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• W1966152110 abstract "The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate. The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate. Mitochondria are temporo-spatial modulators of cytosolic [Ca2+] through their ability to both sequester and release Ca2+ in concert with Ca2+ transport across the endoplasmic reticulum (1Csordas G. Thomas A.P. Hajnoczky G. EMBO J. 1999; 18: 96-108Crossref PubMed Scopus (451) Google Scholar, 2Simpson P.B. Russell J.T. J. Biol. Chem. 1996; 271: 33493-33501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Physiological mitochondrial Ca2+ efflux, including that caused by Ca2+-induced Ca2+ release (mCICR), 1The abbreviations used are: mCICR, mitochondrial Ca2+-induced Ca2+ release; PTP, permeability transition pore; 2-APB, 2-aminoethoxydiphenyl borate; TMRE, tetramethylrhodamine ethyl ester perchlorate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; ΔΨ, mitochondrial membrane potential; SOCE, store-operated Ca2+ entry; TRP, transient receptor potential.1The abbreviations used are: mCICR, mitochondrial Ca2+-induced Ca2+ release; PTP, permeability transition pore; 2-APB, 2-aminoethoxydiphenyl borate; TMRE, tetramethylrhodamine ethyl ester perchlorate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; ΔΨ, mitochondrial membrane potential; SOCE, store-operated Ca2+ entry; TRP, transient receptor potential. is attributed to either activation of the permeability transition pore (PTP) (3Evtodienko Y. Teplova V. Khawaja J. Saris N.E. Cell Calcium. 1994; 15: 143-152Crossref PubMed Scopus (42) Google Scholar, 4Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar) or to reversal of the Ca2+ uniporter (5Montero M. Alonso M.T. Albillos A. Garcia-Sancho J. Alvarez J. Mol. Biol. Cell. 2001; 12: 63-71Crossref PubMed Scopus (82) Google Scholar). Opening of the PTP has been implicated as a mediator of apoptotic and necrotic cell death as well as a regulator of normal cell Ca2+ homeostasis (4Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 6Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 468-477Crossref PubMed Scopus (358) Google Scholar, 7Haworth R.A. Hunter D.R. Arch. Biochem. Biophys. 1979; 195: 460-467Crossref PubMed Scopus (622) Google Scholar, 8Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 453-459Crossref PubMed Scopus (592) Google Scholar, 9Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2189) Google Scholar, 10Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Elmore S.P. Nishimura Y. Crowe R.A. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1223) Google Scholar, 11Gunter T.E. Pfeiffer D.R. Am. J. Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar, 12Ichas F. Jouaville L.S. Sidash S.S. Mazat J.P. Holmuhamedov E.L. FEBS Lett. 1994; 348: 211-215Crossref PubMed Scopus (137) Google Scholar). The characteristic traits of the PTP include reversibility (13Minamikawa T. Williams D.A. Bowser D.N. Nagley P. Exp. Cell Res. 1999; 246: 26-37Crossref PubMed Scopus (149) Google Scholar), sensitivity to inhibition by cyclosporin A (14Fournier N. Ducet G. Crevat A. J. Bioenerg. Biomembr. 1987; 19: 297-303Crossref PubMed Scopus (276) Google Scholar), and mitochondrial swelling associated with loss of matrix solutes (15Di Lisa F. Menabo R. Canton M. Barile M. Bernardi P. J. Biol. Chem. 2001; 276: 2571-2575Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 16Huser J. Rechenmacher C.E. Blatter L.A. Biophys. J. 1998; 74: 2129-2137Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 17Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Herman B. Mol. Cell. Biochem. 1997; 174: 159-165Crossref PubMed Scopus (189) Google Scholar). Cyclosporin A is not always effective, however, at inhibiting Ca2+-induced mitochondrial swelling and loss of ΔΨ (18Kristal B.S. Dubinsky J.M. J. Neurochem. 1997; 69: 524-538Crossref PubMed Scopus (205) Google Scholar, 19He L. Lemasters J.J. FEBS Lett. 2002; 512: 1-7Crossref PubMed Scopus (345) Google Scholar, 20Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 8229-8237Crossref PubMed Google Scholar, 21Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 103-113Crossref PubMed Google Scholar, 22Malkevitch N.V. Dedukhova V.I. Simonian R.A. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 412: 173-178Crossref PubMed Scopus (39) Google Scholar). In addition, the observation that normal intracellular concentrations of adenine nucleotides and Mg2+ partially or completely block PTP activation (23Novgorodov S.A. Gudz T.I. Milgrom Y.M. Brierley G.P. J. Biol. Chem. 1992; 267: 16274-16282Abstract Full Text PDF PubMed Google Scholar, 24Novgorodov S.A. Gudz T.I. Brierley G.P. Pfeiffer D.R. Arch. Biochem. Biophys. 1994; 311: 219-228Crossref PubMed Scopus (113) Google Scholar) casts doubt on a physiological role of the PTP. The extent to which mitochondria swell in response to accumulation of large Ca2+ loads also varies considerably with experimental conditions and with mitochondrial tissue type (25Andreyev A. Fiskum G. Cell Death Differ. 1999; 6: 825-832Crossref PubMed Scopus (164) Google Scholar, 26Kristian T. Weatherby T.M. Bates T.E. Fiskum G. J. Neurochem. 2002; 83: 1297-1308Crossref PubMed Scopus (64) Google Scholar, 27Kobayashi T. Kuroda S. Tada M. Houkin K. Iwasaki Y. Abe H. Brain Res. 2003; 960: 62-70Crossref PubMed Scopus (73) Google Scholar). Brain mitochondria are particularly resistant to Ca2+-induced swelling (25Andreyev A. Fiskum G. Cell Death Differ. 1999; 6: 825-832Crossref PubMed Scopus (164) Google Scholar, 28Berman S.B. Watkins S.C. Hastings T.G. Exp. Neurol. 2000; 164: 415-425Crossref PubMed Scopus (104) Google Scholar); moreover, they represent many cell populations, which may explain their heterogeneous response to large Ca2+ loads and to inhibitors of the PTP, e.g. cyclosporin A (26Kristian T. Weatherby T.M. Bates T.E. Fiskum G. J. Neurochem. 2002; 83: 1297-1308Crossref PubMed Scopus (64) Google Scholar, 29Brustovetsky N. Brustovetsky T. Jemmerson R. Dubinsky J.M. J. Neurochem. 2002; 80: 207-218Crossref PubMed Scopus (204) Google Scholar). In this study we report that 2-APB (30Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (773) Google Scholar) prevents Ca2+-induced PTP in non-synaptosomal brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. 2-APB is reported to inhibit Ca2+ efflux from mitochondria in situ in Jurkat T cells following stimulation of cellular Ca2+ influx through activation of capacitative Ca2+ entry (31Prakriya M. Lewis R.S. J. Physiol. 2001; 536: 3-19Crossref PubMed Scopus (426) Google Scholar). Under these conditions, mitochondria transiently accumulate Ca2+, followed by release upon restoration of basal cytosolic Ca2+ levels. The investigators hypothesized that the inhibition of mitochondrial Ca2+ efflux by 2-APB is because of inhibition of the mitochondrial Na+/Ca2+ exchanger (32Gunter T.E. Buntinas L. Sparagna G. Eliseev R. Gunter K. Cell Calcium. 2000; 28: 285-296Crossref PubMed Scopus (316) Google Scholar). Our results do not provide evidence for 2-APB inhibition of the mitochondrial Na+/Ca2+ exchanger but do indicate that mCICR via the PTP is effectively inhibited. This effect of 2-APB on isolated non-synaptosomal brain mitochondria is observed both in the absence of adenine nucleotides, where inhibition of Ca2+-induced swelling is apparent, and in the presence of millimolar concentrations of ATP and magnesium, where swelling is not observed but where Ca2+ induces release of matrix metabolites and cytochrome c. In the presence of ATP, 2-APB-inhibitable Ca2+-induced mitochondrial alterations are not affected by most other PTP inhibitors, including cyclosporin A. Isolation of Mitochondria—Non-synaptic adult rat brain mitochondria were isolated on a Percoll gradient as described previously (33Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (343) Google Scholar) with minor modifications. Male 300–350-g Sprague-Dawley rats were used for this study. All animal procedures were carried out according to the National Institutes of Health and the University of Maryland, Baltimore, animal care and use committee guidelines. Rats were sacrificed, and forebrains were rapidly removed, chopped, and homogenized in ice-cold isolation buffer containing 225 mm mannitol, 75 mm sucrose, 5 mm Hepes, and 1 mm EGTA with the pH adjusted to 7.4 using KOH. The brain homogenate was centrifuged at 1,250 × g for 3 min; the pellet was discarded, and the supernatant was centrifuged at 20,000 × g for 10 min. The pellet was resuspended in 15% Percoll (Sigma) and layered on a preformed Percoll gradient (40 and 23%). After centrifugation at 30,000 × g for 6 min, the mitochondrial fraction located at the interface of the lower two layers was removed, diluted with isolation buffer, and centrifuged at 16,600 × g for 10 min. The supernatant was discarded, and the loose pellet was resuspended in isolation buffer and centrifuged at 6700 × g for 10 min. The resulting pellet was suspended in 100 μl of isolation medium devoid of EGTA. Mitochondrial Ca2+ Uptake—Mitochondrial-dependent removal of medium Ca2+ was followed using the impermeant pentapotassium salt of the ratiometric dye Fura 6F (Molecular Probes, Portland, OR, USA). Fura 6F (250 nm) was added to a medium containing mitochondria (0.25 mg/ml) and 125 mm KCl, 20 mm Hepes, 2 mm KH2PO4, 1 μm EGTA, 4 mm MgCl2, 3 mm ATP, 5 mm malate, and 5 mm glutamate with the pH adjusted to 7.08 with KOH. According to calculations using the Winmaxc software (34Bers D.M. Patton C.W. Nuccitelli R. Methods Cell Biol. 1994; 40: 3-29Crossref PubMed Scopus (496) Google Scholar), the free [Mg2+] under these conditions is ∼1 mm. It was necessary to use Suprapur KCl (Merck) to minimize Ca2+ contamination, thereby also minimizing the amount of added EGTA necessary to eliminate deleterious effects of background Ca2+. All experiments were performed at 37 °C. Fluorescence intensity was measured in a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan) using 340/380-nm excitation and 510-nm emission wavelengths. The [Ca2+]in the medium was calculated using the ratio calibration approach described by Grynkiewicz et al. (35Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). The K d for Fura 6F was estimated to be 2.47 μm using the calcium calibration buffer kit number 3 (Molecular Probes). An essentially identical value was obtained using the calcium calibration buffer kit number 2 with magnesium (Molecular Probes). Mitochondrial Membrane Potential—Mitochondrial membrane potential was qualitatively assessed by TMRE (Molecular Probes) fluorescence intensity measured in a Hitachi F-2500 fluorescence spectrophotometer using 549- and 580-nm wavelengths for excitation and emission, respectively (37 °C). Because of the small Stokes shift, we assessed the effect of light scatter on the fluorescent signal, and it was found to be less than 10% as compared with TMRE fluorescence. 125 nm dye was added to a medium containing mitochondria (0.25 mg/ml) and 125 mm KCl, 20 mm Hepes, 2 mm KH2PO4, 1 μm EGTA, 4 mm MgCl2, 3 mm ATP, 5 mm malate, and 5 mm glutamate with the pH adjusted to 7.08 using KOH. Oxygen Consumption—Mitochondrial respiration was recorded at 37 °C with a Clark-type oxygen electrode (Hansatech, UK). The incubation medium contained 125 mm KCl, 20 mm Hepes, 2 mm KH2PO4, 1 μm EGTA, 1 mm MgCl2, 0.8 mm ADP, 5 mm malate, and 5 mm glutamate (or 5 mm succinate plus 1 μm rotenone) with the pH adjusted to 7.08 with KOH. In some experiments as indicated 5 mm succinate (plus 1 μm rotenone) replaced malate plus glutamate as the oxidizable substrate. State 3 (phosphorylating) respiration was initiated by the addition of mitochondria (0.5 mg/ml) to the incubation medium. State 3 respiration was terminated and State 4 (resting) respiration was initiated by the addition of 2 μm oligomycin. Cytochrome c Release from Mitochondria—Aliquots of mitochondrial suspensions were taken at specified incubation times, and the mitochondria were separated from the suspending medium by centrifugation at 14,000 × g for 3 min. The supernatant was carefully removed, and both the supernatant and mitochondrial pellet fractions were immediately frozen and stored at –20 °C. Cytochrome c immunoreactivity was quantified in both fractions using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). Before measurement, the supernatant and pellet samples were diluted 1:40 and 1:80, respectively. The release of cytochrome c from mitochondria is expressed as the content of cytochrome c in the supernatant as a percentage of the total content of cytochrome c present in the supernatant plus pellet. NAD + + NADH Release from Mitochondria—Aliquots of mitochondrial suspensions were taken at specified incubation times, and mitochondria were separated from the suspending medium by centrifugation at 14,000 × g for 3 min. The supernatant was carefully removed, and both the supernatant and mitochondrial pellet fractions were immediately frozen and stored at –20 °C. NAD+ plus NADH extraction from both fractions was performed according to the method described by Klingenberg (36Klingenberg M. Methods of Enzymatic Analysis. 3rd Ed. VCH Publishers, Inc., Deerfield Beach, FL1985: 251-269Google Scholar). Extracts were transferred to 2 ml of assay medium maintained at 30 °C containing 0.2 mg of alcohol dehydrogenase (Sigma), 50 mm Tris-HCl, 0.6 m ethanol, 50 mm Na4P2O7·10 H2O, pH 7.8. NADH fluorescence was followed in a Hitachi F-2500 fluorescence spectrophotometer using 340- and 460-nm wavelengths for excitation and emission, respectively. Mitochondrial Swelling—Swelling of isolated mitochondria was assessed by measuring light scatter at 660 nm (37 °C) in a Hitachi F-2500 fluorescence spectrophotometer. Mitochondria were added at a final concentration of 0.25 mg/ml to 2 ml of medium containing 125 mm KCl, 20 mm Hepes, 2 mm KH2PO4, 1 μm EGTA, 1 mm MgCl2, 5 mm malate, and5mm glutamate with the pH adjusted to 7.08 with KOH. At the end of each experiment, the non-selective pore-forming peptide alamethicin (80 μg) was added as a calibration standard to cause maximal swelling. Reagents—Standard laboratory chemicals were from Sigma. 2-APB (Sigma), CGP 37157 (Calbiochem), cyclosporin A (Sigma), bongkrekic acid (Calbiochem), alamethicin (Sigma), tamoxifen (Sigma), spermine (Sigma), xestospongin C (Calbiochem), inositol 1,4,5-triphosphate (ICN), thapsigargin (Calbiochem), trifluoperazine (Sigma), bromoenol lactone (Sigma), aristolochic acid (Biomol), bovine serum albumin (Sigma), 7-nitroindazole (Calbiochem), 1400W (Calbiochem), ubiquinone 0 (Sigma), butylated hydroxytoluene (Sigma), N-acetylcysteine (Sigma), catalase (Sigma), and superoxide dismutase (Sigma). 2-APB Protects against Ca2+-induced Mitochondrial Swelling in the Absence of Adenine Nucleotides—Ca2+-induced swelling of brain mitochondria can be demonstrated, but typically, only if adenine nucleotides are omitted from the medium (26Kristian T. Weatherby T.M. Bates T.E. Fiskum G. J. Neurochem. 2002; 83: 1297-1308Crossref PubMed Scopus (64) Google Scholar). Under these conditions, non-synaptosomal brain mitochondria exposed to 20 nmol of Ca2+ (40 nmol mg–1 protein) (Fig. 1a, curve a) exhibited a gradual decline in absorbance at 660 nm greater than that observed in the absence of added Ca2+ (Fig. 1a, curve d). The presence of cyclosporin A (2 μm) completely eliminated the Ca2+-dependent change in light scattering, indicating involvement of the PTP (Fig. 1a, curve b). 2-APB (100 μm) also eliminated the Ca2+-dependent light scattering (Fig. 1a, curve c). Because agents that block mitochondrial Ca2+ uptake also block mitochondrial swelling without necessarily affecting the PTP, the effect of 2-APB on mitochondrial Ca2+ uptake and release was measured. After the addition of 20 nmol of Ca2+ to the mitochondrial suspension, Ca2+ uptake was faster in the presence of 2-APB than in its absence (Fig. 1b, thick versus thin line). Thus, in the absence of adenine nucleotides, both cyclosporin A and 2-APB are effective inhibitors of the Ca2+-induced PTP in brain mitochondria. Dose-dependent Inhibition of Mitochondrial Ca2+-induced Ca2+ Release by 2-APB in the Presence of ATP—After a demonstration that 2-APB is effective at inhibiting PTP in the absence of adenine nucleotides, subsequent experiments were performed in the presence of 3 mm ATP to model more physiologically relevant conditions. In the presence of ATP, brain mitochondria completely accumulated two additions of 400 nmol of Ca2+ and almost sequestered the third addition. This net uptake of more than 2000 nmol of Ca2+ mg–1 mitochondrial protein was followed by a slow release of Ca2+ back to the medium (Fig. 2, curve a). Mitochondria suspended in the presence of either 50 or 100 μm 2-APB accumulated the entire Ca2+ load (2400 nmol mg–1) without releasing Ca2+ during the course of the 20-min experiment (Fig. 2, curves c and d, respectively). The presence of even 10 μm 2-APB resulted in some inhibition of Ca2+-induced Ca2+ release (Fig. 2, curve b). Fura 6F fluorescence responds reliably to [Ca2+] in the range of 0.5–50 μm (not shown). Each addition of Ca2+ to the mitochondrial suspension was a total of 200 μm; however, according to Winmaxc software (34Bers D.M. Patton C.W. Nuccitelli R. Methods Cell Biol. 1994; 40: 3-29Crossref PubMed Scopus (496) Google Scholar), the calculated free [Ca2+] was <100 μm in the presence of Mg2+ and ATP. Therefore, the peak of the calcium signal before the onset of calcium uptake by mitochondria was underestimated. However, the method used (manual additions of calcium in a cuvette system) is limited in the temporal dimension of the millisecond scale, where much of the calcium uptake takes place, given the enormous avidity of the uniporter (V max > 1200 nmol Ca2+/mg of protein/min) and/or the rapid mode of uptake (37Sparagna G.C. Gunter K.K. Sheu S.S. Gunter T.E. J. Biol. Chem. 1995; 270: 27510-27515Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). It is to be noted that even though the upper range of the bulk [Ca2+]i in cells reported by Fura 2 (or higher K d derivatives) fluorescence is in the relatively low micromolar range (38Stout A.K. Reynolds I.J. Neuroscience. 1999; 89: 91-100Crossref PubMed Scopus (79) Google Scholar), free [Ca2+]i may well reach the ∼100 μm range in certain microdomains (39Neher E. Neuron. 1998; 20: 389-399Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar) such as the perimitochondrial environment (40Szalai G. Csordas G. Hantash B.M. Thomas A.P. Hajnoczky G. J. Biol. Chem. 2000; 275: 15305-15313Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), (41Montero M. Alonso M.T. Carnicero E. Cuchillo-Ibanez I. Albillos A. Garcia A.G. Garcia-Sancho J. Alvarez J. Nat. Cell Biol. 2000; 2: 57-61Crossref PubMed Scopus (414) Google Scholar). Comparison of 2-APB to Cyclosporin A and Bongkrekic Acid—Although cyclosporin A was effective at inhibiting the PTP in brain mitochondria in the absence of adenine nucleotides (Fig. 1a), it failed to inhibit mCICR in the presence of ATP (Fig. 3, curve b). Bongkrekic acid, another PTP inhibitor, exhibited an ability to inhibit mCICR in the presence of ATP (Fig. 3, curve c) that was very similar to that of 2-APB (Fig. 3, curve d). Because the vehicle for bongkrekic acid is 1 m NH4OH, an equal volume of vehicle was tested and found to have no effect on Ca2+ uptake and retention (not shown). Although 2-APB inhibited mCICR, it did cause a slight reduction in the rate of Ca2+ uptake, particularly after the third addition of Ca2+ (Fig. 3, curve d). The effects of 2-APB on mitochondrial bioenergetics were further analyzed using measurements of both mitochondrial membrane potential and O2 consumption. Protection by 2-APB against Ca2+-induced Loss of Mitochondrial Membrane Potential— Fig. 4 provides a qualitative evaluation of ΔΨ after the addition of high Ca2+ loads in the absence and presence of 2-APB (100 μm) using the fluorescent dye TMRE. In the absence of 2-APB, TMRE fluorescence stabilized within 2 min after the addition of mitochondria to the medium (Fig. 4a). The subsequent addition of 400 nmol of Ca2+ caused an immediate increase in fluorescence, i.e. decrease in ΔΨ, as expected because of the collapse of ΔΨ during rapid Ca2+ influx. After the ∼100-s period, during which added Ca2+ was accumulated, the TMRE fluorescence decreased but remained much higher than the original fluorescence in the absence of Ca2+. A similar pattern was observed after the second addition of Ca2+. After the third addition, the TMRE fluorescence failed to return toward base line and gradually increased for many minutes thereafter. When 2-APB was added to the mitochondrial suspension (Fig. 4b), TMRE fluorescence increased, indicating some reduction in ΔΨ by this compound. However, unlike what was observed in the absence of 2-APB, the subsequent addition of three pulses of Ca2+ were followed by a return of TMRE fluorescence toward, and eventually to the level maintained before these additions. Thus, at 800 s of incubation, ΔΨ approached complete depolarization in the absence of 2-APB but was at least partially retained in its presence. These observations are consistent with the onset of mCICR in the absence but not in the presence of 2-APB, also observed at ∼800 s of incubation. Although 2-APB by itself causes partial mitochondrial depolarization, this effect was not strong enough to impair net ΔΨ-dependent Ca2+ uptake. It must be emphasized that because of the Nernst equation, data from potentiometric dyes follow a logarithmic, not linear relationship (42Dykens J.A. Stout A.K. Methods Cell Biol. 2001; 65: 285-309Crossref PubMed Google Scholar). The observation that is intended for demonstration on this experiment is that mitochondria depolarize further upon completion of Ca2+ uptake (Fig. 4a) unless they are pre-treated with 2-APB (Fig. 4b). The onset of depolarization upon high Ca2+ loading (∼700 s) coincides exactly with the onset of Ca2+ release (Fig. 2, curve a). The choice of the membrane potential-sensitive probe was critical. Many of the available probes were tested (safranin O, tetramethylrhodamine methylester perchlorate (TMRM), rhodamine 123, JC-1), but they all exhibited serious drawbacks due to the necessity of high calcium loading; i.e. safranin O reduced maximal calcium uptake capacity considerably, as has also been found by other laboratories (43Valle V.G. Pereira-da-Silva L. Vercesi A.E. Biochem. Biophys. Res. Commun. 1986; 135: 189-195Crossref PubMed Scopus (16) Google Scholar), TMRM and rhodamine 123 were very strong respiratory inhibitors and uncouplers even at low concentrations (20 nm, not shown), affecting respiration more than TMRE although the opposite has been observed for rat heart mitochondria (44Scaduto Jr., R.C. Grotyohann L.W. Biophys. J. 1999; 76: 469-477Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar), and JC-1 gave an unsatisfactory signal-to-noise ratio but no suppression of respiratory control (not shown). Inhibition of Ca2+-induced Mitochondrial Cytochrome c and Pyridine Nucleotide Release by 2-APB—Release of the mitochondrial intermembrane protein cytochrome c typically accompanies the osmotic swelling and rupture of the outer membrane evoked by activation of the PTP (45Crompton M. J. Physiol. 2000; 529: 11-21Crossref PubMed Scopus (277) Google Scholar, 46Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (657) Google Scholar, 47Appaix F. Guerrero K. Rampal D. Izikki M. Kaambre T. Sikk P. Brdiczka D. Riva-Lavieille C. Olivares J. Longuet M. Antonsson B. Saks V.A. Biochim. Biophys. Acta. 2002; 1556: 155-167Crossref PubMed Scopus (24) Google Scholar). Loss of mitochondrial matrix pyridine nucleotides is another measure of the PTP, distinguishing it from a mechanism of depolarization mediated by activation of a “low conductance” pore (48Kushnareva Y.E. Sokolove P.M. Arch. Biochem. Biophys. 2000; 376: 377-388Crossref PubMed Scopus (91) Google Scholar). Fig. 5 provides the results of enzyme-linked immunosorbent assay determinations of cytochrome c released into the medium after the addition of Ca2+ and a comparison of the effectiveness of 2-APB and cyclosporin A at inhibiting this release. In the absence of added Ca2+, less than 10% of total mitochondrial cytochrome c was lost to the suspending medium after 1800 s of incubation. After the addition of 2400 nmol of Ca2+ mg–1 protein, ∼58% of total cytochrome c was released at 1800 s. No net release was observed at 700 s, immediately before net Ca2+ release was observed (Fig. 3). 2-APB but not cyclosporin A inhibited Ca2+-induced cytochrome c release, consistent with the differences in the effects of these drugs observed on mCICR. The same pattern was observed for release of matrix pyridine nucleotides (Fig. 6). The appearance of pyridine nucleotides in the medium over that observed in the absence of Ca2+ occurred after the onset of mCICR and was inhibited by 2-APB but not cyclosporin A.Fig. 6NAD + NADH release (% of the total) estimated as described under “Experimental Procedures” from mitochondria incubated in the presence or absence of 2-APB or cyclosporin A and challenged by high calcium loading. –CaCl 2: no calcium added; +CaCl 2 700 s:,400 nmol of CaCl2 was added at 350, 500, and 600 s, and the sample was collected at 700 s; +CaCl 2 1800 s, 400 nmol of CaCl2 was added at 350, 500, and 600 s, and the sample was collected at 1800 s; +CaCl 2 + 2-APB 1800 s, 100 μm 2-APB was added at 50 s, 400 nmol of CaCl2 was added at 350, 500, and 600 s, and the sample was collected at 1800 s; +CaCl 2 + Cys A 1800 s, 2 μm cyclosporin A (Cys A) was added at 50 s, 400 nmol of CaCl2 was added at 350, 500, and 600 s, and the sample was collected at 1800 s. Samples were sedimented at 14,000 × g for 3 min, and both supernatants and pellets were probed for pyridine nucleotides. *, significant, p < 0.05, one-way analysis of variance, Dunnett's post hoc analysis, n = 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effects of 2-APB on Mitochondrial Bioenergetics and Relationship to Inhibition of mCICR—The effects of 2-APB on respiration by isolated non-synaptosomal brain mitochondria are described in Table I. 2-APB exhibited a dose-dependent inhibition of State 3 respiration with the NADH-linked oxidizable substrates malate plus glutamate, with significant inhibition observed at a concentration of 10 μm and a 57% inhibition observed at 100 μm. 2-APB similarly inhibited uncoupler (FCCP) stimulated respiration (not shown). State 4 respiration was significantly" @default.
• W1966152110 created "2016-06-24" @default.
• W1966152110 creator A5020745992 @default.
• W1966152110 creator A5058174886 @default.
• W1966152110 creator A5080399968 @default.
• W1966152110 date "2003-07-01" @default.
• W1966152110 modified "2023-10-16" @default.
• W1966152110 title "Cyclosporin A-insensitive Permeability Transition in Brain Mitochondria" @default.
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