Takao Ohyashiki

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.

Effect of lipid peroxidation on membrane-bound Ca2+-ATPase activity of the intestinal brush-border membranes

We have studied lipid peroxidation and Ca2+-ATPase activity of the porcine intestinal brush-border membranes using a oxygen-radical-generating system consisting of dithiothreitol (DTT)/Fe2+ and tert-butyl hydroperoxide (t-BuOOH). The rates of lipid peroxidation were measured by formation of thiobarbituric acid-reactive substances (TBAR) and conjugated diene. Incubation of the membranes with DTT/Fe2+ in the absence and presence of t-BuOOH resulted in a slight (about 20%) and a marked (about 50%) inhibition of Ca2+-ATPase activity, respectively. The degree of inhibition was dependent on the hydroperoxide concentration. Addition of thiourea effectively protected Ca2+-ATPase activity but catalase and superoxide dismutase showed a slight and no effect on protection of the ATPase activity, respectively. Results of kinetic studies on the ATPase activity with varying ATP and Ca2+ concentrations revealed that the decrease in the enzyme activity by treatment with these oxidizing agents is mainly due to decrease of the Vmax value. Modification of SH groups in the membrane proteins by thiol group reagents such as N-ethylmaleimide, monoiodoacetate and monoiodacetamide did not induce the inhibition of Ca2+-ATPase activity. From these results, it is suggested that inhibition of the ATPase activity of the membranes by treatment with DTT/Fe2+ in the presence and absence of t-BuOOH is dependent on lipid peroxidation and that oxidative modification of SH groups may not be directly involved to the loss of the ATPase activity. In addition, results of the fluorescence anisotropy measurements of pyrene-labeled membranes suggested that change in the Ca2+-ATPase activity is partly related to a decrease in the membrane lipid fluidity.

Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran

Utilization of a fluorescence dye, 1,3-diphenylisobenzofuran (DPBF) as a detector of superoxide anion radical (O2*-) was examined. The fluorescence intensity of DPBF incorporated in phospholipid liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) is effectively quenched by incubation with xanthine/xanthine oxidase system. On the other hand, xanthine or xanthine oxidase alone did not induce quenching of the DPBF fluorescence in the liposomes. Xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes was almost completely protected by the addition of superoxide dismutase (SOD, 1 U/ml), but not by heat-denatured SOD (10 min boiling) at the same concentration. On the other hand, catalase (1 U/ml), and hydroxyl radical and singlet oxygen scavengers (10 mM sodium benzoate, 300 mM mannitol, 1 mM tryptophan and 1 mM sodium azide) did not protect xanthine/xanthine oxidase-induced fluorescence quenching of DPBF-labeled liposomes. The concentration dependence profiles of xanthine oxidase on the DPBF fluorescence quenching and O2*- generation showed that there is a good correlation between these parameters. Under the present experimental conditions, approximately 7 microM H(2)O(2)/30 min were produced, but the addition of H(2)O(2) (1 mM) to DPBF-labeled liposomes did not quench the dye fluorescence in the liposomes. Temperature dependence profiles of the DPBF fluorescence quenching induced by xanthine/xanthine oxidase treatment and the excimer fluorescence formation of pyrene molecules embedded in the liposomal membrane suggested that the quenching efficiency of the DPBF fluorescence is largely dependent on their lipid dynamics. Based on these results, we proposed the possibility that DPBF fluorescence quenching method is able to be used as a simple method for detecting O2*- inside the membrane lipid layer and that DPBF fluorescence quenching by O2*- is controlled by the physical state of membrane lipids.

A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives

The effect of lipid peroxidation on the lipid fluidity of porcine intestinal brush-border membranes was examined by measuring the rotational mobility and the accessibility to fluorescence quenchers (CH3COOT1, CuSO4 and KI) of pyrene or n-(9-anthroyloxy)stearic acid (n = 2 or 12) in the membranes. The harmonic mean of the rotational relaxation times of pyrene increased and the rate constants, kq, of the quenching reaction of pyrene and 2-(9-anthroyloxy)stearic acid incorporated in the membrane lipids decreased upon lipid peroxidation, indicating reduction of the lipid fluidity of the membranes by lipid peroxidation. In addition, the kq value of the reaction of 2-(9-anthroyloxy)stearic acid in the membranes with Cu2+ decreased in proportion to the amount of the products of lipid peroxidation. On the other hand, the kq value of the reaction of 12-(9-anthroyloxy)stearic acid with Cu2+ or I- was unaffected by lipid peroxidation. Based on these results, a localized change in the lipid fluidity of the membranes in association with lipid peroxidation has been discussed.

Increase of the molecular rigidity of the protein conformation in the intestinal brush-border membranes by lipid peroxidation

The effect of lipid peroxidation on the protein conformation of the porcine intestinal brush-border membranes was studied using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM). By a kinetic analysis of the reaction of the membranes with DACM, it was shown that the reaction rate of the SH groups (SHf) of the membrane proteins, whose reaction with the dye is very fast, decreases in proportion to the extent of thiobarbituric acid-reactive substance formation. The difference in the rate of the reaction of the SHf groups for DACM between the control and peroxidized membranes completely disappeared after denaturation of the proteins by treatment with guanidine hydrochloride. The reaction of DACM with the SHf groups of the control membranes accelerated when the temperature was increased with an apparent transition temperature between 25 degrees C and 30 degrees C. On the other hand, no transition was observed in the peroxidized membranes over the temperature range 20-43 degrees C. These results suggest that the conformation around the SHf groups of the proteins in the peroxidized membranes is apparently different from that in the control membranes. A modification of the conformation around the SH groups in the membrane proteins associated with lipid peroxidation was further demonstrated by finding that the quenching efficiency of the fluorescence of the DACM-labeled membranes by Tl+ was markedly decreased after lipid peroxidation. Based on these results, changes in the protein conformation of the porcine intestinal brush-border membranes by lipid peroxidation are discussed.

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.

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

Abstract The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μ M) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe 2+ . Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μ s after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl + . Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.

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.