 102:1793–1798

    Article  PubMed  CAS  Google Scholar 

  46. Jones DP (2006) Disruption of mitochondrial circuitry in oxidative stress. Chem Biol Interact 163:38–53

    Article  PubMed  CAS  Google Scholar 

  47. Raha S, Robinson BH (2000) Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem Sci 25:502–508

    Article  PubMed  CAS  Google Scholar 

  48. Kakkar P, Mehrotra S, Vishwanathan PN (1996) tert-BHP induced in vitro swelling of rat liver mitochondria. Mol Cell Biochem 154:39–45

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The author’s are grateful to Director, IITR for his interest in this work. Author’s are also thankful to Dr. Yogeshwar Shukla for support in flow cytometric analysis. Author’s are also grateful to Dr. Manjula Murari, Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences for support in performing TEM. Mr. Brijesh Kumar Singh is indebted to Council of Scientific and Industrial Research (CSIR) and Ms. Madhulika Tripathi is grateful to Indian Council of Medical Research (ICMR) for grant of Senior Research Fellowship. Authors also thank IITR Publication Review Committee for allocation of manuscript number 2766.

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Authors and Affiliations

  1. Herbal Research Section, Indian Institute of Toxicology Research (CSIR), Formerly-Industrial Toxicology Research Centre, Mahatma Gandhi Marg, P. O. Box-80, Lucknow, 226001, Uttar Pradesh, India

    Brijesh Kumar Singh, Madhulika Tripathi & Poonam Kakkar

  2. Department of Biotechnology, J.C. Bose Institute of Life Sciences, Bundelkhand University, Jhansi, 284128, Uttar Pradesh, India

    Brijesh Kumar Singh & Pramod Kumar Pandey

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  1. Brijesh Kumar Singh
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  2. Madhulika Tripathi
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  4. Poonam Kakkar
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Correspondence to Poonam Kakkar.

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Singh, B.K., Tripathi, M., Pandey, P.K. et al. Alteration in mitochondrial thiol enhances calcium ion dependent membrane permeability transition and dysfunction in vitro: a cross-talk between mtThiol, Ca2+, and ROS. Mol Cell Biochem 357, 373–385 (2011). https://doi.org/10.1007/s11010-011-0908-0

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