Anti-apoptotic Effect of cGMP in Cultured Astrocytes

Abstract

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 the medium, the cells were with and then by the of 100 of The concentration of DiOC6(3) was measured with nm and nm using a and membrane were 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 of the and membrane were determined by a protein and of the was subjected to gel electrophoresis A cytochrome c antibody was used for The activity of in cell was measured using the (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 for the 7-amino-4-methyl-coumarin were measured with nm and nm using a The were as Cells plated on 100-mm were with using a and by for min The was in 100 of and with of a The was for and the resulting of was Brain mitochondria were isolated from Wistar rats by the of J. Neurochem. PubMed Scopus Google Scholar) using a The mitochondrial was in and and on The of the PTP causes mitochondrial swelling that is as a decrease in the of a mitochondrial The Ca2+-induced mitochondrial swelling was as by et al. H. M. F. T. Neurosci. 1998; 18: PubMed Google Scholar). and were min the of to the of the mitochondrial membrane under The treatment of the mitochondrial with cGMP, the cytosolic was carried out for min the of The were from the acid J. 1994; PubMed Scopus Google Scholar). nm was measured using a (Kyoto, Japan). shown is of similar experiments on mitochondrial analysis of the was carried out by using a for Reperfusion after exposure of astrocytes to H2O2-containing medium causes a decrease in MTT reduction DNA ladder formation, cytochrome c release from mitochondria, and caspase-3 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). the effects of membrane-permeable cGMP on the H2O2-induced apoptosis. 8-pCPT-cGMP, and attenuated the H2O2-induced decrease in MTT reduction activity in a the effect of was that of dibutyryl-cGMP and The protective effect of dibutyryl-cGMP was in the presence of the kinase inhibitor and the inhibitor not also attenuated the H2O2-induced DNA ladder and nuclear Reperfusion after the exposure of astrocytes to H2O2 also caused a decrease in DiOC6(3) which mitochondrial membrane The H2O2-induced of mitochondrial membrane was inhibited by dibutyryl-cGMP in a dibutyryl-cGMP attenuated the H2O2-induced cytochrome c release from mitochondria and activation in a of dibutyryl-cGMP on cell injury by in cultured rat astrocytes. A, effect on DNA ladder Cells were exposed to normal 100 H2O2 for 30 min and then incubated with Earle’s solution for was 30 min H2O2 exposure and was present The of experiments is shown effect on nuclear Cells were in the and presence and of 100 H2O2 for 30 min and then incubated with Earle’s solution for (100 μm) was 30 min and was present assay and of dibutyryl-cGMP on of mitochondrial membrane by H2O2 in cultured rat astrocytes. A, of the of mitochondrial membrane Cells were exposed to normal 100 for 30 min and then incubated with Earle’s solution for the indicated for the effect of Cells were exposed to normal 100 for 30 min and then incubated with Earle’s solution for was 30 min H2O2 exposure and was present are S.E. for and were obtained from experiments. from from the dibutyryl-cGMP of dibutyryl-cGMP on cytochromec release from mitochondria and increase in activity by H2O2 in cultured rat astrocytes. A, effect on cytochromec release from mitochondria. Cells were exposed to normal 100 H2O2 for 30 and then incubated with Earle’s solution for was 30 min and was present c in the and mitochondrial is The of experiments are effect on increase in Cells were exposed to normal 100 H2O2 for 30 min and then incubated with Earle’s solution for was 30 min H2O2 exposure and was present are S.E. for and were obtained from experiments. from from the dibutyryl-cGMP The by dibutyryl-cGMP the H2O2-induced decrease in MTT reduction activity was blocked by the inhibitors and also blocked the effect of dibutyryl-cGMP on mitochondrial membrane cytochrome c release and activation A showed that exposure of rat brain mitochondria to Ca2+ caused a decrease in mitochondrial swelling by the PTP not The swelling was on Ca2+ concentration as reported previously H. M. F. T. Neurosci. 1998; 18: PubMed Google Scholar). In this study, we used 100 Ca2+ to mitochondrial swelling. The swelling was inhibited by the mitochondrial PTP inhibitors cyclosporin A K.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and bongkrekic acid M. 1995; PubMed Scopus Google Scholar) cGMP cytosolic extract from astrocytes the Ca2+-induced mitochondrial the of cGMP and the cytosolic extract prevented the swelling in a B and The effect of cGMP the cytosolic extract was also in the swelling by 30 not The ability of the cytosolic extract was inactivated by it and was mimicked by exogenous The similar by cGMP Ca2+-induced swelling was cGMP was with after Ca2+ not the effect of cGMP the cytosolic extract in 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 in cultured astrocytes In this bongkrekic acid was used a concentration because it showed cell not of protein kinase on the by cGMP and cytosolic extract from astrocytes on Ca2+-induced swelling in rat brain mitochondria. was by the of Ca2+ cGMP, the and were min Ca2+ and were present during the was 30 min Ca2+ and was present during the A, effect of cytosolic The extract was for min Ca2+ and 100 Ca2+ and 100 cGMP and Ca2+ and 100 effect of exogenous PKG. Ca2+ and 100 Ca2+ and 100 cGMP cytosolic and Ca2+ and 100 cGMP PKG. effect of the inhibitor KT5823. Ca2+ and 100 Ca2+ and 100 cGMP and and 100 are for and were obtained from of cyclosporin A and bongkrekic acid on the H2O2-induced cell injury and increase in activity in cultured rat astrocytes. Cells were exposed to normal 100 medium for 30 min and then incubated with Earle’s solution for A μm) and bongkrekic acid μm) were 30 min and were present A, MTT are S.E. for and were obtained from experiments. are S.E. for and were obtained from experiments. from the drugs This study was to the mechanism by which cGMP the H2O2-induced apoptosis in cultured astrocytes. The membrane-permeable cGMP dibutyryl-cGMP, 8-pCPT-cGMP, and attenuated the H2O2-induced decrease in cell In of the of mitochondria in the apoptotic the present study on the effect of cGMP on the H2O2-induced mitochondrial We showed that dibutyryl-cGMP prevented the reduction of the mitochondrial membrane and cytochrome c release and caspase-3 nuclear condensation, and DNA in cultured astrocytes. These findings that cGMP inhibits the H2O2-induced mitochondrial dysfunction and prevents apoptosis. The effect of cGMP analog on caspase-3 activation and cytochrome c release was also 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). of the effects of cGMP are by PKG. In this 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 inhibitor the prevention of cytochrome c release and caspase-3 activation in PC12 We also found that almost completely blocked the by dibutyryl-cGMP the H2O2-induced reduction of the mitochondrial membrane potential, cytochrome c release, and activation in astrocytes. These findings that cGMP apoptosis via the activation of PKG, the mechanism by which prevents apoptotic is not 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 as as cGMP apoptosis in However, it is that signaling also a role in the prevention of apoptosis because phosphodiesterase inhibitors not astrocytes the H2O2-induced (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 other we reported that the a role in the protective effect of and H2O2-induced 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 of the studies that cGMP M. T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, P. H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. P. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), it was considered whether the effect of cGMP reported be by the activation of phosphatidylinositol-3 However, the protective effect of dibutyryl-cGMP was in the presence of the kinase inhibitor and the inhibitor of the mitochondrial PTP has been as a of the cell of cell in to and apoptotic T. 1997; PubMed Scopus Google Scholar, G. M. 1998; PubMed Scopus Google Scholar). it that the anti-apoptotic effect of cGMP be by the of the mitochondrial are on the effect of cGMP on the PTP. We demonstrate that cGMP with PKG, as as cyclosporin A and bongkrekic acid, the Ca2+-induced mitochondrial swelling in isolated brain mitochondria. The effect of cGMP is and that cGMP is an inhibitor of the PTP. The effect of cGMP on the mitochondrial PTP the presence of the cytosolic and the ability of the cytosolic extract was inactivated by heat treatment. the effect of cGMP the cytosolic extract was by KT5823, and exogenous mimicked the effect of the cytosolic it is that cGMP inhibits the mitochondrial PTP via the activation of PKG. The mitochondrial PTP is from a of the the and the mitochondrial and It is by as the mitochondrial potential, the concentration of the the and G. M. 1998; PubMed Scopus Google Scholar). 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 of is in the anti-apoptotic effect of cGMP in In the effect of cGMP was it was with after In of the effect of cGMP, it is that be in the effect of cGMP on the mitochondrial PTP. The present study using a system that cGMP with mitochondria, resulting in the of the mitochondrial PTP via the activation of PKG. It is not known how signaling the mitochondrial PTP. for Snyder 1992; PubMed Scopus Google Scholar), and K. Snyder P. S. A. PubMed Scopus Google Scholar), and phosphodiesterase J. Biol. Chem. Full Text PDF PubMed Google Scholar) in the et al. J. A. K. A. G. M. M. F. P. 2000; PubMed Scopus Google Scholar) have recently a protein in However, the role of in the by cGMP of the mitochondrial PTP is not are a of inhibitors of the mitochondrial PTP. The inhibitor is cyclosporin A, which prevents from with of the of inhibitors of the mitochondrial PTP of the bongkrekic acid M. 1995; PubMed Scopus Google Scholar), the inhibitor J. 1995; PubMed Scopus Google Scholar), the Pharmacol. 1998; PubMed Scopus (82) Google Scholar), and K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). with studies in other cells J.W. Res. 2000; PubMed Scopus Google Scholar, R. T. Y. FEBS Lett. 2000; PubMed Scopus Google Scholar, Kim Park Yoo Y.H. Res. 2000; 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 of the the mitochondrial PTP in tissue it has important to to the of the PTP The present study demonstrates that cGMP inhibits the mitochondrial PTP resulting in apoptotic via the activation of PKG. This that signaling is a target for the prevention of mitochondrial cell In with we have shown that cGMP phosphodiesterase inhibitors including astrocytes reperfusion injury via 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).