Neurochemical Research

, Volume 35, Issue 11, pp 1667–1674 | Cite as

Inhibitory Effects of Adenine Nucleotides on Brain Mitochondrial Permeability Transition

  • Ângela Saito
  • Roger F. Castilho


The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca2+ and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca2+ × mg protein−1. ADP or ATP also prevented Ca2+-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K i) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca2+-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.


Apoptosis Brain ischemia Cell death Intracellular calcium homeostasis Mitochondrial permeability transition Neurodegeneration 



The authors would like to thank Edilene S. Santos for her technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Instituto Nacional de Obesidade e Diabetes. A.S. was supported by a FAPESP fellowship.


  1. 1.
    Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341:233–249CrossRefPubMedGoogle Scholar
  2. 2.
    Kowaltowski AJ, Castilho RF, Vercesi AE (2001) Mitochondrial permeability transition and oxidative stress. FEBS Lett 495:12–15CrossRefPubMedGoogle Scholar
  3. 3.
    Kim JS, He L, Lemasters JJ (2003) Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304:463–470CrossRefPubMedGoogle Scholar
  4. 4.
    Albin RL, Greenamyre JT (1992) Alternative excitotoxic hypotheses. Neurology 42:733–738PubMedGoogle Scholar
  5. 5.
    Fiskum G, Murphy AN, Beal MF (1999) Mitochondria in neurodegeneration: acute ischemia and chronic neurodegenerative diseases. J Cereb Blood Flow Metab 19:351–369CrossRefPubMedGoogle Scholar
  6. 6.
    Fernandes AM, Landeira-Fernandez AM, Souza-Santos P, Carvalho-Alves PC, Castilho RF (2008) Quinolinate-induced rat striatal excitotoxicity impairs endoplasmic reticulum Ca2+-ATPase function. Neurochem Res 33:1749–1758CrossRefPubMedGoogle Scholar
  7. 7.
    Mirandola SR, Melo DR, Saito A, Castilho RF (2010) 3-nitropropionic acid-induced mitochondrial permeability transition: comparative study of mitochondria from different tissues and brain regions. J Neurosci Res 88:630–639PubMedGoogle Scholar
  8. 8.
    Nicholls DG, Budd SL (2000) Mitochondria and neuronal survival. Physiol Rev 80:315–360PubMedGoogle Scholar
  9. 9.
    Gunter TE, Sheu SS (2009) Characteristics and possible functions of mitochondrial Ca2+ transport mechanisms. Biochim Biophys Acta 1787:1291–1308CrossRefPubMedGoogle Scholar
  10. 10.
    Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE (2009) Mitochondria and reactive oxygen species. Free Radic Biol Med 47:333–343CrossRefPubMedGoogle Scholar
  11. 11.
    Petronilli V, Cola C, Massari S, Colonna R, Bernardi P (1993) Physiological effectors modify voltage sensing by the cyclosporin A-sensitive permeability transition pore of mitochondria. J Biol Chem 268:21939–21945PubMedGoogle Scholar
  12. 12.
    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 272:3346–3354CrossRefPubMedGoogle Scholar
  13. 13.
    Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP, MacGregor GR, Wallace DC (2004) The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 427:461–465CrossRefPubMedGoogle Scholar
  14. 14.
    Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55:698–707CrossRefPubMedGoogle Scholar
  15. 15.
    Kristián T, Weatherby TM, Bates TE, Fiskum G (2002) Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria. J Neurochem 83:1297–1308CrossRefPubMedGoogle Scholar
  16. 16.
    Murphy AN, Bredesen DE, Cortopassi G, Wang E, Fiskum G (1996) Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc Natl Acad Sci USA 93:9893–9898CrossRefPubMedGoogle Scholar
  17. 17.
    Fornazari M, de Paula JG, Castilho RF, Kowaltowski AJ (2008) Redox properties of the adenoside triphosphate-sensitive K+ channel in brain mitochondria. J Neurosci Res 86:1548–1556CrossRefPubMedGoogle Scholar
  18. 18.
    Fabiato A, Fabiato F (1978) Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J Physiol 276:233–255PubMedGoogle Scholar
  19. 19.
    Andreyev AY, Fahy B, Fiskum G (1998) Cytochrome c release from brain mitochondria is independent of the mitochondrial permeability transition. FEBS Lett 439:373–376CrossRefPubMedGoogle Scholar
  20. 20.
    Maciel EN, Kowaltowski AJ, Schwalm FD, Rodrigues JM, Souza DO, Vercesi AE, Wajner M, Castilho RF (2004) Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition. J Neurochem 90:1025–1035CrossRefPubMedGoogle Scholar
  21. 21.
    Chudapongse P (1976) Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria. Biochim Biophys Acta 423:196–202CrossRefPubMedGoogle Scholar
  22. 22.
    Leyssens A, Nowicky AV, Patterson L, Crompton M, Duchen MR (1996) The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes. J Physiol 496:111–128PubMedGoogle Scholar
  23. 23.
    Uchino H, Elmér E, Uchino K, Li PA, He QP, Smith ML, Siesjö BK (1998) Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat. Brain Res 812:216–226CrossRefPubMedGoogle Scholar
  24. 24.
    Matsumoto S, Friberg H, Ferrand-Drake M, Wieloch T (1999) Blockade of the mitochondrial permeability transition pore diminishes infarct size in the rat after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 19:736–741CrossRefPubMedGoogle Scholar
  25. 25.
    Schinzel AC, Takeuchi O, Huang Z, Fisher JK, Zhou Z, Rubens J, Hetz C, Danial NN, Moskowitz MA, Korsmeyer SJ (2005) Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc Natl Acad Sci USA 102:12005–12010CrossRefPubMedGoogle Scholar
  26. 26.
    Liu RR, Murphy TH (2009) Reversible cyclosporine A sensitive mitochondrial depolarization occurs within minutes of stroke onset in mouse somatosensory cortex in vivo, a two-photon imaging study. J Biol Chem 284:36109–36117CrossRefPubMedGoogle Scholar
  27. 27.
    Nicholls DG, Scott ID (1980) The regulation of brain mitochondrial calcium-ion transport. The role of ATP in the discrimination between kinetic and membrane-potential-dependent calcium-ion efflux mechanisms. Biochem J 186:833–839PubMedGoogle Scholar
  28. 28.
    Kristián T, Pivovarova NB, Fiskum G, Andrews SB (2007) Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention. J Neurochem 102:1346–1356CrossRefPubMedGoogle Scholar
  29. 29.
    Maciel EN, Kaminski Schierle GS, Hansson O, Brundin P, Castilho RF (2003) Cyclosporin A and Bcl-2 do not inhibit quinolinic acid-induced striatal excitotoxicity in rodents. Exp Neurol 183:430–437CrossRefPubMedGoogle Scholar
  30. 30.
    Vergun O, Reynolds IJ (2005) Distinct characteristics of Ca2+-induced depolarization of isolated brain and liver mitochondria. Biochim Biophys Acta 1709:127–137CrossRefPubMedGoogle Scholar
  31. 31.
    Shalbuyeva N, Brustovetsky T, Brustovetsky N (2007) Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome c release. J Biol Chem 282:18057–18068CrossRefPubMedGoogle Scholar
  32. 32.
    Rottenberg H, Marbach M (1989) Adenine nucleotides regulate Ca2+ transport in brain mitochondria. FEBS Lett 247:483–486CrossRefPubMedGoogle Scholar
  33. 33.
    Rottenberg H, Marbach M (1990) Regulation of Ca2+ transport in brain mitochondria. II. The mechanism of the adenine nucleotides enhancement of Ca2+ uptake and retention. Biochim Biophys Acta 1016:87–98CrossRefPubMedGoogle Scholar
  34. 34.
    Broekemeier KM, Pfeiffer DR (1989) Cyclosporin A-sensitive and insensitive mechanisms produce the permeability transition in mitochondria. Biochem Biophys Res Commun 163:561–566CrossRefPubMedGoogle Scholar
  35. 35.
    Maciel EN, Vercesi AE, Castilho RF (2001) Oxidative stress in Ca2+-induced membrane permeability transition in brain mitochondria. J Neurochem 79:1237–1245CrossRefPubMedGoogle Scholar
  36. 36.
    Williams SP, Fulton AM, Brindle KM (1993) Estimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo. Biochemistry 32:4895–4902CrossRefPubMedGoogle Scholar
  37. 37.
    Vercesi AE, Kowaltowski AJ, Oliveira HC, Castilho RF (2006) Mitochondrial Ca2+ transport, permeability transition and oxidative stress in cell death: implications in cardiotoxicity, neurodegeneration and dyslipidemias. Front Biosci 11:2554–2564CrossRefPubMedGoogle Scholar
  38. 38.
    Friberg H, Ferrand-Drake M, Bengtsson F, Halestrap AP, Wieloch T (1998) Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death. J Neurosci 18:5151–5159PubMedGoogle Scholar
  39. 39.
    Ha HC, Snyder SH (2000) Poly(ADP-ribose) polymerase-1 in the nervous system. Neurobiol Dis 7:225–239CrossRefPubMedGoogle Scholar
  40. 40.
    Alano CC, Ying W, Swanson RA (2004) Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition. J Biol Chem 279:18895–18902CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Departamento de Patologia Clínica, Faculdade de Ciências MédicasUniversidade Estadual de Campinas (UNICAMP)CampinasBrazil

Personalised recommendations