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Anti-apoptotic Effect of cGMP in Cultured Astrocytes

2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês

10.1074/jbc.m108622200

ISSN

1083-351X

Autores

Kazuhiro Takuma, Patamawan Phuagphong, Eibai Lee, Kōichi Mori, Akemichi Baba, Toshio Matsuda,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Reperfusion of cultured astrocytes with normal medium after exposure to H2O2-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H2O2-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H2O2-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca2+-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H2O2-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect. Reperfusion of cultured astrocytes with normal medium after exposure to H2O2-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H2O2-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H2O2-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca2+-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H2O2-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect. extracellular signal-regulated kinase permeability transition pore cGMP-dependent protein kinase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 3,3′-dihexyloxacarbocyanine 8-(4-chlorophenylthio)-cGMP Rp-8-[(4-chlorophenyl)thio]-guanosine-3′,5′-cyclic monophosphorothioate Sp-8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate We previously showed that reperfusion of cultured rat astrocytes with Ca2+-containing medium after exposure to Ca2+-free medium caused an increase in intracellular Ca2+ concentration followed by delayed cell death, including apoptosis (1Matsuda T. Takuma K. Nishiguchi E. Hashimoto H. Azuma J. Baba A. Eur. J. Neurosci. 1996; 8: 951-958Crossref PubMed Scopus (82) Google Scholar, 2Matsuda T. Takuma K. Baba A. Jpn. J. Pharmacol. 1997; 74: 1-20Crossref PubMed Scopus (96) Google Scholar, 3Takuma K. Lee E. Kidawara M. Mori K. Kimura Y. Baba A. Matsuda T. Eur. J. Neurosci. 1999; 11: 4204-4212Crossref PubMed Scopus (62) Google Scholar). This injury is considered to be an in vitro model of ischemia/reperfusion injury because a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue (4Siemkowicz E. Hansen A.J. Stroke. 1981; 12: 236-240Crossref PubMed Scopus (228) Google Scholar, 5Silver I.A. Erecinska M. J. Cereb. Blood Flow Metab. 1992; 12: 759-772Crossref PubMed Scopus (216) Google Scholar, 6Kristian T. Katsura K. Gido G. Siesjo B.K. Brain Res. 1994; 641: 295-302Crossref PubMed Scopus (74) Google Scholar). Subsequently, we have found that the Ca2+ reperfusion injury was mimicked by reperfusion after exposure to H2O2 (3Takuma K. Lee E. Kidawara M. Mori K. Kimura Y. Baba A. Matsuda T. Eur. J. Neurosci. 1999; 11: 4204-4212Crossref PubMed Scopus (62) Google Scholar). Our previous studies using the astrocytic injury models show that heat shock protein (7Takuma K. Matsuda T. Kishida Y. Asano S. Seong Y.H. Baba A. Brain Res. 1996; 735: 265-270Crossref PubMed Scopus (16) Google Scholar), calcineurin (8Matsuda T. Takuma K. Asano S. Kishida Y. Nakamura H. Mori K. Maeda S. Baba A. J. Neurochem. 1998; 70: 2004-2011Crossref PubMed Scopus (49) Google Scholar), mitogen-activated protein/extracellular signal-regulated kinase (ERK)1 kinase (9Takuma K. Fujita T. Kimura Y. Tanabe M. Yamamuro A. Lee E. Mori K. Koyama Y. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 399: 1-8Crossref PubMed Scopus (33) Google Scholar), phosphatidylinositol-3 kinase (9Takuma K. Fujita T. Kimura Y. Tanabe M. Yamamuro A. Lee E. Mori K. Koyama Y. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 399: 1-8Crossref PubMed Scopus (33) Google Scholar, 10Takuma K. Yoshida T. Lee E. Mori K. Kishi T. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 406: 333-339Crossref PubMed Scopus (21) Google Scholar), and cGMP phosphodiesterase (11Takuma K. Lee E. Enomoto R. Mori K. Baba A. Matsuda T. Br. J. Pharmacol. 2001; 133: 841-848Crossref PubMed Scopus (41) Google Scholar) are target molecules for anti-apoptotic drugs in astrocytic apoptosis. The role of cGMP in preventing apoptosis is also reported in B lymphocytes (12Genaro A.M. Hortelano S. Alvarez A. Martinez C. Bosca L. J. Clin. Invest. 1995; 95: 1884-1890Crossref PubMed Scopus (306) Google Scholar), T lymphocytes (13Sciorati C. Rovere P. Ferrarini M. Heltai S. Manfredi A.A. Clementi E. J. Biol. Chem. 1997; 272: 23211-23215Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), eosinophils (14Beauvais F. Michel L. Debertret L. FEBS Lett. 1995; 361: 229-232Crossref PubMed Scopus (122) Google Scholar), motor and sympathetic neurons (15Farinelli S.E. Park D.S. Greene L.A. J. Neurosci. 1996; 16: 2325-2334Crossref PubMed Google Scholar, 16Estevez A.G Spear N. Thompson J.A. Cornwell T.L. Radi R. Barbeito L. Beckman J.S. J. Neurosci. 1998; 18: 3708-3714Crossref PubMed Google Scholar), hepatocytes (17Kim Y.M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: 31138-31148Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar, 18Li J. Yang S. Billiar T.R. J. Biol. Chem. 2000; 275: 13026-13034Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), PC12 cells (15Farinelli S.E. Park D.S. Greene L.A. J. Neurosci. 1996; 16: 2325-2334Crossref PubMed Google Scholar,19Kim Y.M. Chung H.T. Kim S.S. Han J.A. Yoo Y.M. Kim K.M. Lee G.H. Yun H.Y. Green A. Li J. Simmons R.L. Billiar T.R. J. Neurosci. 1999; 19: 6740-6747Crossref PubMed Google Scholar), and ovarian follicles (20Chun S.Y. Eisenhauer K.M. Kubo M. Hsueh A.J. Endocrinology. 1995; 136: 3120-3127Crossref PubMed Scopus (0) Google Scholar). However, it is not known how cGMP prevents apoptosis signaling and supports survival. It is considered that the mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are important in the apoptotic process. Kim et al. (19Kim Y.M. Chung H.T. Kim S.S. Han J.A. Yoo Y.M. Kim K.M. Lee G.H. Yun H.Y. Green A. Li J. Simmons R.L. Billiar T.R. J. Neurosci. 1999; 19: 6740-6747Crossref PubMed Google Scholar) and Li et al. (18Li J. Yang S. Billiar T.R. J. Biol. Chem. 2000; 275: 13026-13034Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) have recently reported that cGMP inhibits caspase activation and cytochrome c release. We (11Takuma K. Lee E. Enomoto R. Mori K. Baba A. Matsuda T. Br. J. Pharmacol. 2001; 133: 841-848Crossref PubMed Scopus (41) Google Scholar) found that ibudilast, which has been clinically used for bronchial asthma and cerebrovascular disorders, inhibited the H2O2-induced cytochrome c release and caspase-3 activation in a cGMP-dependent mechanism. It is not known whether cGMP acts on the mitochondrial PTP. In this study, we examined the effects of cGMP on mitochondrial dysfunction, resulting in astrocytic apoptosis, and on mitochondrial PTP in isolated brain mitochondria. The present study demonstrates that dibutyryl-cGMP prevents the H2O2-induced reduction of the mitochondrial membrane potential, cytochrome c release, and caspase activation. In addition, we demonstrate in isolated rat brain mitochondria that cGMP inhibits the mitochondrial PTP in a cGMP-dependent protein kinase (PKG)-mediated mechanism as determined by monitoring Ca2+-induced mitochondrial swelling. Drugs were obtained from the following sources: mouse anti-glial fibrilliary acidic protein antiserum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cGMP, dibutyryl-cGMP, 8-pCPT-cGMP, Rp-8-pCPT-cGMPS, isolectin B4 (Biotin-labeled), and cyclosporin A, Sigma; Sp-8-pCPT-cGMPS, Biomol Research Laboratories, Inc. (Plymouth Meeting, PA); PKG, KT5823, 2′-amino-3′-methoxyflavone (PD98059), and bongkrekic acid, Calbiochem; horseradish peroxidase-labeled anti-mouse Ig, Amersham Biosciences, Inc.; Hoechst 33342 and 3,3′-dihexyloxacarbocyanine (DiOC6(3)), Molecular Probes, Inc. (Eugene, OR); 7-amino-4-methyl-coumarin and acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartic acid α-(4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA), Peptide Institute, Inc. (Osaka, Japan); anti-cytochrome c antibody (clone 7H8.2C12), PharMingen (San Diego, CA); wortmannin, Nacalai Tesque (Kyoto, Japan); Eagle's minimum essential medium, Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan); and tissue culture wares, Iwaki Glass Co., Ltd. (Tokyo, Japan). All other chemicals used were of the highest purity commercially available. Astrocytes were isolated from the cerebral cortices of 1-day-old Wistar rats as reported previously (21Takuma K. Matsuda T. Hashimoto H. Asano S. Baba A. Glia. 1994; 12: 336-342Crossref PubMed Scopus (95) Google Scholar, 22Takuma K. Matsuda T. Asano S. Baba A. J. Neurochem. 1995; 64: 1536-1540Crossref PubMed Scopus (27) Google Scholar, 23Takuma K. Matsuda T. Hashimoto H. Kitanaka J. Asano S. Kishida Y. Baba A. J. Neurochem. 1996; 67: 1840-1845Crossref PubMed Scopus (58) Google Scholar). Cells were plated in 75-ml tissue culture flasks, split once upon confluence, and plated in 24-well (for MTT assay) and 96-well (for mitochondria energization and caspase assays) plastic tissue culture plates, 60-mm (for DNA ladder) and 100-mm (for cytochromec release analysis and cell extract preparation) plastic tissue culture dishes, and 4-well chamber slides (for fluorescence microscopy). The second cultures were grown for 14–20 days in all experiments. The cells were routinely >95% positive for glial fibrillary acidic protein, and ∼2% of the cells showed microglia based on positive isolectin B4 staining. Reperfusion experiments were carried out as reported previously (9Takuma K. Fujita T. Kimura Y. Tanabe M. Yamamuro A. Lee E. Mori K. Koyama Y. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 399: 1-8Crossref PubMed Scopus (33) Google Scholar, 10Takuma K. Yoshida T. Lee E. Mori K. Kishi T. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 406: 333-339Crossref PubMed Scopus (21) Google Scholar). Cells were exposed to H2O2-containing Earle's solution (100 μm) for 30 min and then incubated with normal Earle's solution for the indicated times. MTT reduction activity was measured by a colorimetric assay as reported previously (1Matsuda T. Takuma K. Nishiguchi E. Hashimoto H. Azuma J. Baba A. Eur. J. Neurosci. 1996; 8: 951-958Crossref PubMed Scopus (82) Google Scholar, 3Takuma K. Lee E. Kidawara M. Mori K. Kimura Y. Baba A. Matsuda T. Eur. J. Neurosci. 1999; 11: 4204-4212Crossref PubMed Scopus (62) Google Scholar). MTT reduction activity is expressed as a percentage of control. DNA was extracted and subjected to 1.8% agarose gel electrophoresis as reported previously (9Takuma K. Fujita T. Kimura Y. Tanabe M. Yamamuro A. Lee E. Mori K. Koyama Y. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 399: 1-8Crossref PubMed Scopus (33) Google Scholar, 10Takuma K. Yoshida T. Lee E. Mori K. Kishi T. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 406: 333-339Crossref PubMed Scopus (21) Google Scholar). DNA in the gel was stained with ethidium bromide and photographed with a Polaroid (type 667) under UV light. To observe individual nuclei, the cells plated on a chamber slide were fixed with 4% formaldehyde and stained with Hoechst 33342 as reported previously (3Takuma K. Lee E. Kidawara M. Mori K. Kimura Y. Baba A. Matsuda T. Eur. J. Neurosci. 1999; 11: 4204-4212Crossref PubMed Scopus (62) Google Scholar, 10Takuma K. Yoshida T. Lee E. Mori K. Kishi T. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 406: 333-339Crossref PubMed Scopus (21) Google Scholar). An Olympus IX70 inverted fluorescence microscope was equipped with a cooled CCD camera system (Roper Scientific, Photometrics CoolSNAP) to scan the staining nuclear images. Mitochondria energization was determined as the retention of the mitochondria-specific dye DiOC6(3) (24Pastorino J.G. Chen S.T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Cells were loaded with 100 nm DiOC6(3) during the last 30 min of treatment. After removing the medium, the cells were washed twice with phosphate-buffered saline and then lysed by the addition of 100 μl of deionized water. The concentration of retained DiOC6(3) was measured with excitation at 485 nm and emission at 510 nm using a Wallac Multilabel counter. Cytosol and membrane fractions were prepared as reported previously (11Takuma K. Lee E. Enomoto R. Mori K. Baba A. Matsuda T. Br. J. Pharmacol. 2001; 133: 841-848Crossref PubMed Scopus (41) Google Scholar). The protein contents of the cytosol and membrane fractions were determined by a Bio-Rad DC protein assay, and 15 μg of the sample was subjected to SDS-polyacrylamide gel electrophoresis (15% polyacrylamide). A cytochrome c antibody (1:1000) was used for immunoblotting. The activity of caspase-3-like protease in cell lysates was measured using the synthetic substrate Ac-DEVD-MCA (11Takuma K. Lee E. Enomoto R. Mori K. Baba A. Matsuda T. Br. J. Pharmacol. 2001; 133: 841-848Crossref PubMed Scopus (41) Google Scholar). After reacting for 1 h at 37 °C, the released 7-amino-4-methyl-coumarin levels were measured with excitation at 355 nm and emission at 460 nm using a Wallac Multilabel counter. The cytosol extracts were prepared as follows. Cells plated on 100-mm dishes were washed twice with cold phosphate-buffered saline, scraped off using a policeman, and collected by centrifugation at 300 × g for 10 min at 4 °C. The pellet was suspended in 100 μl of lysis buffer (320 mm sucrose, 10 mm Tris-base, pH 7.4) and disrupted with 10 strokes of a Dounce homogenizer. The homogenate was centrifuged at 105,000 × g for 1 h at 4 °C, and the resulting supernatant (∼10 mg of protein/ml) was kept at −80 °C. Brain mitochondria were isolated from male Wistar rats (120–140 g) by the method of Sims (25Sims N.R. J. Neurochem. 1991; 56: 1836-1844Crossref PubMed Scopus (114) Google Scholar) using a discontinuous Percoll gradient centrifugation. The mitochondrial fraction was resuspended in buffer containing sucrose (320 mm) and Tris-base (10 mm, pH 7.4) and kept on ice until use. The opening of the PTP causes mitochondrial swelling that is conveniently assayed as a decrease in the light scattering (and thus absorbance) of a mitochondrial suspension. The Ca2+-induced mitochondrial swelling was assayed essentially as described by Friberg et al. (26Friberg H. Ferrand-Drake M. Bengtsson F. Halestrap A.P. Wieloch T. J Neurosci. 1998; 18: 5151-5159Crossref PubMed Google Scholar). Rotenone, antimycin, and A23187 were added 5 min before the addition of CaCl2solution to ensure the complete equilibration of calcium ions across the mitochondrial membrane under de-energized conditions. The treatment of the mitochondrial suspension (0.5 mg/ml) with cGMP, the cytosolic extract, or PKG was carried out for 15 min before the addition of CaCl2 solution. The calcium ion concentrations were calculated from the nitrilotriacetic acid buffering (27Connern C.P. Halestrap A.P. Biochem. J. 1994; 302: 321-324Crossref PubMed Scopus (220) Google Scholar). Absorbance at 540 nm was measured using a Shimadzu UV1200 spectrophotometer (Kyoto, Japan). Each experiment shown is representative of at least three similar experiments performed on separate mitochondrial preparations. Statistical analysis of the experimental data was carried out by Student-Newman-Keuls test, Dunnett's t test, or Tukey HSD test using a software package (Stat View 5.0) for Apple Macintosh. Reperfusion after exposure of astrocytes to H2O2-containing medium causes a decrease in MTT reduction activity, DNA ladder formation, cytochrome c release from mitochondria, and caspase-3 like protease activation (3Takuma K. Lee E. Kidawara M. Mori K. Kimura Y. Baba A. Matsuda T. Eur. J. Neurosci. 1999; 11: 4204-4212Crossref PubMed Scopus (62) Google Scholar). Fig. 1 shows the effects of membrane-permeable cGMP analogs on the H2O2-induced apoptosis. Dibutyryl-cGMP, 8-pCPT-cGMP, and Sp-8-pCPT-cGMPS attenuated significantly the H2O2-induced decrease in MTT reduction activity in a dose-dependent manner; the effect of Sp-8-pCPT-cGMPS was more potent than that of dibutyryl-cGMP and 8-pCPT-cGMP. The protective effect of dibutyryl-cGMP was observed even in the presence of the ERK kinase inhibitor PD98059 and the phosphoinositide 3-kinase inhibitor wortmannin (data not shown). Dibutyryl-cGMP also attenuated the H2O2-induced DNA ladder formation (Fig.2 A) and nuclear condensation (Fig. 2 B). Reperfusion after the exposure of astrocytes to H2O2 also caused a decrease in DiOC6(3) retention, which reflects mitochondrial membrane potential (Fig. 3 A). The H2O2-induced loss of mitochondrial membrane potential was inhibited by dibutyryl-cGMP in a dose-dependent manner (Fig. 3 B). Furthermore, dibutyryl-cGMP attenuated the H2O2-induced cytochrome c release from mitochondria and caspase-3-like protease activation in a dose-dependent manner (Fig.4).Figure 2Effect of dibutyryl-cGMP on apoptosis-like cell injury induced by H2O2exposure/reperfusion in cultured rat astrocytes. A, effect on DNA ladder formation. Cells were exposed to normal (control) or 100 μm H2O2 for 30 min and then incubated with Earle's solution for 5 days. Dibutyryl-cGMP was added 30 min before H2O2 exposure and was present until assay. The typical result of 3 independent experiments is shown (M, 100-bp marker). B, effect on nuclear condensation. Cells were preincubated in the absence (a and b) or presence (c and d) of 100 μm H2O2 for 30 min and then incubated with Earle's solution for 3 days. Dibutyryl-cGMP (100 μm) was added 30 min before H2O2exposure and was present until assay (b and d).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of dibutyryl-cGMP on loss of mitochondrial membrane potential induced by H2O2 exposure/reperfusion in cultured rat astrocytes. A, time course of the loss of mitochondrial membrane potential. Cells were exposed to normal (open circle) or 100 μm H2O2(closed circle) for 30 min and then incubated with Earle's solution for the indicated time. B, dose response for the effect of dibutyryl-cGMP. Cells were exposed to normal (open circle) or 100 μm H2O2(closed circle) for 30 min and then incubated with Earle's solution for 23.5 h. Dibutyryl-cGMP was added 30 min before H2O2 exposure and was present until assay. Results are means ± S.E. for 8–10 wells and were obtained from 2 separate experiments. *, p < 0.01, significantly different from control (Student-Newman-Keuls test); †,p < 0.01, significantly different from the values without dibutyryl-cGMP (Dunnett's t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effects of dibutyryl-cGMP on cytochromec release from mitochondria and increase in DEVDase activity induced by H2O2 exposure/reperfusion in cultured rat astrocytes. A, effect on cytochromec release from mitochondria. Cells were exposed to normal (control) or 100 μm H2O2 for 30 min, and then incubated with Earle's solution for 23.5 h. Dibutyryl-cGMP was added 30 min before H2O2exposure and was present until assay. Cytochrome c (arrow) in the cytosol (upper) and mitochondrial (lower) fractions is shown. The typical results of 3 independent experiments are shown. B, effect on increase in DEVDase activity. Cells were exposed to normal (open circle) or 100 μm H2O2 (closed circle) for 30 min and then incubated with Earle's solution for 23.5 h. Dibutyryl-cGMP was added 30 min before H2O2 exposure and was present until assay. Results are means ± S.E. for 6 wells and were obtained from 2 separate experiments. *, p < 0.01, significantly different from control (Student-Newman-Keuls test); †,p < 0.01, significantly different from the values without dibutyryl-cGMP (Dunnett's t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The protection by dibutyryl-cGMP against the H2O2-induced decrease in MTT reduction activity was blocked by the PKG inhibitors KT5823 and Rp-8-pCPT-cGMPS (Fig.5 A). KT5823 also blocked the effect of dibutyryl-cGMP on mitochondrial membrane potential (Fig.5 B), cytochrome c release (Fig. 5 C), and caspase-3-like protease activation (Fig. 5 D). A pilot experiment showed that exposure of purified rat brain mitochondria to 30–100 μm Ca2+ caused a significant decrease in absorbance, indicating mitochondrial swelling by the PTP opening (data not shown). The swelling was dependent on Ca2+ concentration as reported previously (26Friberg H. Ferrand-Drake M. Bengtsson F. Halestrap A.P. Wieloch T. J Neurosci. 1998; 18: 5151-5159Crossref PubMed Google Scholar). In this study, we used 100 μm Ca2+ to induce mitochondrial swelling. The swelling was inhibited by the mitochondrial PTP inhibitors cyclosporin A (28Broekemeier K.M. Dempsey M.E. Pfeiffer D.R. J. Biol. Chem. 1989; 264: 7826-7830Abstract Full Text PDF PubMed Google Scholar) and bongkrekic acid (29Zoratti M. Szabò I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar) (Fig.6 A). Neither cGMP nor cytosolic extract from astrocytes affected the Ca2+-induced mitochondrial swelling, but the simultaneous addition of cGMP and the cytosolic extract prevented the swelling in a dose-dependent manner (Fig. 6, B and C). The effect of cGMP plus the cytosolic extract was also observed in the swelling induced by 30 μmCa2+ (data not shown). The ability of the cytosolic extract was inactivated by heat-treating it (Fig.7 A) and was mimicked by exogenous PKG (Fig. 7 B). The similar protection by cGMP against Ca2+-induced swelling was observed even when cGMP was added together with or after Ca2+ (data not shown). KT5823 antagonized the effect of cGMP plus the cytosolic extract (Fig. 7 C) or PKG (Fig. 7 B) in inhibiting the Ca2+-induced swelling. The mitochondrial PTP inhibitors cyclosporin A and bongkrekic acid inhibited the H2O2-induced decrease in MTT reduction activity and activation of caspase-3-like protease in cultured astrocytes (Fig.8). In this experiment, bongkrekic acid was used at a relatively low concentration because at 50 μm, it showed cell toxicity (data not shown).Figure 7Involvement of protein kinase G on the inhibition by cGMP and cytosolic extract from astrocytes on Ca2+-induced swelling in purified rat brain mitochondria. Swelling was induced by the addition of Ca2+ at 0 time (arrow) (b –e). cGMP, the extract, and PKG were added 15 min before Ca2+ addition and were present during the incubation. KT5823 was added 30 min before Ca2+ and was present during the incubation. A, effect of heat-treated cytosolic extract. The extract was heated at 90 °C for 20 min before use. a, none; b, Ca2+; c, Ca2+ and 100 μm cGMP; d, Ca2+ and 100 μm cGMP plus 10 μg/ml fresh extract; and e, Ca2+ and 100 μmcGMP plus 10 μg/ml heated extract. B, effect of exogenous PKG. a, none; b, Ca2+; c, Ca2+ and 100 μm cGMP; d, Ca2+ and 100 μm cGMP plus 10 μg/ml cytosolic extract; and e, Ca2+ and 100 μm cGMP plus 10,000 units/ml PKG. C, effect of the PKG inhibitor KT5823. a, none; b, Ca2+; c, Ca2+ and 100 μm cGMP; d, Ca2+ and 100 μm cGMP plus 10 μg/ml extract; and e, Ca2+, 2 μm KT5823 and 100 μmcGMP plus 10 μg/ml extract. Results are means for 4–9 measurements and were obtained from 2 or 3 separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Effects of cyclosporin A and bongkrekic acid on the H2O2-induced cell injury and increase in DEVDase activity in cultured rat astrocytes. Cells were exposed to normal (open columns) or 100 μmH2O2-containing medium (black columns) for 30 min and then incubated with Earle's solution for 23.5 h. Cyclosporin A (1 μm) and bongkrekic acid (5 μm) were added 30 min before H2O2exposure and were present until assay. A, MTT assay. Results are means ± S.E. for 9–19 wells and were obtained from 2 or 3 separate experiments. B, DEVDase activity. Results are means ± S.E. for 10 wells and were obtained from 2 separate experiments. *, p < 0.05, significant from the values without drugs (Tukey-HSD test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) This study was undertaken to characterize the mechanism by which cGMP suppresses the H2O2-induced apoptosis in cultured astrocytes. The membrane-permeable cGMP analogs dibutyryl-cGMP, 8-pCPT-cGMP, and Sp-8-pCPT-cGMPS attenuated the H2O2-induced decrease in cell viability. In view of the importance of mitochondria in the apoptotic process, the present study focused on the effect of cGMP on the H2O2-induced mitochondrial dysfunction. We showed that dibutyryl-cGMP prevented the reduction of the mitochondrial membrane potential and cytochrome c release and consequently abolished caspase-3 activation, nuclear condensation, and DNA fragmentation in cultured astrocytes. These findings suggest that cGMP inhibits the H2O2-induced mitochondrial dysfunction and prevents astrocyte apoptosis. The inhibitory effect of cGMP analog on caspase-3 activation and cytochrome c release was also observed in hepatocytes (17Kim Y.M. Talanian R.V. Billiar T.R. J. Biol. Chem. 1997; 272: 31138-31148Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar, 18Li J. Yang S. Billiar T.R. J. Biol. Chem. 2000; 275: 13026-13034Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and PC12 cells (19Kim Y.M. Chung H.T. Kim S.S. Han J.A. Yoo Y.M. Kim K.M. Lee G.H. Yun H.Y. Green A. Li J. Simmons R.L. Billiar T.R. J. Neurosci. 1999; 19: 6740-6747Crossref PubMed Google Scholar). Many of the cellular effects of cGMP are mediated by PKG. In this respect, Kimet al. (19Kim Y.M. Chung H.T. Kim S.S. Han J.A. Yoo Y.M. Kim K.M. Lee G.H. Yun H.Y. Green A. Li J. Simmons R.L. Billiar T.R. J. Neurosci. 1999; 19: 6740-6747Crossref PubMed Google Scholar) reported that the PKG inhibitor KT5823 reversed the 8-bromo-cGMP-mediated prevention of cytochrome c release and caspase-3 activation in PC12 cells. We also found that KT5823 almost completely blocked the protection provided by dibutyryl-cGMP against the H2O2-induced reduction of the mitochondrial membrane potential, cytochrome c release, and caspse-3 activation in astrocytes. These findings suggest that cGMP analogs prevent astrocyte apoptosis via the activation of PKG, although the exact mechanism by which cGMP/PKG prevents apoptotic events is not known. Li et al. (18Li J. Yang S. Billiar T.R. J. Biol. Chem. 2000; 275: 13026-13034Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) reported that cAMP as well as cGMP analogs suppressed tumor necrosis factor-α plus actinomycin D-induced apoptosis in hepatocytes. However, it is unlikely that cAMP signaling also plays a role in the prevention of astrocyte apoptosis because cAMP phosphodiesterase inhibitors do not protect astrocytes against the H2O2-induced cytotoxicity (11Takuma K. Lee E. Enomoto R. Mori K. Baba A. Matsuda T. Br. J. Pharmacol. 2001; 133: 841-848Crossref PubMed Scopus (41) Google Scholar). On the other hand, we reported that the ERK signal plays a role in the protective effect of T-588 and NGF against H2O2-induced astrocyte apoptosis (9Takuma K. Fujita T. Kimura Y. Tanabe M. Yamamuro A. Lee E. Mori K. Koyama Y. Baba A. Matsuda T. Eur. J. Pharmacol. 2000; 399: 1-8Crossref PubMed Scopus (33) Google Scholar). In view of the recent studies that cGMP activates ERK (30Silberbach M. Gorenc T. Hershberger R.E. Stork P.J. Steyger P.S. Roberts C.T. J. Biol. Chem. 1999; 274: 24858-24864Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 31Komalavilas P. Shah P.K. Jo H. Lincoln T.M. J. Biol. Chem. 1999; 274: 34301-34309Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 32Gu M. Lynch J. Brecher P. J. Biol. Chem. 2000; 275: 11389-11396Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), it was considered whether the effect of cGMP reported here may be mediated by the activation of ERK or phosphatidylinositol-3 kinase. However, the protective effect of dibutyryl-cGMP was observed even in the presence of the ERK kinase inhibitor PD98059 and the phosphoinositide 3-kinase inhibitor wortmannin. Activation of the mitochondrial PTP has been identified as a possible common effector of the cell death of numerous cell types in response to both necrotic and apoptotic stimuli (33Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Herman B. Mol. Cell. Biochem. 1997; 174: 159-165Crossref PubMed Scopus (190) Google Scholar, 34Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1762) Google Scholar). Thus, it seems likely that the anti-apoptotic effect of cGMP may be mediated by the inhibition of the mitochondrial PTP; however there are no reports on the effect of cGMP on the PTP. We demonstrate here that cGMP together with PKG, as well as cyclosporin A and bongkrekic acid, inhibit the Ca2+-induced mitochondrial swelling in isolated brain mitochondria. The effect of cGMP is dose-dependent and observed even at 1 μm, suggesting that cGMP is an endogenous potent inhibitor of the PTP. The effect of cGMP on the mitochondrial PTP required the presence of the cytosolic extract, and the ability of the cytosolic extract was inactivated by heat treatment. Furthermore, the effect of cGMP plus the cytosolic extract was antagonized by KT5823, and exogenous PKG mimicked the effect of the cytosolic extract. Taken together, it is likely that cGMP inhibits the mitochondrial PTP via the activation of PKG. The mitochondrial PTP is formed from a complex of the voltage-dependent anion channel, the adenine nucleotide translocase, and cyclophilin-D at contact sites between the mitochondrial outer and inner membranes. It is regulated by numerous endogenous physiological effectors such as ions, protons, the mitochondrial transmembrane potential, the concentration of adenine nucleotides, the pyrimidine redox state, the thiol redox state, reactive oxygen species, and Bcl-2 family proteins (34Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1762) Google Scholar). Genaroet al. (12Genaro A.M. Hortelano S. Alvarez A. Martinez C. Bosca L. J. Clin. Invest. 1995; 95: 1884-1890Crossref PubMed Scopus (306) Google Scholar) reported that an induction of Bcl-2 is involved in the anti-apoptotic effect of cGMP in splenocytes. In contrast, the effect of cGMP was observed even when it was added together with or after Ca2+. In view of the rapid effect of cGMP, it is unlikely that these endogenous effectors may be involved in the effect of cGMP on the mitochondrial PTP. The present study using a cell-free system suggests that cGMP interacts directly with mitochondria, resulting in the inhibition of the mitochondrial PTP via the activation of PKG. It is not known how cGMP/PKG signaling affects the mitochondrial PTP. Known substrates for PKG include inositol 1,4,5-trisphosphate receptor (35Ferris C.D. Snyder S.H. Annu. Rev. Physiol. 1992; 54: 469-488Crossref PubMed Scopus (197) Google Scholar), dopamine- and cAMP-regulated phosphoprotein (36Tsou K. Snyder G.L. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3462-3465Crossref PubMed Scopus (102) Google Scholar), and cGMP-binding phosphodiesterase (37Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14971-14978Abstract Full Text PDF PubMed Google Scholar) in the central nervous system. Schlossmann et al. (38Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.X. Allescher H.D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (385) Google Scholar) have recently identified a PKG substrate protein in smooth muscle. However, the role of these substrates in the inhibition by cGMP of the mitochondrial PTP is not clear. There are a limited number of inhibitors of the mitochondrial PTP. The most specific inhibitor is cyclosporin A, which competitively prevents cyclophilin from interacting with specific cyclophilin-dependent binding domains of the pore. Other classes of inhibitors of the mitochondrial PTP consist of the adenine nucleotide translocase ligand bongkrekic acid (29Zoratti M. Szabò I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2194) Google Scholar), the phospholipase inhibitor trifluoperazine (39Nieminen A.L. Saylor A.K. Tesfai S.A. Herman B. Lemasters J.J. Biochem. J. 1995; 307: 99-106Crossref PubMed Scopus (354) Google Scholar), the antiestrogen drug tamoxifen (40Custodio J.B.A. Moreno A.J.M. Wallace K.B. Toxicol. Appl. Pharmacol. 1998; 152: 10-17Crossref PubMed Scopus (82) Google Scholar), and nitric oxide (41Brookes P.S. Salinas E.P. Darley-Usmar K. Eiserich J.P. Freeman B.A. Darley-Usmar V.M. Anderson P.G. J. Biol. Chem. 2000; 275: 20474-20479Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Consistent with recent studies in other cells (42Adams J.W. Pagel A.L. Means C.K. Oksenberg D. Armstrong R.C. Brown J.H. Circ. Res. 2000; 87: 1180-1187Crossref PubMed Scopus (99) Google Scholar, 43Moriya R. Uehara T. Nomura Y. FEBS Lett. 2000; 484: 253-260Crossref PubMed Scopus (71) Google Scholar, 44Yoon H.S. Moon S.C. Kim N.D. Park B.S. Jeong M.H. Yoo Y.H. Biochem. Biophys. Res. Commun. 2000; 276: 151-156Crossref PubMed Scopus (96) Google Scholar), we found that the PTP inhibitors cyclosporin A and bongkrekic acid prevented the H2O2-induced reduction in cell viability and caspase-3 activation in astrocytes. In view of the evidence implicating the mitochondrial PTP in ischemia-related tissue damage, it has become important to identify strategies to inhibit the induction of the PTP opening. The present study demonstrates that cGMP inhibits the mitochondrial PTP opening, resulting in apoptotic events via the activation of PKG. This suggests that cGMP/PKG signaling is a novel target for the prevention of mitochondrial dysfunction-mediated cell death. In line with this, we have shown that cGMP phosphodiesterase inhibitors including ibudilast protect astrocytes against reperfusion injury via cGMP/PKG signaling (11Takuma K. Lee E. Enomoto R. Mori K. Baba A. Matsuda T. Br. J. Pharmacol. 2001; 133: 841-848Crossref PubMed Scopus (41) Google Scholar).

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