Glutathionylation of adenine nucleotide translocase induced by carbon monoxide prevents mitochondrial membrane permeabilization and apoptosis

Abstract

The present work demonstrates the ability of CO to prevent apoptosis in a primary culture of astrocytes. For the first time, the antiapoptotic behavior can be clearly attributed to the inhibition of mitochondrial membrane permeabilization (MMP), a key event in the intrinsic apoptotic pathway. In isolated non-synaptic mitochondria, CO partially inhibits (i) loss of potential, (ii) the opening of a nonspecific pore through the inner membrane, (iii) swelling, and (iv) cytochrome c release, which are induced by calcium, diamide, or atractyloside (a ligand of ANT). CO directly modulates ANT function by enhancing ADP/ATP exchange and prevents its pore-forming activity. Additionally, CO induces reactive oxygen species (ROS) generation, and its prevention by beta-carotene decreases CO cytoprotection in intact cells as well as in isolated mitochondria, revealing the key role of ROS. On the other hand, CO induces a slight increase in mitochondrial oxidized glutathione, which is essential for apoptosis modulation by (i) delaying astrocytic apoptosis, (ii) decreasing MMP, and (iii) enhancing ADP/ATP translocation activity of ANT. Moreover, CO and GSSG trigger ANT glutathionylation, a post-translational process regulating protein function in response to redox cellular changes. In conclusion, CO protects astrocytes from apoptosis by preventing MMP, acting on ANT (glutathionylation and inhibition of its pore activity) via a preconditioning-like process mediated by ROS and GSSG.

FIGURE 1.

Carbon monoxide confers protection against apoptosis. Primary cultures of astrocytes cultured in 24-well plates were pretreated with 50 μm CO for 3 h, following apoptosis induction by 18-h exposure to the thiol cross-linker diamide (Dia; 0–250 μm) (A and B) and to the pro-oxidant t-BHP (0–280 μm) (C and D). The apoptotic hallmarks were assessed by flow cytometry. In A and C, the percentage of cells presenting high mitochondrial potential, detected by DiOC₆(3), is expressed. In B and D, the percentage of cells containing intact plasma membrane (viable cells) is presented, assessed with PI fluorochrome. All values are mean ± S.D. (error bars), n = 4. *, p < 0.05 compared with control and CO-treated cells for each concentration of diamide (A and B); #, p < 0.05 compared with control and CO-treated cells for each concentration of t-BHP (C and D). E, immunodetection of caspase-3 activation by its cleavage into 12-kDa fractions. The first lane corresponds to astrocytes treated with diamide at 200 μm (18 h); the second lane shows astrocytes pretreated with 50 μm CO (3 h), followed by diamide induction of apoptosis; and the third lane shows control astrocytes.

FIGURE 2.

Relevance of ROS to a carbon monoxide protective role. A, CO induces intracellular ROS generation, and it is prevented by the antioxidant β-carotene. Primary cultures of astroglial cells were pretreated for 1 h with 1 μm β-carotene, followed by the addition of 50 μm CO for 3 h. ROS quantification was performed using H₂DCFDA. B and C, astrocytes were subjected to 1 μm β-carotene for 1 h, followed by treatment with 50 μm CO for 3 h, and apoptosis was induced with diamide (B) or t-BHP (C) for 18 h. Mitochondrial potential and viability were assessed by flow cytometry, using DiOC₆(3) and PI, respectively. All values are mean ± S.D. (error bars), n = 3. A, *, p < 0.05 compared with control or with β-carotene and CO-treated cells. B, *, p < 0.05 compared with control or with β-carotene and CO-treated cells for high ΔΨm; **, p < 0.05 compared with control or with β-carotene and CO-treated cells for viability. C, *, p < 0.05 compared with control or with β-carotene and CO-treated cells for high ΔΨm; **, p < 0.05 compared with control or with β-carotene and CO-treated cells for viability.

FIGURE 3.

Carbon monoxide effect on the mitochondrial membrane depolarization, inner membrane permeabilization, mitochondrial swelling, and cytochrome c release. All four experimental assays were performed using isolated non-synaptic mitochondria in modified brain buffer. A, representative micrograph for rhodamine 123 fluorescence change (λ_ex, 485 nm; λ_em, 535 nm), measured for 30 min at 37 °C, in the absence or presence of CO at 10 μm and atractyloside at 300 μm or Ca²⁺ at 5 μm. B, quantitative expression of rhodamine 123 fluorescent measurements at 15 min of incubation. Isolated mitochondria were pretreated with CO at 10 μm or cyclosporine A (CsA) at 1 μm, and then Ca²⁺ at 0, 5, or 7.5 μm, atractyloside at 300 μm, or diamide at 250 μm was added. The values are expressed in relative percentage to 5 μm Ca²⁺ (100%). All values are mean ± S.D. (error bars), n = 3. *, p < 0.05 compared with control mitochondria for each inducer. C, an enzymatic assay based on citrate synthase activity was used to follow inner membrane permeabilization. Measurements were performed at 412 nm in the absence or presence of 10 μm CO and 300 μm atractyloside, at 37 °C for 20 min. All values are mean ± S.D., n = 3. D, mitochondrial swelling was measured by absorbance at 540 nm at 37 °C for 30 min, and the effect of calcium at 15 μm was normalized to 100% of swelling. Mitochondria were treated in the presence or absence of Ca²⁺ at 5 or 15 μm and/or CO at 10 μm. Experiments were done in triplicate and repeated three times. All values are mean ± S.D. (error bars), n = 3. *, p < 0.05 compared with control and CO-treated mitochondria. E, mitochondria, in the absence or presence of 10 μm CO, were treated with Ca²⁺ at 15 μm at 37 °C for 30 min, followed by centrifugation to separate mitochondrial pellet for immunodetection of cytochrome c release.

FIGURE 4.

Influence of ROS on CO effect at mitochondrial level. In A, mitochondria were treated with 10, 50, or 250 μm CO in the presence or absence of 1 μm β-carotene, followed by ROS quantification using H₂DCFDA (λ_ex, 485 nm; λ_em, 530 nm). The values are expressed in percentage relative to control (100%). All values are mean ± S.D., n = 4. *, p < 0.05 compared with control; **, p < 0.05 compared with control; #, p < 0.05 compared with control; ##, p < 0.05 compared with 10 μm CO; ***, p < 0.05 compared with 50 μm CO. B, mitochondria were pretreated with 1 μm β-carotene and 10 μm CO, and then atractyloside at 300 μm or diamide at 250 μm was added. The fluorescent measurements (λ_ex, 485 nm; λ_em, 535 nm) are expressed in relative percentage to 5 μm Ca²⁺ (100%) at 15 min of incubation. All values are mean ± S.D., n = 3; *, p < 0.05 compared with control and with β-carotene and CO-treated mitochondria. C, inner membrane permeabilization was assessed according to Ref. . Measurements were performed at 412 nm in the absence or presence of 10 μm CO and 300 μm atractyloside for 20 min at 37 °C. All values are mean ± S.D. (error bars), n = 3.

FIGURE 5.

Carbon monoxide effect on ADP/ATP translocase activity of ANT. The results were obtained using isolated non-synaptic mitochondria treated with 10 μm CO, and ADP/ATP translocation was assessed according to Ref. . ADP was added to mitochondria and diffused into the intermembrane space through the voltage-dependent anion channel. Once into the intermembrane space, ADP can be transformed by adenylate kinase (AK) in AMP and ATP or exchanged against ATP by adenine nucleotide translocator (ANT). The values are expressed in relative percentage to control (100%) at 15 min of incubation and are mean ± S.D. (error bars), n = 3. *, p < 0.05 compared with control mitochondria.

FIGURE 6.

Carbon monoxide effect on mitochondrial GSSG/GSH ratio. After CO treatment (10 or 50 μm) in the presence or absence of β-carotene (1 μm), oxidized and reduced glutathione quantification was performed using a microtiter plate assay, as described under “Experimental Procedures.” The values are mean ± S.D. (error bars), n = 3. *, p < 0.05 compared with control; **, p < 0.05 compared with mitochondria treated with 10 μm CO; #, p < 0.05 compared with mitochondria without β-carotene treatment and 10 μm CO treatment; ##, p < 0.05 compared with mitochondria without β-carotene treatment and 50 μm CO treatment.

FIGURE 7.

Role of ANT glutathionylation in MMP modulation. A, ADP/ATP translocation was followed in isolated mitochondria in the presence of 1 or 100 μm GSSG or 10 μm EA. The values are expressed in relative percentage to control (100%) at 15 min of incubation at 37 °C and are mean ± S.D. (error bars), n = 3. *, p < 0.05 compared with control. B and C, isolated non-synaptic mitochondria were treated with GSSG at 1 μm (B) or with EA at 25 μm (C) for 10 min, followed by atractyloside (Atra; 300 μm) or Ca²⁺ (5 μm) addition in order to induce inner membrane permeabilization, which was assessed according to Ref. . Measurements were performed at 412 nm for 20 min at 37 °C. All values are mean ± S.D., n = 3. D, primary cultures of astrocytes were treated with 0, 10, or 50 μm CO following mitochondria isolation; additionally, 1 μm GSSG was added to mitochondria isolated from control astrocytes. Glutathionylated proteins (α-GSH) were immunoprecipitated in mitochondria isolated from astrocytes, and ANT was immunodetected by Western blot from the immunoprecipitated proteins. The area and intensity of bands were quantified by densitometry analysis (GraphPad Prism 4) and are presented as relative percentage to the positive control (100%). This experiment was repeated three times with similar results. E, isolated non-synaptic mitochondria were treated in the presence or absence of diamide at 100 μm for 15 min, followed by CO (10 μm) incubation for 15 min, and then glutathionylated proteins were immunoprecipitated, and ANT was immunodetected by Western blot from the immunoprecipitated proteins. This experiment was repeated three times with similar results.

FIGURE 8.

Effect of CO in ANT-GST interaction. Primary cultures of astrocytes were treated with 0, 10, or 50 μm CO following mitochondria isolation; ANT (α-ANT) was immunoprecipitated in mitochondria isolated from astrocytes, and GST was immunodetected by Western blot from the immunoprecipitated proteins. This experiment was repeated three times with similar results.

FIGURE 9.

Effect of mitochondrial GSSG on cell death, MMP, and post-translational ANT modifications. A, the percentage of astrocytic survival when subjected to 50 μm EA or 100 μm BCNU for 1 h, followed by medium exchange and treatment with t-BHP (200 μm) for 18 h. Mitochondrial potential and viability were assessed by flow cytometry, using DiOC₆(3) and PI, respectively. All values are mean ± S.D. (error bars), n = 4. *, p < 0.05 compared with control cells treated with t-BHP for ΔΨm high. **, p < 0.05 compared with control cells treated with t-BHP for viability. B, depolarization assay. Isolated mitochondria were pretreated with 10 μm EA for 10 min, followed by 300 μm atractyloside or 5 μm Ca²⁺ treatment at 37 °C. The fluorescent measurements (λ_ex, 485 nm; λ_em, 535 nm) are expressed in relative percentage to 5 μm Ca²⁺ (100%) at 15 min of incubation. All values are mean ± S.D., n = 4. *, p < 0.05 compared with atractyloside-treated mitochondria. C, primary cultures of astrocytes were treated with 0 or 50 μm EA following mitochondria isolation; glutathionylated proteins (α-GSH) were immunoprecipitated in mitochondria isolated from astrocytes, and ANT was immunodetected by Western blot from the immunoprecipitated proteins. This experiment was repeated three times with similar results.