Benzyl alcohol inhibits N-methyl-D: -aspartate receptor-mediated neurotoxicity and calcium accumulation in cultured rat cortical neurons.

The purpose of this study is to examine whether benzyl alcohol affects N-methyl-D-aspartate (NMDA) receptor in cortical cells. Benzyl alcohol (0.5-2 mM) inhibited NMDA-induced cytotoxicity. The protective effect of benzyl alcohol on NMDA-induced toxicity disappeared by washing cells with buffer to remove benzyl alcohol. Benzyl alcohol reduced NMDA receptor-mediated calcium accumulation, indicating that benzyl alcohol inhibits NMDA receptor activity.

Involvement of NO generation in aluminum-induced cell death.

Previously, we have reported that the exposure of PC12 cells to the aluminum-maltolate complex (Al(maltol)(3)) results in decreased cell viability via the apoptotic cell death pathway. In this study, we have used several nitric oxide synthase (NOS) inhibitors and the NO generator diethylenetriamine NONOate (DETA NONOate) to examine whether or not intracellular nitric oxide (NO) generation is involved in the onset mechanism of Al(maltol)(3)-induced cell death. Cell viability was assessed by measuring lactate dehydrogenase (LDH) release and caspase-3 activity. Treatment of the cells with 150 microM Al(maltol)(3) for 48 h resulted in intracellular NO generation. Exposure of the cells to DETA NONOate also induced a marked decrease in cell viability. Pre-treatment of the cells with a general NOS inhibitor or with a selective inducible NOS (iNOS) inhibitor effectively prevented Al(maltol)(3)-induced cell death. However, a neuronal NOS (nNOS) inhibitor did not exhibit any protective effect against Al(maltol)(3)-induced cell death. In addition, ascorbic acid markedly inhibited Al(maltol)(3)- and DETA NONOate-induced cell death. Based on these results, we discussed the involvement of intracellular NO generation in the onset mechanisms of Al(maltol)(3)-induced cell death.

Temperature-dependent N-methyl-D-aspartate receptor-mediated cytotoxicity in cultured rat cortical neurons.

Hypothermia protects against hypoxic or ischemic damage. However, the mechanisms by which brain cooling prevents hypoxic or ischemic damage are not clear. We examined whether hypothermia protects against excitotoxicity in cultured cortical cells. Exposure of cortical cell culture to 500 microM N-methyl-D-aspartate (NMDA) for 15 min at 32 degrees C or 37 degrees C did not induce neurotoxicity. On the other hand, reduction of temperature to 20 degrees C resulted in widespread neuronal disintegration by the following day. Moreover, intracellular calcium concentration increased markedly by adding NMDA to cells at 20 degrees C. These results suggest that profound hypothermia does not protect neurons from excitotoxicity by inhibiting NMDA receptor activity.

Calmodulin inhibitor-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells.

Calmodulin is known to transduce Ca(2+) signals by interacting with specific target proteins. In order to determine the role of calmodulin in regulating neuronal survival and death, we examined, whether calmodulin inhibitors induce caspase-dependent apoptotic cell death, and whether glycogen synthase kinase-3 is involved in calmodulin inhibitor-induced cell death in PC12 cells. W13, a calmodulin specific inhibitor increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation of fragmentation. Glycogen synthase kinase-3 inhibitors prevented calmodulin inhibitor-induced apoptosis. In addition, nerve growth factor and cycloheximide, a protein synthesis inhibitor, completely blocked cell death. Moreover, caspase-3 activation was accompanied by calmodulin inhibitor-induced cell death and inhibited by nerve growth factor. These results suggest that calmodulin inhibitors induce caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.

Caspase-dependent apoptosis induced by thapsigargin was prevented by glycogen synthase kinase-3 inhibitors in cultured rat cortical neurons.

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca(2+)-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9-10). Blockade of N-methyl-D-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3beta activation in rat cortical neurons.

Caspase-dependent apoptosis induced by calcineurin inhibitors was prevented by glycogen synthase kinase-3 inhibitors in cultured rat cortical cells.

Calcineurin is selectively enriched within neurons of the central nervous system. The mechanism of calcineurin inhibitor-induced neurotoxicity remains poorly understood. The purpose of this study is to examine whether glycogen synthase-3 (GSK-3) is involved in calcineurin inhibitor-induced apoptosis. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage, nuclear condensation of fragmentation, and internucleosomal DNA fragmentation. Alsteropaullone and 1-azakenpaullone, GSK-3 inhibitors, prevented calcineurin inhibitor-induced apoptosis. In addition, insulin growth factor-I (IGF-I) and cycloheximide completely blocked cell death. Moreover, caspase-3 activation was accompanied by calcineurin inhibitor-induced cell death. These results suggest that calcineurin inhibitors induce caspase-dependent apoptosis and activation of GSK-3 is involved in cell death in rat cortical neurons.

Thapsigargin-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in PC12 cells.

Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases, including ischemia, excitotoxicity and Alzheimer's disease. Thapsigargin, which increases [Ca2+]i, induces apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by caspase-3 activation in PC12 cells. We examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors in PC12 cells. Cells treated with 0.1 microM thapsigargin for 24h shrank. The injured cells underwent chromatin condensation and nuclear fragmentation, indicating apoptotic cell death. We assayed the effects of selective GSK-3 inhibitors, SB216763, azakenpaullone and alsteropaullone on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. Alsterpaullone did not reduce the GRP78 protein expression induced by thapsigargin, suggesting that GSK-3 activation is not involved in induction of GRP78. In addition, GSK-3 inhibitors inhibited caspase-3 activation accompanied by thapsigargin-induced apoptosis. We showed in this report that thapsigargin-induced apoptosis is prevented by GSK-3 inhibitors, suggesting that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3 activation in PC12 cells.

Prevention of rat cortical neurons from prostaglandin E2-induced apoptosis by glycogen synthase kinase-3 inhibitors.

Cyclooxygenase-2 (COX-2) induction and prostaglandin E(2) (PGE(2)) elevation have been reported to occur after cerebral ischemic insult. PGE(2) induces apoptosis through the PGE(2) EP2 receptor by a cAMP-dependent pathway. Glycogen synthase kinase-3 (GSK-3) affects many fundamental cellular functions. We examined whether GSK-3 is involved in PGE(2)-induced cell death by using GSK-3 inhibitors in rat cultured cortical neurons. Cells treated with 12.5 microM PGE(2) for 2 days shrank. The injured cells underwent chromatin condensation and nuclear fragmentation detected by staining with Hoechst33258, indicating apoptotic cell death. We assayed the effects of selective GSK-3 inhibitors SB216763 and alsteropaullone on PGE(2)-induced apoptosis. These inhibitors completely protected the cells from apoptosis induced by PGE(2). Moreover, dibutyryl cAMP (a cell permeable cAMP)-induced apoptosis was also prevented by alsteropaullone. In addition, GSK-3 inhibitors inhibited caspase-3 activation accompanied by PGE(2)-induced apoptosis. We showed in this report that PGE(2)-induced apoptosis is prevented by GSK-3 inhibitors, suggesting that PGE(2) induces caspase-dependent apoptosis mediated through GSK-3 activation in rat cultured cortical neurons.

Ketamine-induced apoptosis in cultured rat cortical neurons.

Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cell death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons.

Prostaglandin E2 deteriorates N-methyl-D-aspartate receptor-mediated cytotoxicity possibly by activating EP2 receptors in cultured cortical neurons.

The activation of glutamate receptors, particularly N-methyl-D-aspartate (NMDA) receptors, initiates ischemic cascade in the early stages of cerebral ischemia. Postischemia, cerebral ischemia is also associated with an inflammatory reaction that contributes to tissue damage. The up-regulation of neuronal cyclooxygenase-2 (COX-2) and elevation of prostaglandin E2 (PGE2) have been reported to occur after cerebral ischemic insult. We therefore studied whether the COX-2 reaction product PGE2 affects glutamate receptor-mediated cell death in cultured rat cortical cells. PGE2 was found to augment NMDA-mediated cell death. The transcription of EP1, EP2, EP3 and EP4 PGE2 receptor genes was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). EP1, EP2 and EP3 receptor genes were found in cortical cells. Butaprost (an EP2 agonist) markedly enhanced NMDA-mediated cell death, whereas 17-phenyl trinor-PGE2 (an EP1 agonist) and sulprostone (an EP3 agonist) had little effect. Both PGE2 and butaprost elevated cAMP intracellular levels in the cortical cells; moreover, forskolin, an activator of adenylate cyclase, enhanced NMDA-mediated cell death. These results suggest that PGE2, acting via EP2 receptors, aggravates excitotoxic neurodegeneration by a cAMP-dependent mechanism.

Glutathione depletion promotes aluminum-mediated cell death of PC12 cells.

Exposure of rat phenochromocytoma cells (PC12 cells) to aluminum maltolate complex, Al(maltol)3, induced a decrease in intracellular glutathione (GSH) concentration, resulting in a facilitated release of lactate dehydrogenase (LDH) from the cell and an increase in trypan blue-stained cells. Similar phenomena were observed as the cells were treated with L-buthione-[S,R]-sulfoximine (BSO) in the presence of Al(maltol)3. On the other hand, treatment of PC 12 cells with BSO alone in the absence of Al(maltol)3 did not affect the cell viability. Pre-treatment of PC12 cells with N-acetylcysteine (NAC) for 30 min before a 48 h-exposure to Al(maltol)3 effectively protected the cells from Al(maltol)3 toxicity by increasing intracellular GSH concentration. NAC also effectively inhibited reactive oxygen species (ROS) generation induced by treatment of the cells with Al(maltol)3. However, several lipophilic radical scavengers such as alpha-tocopherol and 3(2)-tert-butyl-4-hydroxyanisole, and an iron chelator, desferrioxamine, did not prevent Al(maltol)3-mediated ROS production or the decrease of cell viability. Based on these results, we discussed the role of intracellular GSH against the onset of aluminum toxicity in the context of ROS production.

Glycogen synthase kinase-3 inhibitors prevent caspase-dependent apoptosis induced by ethanol in cultured rat cortical neurons.

The effect of ethanol on cell viability was examined in rat cultured cortical neurons. Ethanol induced apoptosis, which was characterized by cell shrinkage, nuclear condensation or fragmentation and internucleosomal DNA fragmentation. Ethanol-induced apoptosis was prevented by N-methyl-d-aspartate (NMDA), an agonist of the NMDA receptor, which is a subtype of ionotropic glutamate receptors. Incubation with glycogen synthase kinase-3 (GSK-3) inhibitors 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) and alsteropaullone, but not a cyclin-dependent protein kinase 5 inhibitor roscovitine, completely protected the neurons from ethanol-induced apoptosis. Apoptosis was accompanied by the activation of caspase-3 and prevented by a caspase-3 inhibitor. These results suggest that ethanol induces caspase-dependent apoptosis mediated by glycogen synthase kinase-3 activation in cultured rat cortical neurons.

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.

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.

Increase in molecular rigidity of the protein conformation of brain Na+-K+-ATPase by modification with 4-hydroxy-2-nonenal.

The effect of lipid peroxidation product 4-hydroxy-2-nonenal (HNE) on the protein conformation of porcine cerebral cortex Na(+)-K(+)-ATPase was examined in term of the intrinsic tryptophanyl fluorescence measurement. Treatment of ATPase with HNE resulted in a decrease in the fluorescence intensity and an increase in the fluorescence anisotropy in a concentration-dependent manner. The difference in the fluorescence intensity and fluorescence anisotropy observed between the control and HNE-modified ATPase completely disappeared after treatment of the protein with guanidine hydrochloride (1 M). These results suggest that HNE-modification of the Na(+)-K(+)-ATPase induces alterations in the conformation of the enzyme molecule. This interpretation was further supported by a decrease in fluorescence quenching efficiency with acrylamide and sulfhydryl (SH) content. The decrease in quenching efficiency suggests that the proximity of the quencher molecule to the fluorophores located in the enzyme is suppressed. Modification of the enzyme with N-ethylmaleimide (NEM) also resulted in a decrease in quenching efficiency with the loss of SH groups. Furthermore, a good relationship between the SH content and these fluorescence parameters (fluorescence anisotropy and quenching efficiency) were observed. On the other hand, treatment of the Na(+)-K(+)-ATPase with other aldehydes such as malondialdehyde (MDA), 1-hexanal and nonanal did not affect either the quenching efficiency or SH content. Based on these results, the possibility of alterations in the physical properties of the Na(+)-K(+)-ATPase associated with modification by HNE has been discussed.

Contribution of lipid dynamics on the inhibition of bovine brain synaptosomal Na+-K+-ATPase activity induced by 4-hydroxy-2-nonenal.

The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na(+)-K(+)-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na+-K+-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca2+- and Mg2+-ATPase activities. The results of the kinetic analysis of the Na+-K+-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na+-K+-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na+-K+-ATPase activity associated with alterations in the membrane lipid organization.

Nerve growth factor protects against aluminum-mediated cell death.

In the present study, we examined the effect of two salts of aluminum (Al), aluminum maltolate (Almal) and aluminum chloride (AlCl(3)), on the cell viability of PC12 cells in the absence and presence of nerve growth factor (NGF). A 72-h exposure of PC12 cells to Almal (300 microM) resulted in a marked increase of lactic dehydrogenase (LDH) release from the cells and a decrease of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) activity. These results indicate that Almal induces a decrease in the cell viability. Under the same conditions, Almal also caused DNA ladder formation and chromatin condensation. In contrast, AlCl(3) did not showed an increased LDH release and a decreased MTT activity in the concentration range of the salt tested (0.1-1 mM). The extent of LDH release and MTT activity decrease induced by Almal treatment closely depended on the amount of Almal incorporated into the cells. An increase in the fluorescence intensity of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) which was loaded into the cell by Almal treatment and its prevention by pyrrolodine dithiocarbamate, a potent antioxidant, suggested that Almal-induced cell death partly proceeds via reactive oxygen species (ROS) production. NGF effectively inhibited the increase of LDH release and the decrease of MTT activity, as well as DNA fragmentation and chromatin condensation. However, NGF did not inhibit the increase of C-DCDHF-DA fluorescence in the cells induced by Almal treatment. From these results, it is suggested that ROS production associated with accumulation of Al is one possible important factor in the onset of Al neurotoxicity via apoptotic cell death and that NGF protects against cell degeneration associated with Al accumulation, but independently of ROS production.

The role of Fe3+ on Fe2+-dependent lipid peroxidation in phospholipid liposomes.

Fe2+-dependent lipid peroxidation in phosphatidylcholine (PC) liposomes, assessed by thiobarbituric acid-reactive substances (TBARS) production, was stimulated in the presence of Fe3+ in a concentration-dependent manner. The rates of nitroblue tetrazolium (NBT) reduction and Fe2+ oxidation (Fe2+ disappearance and Fe3+ formation) were also enhanced by the addition of Fe3+ to the reaction mixture, and there is a good linear relationship between these parameters. These results suggest that the facilitation of reactive oxygen species (ROS) production via Fe2+ oxidation is closely related to the onset of the stimulatory effect of Fe3+ on Fe2+-dependent lipid peroxidation. On the other hand, results using the liposomes containing various concentrations of endogenous lipid hydroperoxides (LOOH) indicated that endogenous LOOH is not directly involved in the onset of the Fe3+ stimulatory effect on Fe2+-dependent TBARS production and ROS production. This hypothesis was further confirmed by the evidence that Fe2+-dependent ROS production and Fe2+ oxidation of dipalmitoylphosphatidylcholine liposomes were also stimulated by the addition of Fe3+. The results with several antioxidants and radical scavengers suggested that ROS related to Fe2+-dependent lipid peroxidation and its stimulation by Fe3+ are ferrous-oxygen complexes rather than superoxide anion, hydrogen peroxide and hydroxyl radicals. Based on these results, we proposed a possible mechanism for the onset of the Fe3+ stimulation in Fe2+-dependent lipid peroxidation.

Prostaglandin E(2) 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.