Tsuneo Takadera

Prostaglandin E2 induces caspase-dependent apoptosis in rat cortical cells

Up-regulation of neuronal cyclooxygenase-2 (COX-2) and the elevation in prostaglandin E(2) (PGE(2)) have been reported to occur after cerebral ischemic insult. To evaluate whether the COX-2 reaction product PGE(2) is directly related to induction of apoptosis in neuronal cells, the effect of PGE(2) on cell viability was examined in rat cortical cells. PGE(2) induced apoptosis in a dose-dependent manner (5-25 microM) 48 h after addition to the cells, which was characterized by cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Neither 17-phenyl trinor-prostaglandin E(2) (an EP1 agonist) or sulprostone (an EP3 agonist) induced cell death, whereas butaprost (an EP2 agonist) induced apoptotic cell death. In addition, PGE(2) activated caspase-3 in a time-dependent manner until 24 h after treatment. The apoptosis induced by PGE(2) was prevented by a caspase-3 inhibitor in a dose-dependent manner. In contrast, dibutyryl cyclic adenosine monophosphate also induced apoptotic cell death in a dose-dependent manner (20-100 microM). These results suggest that PGE(2), acting via an EP2-like receptor, induces apoptosis in neurons.

Prostaglandin E2 induced caspase-dependent apoptosis possibly through activation of EP2 receptors in cultured hippocampal neurons

Cyclooxygenase-2 (COX-2) induction and prostaglandin E2 elevation have been reported to occur after cerebral ischemic insult. To evaluate whether the cyclooxygenase-2 reaction product prostaglandin E2 is directly related to induction of apoptosis in neuronal cells, the effect of prostaglandin E2 on cell viability was examined in hippocampal cells. Prostaglandin E2 (5-25 microM) induced apoptosis in a dose-dependent manner 48 h after addition to the cells, which was characterized by cell shrinkage, nuclear condensation or fragmentation and attenuated by a protein synthesis inhibitor, cycloheximide. Neither 17-phenyl trinor-prostaglandin E2 (an EP1 agonist) nor sulprostone (an EP3 agonist) induced cell death, whereas butaprost (an EP2 agonist) induced apoptosis. Prostaglandin E2 increased the intracellular concentration of cAMP, and the selective EP2 agonist butaprost also induced apoptosis accompanied by increasing cAMP levels in hippocampal cells. Moreover, a cell permeable cAMP analog, dibutyryl cAMP also induced apoptosis in hippocampal cells. These findings suggest that prostaglandin E2-induced apoptosis was mediated through a mechanism involving the cAMP-dependent pathway. In addition, prostaglandin E2 activated caspase-3 activity in a dose-dependent manner and a caspase-3 inhibitor prevented the prostaglandin E2-induced apoptosis. We showed in this report that prostaglandin E2 directly induced apoptosis in hippocampal neurons. Moreover, it is likely that the direct effects of prostaglandin E2 on hippocampal neurons were mediated by activation of EP2 receptors followed by elevation of the intracellular cAMP levels.

Apoptotic cell death and caspase-3 activation induced by N-methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I.

Abstract : The effect of N‐methyl‐d‐aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK‐801 and 2‐amino‐5‐phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK‐801 (an NMDA antagonist) for 1‐2 days induced apoptotic cell death in a dose‐dependent manner (1 nM to 10 μM). NMDA (25 μM), however, inhibited the MK‐801 (0.1 μM)‐induced apoptotic cell death. MK‐801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase‐3 was accompanied by MK‐801‐induced cell death in a dose‐dependent manner, and an inhibitor of caspase‐3 reduced the cell death. Further, cycloheximide (0.2 μg/ml) completely protected the cells from MK‐801‐induced apoptotic cell death and caspase‐3 activation. Insulin‐like growth factor I completely attenuated MK‐801‐induced apoptotic cell death and caspase‐3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.

Toxic effect of a β-amyloid peptide (β22–35) on the hippocampal neuron and its prevention

A synthetic truncated beta-amyloid peptide, beta 22-35, was shown to have a cytotoxic effect on cultured neurons from the rat hippocampus in serum-free medium. The peptide formed aggregates and typical amyloid fibrils resembling those of the beta-amyloid protein (AP) in neutral buffer solution and showed characteristic staining with Congo red and thioflavin-S. The neurotoxicity of beta 22-35 was suppressed by addition of calf serum, dibutyryl cAMP or insulin to culture medium, but not by addition of NGF or substance P. beta 22-35 had no effect on the glial cells. These results suggest that the AP can induce neurotoxicity in the hippocampal cells in vitro and the toxicity may involve a disorder in the intracellular signal transduction.

Protection by ethanol of cortical neurons from N-methyl-d-aspartate-induced neurotoxicity is associated with blocking calcium influx

Effect of ethanol on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in rat dissociated cortical cells (8-12 day cultures) was studied. Treatment of cells with NMDA (50 and 500 microM) for 15 min caused cytotoxic effects on the cells, as examined by microscopic observations and lactate dehydrogenase release from cells 18 h after the treatment. Ca2+ is essential for these effects in medium during treatment. Presence of ethanol (50-300 mM) simultaneously with NMDA protected cells from the cytotoxicity depending on the concentration of ethanol. Calcium accumulation in cells on addition of NMDA, as monitored by fluorescence ratio (F405/F485) of Indo-1-preloaded cortical cells, was also decreased depending on the concentration of added ethanol. APV (200 microM) and ketamine (100 microM) blocked both the cytotoxicity and cellular calcium accumulation due to NMDA. These results suggest that ethanol effects its protection of neurons from NMDA-induced cytotoxicity by blocking the receptor-mediated calcium influx.

Reactive oxygen species involved in the glutamate toxicity of C6 glioma cells via xc antiporter system.

We recently demonstrated that continuous L-glutamate exposure led to cell death in C6 glioma cells over a period of 24-36 h, due to inhibition of cystine uptake through the cystine/glutamate (XC) antiporter. The antioxidant vitamin E provided protection against this effect, supporting the hypothesis that depletion of glutathione might be responsible, resulting from insufficient cystine uptake. To clarify the content of oxidative stress after glutathione depletion, the present study was done to investigate accumulation and target molecules of reactive oxygen species induced by glutamate treatment. The accumulation of reactive oxygen species was increased three-fold as compared to a control culture. Membrane oxidation, as judged by lipid peroxidation, was increased two-fold after glutamate treatment. Cellular ATP content was significantly reduced by glutamate exposure. For the two cytosolic enzymes examined, activity of glyceraldehyde 3-phosphate dehydrogenase was slightly enhanced by glutamate treatment, while activity of glutamine synthetase was not changed. Impairment of nuclear DNA after glutamate exposure was also revealed by nuclear chromatin condensation with DNA fragmentation. Thus, the multiple targets (membrane, cytoplasm and nuclei) of oxygen radicals in glutamate toxicity through the xc antiporter system were evaluated for the first time. Furthermore, prevention from cell death and from cellular toxicity induced by oxygen radicals could be seen using three specific oxygen radical scavengers, catalase, 3,3,5,5-tetramethyl-pyrroline N-oxide and alpha-phenyl-N-t-butylnitrone, without restoring the glutathione deficit. This indicates that radical scavengers did not interact with the xc antiporter system, but directly scavenged the oxygen radicals. Taken together, the data strongly suggest that O2-, H2O2 and OH accumulate in response to oxidative stress after glutathione depletion, resulting in glutamate cell death of C6 glioma cells.

The quaternary structure of carp muscle alkaline protease

Abstract Carp muscle alkaline protease consists of four kinds of subunits, and its composition was assumed to be ( αβγ 2 δ 2 ) 4 . It dissociated in the presence of 2-mercaptoethanol into an enzyme and α-subunits which upon removal of 2-mercaptoethanol rapidly aggregated to form a precipitate. The composition of the 2-mercaptoethanol-treated enzyme was ( βμ 2 δ 2 ) 4 . The pH of a 2-mercaptoethanol-treated enzyme solution was lowered to 4.5 by the addition of acetic acid in the presence of 0.4 M LiCl and centrifuged to separate the precipitate formed; this exhibited little activity and was mainly composed of β-subunits. The supernatant fluid recovered 53% of activity and contained an enzyme, whose composition was ( γ 4 δ 4 ) 4 . The temperature-activity curve of the native enzyme was the same as that of the 2-mercaptoethanol-treated enzyme and both were unable to hydrolyze casein at all below 55°C. However, the temperature dependence for activity of the LiCl-treated enzyme was ordinary: it hydrolyzed casein at physiological temperatures. When the 2-mercaptoethanol-treated enzyme was incubated with 4.5 M urea at 45°C for 20 min and this was followed by column chromatography, a little activity was recovered and the amount of recovery was parallel with the amount of δ-subunit in the fractions. These findings suggest; (1) the α-subunit does not take any part in activity but is a protein necessary for binding between subunits or between the enzyme and some functional proteins in the cells, (2) the β-subunit is used as inhibitor in the quaternary structure of the enzyme, (3) the δ-subunit is the catalytic one, and (4) binding with the γ-subunit is necessary for the δ-subunit to retain its active comformation.

NMDA receptor 2B-selective antagonist ifenprodil-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in cultured rat cortical neurons

The N-methyl-d-aspartate (NMDA) receptor 2B-selective antagonist ifenprodil induced morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation in rat cultured cortical cells. Ifenprodil increased the apoptotic cell death in a dose-dependent manner (0.5-10 microM). In addition, the protein synthesis inhibitor cycloheximide completely blocked ifenprodil-induced apoptotic cell death. The selective inhibitors of glycogen synthase kinase-3 (GSK-3) prevented the ifenprodil-induced apoptosis. Moreover, activation of caspase-3 was accompanied by cell death induced by ifenprodil in a dose-dependent manner. The ifenprodil-induced apoptosis was prevented by a caspase-3 inhibitor. These results suggested that activation of GSK-3 involves in the apoptosis induced by blocking of trophic effect of NMDA receptor consisting of NR2B subunit in rat cortical neurons.

Extracellular pH modulates N-methyl-D-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures

The effect of extracellular pH (pHo) on the excitotoxicity of N-methyl-D-aspartate (NMDA) in cultured rat cortical cells was studied. Treatment of cells with 500 microM NMDA for 15 min at various pHs in a range from 6.5 to 8.0 progressively enhanced staining with Trypan blue and release of lactate dehydrogenase with increased pH after 18 h of culture following treatment. The cytotoxic effect of high concentration of K+ (40 mM) or veratridine (10 microM) was also directly related to the increase in pHo. Free calcium accumulation in cells on addition of NMDA increased parallel to pHo. Changes in intracellular pH were estimated to be minor compared with extracellular changes. Specific NMDA antagonists could block both the NMDA- and membrane depolarization-induced neurotoxicity and calcium accumulation completely. These results suggest that the proton concentration outside of cells attenuates NMDA-induced neurotoxicity by blocking calcium accumulation.

Apoptotic cell death and CPP32-like activation induced by thapsigargin and their prevention by nerve growth factor in PC12 cells

Thapsigargin, an endoplasmic reticular Ca2+-ATPase inhibitor, induced apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by the activation of CPP32-like protease, a member of the interleukin-1beta converting enzyme protease (ICE) family, but not the activation of ICE-like protease. Nerve growth factor (NGF) completely inhibited the cell death and CPP32-like activation induced by thapsigargin while Ac-Asp-Glu-Val-Asp-CHO, an inhibitor of CPP32-like protease, reduced the cell death. PD98059, a specific inhibitor of Map kinase kinase, did not reduce the protective effect of NGF on thapsigargin-induced cell death. These results suggest that calcium ion-induced apoptotic cell death was mediated by CPP32-like, but not ICE-like, protease and was regulated by a neurotrophic factor possibly, through the Map kinase cascade independent pathway.