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• W2167574366 abstract "Ischemic injuries are associated with several pathological conditions, including stroke and myocardial infarction. Several studies have indicated extensive apoptotic cell death in the infarcted area as well as in the penumbra region of the infarcted tissue. Studies with transgenic animals suggest that the mitochondrion-mediated apoptosis pathway is involved in ischemia-related cell death. This pathway is triggered by activation of pro-apoptotic Bcl-2 family members such as Bax. Here, we have identified and synthesized two low molecular weight compounds that block Bax channel activity. The Bax channel inhibitors prevented cytochrome c release from mitochondria, inhibited the decrease in the mitochondrial membrane potential, and protected cells against apoptosis. The Bax channel inhibitors did not affect the conformational activation of Bax or its translocation and insertion into the mitochondrial membrane in cells undergoing apoptosis. Furthermore, the compounds protected neurons in an animal model of global brain ischemia. The protective effect in the animal model correlated with decreased cytochrome c release in the infarcted area. This is the first demonstration that Bax channel activity is required in apoptosis. Ischemic injuries are associated with several pathological conditions, including stroke and myocardial infarction. Several studies have indicated extensive apoptotic cell death in the infarcted area as well as in the penumbra region of the infarcted tissue. Studies with transgenic animals suggest that the mitochondrion-mediated apoptosis pathway is involved in ischemia-related cell death. This pathway is triggered by activation of pro-apoptotic Bcl-2 family members such as Bax. Here, we have identified and synthesized two low molecular weight compounds that block Bax channel activity. The Bax channel inhibitors prevented cytochrome c release from mitochondria, inhibited the decrease in the mitochondrial membrane potential, and protected cells against apoptosis. The Bax channel inhibitors did not affect the conformational activation of Bax or its translocation and insertion into the mitochondrial membrane in cells undergoing apoptosis. Furthermore, the compounds protected neurons in an animal model of global brain ischemia. The protective effect in the animal model correlated with decreased cytochrome c release in the infarcted area. This is the first demonstration that Bax channel activity is required in apoptosis. Apoptosis is a conserved cell death mechanism essential for normal development and tissue homeostasis in multicellular organisms. Although apoptosis presumably participates in the development of all cell lineages, aberrations in the expression of pro- or anti-apoptotic proteins have been implicated in the initiation of a variety of human diseases, including arteriosclerosis, heart failure, infertility, autoimmunity, immunodeficiency, and cancer, in addition to diseases affecting the nervous system such as neurodegeneration and ischemia (1Yin X.M. Luo Y. Cao G. Bai L. Pei W. Kuharsky D.K. Chen J. J. Biol. Chem. 2002; 277: 42074-42081Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 2Narula J. Pandey P. Arbustini E. Haider N. Narula N. Kolodgie F.D. Dal Bello B. Semigran M.J. Bielsa-Masdeu A. Dec G.W. Israels S. Ballester M. Virmani R. Saxena S. Kharbanda S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8144-8149Crossref PubMed Scopus (516) Google Scholar, 3Green D.R. Bissonnette R.P. Cotter T.G. Important Adv. Oncol. 1994; : 37-52PubMed Google Scholar). Several intracellular apoptosis signaling pathways have been identified, including the death receptor pathway and the mitochondrial pathway (4Schmitz I. Kirchhoff S. Krammer P.H. Int. J. Biochem. Cell Biol. 2000; 32: 1123-1136Crossref PubMed Scopus (230) Google Scholar, 5Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 6Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3245) Google Scholar). The induction of apoptosis ultimately converges upon the activation of cysteine proteases of the caspase family. The Bcl-2 family proteins are located upstream at organelle membranes and control the activation of downstream caspases, representing a critical proximal intracellular checkpoint in the mitochondrial apoptosis pathway. The Bcl-2 family is composed of pro- and anti-apoptotic members. Anti-apoptotic Bcl-2 family members display sequence homology in four α-helical domains called BH1–BH4. 3The abbreviations used are: BHBcl-2 homologyPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidVDACvoltage-dependent anion channelCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBci1Bax channel inhibitor-1Bci2Bax channel inhibitor-2HEKhuman embryonic kidneyFACSfluorescence-activated cell sorterMEFmurine embryonic fibroblast.3The abbreviations used are: BHBcl-2 homologyPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidVDACvoltage-dependent anion channelCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidBci1Bax channel inhibitor-1Bci2Bax channel inhibitor-2HEKhuman embryonic kidneyFACSfluorescence-activated cell sorterMEFmurine embryonic fibroblast. Pro-apoptotic Bcl-2 members can be further subdivided into more fully conserved, “multidomain” members with homology in the BH1–BH3 domains (e.g. Bax and Bak) or the “BH3-only” members (e.g. Bid, Bad, and Bim). Genetic and biochemical analyses indicate that the multidomain proteins Bax and Bak function as the essential gateway to the intrinsic cell death pathway operating at the mitochondria. The upstream BH3-only members respond to particular apoptotic signals and subsequently, either directly or indirectly, trigger the conformational activation of Bax and/or Bak. Overexpression of the anti-apoptotic protein Bcl-2 or deletion of the pro-apoptotic protein Bax increases resistance to ischemic insults, indicating that the mitochondrial pathway is involved in ischemia-related apoptosis (7Martinou J.-C. Dubois-Dauphin M. Staple J.K. Rodriguez I. Frankowski H. Missotten M. Albertini P. Talabot D. Catsicas S. Pietra C. Neuron. 1994; 13: 1017-1030Abstract Full Text PDF PubMed Scopus (1013) Google Scholar, 8Gibson M.E. Han B.H. Choi J. Knudson C.M. Korsmeyer S.J. Parsadanian M. Holtzman D.M. Mol. Med. 2001; 7: 644-655Crossref PubMed Google Scholar). Further evidence for the crucial role of Bax in neuronal cell death was provided by a recent study showing that cerebellum granule neurons are protected against prion-induced apoptosis in Bax–/– mice (9Chiesa R. Piccardo P. Dossena S. Nowoslawski L. Roth K.A. Ghetti B. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 238-243Crossref PubMed Scopus (80) Google Scholar). In normal cells, Bax is present as a soluble monomeric protein in the cytosol. When cells are exposed to various apoptotic stimuli, including hypoxia, the protein translocates specifically to the mitochondria (10Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (966) Google Scholar, 11Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1025) Google Scholar, 12Saikumar P. Dong Z. Patel Y. Hall K. Hopfer U. Weinberg J.M. Venkatachalam M.A. Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (259) Google Scholar). Bid activation has been shown to be important in hypoxia-induced apoptosis (13Plesnila N. Zinkel S. Le D.A. Amin-Hanjani S. Wu Y. Qiu J. Chiarugi A. Thomas S.S. Kohane D.S. Korsmeyer S.J. Moskowitz M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15318-15323Crossref PubMed Scopus (227) Google Scholar). At the mitochondria, Bax forms oligomers, which are inserted into the outer mitochondrial membrane, permeabilizing the membrane and triggering the release of proteins, including cytochrome c, from the mitochondrial intermembrane space (11Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1025) Google Scholar, 14Antonsson B. Montessuit S. Sanchez B. Martinou J.-C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). In the cytosol, cytochrome c forms a complex with the cytosolic proteins Apaf-1 and procaspase-9 (15Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6199) Google Scholar). Upon complex formation, caspase-9 is proteolytically modified, leading to its activation. The active caspase-9 activates downstream executor caspases, which subsequently cleave several substrates, ultimately leading to apoptotic cell death. Bcl-2 homology phosphate-buffered saline 4-morpholineethanesulfonic acid voltage-dependent anion channel 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Bax channel inhibitor-1 Bax channel inhibitor-2 human embryonic kidney fluorescence-activated cell sorter murine embryonic fibroblast. Bcl-2 homology phosphate-buffered saline 4-morpholineethanesulfonic acid voltage-dependent anion channel 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Bax channel inhibitor-1 Bax channel inhibitor-2 human embryonic kidney fluorescence-activated cell sorter murine embryonic fibroblast. Recombinant oligomeric Bax possesses channel-forming activity in artificial lipid membranes and triggers cytochrome c release from liposomes and purified mitochondria (16Saito M. Korsmeyer S.J. Schlesinger P.H. Nat. Cell Biol. 2000; 2: 553-555Crossref PubMed Scopus (409) Google Scholar, 17Schlesinger P.H. Gross A. Yin X.M. Yamamoto K. Saito M. Waksman G. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (442) Google Scholar, 18Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (925) Google Scholar). Although it is clear that Bax has a central function in the regulation of the mitochondrial apoptosis pathway, it remains unclear how the protein executes its pro-apoptotic activity at the molecular level and whether its channel-forming activity is required. To elucidate the function of Bax, we have identified low molecular weight compounds that function as Bax channel blockers and that do not alter Bax homo-oligomerization. Using this pharmacological tool, we show that Bax channel activity is required for cytochrome c release from mitochondria and that blocking Bax channel activity inhibits mitochondrion-mediated apoptosis in cells in vitro. In addition, the compounds protected neurons against apoptotic cell death in an animal model of global brain ischemia. The effect in vitro is Bax-specific because the channel inhibitors did not prevent apoptosis in Bax-deficient cells, whereas they did in Bak-deficient and wild-type cells. These findings strongly suggest that the mechanism by which these compounds inhibit apoptosis in vivo is through blockage of Bax channel activity and thus, for the first time, demonstrate involvement of Bax channel activity in the regulation of apoptotic cell death. Recombinant Proteins—C-terminally truncated oligomeric Bax was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified as described previously (19Lewis S. Bethell S.S. Patel S. Martinou J.-C. Antonsson B. Protein Expression Purif. 1998; 13: 120-126Crossref PubMed Scopus (27) Google Scholar). Full-length oligomeric Bax with an N-terminal His tag was expressed in and purified from E. coli (20Montessuit S. Mazzei G. Magnenat E. Antonsson B. Protein Expression Purif. 1999; 15: 202-206Crossref PubMed Scopus (39) Google Scholar). Bid with an N-terminal His tag was expressed in E. coli and purified on nickel-nitrilotriacetic acid-agarose as described (21Kudla G. Montessuit S. Eskes R. Berrier C. Martinou J.-C. Ghazi A. Antonsson B. J. Biol. Chem. 2000; 275: 22713-22718Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). tcBid was generated through cleavage of purified Bid with caspase-8. Liposome Channel Assay—Liposomes containing 20 mm 5,6-carboxyfluorescein in phosphate-buffered saline (PBS) were prepared as described (18Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (925) Google Scholar). The liposomes were diluted in PBS to give a suitable fluorescence value. The channel activity assay was performed in 96-well plates on a fluorescence plate reader (FLIPR™, Novel Tech Systems Inc.). For the assay, 70 ml of PBS, 15 ml of oligomeric Bax (1 μm) in PBS, and 10 ml of compound in eight dilutions (final concentrations of 22 nm to 3.8 μm) in 10% Me2SO in PBS were mixed in a 96-well plate and incubated at room temperature for 1 min. At the end of the incubation, 20 μl of liposomes in PBS was added, and the fluorescence was monitored in the FLIPR™ every 3 s for 3 min. The fluorescence values at 120 s were used to calculate the IC50 values. Electrophysiological Recordings—Planar lipid membranes were formed from monolayers made from 1% (w/v) lipids in hexane on 70–80-μm diameter orifices in a 15-μm-thick Teflon partition that separated the two chambers as described (22Montal M. Mueller P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3561-3566Crossref PubMed Scopus (1590) Google Scholar, 23Rostovtseva T.K. Bezrukov S.M. Biophys. J. 1998; 74: 2365-2373Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The lipid-forming solutions contained 90% asolectin (soybean phospholipids) and 10% cholesterol or 42% asolectin, 42% diphytanoylphosphatidylcholine, 8% cardiolipin, and 8% cholesterol. Asolectin, diphytanoylphosphatidylcholine, and cardiolipin were purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol was purchased from Sigma. The membranes were made in aqueous solutions containing 250 mm KCl and 1 mm MgCl2 or CaCl2 buffered with 5 mm HEPES or MES at pH 7.0 or 5.5, correspondingly. VDAC channels were isolated from rat liver mitochondrial outer membranes and purified according to the standard method (24Parsons D.F. Williams G.R. Chance B. Ann. N. Y. Acad. Sci. 1966; 137: 643-666Crossref PubMed Scopus (232) Google Scholar, 25Blachly-Dyson E. Peng S. Colombini M. Forte M. Science. 1990; 247: 1233-1236Crossref PubMed Scopus (235) Google Scholar). Conductance measurements were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) in the voltage clamp mode. Data were filtered by a low pass 8-pole Butterworth filter (Model 9002, Frequency Devices, Inc., Haverhill, MA) at 15 kHz, recorded on a chart recorder, and directly saved in the computer memory with a sampling frequency of 50 kHz. The inhibitory effect of the compounds was determined as follows. After 20 min of continuous recording of Bax-induced channel activity, the inhibitors (50–60 nm) were added either to the trans-side or to both sides of the membrane under constant stirring for 2 min. The currents were recorded for 10 min, followed by a series of additions of the inhibitor until a significant decrease or complete inhibition of the conductance was obtained. Mitochondrial Cytochrome c Release Assay—Mitochondria were isolated from HeLa cells as described (14Antonsson B. Montessuit S. Sanchez B. Martinou J.-C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). In summary, the cells were suspended in buffer A (10 mm HEPES-NaOH, 210 mm mannitol, 70 mm sucrose, and 1 mm EDTA, pH 7.4) and disrupted by passage through a 25 G1 0.5 × 25 needle, and the mitochondria were isolated by differential centrifugation. The mitochondria were diluted to 0.4 mg/ml in buffer B (10 mm HEPES-NaOH, 125 mm KCl, 4 mm MgCl2, 5 mm NaH2PO4, 0.5 mm EGTA, and 5 mm succinate, pH 7.5). The compound was added to 1 ml of mitochondrial suspension, and the samples were incubated for 5 min at room temperature. tcBid was subsequently added to a final concentration of 10 nm, and the samples were incubated at 30 °C for 15 min and centrifuged twice at 12,000 × g for 10 min. The supernatant was removed and analyzed by Western blotting with an in house-raised rabbit anti-cytochrome c polyclonal antibody. The blots were developed with an ECL detection kit (Amersham Biosciences), and the intensity of the cytochrome c bands was determined by densitometry. Mitochondria incubated without addition of tcBid were taken as the blank value, and 100% cytochrome c release corresponds to mitochondria treated with tcBid in the absence of inhibitor. Quaternary Structure of Purified Bax—Purified recombinant monomeric Bax at a concentration of 0.4 mg/ml was incubated in the presence and absence of the compounds (10 μm) for 1 h at 37°C. Alternatively, Bax was preincubated as described above; at the end of the incubation 2% octyl glucoside was added to induce Bax oligomerization; and the samples were further incubated for 2 h. At the end of the incubation periods, the samples were analyzed by gel filtration on a Superdex 200 column (PC 3.2/30) equilibrated in 25 mm HEPES-NaOH, 300 mm NaCl, 0.2 mm dithiothreitol, and 2% (w/v) CHAPS, pH 7.5, in the SMART system (Amersham Biosciences). 50 μl of the sample was loaded and eluted at 50 μl/min; 50-μl fractions were collected; and the eluate was monitored at 280 nm. Quaternary Structure of Bax in Cells—HeLa cells were seeded and grown to confluence in 15-cm plates. The cells were then treated with 1 μm staurosporine in the presence and absence of 2 μm Bax channel inhibitor-1 (Bci1) and Bax channel inhibitor-2 (Bci2) for 16 h. In addition, cells were also treated with the compounds in the absence of staurosporine. Untreated cells were used as a negative control. At the end of the incubation, all cells were collected and washed, and the mitochondria were isolated as described above. The mitochondria isolated from the staurosporine-treated cells were washed with 0.1 m Na2CO3, pH 12, to remove non-membrane integrated proteins. Untreated cells and cells treated with the compounds alone were not because this would have removed non-activated Bax completely (14Antonsson B. Montessuit S. Sanchez B. Martinou J.-C. J. Biol. Chem. 2001; 276: 11615-11623Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). The washed mitochondria were solubilized in 25 mm HEPES-NaOH, 300 mm NaCl, 0.2 mm dithiothreitol, and 2% (w/v) CHAPS, pH 7.5. After a 1-h incubation at 4 °C, the samples were centrifuged at 100,000 × g for 30 min. The supernatant containing the solubilized mitochondrial proteins was analyzed by gel filtration as described above. The fractions eluted from the column were analyzed by Western blotting with anti-Bax antibodies (anti-Bax NT, catalog no. 06-499, Upstate Biotechnology, Inc.). Alternatively, Bax activation was monitored by immunofluorescence in cells treated with staurosporine using anti-Bax NT antibodies (which do not react with non-activated Bax) following the protocol described previously (26Hetz C. Russelakis-Carneiro M. Walchli S. Carboni S. Vial-Knecht E. Maundrell K. Castilla J. Soto C. J. Neurosci. 2005; 25: 2793-2802Crossref PubMed Scopus (179) Google Scholar). Induction of Apoptosis in Cell Lines—HeLa cells or SV40 immortalized murine embryonic fibroblasts from wild-type or knockout cells were seeded in 6-well plates; and after 24 h, the cells were treated with 2 μm staurosporine for 3 h with and without a 30-min pretreatment with the inhibitors (10 μm). At the end of the incubation, the mitochondrial membrane potential and cell volume were analyzed by flow cytometry (FACSCalibur, BD Biosciences) after staining the cells with 3,3′-dihexyloxacarbocyanine iodide (Molecular Probes) as described (27Hetz C. Bono M.R. Barros L.F. Lagos R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2696-2701Crossref PubMed Scopus (124) Google Scholar). Analysis was performed using the CellQuest program. In parallel, cells were treated with 2 μm staurosporine for 6 h with and without pretreatment with the inhibitors (2 μm), and nuclear morphology was analyzed by staining with Hoechst 33342 (Molecular Probes). Alternatively, cell viability was quantified using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and phenazine methosulfate (CellTiter 96® AQueous, Promega Corp., Madison, WI) according to the recommendations of the supplier. For Bax or Bak overexpression, human embryonic kidney (HEK) cells were transfected using SuperFect reagent (Qiagen Inc.) following the manufacturer's instructions. After 16 h in the presence or absence of the compounds (2 μm), the cells were collected, and hypodiploid cells were quantified by fluorescence-activated cell sorter (FACS) analysis after propidium iodide staining as described previously (27Hetz C. Bono M.R. Barros L.F. Lagos R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2696-2701Crossref PubMed Scopus (124) Google Scholar). Alternatively, apoptosis was induced by overexpression of tcBid in wild-type, Bax–/–, and Bak–/– murine embryonic fibroblast (MEF) cells. tcBid was expressed by retroviral delivery as described previously, and cell viability was determined after 24 h by FACS analysis after propidium iodide staining (28Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3332) Google Scholar). Global Brain Ischemia in Gerbils—Adult male Mongolian gerbils (Elevage Janvier, Le Genest St. Isle, France) weighing 60–80 g were kept in a temperature-controlled (23 ± 1 °C) and light/dark cycle-controlled animal room (lights on at 7 a.m. and off at 7 p.m.). All experiments (eight animals/group) were performed under spontaneous respiration. Anesthesia was induced with 4% isoflurane in a gas mixture of medical air administered via facemask and maintained at 2% isoflurane in the same gas mixture. Bilateral common carotid arteries were dissected and occluded with bulldog clamps for 5 min. To examine the effect of the Bax channel inhibitor on brain ischemia, Bci1 (3 and 30 mg/kg intraperitoneally) and Bci2 (30 mg/kg intraperitoneally) solubilized in saline were injected 15 min, 24 h, and 48 h after reperfusion. Orotic acid (300 mg/kg intraperitoneally) was used as the reference compound. 7 days after the onset of occlusion, the gerbils were killed by decapitation. The brains were frozen in isopentane (–20 °C) and cut into 20-μm-thick sections in a microtome. The sections were stained with cresyl violet, and infarcts in the right and left parts of the hippocampus were scored with a 4-point scale. Pyramidal cells showing atrophy, shrinkage, nuclear pyknosis, dark cytoplasmic coloration, and vacuolation and the disappearance of the radial striated zone indicated cell degeneration: 0, no loss of CA1 neurons; 1, weak damage of CA1 (CA1/subiculum or CA1/CA3 border); 2, loss of CA1 neurons (less than half); 3, loss of CA1 neurons (more than half); and 4, total loss of CA1 neurons and expansion into other areas (CA3, dentate gyrus, and cortex). The total score is the sum of the scores for the left and right hemispheres (29Gerhardt S.C. Boast C.A. Behav. Neurosci. 1988; 102: 301-303Crossref PubMed Scopus (95) Google Scholar, 30Gronborg M. Johansen T.E. Peters D. Ahring P.K. Drejer J. Moller A. Jensen L.H. J. Pharmacol. Exp. Ther. 1999; 290: 348-353PubMed Google Scholar). Global Brain Ischemia-induced Cytochrome c Release in Gerbil Hippocampus—Gerbils were submitted to a 5-min bilateral occlusion of the common carotid arteries as described above. The ischemic gerbils (n = 2–6) were killed 2, 4, 24, 48, 96, 120, or 168 h after the onset of ischemia. To evaluate the effects of the Bax inhibitor on the global ischemia-induced cytochrome c release in gerbil hippocampus, the animals were treated with Bci1 (30 mg/kg intraperitoneally) 15 min, 24 h, and 48 h after reperfusion. They were killed 5 days after the onset of ischemia. The hippocampi were excised and kept on ice. They were cut into small pieces and subsequently suspended in 10 mm HEPES-NaOH, 210 mm mannitol, 70 mm sucrose, 1 mm EDTA, 5 mm EGTA, and protease inhibitor mixture (Roche Applied Science), pH 7.4. The cells were disrupted by passage through a 25-gauge 1-inch needle, and the mitochondria were removed by differential centrifugation as described above for isolation of mitochondria from HeLa cells. Three breaking cycles through the needle were performed, and the supernatant fraction from each one was treated and analyzed separately. The supernatant fractions corresponding to the cytosolic cell fraction were analyzed by Western blotting with anti-cytochrome c antibody. Most cytochrome c was detected in the first and second fractions. The Western blots were scanned with a densitometer, and the intensity of the band corresponding to cytochrome c was determined. All cytochrome c values were normalized to the cytochrome c content in the cytosolic fraction from non-ischemic animals, which corresponded to a value of 1. Statistical Analysis—All data are expressed as means ± S.E. Statistical analysis was performed using one-way analysis of variance, followed by Dunnett's t test. Significance was considered as p < 0.05 versus the control group. Bax Channel Inhibitors in the Liposome Channel Assay—To elucidate the molecular mechanism by which Bax exerts its pro-apoptotic activity, a large collection of low molecular weight compounds was screened for Bax channel inhibitors. The results enabled us to identify and synthesize compounds possessing specific Bax channel inhibitory activity. Here, we describe the mechanism of two of these inhibitors, Bci1 and Bci2 (Fig. 1A). Bax channel-forming activity was first studied using liposomes charged with the fluorescent dye 5,6-carboxyfluorescein. When the liposomes were incubated with either C-terminally truncated or full-length oligomeric Bax, its channel-forming activity induced the release of 5,6-carboxyfluorescein from the liposomes, which was measured as an increase in fluorescence over time. Addition of Bci1 or Bci2 to the assay solution inhibited Bax channel-forming activity in a concentration-dependent manner (Fig. 1, B and D). The IC50 values were determined to be 0.81 ± 0.22 and 0.89 ± 0.29 μm, respectively (Fig. 1, C and E). At 2.4 μm, the activity was completely inhibited in both cases. Electrophysiological Characterization of the Bax Inhibitors—Because the liposome assay cannot distinguish between various possible mechanisms of inhibition such as inhibition of Bax insertion into the lipid membrane, the prevention of Bax channel assembly, or a genuine blockage of the Bax channels, we characterized the compounds electrophysiologically. Two typical experiments are illustrated in Fig. 2. Fig. 2A shows an example of Bax channel activity 15 min after addition of 20 nm full-length oligomeric Bax. Addition of 0.15 μm Bci1 caused a gradual decrease in Bax-induced conductance from ∼9 to 0.7 nanosiemens over 10 min (Fig. 2B). The conductance was completely inhibited after addition of 0.20 μm Bci1 (Fig. 2C). The inhibitory effect of Bci2 is illustrated in Fig. 2 (D–F). To avoid any possible interaction between the inhibitor and Bax other than on the Bax-formed channels, the protein and the compound were added to the opposite sides of the membrane. Fig. 2D shows Bax-induced channels 5 min after addition of 30 nm full-length oligomeric Bax to the cis-compartment. Addition of 0.15 μm Bci2 to the trans-compartment caused a gradual decrease in Bax-induced conductance (Fig. 2E), with complete inhibition after addition of 0.2 μm Bci2 (Fig. 2F). The inhibitory effects of both Bci1 and Bci2 were seen in the 0.10–0.22 μm range. We previously demonstrated that full-length oligomeric Bax channels have a wide distribution of conductances, ranging from 100 picosiemens to tens of nanosiemens (18Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.-C. Science. 1997; 277: 370-372Crossref PubMed Scopus (925) Google Scholar, 31Roucou X. Rostovtseva T. Montessuit S. Martinou J.-C. Antonsson B. Biochem. J. 2002; 368: 915-921Crossref PubMed Scopus (170) Google Scholar). When the Bax-induced conductance was large (more than ∼20 nanosiemens), addition of Bci1 up to 2.6 μm did not inhibit the channels. Bax channel activity is enhanced at acidic pH. The possibility that acidic pH might influence the interaction between Bax channels and the inhibitor was tested by performing experiments at both neutral (pH 7.0, n = 4) and acidic (pH 5.5, n = 9) pH. No difference in inhibition was detected (data not shown). VDAC is one of the major proteins in the mitochondrial outer membrane. It has been suggested that VDAC could play a role in cytochrome c release during apoptosis (32Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1910) Google Scholar). To test the specificity of the inhibitors, we performed control experiments with VDAC channels. No effect on either VDAC channel conductance or gating properties was observed in the presence of 0.20–0.75 μm Bci1 (n = 5) or 0.1–2.0 μm Bci2 (n = 2) (data not shown). Bid is another pro-apoptotic member of the Bcl-2 protein family. We have found that Bid forms stable pores in planar lipid membranes containing cardiolipin after cleavage with caspase-8 (tcBid) (33Rostovtseva T.K. Antonsson B. Suzuki M. Youle R.J. Colombini M. Bezrukov S.M. J. Biol. Chem. 2004; 279: 13575-13583Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We tested the inhibitory effect of Bci1 on tcBid-induced conductance. Here again, there was no" @default.
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• W2167574366 date "2005-12-01" @default.
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• W2167574366 title "Bax Channel Inhibitors Prevent Mitochondrion-mediated Apoptosis and Protect Neurons in a Model of Global Brain Ischemia" @default.
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