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• W1974465178 abstract "Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Δψm) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to “state 4” could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na+ cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Δψm collapse followed by cytoplasmic Ca2+ deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction. Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Δψm) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to “state 4” could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na+ cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Δψm collapse followed by cytoplasmic Ca2+ deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction. A balance between the formation of reactive oxygen species, as normal byproducts of mitochondrial respiration (1.Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7276) Google Scholar), and the actions of antioxidants prevents oxidative stress and is crucial to neuronal survival (2.Andersen J.K. Nat. Med. 2004; 10: 18-25Crossref PubMed Scopus (1416) Google Scholar). Together with the overactivation of glutamate receptors (excitotoxicity), oxidative stress is a result of the bioenergetic crisis that characterizes ischemia and plays a central role in the pathophysiology of the consequent neuronal damage (3.Fiskum G. Rosenthal R.E. Vereczki V. Martin E. Hoffman G.E. Chinopoulos C. Kowaltowski A. J. Bioenerg. Biomembr. 2004; 36: 347-352Crossref PubMed Scopus (137) Google Scholar). Neurons are particularly sensitive to oxidative damage and can be strongly sensitized to other injurious stimuli by levels of oxidative stress that are nontoxic per se (4.Arundine M. Aarts M. Lau A. Tymianski M. J. Neurosci. 2004; 24: 8106-8123Crossref PubMed Scopus (98) Google Scholar). Similarly, Ca2+ homeostasis is lost more quickly in cerebellar granule neurons (CGNs) 2The abbreviations used are: CGNs, cerebellar granule neurons; PTP, permeability transition pore; ANT, adenine nucleotide translocator; mBCl, monochlorobimane; GST, glutathione S-transferase; [Ca2+]c, cytoplasmic free Ca2+ concentration; Δψm, mitochondrial membrane potential; TMRM+, tetramethylrhodamine methyl ester; roGFP2, redox-sensitive green fluorescent protein-2; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.2The abbreviations used are: CGNs, cerebellar granule neurons; PTP, permeability transition pore; ANT, adenine nucleotide translocator; mBCl, monochlorobimane; GST, glutathione S-transferase; [Ca2+]c, cytoplasmic free Ca2+ concentration; Δψm, mitochondrial membrane potential; TMRM+, tetramethylrhodamine methyl ester; roGFP2, redox-sensitive green fluorescent protein-2; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone., which show higher superoxide levels prior to the application of toxic concentrations of glutamate (5.Vesce S. Kirk L. Nicholls D.G. J. Neurochem. 2004; 90: 683-693Crossref PubMed Scopus (56) Google Scholar). The tripeptide glutathione is a key antioxidant that maintains protein thiols in a reduced state and scavenges H2O2 in a reaction catalyzed by glutathione peroxidase (6.Dringen R. Kussmaul L. Gutterer J.M. Hirrlinger J. Hamprecht B. J. Neurochem. 1999; 72: 2523-2530Crossref PubMed Scopus (190) Google Scholar, 7.Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3551) Google Scholar). In vivo, mitochondrial glutathione is partially lost during ischemia (8.Anderson M.F. Sims N.R. J. Neurochem. 2002; 81: 541-549Crossref PubMed Scopus (75) Google Scholar), and its supplementation in the form of glutathione ethyl ester can reduce the infarct size (9.Anderson M.F. Nilsson M. Eriksson P.S. Sims N.R. Neurosci. Lett. 2004; 354: 163-165Crossref PubMed Scopus (64) Google Scholar). Although mitochondria within cells deprived of GSH can eventually release cytochrome c and undergo opening of the permeability transition pore (PTP) (10.Ghibelli L. Coppola S. Fanelli C. Rotilio G. Civitareale P. Scovassi A.I. Ciriolo M.R. FASEB J. 1999; 13: 2031-2036Crossref PubMed Scopus (133) Google Scholar, 11.Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 2001; 495: 12-15Crossref PubMed Scopus (702) Google Scholar, 12.Armstrong J.S. Yang H.Y. Duan W. Whiteman M. J. Biol. Chem. 2004; 279: 50420-50428Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), the initial bioenergetic consequences of GSH depletion remain unclear. Thermodynamic and kinetic factors predict that the steady-state thiol redox potential will be very sensitive to changes in total glutathione pool size (7.Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3551) Google Scholar, 13.Nicholls D.G. Curr. Mol. Med. 2004; 4: 149-177Crossref PubMed Scopus (221) Google Scholar). This may lead to the oxidative damage of key mitochondrial proteins. Indeed, either an inhibitor of GSH synthesis or the direct removal of reduced GSH with ethacrynic acid causes the inactivation of complexes I (14.Hsu M. Srinivas B. Kumar J. Subramanian R. Andersen J. J. Neurochem. 2005; 92: 1091-1103Crossref PubMed Scopus (99) Google Scholar) and II/III and IV (15.Heales S.J. Davies S.E. Bates T.E. Clark J.B. Neurochem. Res. 1995; 20: 31-38Crossref PubMed Scopus (194) Google Scholar, 16.Seyfried J. Soldner F. Schulz J.B. Klockgether T. Kovar K.A. Wüllner U. Neurosci. Lett. 1999; 264: 1-4Crossref PubMed Scopus (69) Google Scholar) that becomes apparent several hours from the beginning of treatment. An intriguing target for thiol oxidation is represented by the adenine nucleotide translocator (ANT), which functions as a dimer and can be progressively inhibited as the intermolecular oxidation of thiol groups increases (17.Majima E. Ikawa K. Takeda M. Hashimoto M. Shinohara Y. Terada H. J. Biol. Chem. 1995; 270: 29548-29554Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). This loss of ADP/ATP exchange activity precedes the formation of intramolecular cross-linking, which seems to require stronger oxidative conditions and may be related to mitochondrial PTP opening (18.Costantini P. Belzacq A.S. Vieira H.L. Larochette N. de Pablo M.A. Zamzami N. Susin S.A. Brenner C. Kroemer G. Oncogene. 2000; 19: 307-314Crossref PubMed Scopus (258) Google Scholar, 19.McStay G.P. Clarke S.J. Halestrap A.P. Biochem. J. 2002; 367: 541-548Crossref PubMed Scopus (302) Google Scholar). In this study, we rapidly depleted neuronal glutathione by conjugation with monochlorobimane (mBCl), a glutathione S-transferase (GST) substrate that is extensively used to assay glutathione pools (20.Barhoumi R. Bailey R.H. Burghardt R.C. Cytometry. 1995; 19: 226-234Crossref PubMed Scopus (54) Google Scholar, 21.Kamencic H. Lyon A. Paterson P.G. Juurlink B.H. Anal. Biochem. 2000; 286 (H. J.): 35-37BCrossref PubMed Scopus (211) Google Scholar). The intracellular thiol redox potential was detected with a redox-sensitive green fluorescent protein variant (22.Dooley C.M. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar), whereas population cell respiratory rates were monitored in parallel with cytoplasmic free Ca2+ concentration ([Ca2+]c) and mitochondrial membrane potential (Δψm) (23.Jekabsons M.B. Nicholls D.G. J. Biol. Chem. 2004; 279: 32989-33000Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), allowing glutathione depletion to be correlated with a rapid inhibition of ATP turnover that became evident within the first hour of treatment. The data suggest that a restriction of mitochondrial ATP export to the cytoplasm, consistent with ANT inhibition, is the first event occurring in this model of GSH depletion. Reagents—Tetramethylrhodamine methyl ester (TMRM+), Fluo-4FF, Fluo-5F, and mBCl were from Molecular Probes (Eugene, OR). Redox-sensitive green fluorescent protein-2 (roGFP2) cDNA was a kind gift of S. James Remington (University of Oregon, Eugene). Lipofectamine 2000 was from Invitrogen. All other reagents were from Sigma. Preparation of CGNs—CGNs were prepared from 7-day-old Wistar rats as described previously (24.Courtney M.J. Lambert J.J. Nicholls D.G. J. Neurosci. 1990; 10: 3873-3879Crossref PubMed Google Scholar), with modifications. Cells were plated onto Lab-Tek 8-well chambered coverglasses at a density of 380,000 cells/well or onto 22 × 40-mm coverslips at a density of 3 × 106 cells/coverslip. Coverslips had previously been coated with 33 μg/ml polyethyleneimine. Cultures were maintained in minimal essential medium supplemented with 10% fetal bovine serum, 30 mm glucose, 20 mm KCl, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. 24 h after plating, 10 μm cytosine arabinoside was added to inhibit growth of non-neuronal cells. Cell cultures were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO2 and 95% air and used for experiments at 7-9 days in vitro. Experimental Buffers—Standard “high KCl” buffer consisted of 100 mm NaCl, 25 mm KCl, 20 mm TES, 15 mm glucose, 1.3 mm MgCl2, 1.3 mm CaCl2, 1.2 mm Na2SO4, 0.4 mm KH2PO4, and 0.2 mm NaHCO3 (pH 7.3) at 37 °C. Some experiments were conducted in “low KCl” buffer containing 120 mm NaCl, 3.5 mm KCl, 20 mm TES, 15 mm glucose, 1.3 mm CaCl2, 1.2 mm Na2SO4, 0.4 mm KH2PO4, and 5 mm NaHCO3 (pH 7.3) at 37 °C. Functional Confocal Microscopy—Imaging of single neurons plated onto Lab-Tek 8-well chambered coverglasses was performed in a Pascal confocal system (Carl Zeiss AG, Oberkochen, Germany) using an Axio-vert 100M inverted microscope with a ×20 air objective and argon (488 nm) and helium/neon (543 nm) lasers. Control and experimental wells were imaged in parallel using a computer-controlled motorized stage and Physiology software, which allows parallel or sequential acquisition of time courses. For simultaneous detection of [Ca2+]c and Δψm (see Fig. 7), neurons were loaded with the low affinity Ca2+ indicator Fluo-4FF (0.5 μm; as acetoxymethyl ester; Ca2+ Kd = 9.7 μm) and TMRM+ (5 nm) for 30 min at 37 °C. This concentration of TMRM+ was insufficient for aggregation in the matrix (i.e. the experiment was performed in “non-quench mode”) (25.Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar), meaning that a decrease in either the plasma membrane or mitochondrial membrane potential was reflected in a reduction in whole cell fluorescence. In experiments in which [Ca2+]c transients following KCl-induced plasma membrane depolarization were detected, neurons were loaded with 0.5 μm Fluo-5F (Ca2+ Kd = 2.3 μm). Fluo-4FF and Fluo-5F were excited at 488 nm, and emission was collected between 505 and 530 nm. TMRM+ was excited at 543 nm, and the emitted fluorescence was collected between 560 and 615 nm. Under both conditions, TMRM+ was always present in the buffer throughout the experiment (25.Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar). Cell Respirometer—Respiration of intact neurons in situ was measured as described previously (23.Jekabsons M.B. Nicholls D.G. J. Biol. Chem. 2004; 279: 32989-33000Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). A 22 × 40-mm glass coverslip with attached cells was assembled in a closed RC-30 imaging chamber (Warner Instruments, Hamden, CT) and then mounted on an Olympus IX81 inverted fluorescence microscope equipped with ×20 and ×40 objectives. A miniature polarographic Clark-type oxygen electrode for perfusion systems with 116-inch fittings (Microelectrodes Inc., Bedford, NH) was used to monitor the oxygen tension in the eluted buffer. The flow rate was typically kept at 40-60 μl/min to provide sufficient oxygen depletion for detection. In experiments in which brevetoxin was employed, the buffer was also supplemented with 1 μm MK-801 to ensure that the results observed did not originate from the activation of N-methyl-d-aspartate receptors. 3 nm TMRM+ was present in the buffer (non-quench mode conditions). A chart recorder was wired to an OM-4 oxygen meter (Microelectrodes, Inc.) to provide a continuous trace of the oxygen depletion (and hence respiration) of the cells. The microscope was equipped with a CoolSNAPHQ CCD camera (Roper Scientific, Tucson, AZ) and MetaFluor and MetaMorph imaging software (Universal Imaging Corp., Downingtown, PA). TMRM+ and mBCl were excited via S555/20x and D380/13x filters, respectively (Chroma Technology Corp., Rockingham, VT). The emission from both fluorophores was collected through a 73101 dual band emission filter. roGFP2 was excited via a D480/30x filter, and the emission was collected through the 73101 filter. Transfection of roGFP2—Cultured neurons were transfected following a published protocol (26.Dalby B. Cates S. Harris A. Ohki E.C. Tilkins M.L. Price P.J. Ciccarone V.C. Methods. 2004; 33: 95-103Crossref PubMed Scopus (412) Google Scholar), with modifications. On day 0, following attachment to their coverslips, CGNs were transferred to culture medium without penicillin and streptomycin and incubated with 0.8 μg of roGFP2 cDNA and the transfection reagent Lipofectamine 2000. After 2 h, neurons were returned to the usual culture medium and used for experiments after day 6. Monitoring Δψm with Rhodamine 123—Rhodamine 123 in quench mode is the most sensitive means of detecting small changes in Δψm (25.Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar). Neurons were incubated in low KCl buffer and exposed to 2.2 μm rhodamine 123 for 15 min at 37 °C (to attain quench mode conditions). Cells were then rinsed with fresh buffer and imaged with the Olympus IX81 microscope. Rhodamine 123 was excited via a D480/20x filter, and the emitted fluorescence was collected through a 51000m filter (Chroma Technology Corp., Rockingham, VT). Given the relatively low membrane permeability, rhodamine 123 leaks out of the cells very slowly and therefore was not present in the media during the experiments. Determination of ATP/ADP Ratios—Total cellular ATP/ADP ratios were determined using a luciferase chemiluminescence assay (Calbiochem). Cells were incubated in low KCl buffer under the relevant conditions and lysed, and extracts were assayed for ATP according to the manufacturer's instructions. Pyruvate kinase (2 units/assay) was added to the buffer supplied with the kit. The increase in chemiluminescence was recorded in a TD-20/20 luminometer (Turner BioSystems, Sunny-vale, CA). Once the ATP signal was detected, 0.5 mm phosphoenolpyruvate was added, and the further increase in chemiluminescence due to the conversion of ADP to ATP was determined. Immunocytochemistry—Neurons plated onto Lab-Tek 4-well chambered slides were washed with 50 mm Tris and 150 mm NaCl (pH 7.5), fixed with 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100. A purified mouse anti-cytochrome c monoclonal antibody (Pharmingen) that recognizes the native form of cytochrome c (1:100 dilution) was added to the coverslips (overnight, 4 °C). The cells were then incubated with a fluorescein isothiocyanate-conjugated anti-mouse secondary antibody. Nuclei were stained with TOTO-3 before mounting the slides on ProLong Gold medium (both reagents from Molecular Probes). The samples were imaged with a Nikon PCM 2000 confocal system equipped with argon (488 nm) and helium/neon (633 nm) lasers. Statistical Analysis—All data analyzed were collected from at least three independent experiments and are expressed as means ± S.E. Data from two populations were compared with the unpaired two-tailed Student's t test. One-way analysis of variance followed by Tukey's test was used in the analysis of three or more experimental groups. p values <0.05 were considered significant. Conjugation of Endogenous GSH with mBCl Oxidizes the Thiol Redox Potential—mBCl becomes fluorescent when conjugated to GSH by intracellular GST. The reaction is highly selective for glutathione over other intracellular thiols (27.Fernandez-Checa J.C. Kaplowitz N. Anal. Biochem. 1990; 190: 212-219Crossref PubMed Scopus (200) Google Scholar), but is not a good indicator of subcellular distribution because GSTs are present in both the cytoplasm and the mitochondrion (28.Raza H. Robin M.A. Fang J.K. Avadhani N.G. Biochem. J. 2002; 366: 45-55Crossref PubMed Google Scholar), and the probe eventually accumulates in the nucleus (Fig. 1) (29.Keelan J. Allen N.J. Antcliffe D. Pal S. Duchen M.R. J. Neurosci. Res. 2001; 66: 873-884Crossref PubMed Scopus (111) Google Scholar). Another substrate for GST, ethacrynic acid, has been frequently used to deplete cells of GSH (16.Seyfried J. Soldner F. Schulz J.B. Klockgether T. Kovar K.A. Wüllner U. Neurosci. Lett. 1999; 264: 1-4Crossref PubMed Scopus (69) Google Scholar, 30.Muyderman H. Nilsson M. Sims N.R. J. Neurosci. 2004; 24: 8019-8028Crossref PubMed Scopus (75) Google Scholar). However, ethacrynic acid also directly inhibits mitochondrial complex II (31.Manuel M.A. Weiner M.W. J. Pharmacol. Exp. Ther. 1976; 198: 209-221PubMed Google Scholar) and, in our hands, caused a partial drop in Δψm when added to CGNs. An additional advantage of mBCl is that the reaction course can be followed by the development of fluorescence, as mBCl depletes GSH by forming the adduct GSH-mBCl (Fig. 1A), with the reaction usually reaching completion within 60 min. A major advance in the ability to monitor thiol redox potentials has come as a result of the development of roGFP analogs by substituting surface-exposed residues of the chromophore with cysteines (22.Dooley C.M. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Notably, an analog of roGFP2 (redox-sensitive yellow fluorescent protein) expressed in the cytoplasm of yeast cells appears to directly equilibrate with the cytoplasmic glutathione couple (32.Ostergaard H. Tachibana C. Winther J.R. J. Cell Biol. 2004; 166: 337-345Crossref PubMed Scopus (249) Google Scholar). Although the redox state of roGFPs can be monitored ratiometrically by excitation at 400/480 nm (22.Dooley C.M. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar), the presence of the mBCl adduct interferes with the emission from 400 nm excitation, and so only 480 nm excitation was employed. When excited at 480 nm, oxidation of roGFP2 will result in a lower fluorescence emission, whereas its reduction will cause fluorescence enhancement. The validity and sensitivity of the construct in CGNs were established by applying 500 μm H2O2 to elicit exogenous oxidative stress. The treatment resulted in a rapid decrease in roGFP2 emission (Fig. 1B) that was reversed by the thiol-reducing agent dithiothreitol. The endogenous oxidative stress induced by 100 μm mBCl caused a slower but equally extensive oxidation of roGFP2 that was reversed by dithiothreitol, confirming the ability of GSH depletion to cause an oxidative intracellular thiol redox potential shift. Depletion of GSH Affects Mitochondrial ATP Synthesis—Because the active synthesis of ATP by the mitochondrial ATP synthase slightly decreases Δψm by utilizing the proton current, it follows that addition of the ATP synthase inhibitor oligomycin will acutely hyperpolarize the mitochondria. Conversely, mitochondria that have inhibited respiratory chains or that have leaky inner membranes can reverse the ATP synthase to maintain a suboptimal Δψm by utilizing glycolytic ATP. In this case, oligomycin will depolarize the inner membrane. This “oligomycin null point” assay (33.Rego A.C. Vesce S. Nicholls D.G. Cell Death Differ. 2001; 8: 995-1003Crossref PubMed Scopus (74) Google Scholar, 34.Rego A.C. Ward M.W. Nicholls D.G. J. Neurosci. 2001; 21: 1893-1901Crossref PubMed Google Scholar) can therefore be used to investigate the bioenergetic status of the mitochondrial population within a single cell body. Fig. 2A (panels i-iii) shows responses of representative neurons whose Δψm hyperpolarized, was unchanged, or depolarized in response to the inhibitor, respectively. Because rhodamine 123 is in “quench mode,” hyperpolarization will cause further sequestration and quenching of the dye inside the matrix and decrease the whole cell fluorescence. The final addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to totally release the dye into the cytoplasm confirms that the experiment is performed under quench mode conditions. As expected, oligomycin decreased the fluorescence in a majority of control neurons (53%) (Fig. 2A, panel i). By contrast, the large majority of mBCl-treated cells (83%) did not undergo a detectable change in fluorescence following oligomycin addition, whereas 15% of the cells showed mitochondrial depolarization upon addition of oligomycin, indicating that, in some mitochondria, the membrane potential was sustained by the ATP synthase running in reverse. The unchanged rhodamine 123 fluorescence observed in most mBCl-treated cells upon oligomycin addition suggests that the mitochondria are close to respiratory state 4 (35.Nicholls D.G. Ferguson S.J. Bioenergetics 3. Academic Press Ltd., London2002Google Scholar), i.e. with little ATP turnover. This could be due either to a decreased cellular ATP demand or to an inhibition of the generation or export of ATP from the mitochondrion to the cytoplasm. The former would result in an increased total cellular ATP/ADP ratio, whereas the latter would decrease the ratio. A 75-min exposure to mBCl caused a significant decrease in the ATP/ADP ratio compared with control cells (2.76 ± 0.29 versus 4.83 ± 0.36) (Fig. 2B). This decrease was similar to that induced by oligomycin (2.54 ± 0.18). ATP synthase reversal induced by the inhibition of complex I with rotenone and by the protonophore FCCP caused a greater reduction in the ATP/ADP ratio (to 1.9 ± 0.16 and 1.2 ± 0.23, respectively). The decreased ratio is thus consistent with inhibited generation or export of ATP from the matrix. Mitochondria in CGNs Exposed to mBCl Approach State 4—To confirm and quantify the above observations, the O2 consumption of intact neurons attached to their coverslips was determined (Fig. 3) with a cell respirometer assembly as described previously (23.Jekabsons M.B. Nicholls D.G. J. Biol. Chem. 2004; 279: 32989-33000Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Cells were either perfused with standard high KCl buffer + 0.1% Me2SO (control) until mitochondrial inhibitors were added or switched to buffer additionally containing 100 μm mBCl after basal respiration was assessed. Rates of respiration were calculated from the perfusion rate times the difference between the downstream oxygen tension during chamber perfusion and that recorded when the chamber was bypassed with a shunt. The basal respiration of control neurons was stable until oligomycin-containing medium was perfused. The inhibitor decreased respiration to 44 ± 6% of the basal level, the residual value reflecting the inherent proton leak across the inner mitochondrial membrane (Fig. 3A). Thus, almost half of the basal respiration was used to compensate for the endogenous mitochondrial proton leak, whereas the remainder reflected basal ATP turnover. The effect of 100 μm mBCl was to initiate within 15 min a slowing of the basal respiration that progressed as GSH was conjugated to mBCl over a 60-min period (Fig. 3B). At that point, ATP turnover in the cells (calculated as the oligomycin-sensitive component of respiration) had decreased to <50% of that in control cells. Notably, mBCl caused no significant change in state 4 respiration (following oligomycin addition); thus, there was no uncoupling or permeability transition induction following 60 min of mBCl exposure, and the decrease in total respiration could be ascribed to an inhibition of mitochondrial ATP synthesis. The respirometer also allows maximal respiratory capacity to be quantified. Following the addition of the protonophore FCCP, mBCl-exposed neurons increased their respiration to the same extent as control cells (203 ± 42% over the state 4 rate in control cells versus 180 ± 30% in mBCl-treated cells) (Fig. 3D), demonstrating that this acute GSH depletion did not detectably restrict substrate delivery or electron transfer activity. This contrasts with long-term partial glutathione depletion that manifests as an inhibition of complex I activity in PC12 cells (36.Jha N. Jurma O. Lalli G. Liu Y. Pettus E.H. Greenamyre J.T. Liu R.M. Forman H.J. Andersen J.K. J. Biol. Chem. 2000; 275: 26096-26101Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). The respirometer was mounted on an epifluorescence microscope, which allows imaging of multiple fluorescence indicators at a single cell level (see “Materials and Methods”). Thus, when 100 μm mBCl was added, we could confirm that the fluorescence of the intracellular adduct GSH-mBCl reached a plateau within 1 h (see Fig. 1A). Also the fluorescence from cells equilibrated with 3 nm TMRM+ indicated that there was no detectable depolarization of either plasma or mitochondrial membrane potentials during this period (Fig. 4). Because respiratory capacity is not rate-limiting, the approach to state 4 following mBCl addition could be due to a decreased capacity for mitochondrial ATP synthesis (ATP synthase inhibition) or to inhibited export to the cytoplasm (ANT inhibition), whereas a decreased cellular ATP demand (e.g. inhibited ion pump activity) would be inconsistent with the decreased ATP/ADP ratio observed (Fig. 2B). Because of reports showing ANT sensitivity to thiol oxidation (18.Costantini P. Belzacq A.S. Vieira H.L. Larochette N. de Pablo M.A. Zamzami N. Susin S.A. Brenner C. Kroemer G. Oncogene. 2000; 19: 307-314Crossref PubMed Scopus (258) Google Scholar, 19.McStay G.P. Clarke S.J. Halestrap A.P. Biochem. J. 2002; 367: 541-548Crossref PubMed Scopus (302) Google Scholar, 37.Majima E. Koike H. Hong Y.M. Shinohara Y. Terada H. J. Biol. Chem. 1993; 268: 22181-22187Abstract Full Text PDF PubMed Google Scholar), a partial inhibition of this protein appears a plausible mechanism for the observed effects. In support of this view, incubation with the cell-permeant ANT inhibitor bongkrekic acid (10 μm) induced a decline in basal respiration; but, as with 60 min mBCl exposure, the approach to state 4 was incomplete, and an additional effect of oligomycin was apparent (Fig. 3C). If bongkrekic acid and mBCl were affecting cell respiration via alternative mechanisms, their combined effects should be additive and produce a greater decrease in respiration. To investigate this, we perfused cells with mBCl for 30 min, switched to buffer containing mBCl + bongkrekic acid for an additional 30 min, and then assessed the oligomycin-ins" @default.
• W1974465178 created "2016-06-24" @default.
• W1974465178 creator A5004270076 @default.
• W1974465178 creator A5016347084 @default.
• W1974465178 creator A5020342330 @default.
• W1974465178 creator A5041537888 @default.
• W1974465178 date "2005-11-01" @default.
• W1974465178 modified "2023-10-15" @default.
• W1974465178 title "Acute Glutathione Depletion Restricts Mitochondrial ATP Export in Cerebellar Granule Neurons" @default.
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