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1212 Apoptotic DNA fragmentation and upregulation of bax induced by transient ischemia of the rat retina
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1212
Apoptotic DNA fragmentation and upregulation of bcz induced by transient ischemia of the rat retina Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan’, Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan2 Katsuyuki Kaneda’ , Takehiko Maeda’ , Shuji Kaneko’ , Akinori Akaike’ , Masabumi Minami2, Masamichi Satoh This study was performed to examine the involvement of apoptosis and Bax in ischemia-induced retinal injury. Retinal ischemia was induced by intraocular pressure elevation for 45 min in the adult rats. A selective damage of the inner retina was observed 7 days after ischemia. No TUNEL positive cells were observed in the normal retina, but there was a significant number of TUNEL positive cells 6-168 hr after transient ischemia. The number of TUNEL positive cells reached a maximum at 24 hr after ischemia. DNA laddering was observed on agarose gel electrophoresis with the retina 24 hr after ischemia but not in the normal retina. Semiquantitative PCR revealed that bax gene expression did not change immediately after cessation of ischemia, and gradually increased as early as 6 hr, reached a peak at 24 hr, then decreased to near baseline levels at 168 hr. These results suggest that apoptosis associated with expression of Bax is involved in retinal cell loss after ischemic insult.
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Protective effects of CP-ceramide against glutamate-induced neurotoxicity in cultured cortical neurons. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University’, Department of Neurology, Faculty of Medicine, Kyoto University2 Toshiaki Kumel , Takehiko Maeda’ , Shuji Kaneko’ , Akinori Akaike’ , Takeshi Kihara2, Shun Shimohama2, Jun Kimura2 This study was performed to investigate the role of ceramide, which is involved in the signal transduction pathway of the low affinity NGF receptor (p75), in the neuroprotective action of NGF. Delayed cytotoxicity was induced by brief exposure of cells to glutamate, ionomycin, a calcium ionophore, or S-nitrosocysteine (SNOC), an NO-generating agent. Exposure of the cultures to CP-ceramide, a membrane-permeable analogue of ceramide, for 24 hr prior to glutamate or ionomycin exposure reduced cytotoxicity induced by glutamate or ionomycin. By contrast, CZceramide did not affect SNOC-induced cytotoxicity. Marked reduction of cell viability was elicited by depriving serum from incubating medium for 48 hr. CZ-ceramide exacerbated neuronal death induced by serum deprivation. These results suggest that ceramide is partially concerned in the protective action of NGF against glutamate-induced neurotoxicity.
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MECHANISMS OF DOPAMINE DZ-INDUCED NEUROPROTECTION AGAINST TAMATE NEUROTOXICITY IN RAT MESENCEPHALON l, Dept. of Pharm., Fat. of Pharm. Sci., Kyoto Univ.2 Dept. of Neurol., Fat. of Med., Kyoto Univ.
GLU-
HIDEYUKI SAWADA’, TAKESHI KIHARA’ , MAKOTO URUSHITANI’, SHUN SHIMOHAMA’ , JUN KIMURA’, MASAKAZU IB12, AKINORI AKAIKE2 Using the rat cultured mesencephalon, we investigated the mechanisms of protecting effects of D2 agonists against glutamate-induced neuronal death. Brief exposure to glutamate reduced the surviving rate of both dopaminergic and non-dopaminergic neurons. Preincubation with D2 agonists for 24 hours prior to glutamate exposure protects both dopaminergic and non-dopaminergic neurons from glutamate-induced neuronal death. The protecting effects were significantly blocked by D2 antagonists or an inhibitor of protein synthesis. Furthermore, preincubation with D2 agonists showed protective effects against neurotoxicity induced by superoxide anion or calcium ionophore. These results indicate that precedent D2 stimulation protects neurons from neurotoxicity induced by calcium influx or radical formation following glutamate exposure.
1212
Apoptotic DNA fragmentation and upregulation of bcz induced by transient ischemia of the rat retina Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan’, Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606-01, Japan2 Katsuyuki Kaneda’ , Takehiko Maeda’ , Shuji Kaneko’ , Akinori Akaike’ , Masabumi Minami2, Masamichi Satoh This study was performed to examine the involvement of apoptosis and Bax in ischemia-induced retinal injury. Retinal ischemia was induced by intraocular pressure elevation for 45 min in the adult rats. A selective damage of the inner retina was observed 7 days after ischemia. No TUNEL positive cells were observed in the normal retina, but there was a significant number of TUNEL positive cells 6-168 hr after transient ischemia. The number of TUNEL positive cells reached a maximum at 24 hr after ischemia. DNA laddering was observed on agarose gel electrophoresis with the retina 24 hr after ischemia but not in the normal retina. Semiquantitative PCR revealed that bax gene expression did not change immediately after cessation of ischemia, and gradually increased as early as 6 hr, reached a peak at 24 hr, then decreased to near baseline levels at 168 hr. These results suggest that apoptosis associated with expression of Bax is involved in retinal cell loss after ischemic insult.
1213
Protective effects of CP-ceramide against glutamate-induced neurotoxicity in cultured cortical neurons. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University’, Department of Neurology, Faculty of Medicine, Kyoto University2 Toshiaki Kumel , Takehiko Maeda’ , Shuji Kaneko’ , Akinori Akaike’ , Takeshi Kihara2, Shun Shimohama2, Jun Kimura2 This study was performed to investigate the role of ceramide, which is involved in the signal transduction pathway of the low affinity NGF receptor (p75), in the neuroprotective action of NGF. Delayed cytotoxicity was induced by brief exposure of cells to glutamate, ionomycin, a calcium ionophore, or S-nitrosocysteine (SNOC), an NO-generating agent. Exposure of the cultures to CP-ceramide, a membrane-permeable analogue of ceramide, for 24 hr prior to glutamate or ionomycin exposure reduced cytotoxicity induced by glutamate or ionomycin. By contrast, CZceramide did not affect SNOC-induced cytotoxicity. Marked reduction of cell viability was elicited by depriving serum from incubating medium for 48 hr. CZ-ceramide exacerbated neuronal death induced by serum deprivation. These results suggest that ceramide is partially concerned in the protective action of NGF against glutamate-induced neurotoxicity.
1214
MECHANISMS OF DOPAMINE DZ-INDUCED NEUROPROTECTION AGAINST TAMATE NEUROTOXICITY IN RAT MESENCEPHALON l, Dept. of Pharm., Fat. of Pharm. Sci., Kyoto Univ.2 Dept. of Neurol., Fat. of Med., Kyoto Univ.
GLU-
HIDEYUKI SAWADA’, TAKESHI KIHARA’ , MAKOTO URUSHITANI’, SHUN SHIMOHAMA’ , JUN KIMURA’, MASAKAZU IB12, AKINORI AKAIKE2 Using the rat cultured mesencephalon, we investigated the mechanisms of protecting effects of D2 agonists against glutamate-induced neuronal death. Brief exposure to glutamate reduced the surviving rate of both dopaminergic and non-dopaminergic neurons. Preincubation with D2 agonists for 24 hours prior to glutamate exposure protects both dopaminergic and non-dopaminergic neurons from glutamate-induced neuronal death. The protecting effects were significantly blocked by D2 antagonists or an inhibitor of protein synthesis. Furthermore, preincubation with D2 agonists showed protective effects against neurotoxicity induced by superoxide anion or calcium ionophore. These results indicate that precedent D2 stimulation protects neurons from neurotoxicity induced by calcium influx or radical formation following glutamate exposure.