Human platelet aggregation induced by prostaglandin endodisulfide

The effect of human platelet functions of 9,11-dithio analogues of prostaglandin endoperoxide was investigated. Methyl (5z,9α,11α,13e,15S)-9,11-epidithio-15-hydroxyprosta- 5,13-dienoate induced platelet aggregation, while the 9β,11β-epimer was inactive. The platelet aggregation caused by the 9α,11α-dithio analogue was associated with serotonin release from platelets, and was inhibited by methyl ester of prostaglandin I₂ (prostacyclin) but not by indomethacin.     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

1-Carboxyalkylimidazoles inhibited the conversion of prostaglandin H₂ to thromboxane B₂ and 12L-hydroxy-5, 8, 10- heptadecatrienoic acid by a partially purified enzyme (prostaglandin endoperoxide thromboxane isomerase) from bovine platelet microsomes. The degree of the inhibition was dependent on the length of carboxyalkyl chain. 1-Carboxyheptylimidazole was the most potent inhibitor, and an almost complete inhibition was obtained at a concentration on the order of 1 μM. The inhibition, as examined with 1-carboxyheptylimidazole, was of noncompetitive type. These 1-carboxyalkylimidazoles did not affect the formation of prostaglandin H₂ from arachidonic acid. Such a selective inhibition was also demonstrated by the reaction of bovine platelet microsomes with arachidonic acid in the presence of 1-carboxyheptylimidazole, resulting in the accumulation of prostaglandin H₂ as an intermediate. Furthermore, a series of 1-alkylimidazoles with no carboxyl group also inhibited the isomerase at higher concentrations. However, the inhibition was not specific for the isomerase; namely, the prostaglandin H₂ formation from arachidonic acid was also affected.     

Heat Shock Protein 70.1 (Hsp70.1) Affects Neuronal Cell Fate by Regulating Lysosomal Acid Sphingomyelinase

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion.     

Why are hippocampal CA1 neurons vulnerable but motor cortex neurons resistant to transient ischemia?

It is well-known that heat-shock protein 70.1 (Hsp70.1), a major protein of the human Hsp70 family, plays cytoprotective roles by both its chaperone function and stabilization of lysosomal membranes. Recently, we found that calpain-mediated cleavage of carbonylated Hsp70.1 in the hippocampal cornu Ammonis1 (CA1) contributes to neuronal death after transient global ischemia. This study aims to elucidate the differential neuronal vulnerability between the motor cortex and CA1 sector against ischemia/reperfusion. Fluoro-Jade B staining and terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labeling analysis of the monkey brain undergoing 20min whole brain ischemia followed by reperfusion, showed that the motor cortex is significantly resistant to the ischemic insult compared with CA1. Up-regulation of Hsp70.1 but absence of its cleavage by calpain facilitated its binding with NF-κB p65/IκBα complex to minimize NF-κB p65 activation, which contributed to a neuroprotection in the motor cortex. In contrast, because activated μ-calpain cleaved carbonylated Hsp70.1 in CA1, the resultant Hsp70.1 dysfunction not only destabilized lysosomal membrane but also induced a sustained activation of NF-κB p65, both of which resulted in delayed neuronal death. We propose that the cascades underlying lysosomal stabilization and regulating NF-κB activation by Hsp70.1 may influence neuronal survival/death after the ischemia/reperfusion.     

Low density lipoprotein receptor-related protein-mediated membrane translocation of 12/15-lipoxygenase is required for oxidation of low density lipoprotein by macrophages

Oxidation of low density lipoprotein (LDL) is the key step for the development of atherosclerosis. The 12/15-lipoxygenase expressed in macrophages is capable of oxygenating linoleic acid esterified to cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDL oxidation. We recently reported that LDL receptor-related protein (LRP) was required for the enzyme-mediated LDL oxidation by macrophages and suggested the selective uptake of cholesterol ester from LDL to the plasma membrane (Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454-36459). To elucidate precise mechanisms of lipoxygenase-mediated LDL oxidation, we investigated the intracellular localization of 12/15-lipoxygenase. The 12/15-lipoxygenase was predominantly detected in cytosol of resting peritoneal macrophages and of macrophage-like J774A.1 cells permanently transfected with the cDNA for the enzyme. When the cells were treated with LDL and subjected to subcellular fractionation, the 12/15-lipoxygenase was detected in the membranes with a concomitant decrease in cytosol as shown by Western blot analysis. The levels of the enzyme associated with the membrane reached maximum in 15 min after LDL addition and then decreased. However, the enzymatic activity of 12/15-lipoxygenase in the membrane fraction was very weak even after LDL treatment. This fact supports the suicide inactivation of the enzyme by the oxygenation of cholesterol ester transferred from the LDL particle to the plasma membrane. Immunohistochemical analysis using an antibody against 12/15-lipoxygenase revealed that the plasma membrane was the major site of the enzyme translocation by the LDL treatment. LDL-dependent 12/15-lipoxygenase translocation was inhibited by a blocking antibody against LRP. Furthermore, an enzyme translocation inhibitor, L655238, inhibited the LDL oxidation caused by the 12/15-lipoxygenase. We propose that cholesterol ester selectively transferred from the LDL particle to the plasma membrane via LRP is oxygenated by 12/15-lipoxygenase translocated to this membrane.     

Protein tyrosine phosphatase PTP20 induces actin cytoskeleton reorganization by dephosphorylating p190 RhoGAP in rat ovarian granulosa cells stimulated with follicle-stimulating hormone

We identified 25 protein tyrosine phosphatases (PTPs) expressed in rat ovarian granulosa cells. Of these PTPs, the expression levels of at least PTP20, PTP-MEG1, PTPepsilonM, and PTPepsilonC significantly changed during the estrous cycle. We examined the cellular functions of PTP20 in granulosa cells by expressing the wild type, a catalytically inactive CS mutant in which Cys229 of PTP20 was changed to Ser, or a substrate-trapping DA mutant in which Asp197 was mutated to Ala, using an adenovirus vector. Overexpression of the wild type, but not of the CS mutant, induced retraction of the cell body with the extension of long, dendritic-like processes after stimulation with FSH, a critical factor for the survival and differentiation of these cells. In addition, cell adhesion to the substratum decreased in an FSH-dependent manner. Inhibiting Rho GTPase activity with C3 botulinum toxin caused similar morphological changes. The FSH-enhanced phosphotyrosine (p-Tyr) level of p190 RhoGAP was selectively reduced by the overexpressed wild type, but not by mutated PTP20. Although p190 RhoGAP is tyrosine phosphorylated by c-Src via the tyrosine kinase Pyk2, wild-type PTP20 had little effect on p-Tyr418 of c-Src and no effect on p-Tyr402 of Pyk2, which are required for full c-Src activity and for interacting between Pyk2 and c-Src, respectively. The CS and DA mutants as well as the wild type reduced the formation of p190 RhoGAP-p120 RasGAP complexes. Confocal microscopy analysis revealed that PTP20 intracellularly colocalizes with p190 RhoGAP. These results demonstrate that PTP20 regulates the functions of granulosa cells in an FSH-dependent manner by dephosphorylating p190 RhoGAP and subsequently inducing reorganization of the actin cytoskeleton. Moreover, our data suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.     

Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation

Enhanced endoplasmic reticulum (ER) stress has been implicated in various pathological situations including inflammation. During a search for compounds that regulate ER stress, we identified vaticanol B, a tetramer of resveratrol, as an agent that protects against ER stress-induced cell death. Vaticanol B suppressed the induction of unfolded protein response-targeted genes such as glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP) after cells were treated with ER stressors. Analysis in the mouse macrophage cell line RAW 264.7 revealed that vaticanol B also possesses a strong anti-inflammatory activity. Production of a variety of inflammatory modulators such as tumor necrosis factor-, nitric oxide, and prostaglandin E₂ was inhibited by vaticanol B to a much greater extent than by monomeric or dimeric resveratrol after exposure of cells to lipopolysaccharide. Further investigations to determine the common mechanisms underlying the regulation of ER stress and inflammation by vaticanol B disclosed an important role for vaticanol B in regulation of basic gene expression and in prevention of the protein leakage from the ER into the cytosol in both conditions. These results suggest that vaticanol B is a novel anti-inflammatory agent that improves the ER environment by reducing the protein load on the ER and by maintaining the membrane integrity of the ER. gene expression; membrane integrity     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Synthesis of 8,9-leukotriene A4 by murine 8-lipoxygenase

Arachidonate 8-lipoxygenase was identified in phorbol ester induced mouse skin. We expressed the enzyme in an Escherichia coli system using pET-15b carrying an N-terminal histidine-tag sequence. The enzyme, purified by nickel-nitrilotriacetate affinity chromatography, showed specific activity of about 0.1 micromol/min/mg of protein with arachidonic acid as a substrate. When metabolites of arachidonic acid were reduced and analyzed by reverse-phase HPLC, 8-hydroxy derivative was a major product as measured by absorbance at 235 nm. In addition, three polar compounds (I, II, and III) were detected by measuring absorbance at 270 nm. These compounds were also produced when the enzyme was incubated with 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. Neither heat-inactivated enzyme nor mutated enzyme produced these compounds, suggesting that they are enzymatically generated. Ultraviolet spectra of these compounds showed typical triplet peaks around 270 nm, indicating that they have a triene structure. Molecular weight of these compounds was determined to be 336 by liquid chromatography-mass spectrometry, indicating that they carry two hydroxyl groups. Compounds I and III were generated even under anaerobic condition, indicating that oxygenation reaction was not required for their generation from 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. By analogy to the reactions of 5-lipoxygenase pathway where leukotriene A4 is generated, it is suggested that 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid is converted by the 8-lipoxygenase to 8,9-epoxyeicosa-5,10,12,14-tetraenoic acid which degrades to compounds I and III by non-enzymatic reaction. In contrast, compound II was not generated under anaerobic condition, indicating that it was produced by oxygenation reaction. Taken together, 8-lipoxygenase catalyzes both dehydration reaction to yield 8,9-epoxy derivative and oxygenation reaction presumably at 15-position of 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid.