Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria

Lisa Marroquin1, Rachel Swiss1, Yvonne Will1

1 Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc, Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.4
DOI:  10.1002/0471140856.tx2504s60
Online Posting Date:  May, 2014
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Abstract

The mitochondrial permeability transition pore (MPTP) is a protein pore that forms in the inner mitochondrial membrane and allows the membrane to be permeable to all molecules of less than 1500 Da. Ca2+, numerous reactive chemicals, and oxidative stress induce MPTP opening, whereas cyclosporin A (CsA) or bongkrekic acid block it. In addition, several drugs have been shown to induce MPTP opening, leading to the loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, and release of intermembrane space proteins into the cytosol. This ultimately leads to the rupture of the outer mitochondrial membrane and cell demise. Here, we describe an assay using isolated rat liver mitochondria that can detect Ca2+‐dependent drug‐induced opening of the MPTP, providing protocols for screening in both cuvette and 96‐well format. Curr. Protoc. Toxicol. 60:25.4.1:‐25.4.17. © 2014 by John Wiley & Sons, Inc.

Keywords: mitochondria permeability transition pore; mitochondrial toxicity; mitochondrial swelling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of Mitochondrial Swelling of Compounds in a 96‐Well Format
  • Basic Protocol 2: Assessment of Mitochondrial Swelling of Compounds in a Cuvette Format
  • Support Protocol 1: Isolation of Rat Liver Mitochondria
  • Support Protocol 2: Mitochondrial Protein Assay
  • Support Protocol 3: Optimizing Protein Concentration for Mitochondrial Swelling Measurement
  • Support Protocol 4: Optimizing CaCl2 Concentration for the Mitochondrial Swelling Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assessment of Mitochondrial Swelling of Compounds in a 96‐Well Format

  Materials
  • Sprague‐Dawley male rats (150 to 250 g; Charles River) or equivalent strain
  • BCA protein assay kit (Pierce)
  • 1 M succinate stock solution (see recipe)
  • 10 mM CaCl 2 stock solution (see recipe)
  • 10 mM rotenone stock solution (see recipe)
  • 30 mM oligomycin stock solution (see recipe)
  • 1 mM cyclosporin A stock solution (see recipe)
  • 100 mM phosphate stock solution (see recipe)
  • Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested)
  • Dimethylsulfoxide (DMSO)
  • Mitochondrial swelling buffer (see recipe)
  • 15‐ml tubes, polystyrene, disposable (e.g., BD Falcon)
  • 30°C water bath
  • Multichannel pipettors
  • Standard clear‐bottom 96‐well plate
  • Absorbance plate reader (capable of measuring absorbance at kinetic interval)
  • Computer running Microsoft Excel
  • Program capable of calculating IC 50 (e.g., GraphPad Prism)
  • Additional reagents and equipment for isolation of mitochondria from rat liver ( protocol 3), mitochondrial protein assay ( protocol 4), optimizing mitochondrial protein concentration ( protocol 5), and optimizing CaCl 2 concentration ( protocol 6)

Basic Protocol 2: Assessment of Mitochondrial Swelling of Compounds in a Cuvette Format

  Materials
  • Sprague‐Dawley male rats (150 to 250 g; Charles River) or equivalent strain
  • BCA protein assay kit (Pierce)
  • 1 M succinate stock solution (see recipe)
  • 10 mM CaCl 2 stock solution (see recipe)
  • 10 mM rotenone stock solution (see recipe)
  • 30 mM oligomycin stock solution (see recipe)
  • 1 mM cyclosporin A stock solution (see recipe)
  • 100 mM phosphate stock solution (see recipe)
  • Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested)
  • Mitochondrial swelling buffer (see recipe)
  • Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested)
  • 30°C water bath
  • Standard cuvettes for absorbance
  • Absorbance cuvette reader (spectrophotometer capable of measuring absorbance at kinetic intervals)
  • Computer running Microsoft Excel
  • Program capable of calculating IC 50 (e.g., GraphPad Prism)
  • Additional reagents and equipment for isolation of mitochondria from rat liver ( protocol 3), mitochondrial protein assay ( protocol 4), optimizing mitochondrial protein concentration ( protocol 5), and optimizing CaCl 2 concentration ( protocol 6)

Support Protocol 1: Isolation of Rat Liver Mitochondria

  Materials
  • Sprague‐Dawley male rats (150 to 250 g; Charles River) or equivalent strain
  • Rat food
  • Isolation buffer I (see recipe), ice cold
  • Isolation buffer II (see reagents and solutions)
  • BCA protein assay kit (Pierce)
  • Rat housing (21°C with 12‐hr light/dark cycle capabilities)
  • Sterile dissection tools including scissors
  • 100‐ml glass tissue homogenizer with Teflon pestle
  • Power drill (hand‐held or static)
  • Refrigerated high‐speed centrifuge
  • Cheesecloth
  • Additional reagents and equipment for euthanasia of rats (Donovan and Brown, )

Support Protocol 2: Mitochondrial Protein Assay

  Materials
  • Triton X‐100
  • 2 mg/ml albumin stock solution (Pierce, cat. no. 23209)
  • Isolated mitochondria (see protocol 3)
  • BCA protein assay kit (Pierce, cat. no. 23225) containing:
    • Protein reagent A
    • Protein reagent B
  • 2‐ml microcentrifuge tubes
  • Standard clear‐bottom 96‐well plates
  • 37°C heating block
  • Absorbance plate reader

Support Protocol 3: Optimizing Protein Concentration for Mitochondrial Swelling Measurement

  Materials
  • Sprague‐Dawley male rats (150 to 250 g; Charles River) or equivalent strain
  • BCA protein assay kit (Pierce)
  • 1 M succinate stock solution (see recipe)
  • 10 mM CaCl 2 stock solution (see recipe)
  • 10 mM rotenone stock solution (see recipe)
  • 30 mM oligomycin stock solution (see recipe)
  • 1 mM cyclosporin A stock solution (see recipe)
  • 100 mM phosphate stock solution (see recipe)
  • Mitochondrial swelling buffer (see recipe)
  • Test compound stock: 30 mM troglitazone dissolved in DMSO
  • Dimethylsulfoxide (DMSO)
  • 30°C water bath
  • Standard clear bottom 96‐well plate
  • 15‐ml tubes, polystyrene, disposable (e.g., BD Falcon)
  • Absorbance plate reader (capable of measuring absorbance at kinetic interval)
  • Additional reagents and equipment for isolation of mitochondria from rat liver ( protocol 3) and mitochondrial protein assay ( protocol 4)

Support Protocol 4: Optimizing CaCl2 Concentration for the Mitochondrial Swelling Assay

  Materials
  • Sprague‐Dawley male rats (150 to 250 g; Charles River) or equivalent strain
  • BCA protein assay kit (Pierce)
  • 1 M succinate stock solution (see recipe)
  • 10 mM CaCl 2 stock solution (see recipe)
  • 10 mM rotenone stock solution (see recipe)
  • 30 mM oligomycin stock solution (see recipe)
  • 1 mM cyclosporin A stock solution (see recipe)
  • Mitochondrial swelling buffer (see recipe)
  • 30°C water bath
  • 15‐ml tubes, polystyrene, disposable (BD Falcon)
  • Standard clear bottom 96‐well plate
  • Absorbance plate reader (capable of measuring absorbance at kinetic interval)
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Figures

Videos

Literature Cited

Literature Cited
  Beavis, A.D., Brannan, R.D., and Garlid, K.D. 1985. Swelling and contraction of the mitochondrial matrix. I. A structural interpretation of the relationship between light scattering and matrix volume. J. Biol. Chem. 260:13424‐13433.
  Berson, A., Cazanave, S., Descatoire, V., Tinel, M., Grodet, A., Wolf, C., Feldmann, G., and Pessayre, D. 2006. The anti‐inflammatory drug, nimesulide (4‐nitro‐2‐phenoxymethane‐sulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: Protection by albumin. J. Pharmacol. Exp. Ther. 318:444‐454.
  Crompton, M., Ellinger, H., and Costi, A. 1988. Inhibition by cyclosporin A of a Ca2+‐dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem. J. 255:357‐360.
  Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Drahota, Z., Milerovia, M., Endlicher, R., Rychtromoc, D., Cervinkova, Z., and Ostadal, B. 2012. Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress. Physiol. Res. 61:S165‐S172.
  Dykens, J.A. and Will, Y. 2007. The significance of mitochondrial testing in drug development. Drug Discov. Today. 12:777‐785.
  Halestrap, A.P. and Brenner, C. 2003. The adenine nucleotide translocase: A central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 10:1507‐1525.
  Haworth, R.A. and Hunter, D.R. 1979. The Ca2+‐induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch. Biochem. Biophys. 195:460‐467.
  He, L. and Lemasters, J.J. 2002. Regulated and unregulated mitochondrial permeability transition pores: A new paradigm of pore structure and function? FEBS Lett. 512:1‐7.
  Hunter, D.R. and Haworth, R.A. 1979. The Ca2+‐induced membrane transition in mitochondria. I. The protective mechanisms. Arch. Biochem. Biophys. 195:453‐459.
  Ichas, F., Jouaville, L.S., Sidash, S.S., Mazat, J.P., and Holmuhamedov, E.L. 1994. Mitochondrial calcium spiking: A transduction mechanism based on calcium‐induced permeability transition involved in cell calcium signalling. FEBS Lett. 348:211‐215.
  Kim, J.S., He, L., and Lemasters, J.J. 2003. Mitochondrial permeability transition: A common pathway to necrosis and apoptosis. Biochem. Biophys. Res. Commun. 304:463‐470.
  Lapidus, R.G. and Sokolove, P.M. 1993. Spermine inhibition of the permeability transition of isolated rat liver mitochondria: An investigation of mechanism. Arch. Biochem. Biophys. 306:246‐253.
  Masubuchi, Y., Nakayama, S., and Horie, T. 2002. Role of mitochondrial permeability transition in diclofenac‐induced hepatocyte injury in rats. Hepatology 35:544‐551.
  Masubuchi, Y., Kano, S., and Horie, T. 2006. Mitochondrial permeability transition as a potential determinant of hepatotoxicity of antidiabetic thiazolidinediones. Toxicology 222:233‐239.
  National Institutes of Health. 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. http://grants.nih.gov/grants/olaw/Guide‐for‐the‐care‐and‐use‐of‐laboratory‐animals.pdf.
  Okuda, T., Norioka, M., Shitara, Y., and Horie, T. 2010. Multiple mechanisms underlying troglitazone‐induced mitochondrial permeability transition. Toxicol. Appl. Pharmacol. 248:242‐248.
  Panov, A., Dikalov, S., Shalbuyeva, N., Hemendinger, R., Greenamyre, J.T., and Rosenfeld, J. 2006. Species‐ and tissue‐specific relationships between mitochondrial permeability transition and generation of ROS in brain and liver mitochondria of rats and mice. Am. J. Physiol. Cell Physiol. 292:C708‐718.
  Tanveer, A., Virji, S., Andreeva, L., Totty, N.F., Hsuan, J.J., Ward, J.M., and Crompton, M. 1996. Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress. Eur. J. Biochem. 238:166‐172.
  Tay, V.K., Wang, A.S., Leow, K.Y., Ong, M.M., Wong, K.P., and Boelsterli, U.A. 2005. Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro. Free Radic. Biol. Med. 39:949‐959.
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