Involvement of phospholipase A2 and lipoxygenase in lipopolysaccharide-induced inducible nitric oxide synthase expression in glial cells

The present study underlines the importance of phospholipase A2 (PLA2)- and lipoxygenase (LO)-mediated signaling processes in the regulation of inducible nitric oxide synthase (iNOS) gene expression. In glial cells, lipopolysaccharide (LPS) induced the activities of PLA2 (calcium-independent PLA2; iPLA2 and cytosolic PLA2; cPLA2) as well as gene expression of iNOS. The inhibition of cPLA2 by methyl arachidonyl fluorophosphates (MAFP) or antisense oligomer against cPLA2 and inhibition of iPLA2 by bromoenol lactone reduced the LPS-induced iNOS gene expression and NFkappaB activation. In addition, the inhibition of LO by nordihydroguaiaretic acid (NDGA; general LO inhibitor) or MK886 (5-LO inhibitor), but not baicalein (12-LO inhibitor), completely abrogated the LPS-induced iNOS expression. Because NDGA could abrogate the LPS-induced activation of NFkappaB, while MK886 had no effect on it, LO-mediated inhibition of iNOS gene induction by LPS may involve an NFkappaB-dependent or -independent (by 5-LO) pathway. In contrast to LO, however, the cyclooxygenase (COX) may not be involved in the regulation of LPS-mediated induction of iNOS gene because COX inhibition by indomethacin (general COX inhibitor), SC560 (COX-1 inhibitor), and NS398 (COX-2 inhibitor) affected neither the LPS-induced iNOS expression nor activation of NFkappaB. These results indicate a role for cPLA2 and iPLA2 in LPS-mediated iNOS gene induction in glial cells and the involvement of LO in these reactions.     

Blockade of cytosolic phospholipase A2 alpha prevents experimental autoimmune encephalomyelitis and diminishes development of Th1 and Th17 responses

Cytosolic phospholipase A2 alpha (cPLA2 alpha) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2 alpha, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2 alpha prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2 alpha from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2 alpha represents a potential therapeutic target for treatment of MS.     

Chronic lithium downregulates cyclooxygenase-2 activity and prostaglandin E(2) concentration in rat brain

Rats treated with lithium chloride for 6 weeks have been reported to demonstrate reduced turnover of arachidonic acid (AA) in brain phospholipids, and decreases in mRNA and protein levels, and enzyme activity, of AA-selective cytosolic phospholipase A(2)(cPLA(2)). We now report that chronic lithium administration to rats significantly reduced the brain protein level and enzyme activity of cyclooxygenase-2 (COX-2), without affecting COX-2 mRNA. Lithium also reduced the brain concentration of prostaglandin E(2) (PGE(2)), a bioactive product of AA formed via the COX reaction. COX-1 and the Ca(2+)-independent iPLA(2) (type VI) were unaffected by lithium. These and prior results indicate that lithium targets a part of the AA cascade that involves cPLA(2) and COX-2. This effect may contribute to lithium's therapeutic action in bipolar disorder.     

Regulation of the cyclooxygenase-2 system by interleukin-1beta through mitogen-activated protein kinase signaling pathways: a comparative study of human neuroglioma and neuroblastoma cells

Glial activation and inflammation following brain injury may initiate and maintain the process of neurodegeneration. Both glia and neurons synthesize proinflammatory mediators such as interleukin 1 beta (IL-1beta), cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and prostaglandins. The molecular mechanisms by which IL-1beta regulates inflammatory genes such as cPLA2 and COX-2 in glial and neuronal cells are poorly understood. We have studied IL-1beta-mediated gene regulation in an established glial and neuronal human cell lines. We report that IL-1beta induced cPLA2 and COX-2 mRNA and protein expression and subsequent prostaglandin E2 (PGE2) release in a time-dependent manner in H4 neuroglioma cells. Both SB203580 and PD98059 [p38 and p42/44 mitogen-activated protein kinase (MAPKs) inhibitors, respectively] reduced IL-1beta-induced PGE2 production, while only SB203580 reduced both cPLA2 and COX-2 expression. Similarly, in SKNSH neuroblastoma cells, both SB203580 and PD98059 reduced IL-1beta-induced PGE2 release, with no detectable COX-2 and cPLA2 protein expression in these cells. Our results indicate that the signaling mechanisms of p38 and p42/44 MAPKs play a role in IL-1beta-mediated PGE2 release in both of these cell lines, with differences upstream at the level of cPLA(2)/COX-2 expression. IL-1beta-induced cPLA2 and COX-2 gene expression is modulated through the p38 MAPK pathway in both neuroglioma and neuroblastoma cells. Understanding the signaling mechanisms involved in IL-1beta-mediated inflammatory processes in both glia and neuronal cells may provide potential targets for therapeutic intervention for neurological disorders.     

Eicosanoid levels in the neocortex of drug-resistant epileptic patients submitted to epilepsy surgery

There is an increasing body of evidence implicating eicosanoids (arachidonic acid metabolites) in the experimental generation of epileptic seizures and the development of epilepsy. Our purpose was to measure the synthesis of eicosanoids from the cyclooxygenase and lipoxygenase pathways in human brain neocortex tissue samples obtained from epileptic patients, and to compare them with non-epileptic control subjects. Epileptic neocortex specimens demonstrated a significant increase (P<0.001) in the levels of three eicosanoids derived from the cyclooxygenase pathway: Prostaglandin E(2) (PGE(2)), Thromboxane A(2) (TXA(2)), and Prostacyclin (PGI(2)), compared to controls. In the epileptic samples the level of TXA(2) was twice as much the levels of PGI(2), while in the control samples the levels of PGI(2) were slightly higher than TXA(2). Conversely, there were no detectable levels of eicosanoids derived from the lipoxygenase pathway: Leukotriene B(4) (LTB(4)) and Leukotriene C(4) (LTC(4)). The lack of leukotrienes synthesis illustrates that COX pathway is dominant in neocortex of epileptic patients. Our human data are consistent with the results obtained in experimental animal models of epilepsy. The important increase in PGE(2) and TXA(2) suggests that selective inhibition of prostanoid synthesis or blockage of prostanoid receptors might provide novel antiepileptic strategies in human epilepsy.     

Effects of aminoguanidine and cyclooxygenase inhibitors on nitric oxide and prostaglandin production, and nitric oxide synthase and cyclooxygenase expression induced by lipopolysaccharide in the estrogenized rat uterus

BACKGROUND/OBJECTIVE: The aim of our study was first to investigate if there exists an interaction between nitric oxide (NO) and prostaglandin (PG) generation in the estrogenized rat uterus challenged by lipopolysaccharide (LPS), and, secondly, which isoforms of nitric oxide synthase (NOS) and cyclooxygenase (COX) participate in this process. METHODS: To study the effect of LPS and to characterize the isoenzymes involved in the process, specific inhibitors of iNOS (aminoguanidine) and COX-II (meloxicam, nimesulide) and non-specific of COX (indomethacin) were injected intraperitoneally to determine their effect on NO and PG production, and on NOS and COX expression induced by LPS in estrogenized rat uterus. NO production was measured by arginine-citrulline conversion assay and PGE(2)/PGF(2alpha,)by radioconversion. Enzyme expression was evaluated by Western blot analysis. RESULTS: The present work shows that iNOS inhibitor, aminoguanidine, reduced NO and PGE(2)/PGF(2alpha) production induced by LPS injection. Aminoguanidine exerts its effect over the PG metabolism by inhibiting COX-II activity and expression. On the other hand, both indomethacin, a non-selective PG inhibitor, and meloxicam, a COX-II inhibitor, stimulated NO production and reduced PGE(2)/PGF(2alpha) generation. Indomethacin also reduced COX-II and iNOS expression. CONCLUSION: These results indicate that in the estrogenized rat uterus challenged with LPS, PG and NO interact affecting each other's metabolic pathways. The above findings indicate that the interaction between NOS and COX might be important in the regulation of physiopathologic events during pregnancy.     

The effect of chronic lithium on arachidonic acid release and metabolism in rat brain does not involve secretory phospholipase A2 or lipoxygenase/cytochrome P450 pathways

The mood-stabilizer lithium, when chronically administered to rats at therapeutic concentrations, has been shown to downregulate brain arachidonic acid (AA) turnover and total phospholipase A2 (PLA2) activity, as well as protein and mRNA levels of cytosolic cPLA2. These effects are accompanied by a decrease in cyclooxygenase (COX)-2 protein level, COX activity, and brain prostaglandin E2 (PGE2) concentration. The involvement of Ca2+-dependent secretory PLA2 (sPLA2) in the mechanism of action of lithium has not been investigated. The purpose of this study was to examine, whether the effect of lithium is selectively directed to cPLA2 or it also affects sPLA2 protein and enzyme activity and whether other AA metabolizing enzymes (5-lipoxygenase and cytochrome P450 epoxygenase) were also altered. Furthermore, to determine if the reduction of brain PGE2 concentration was due only to downregulation of COX-2 protein or if it also involves the terminal PGE synthase, we determined brain microsomal PGE synthase protein level. Male Fischer-344 rats were fed lithium chloride for 6 weeks, whereas, control rats were fed lithium-free chow under parallel conditions. We found that chronic lithium did not significantly change sPLA2 activity or protein level. 5-Lipoxygenase and cytochrome P450 epoxygenase protein levels were unchanged, as were levels of the terminal PGE synthase. These results indicate that the effect of lithium selectively involves the cPLA2/COX-2 pathway, which might be responsible for the therapeutic effect in bipolar disorder.     

The role of cyclo-oxygenase 1 and 2 activity in prostaglandin E(2) secretion by cultured human adult microglia: implications for Alzheimer's disease

Microglial cyclo-oxygenase (COX) expression is considered to be important in the pathogenesis of Alzheimer's disease (AD) and, therefore, constitutes a key target for therapeutic intervention. We investigated the influence of AD plaque associated factors on COX-1 and COX-2 expression and activity in adult human microglial cells in vitro. COX-2 immunoreactivity and mRNA were induced by lipopolysaccharide (LPS), not by AD plaque associated cytokines interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, or amyloid (A)beta(1-42). To assess functional COX activity, the release of PGE(2) into the culture medium was determined. LPS and also arachidonic acid (AA) dose-dependently stimulated PGE(2) release. The effects of AA are independent from induction of COX mRNA expression, or of de novo protein synthesis. No effects of either plaque-associated cytokines or Abeta(1-42) on PGE(2) secretion were seen, even when cells were co-stimulated with AA, to provide enough substrate. COX isotype selective inhibitors were used to discern relative contributions of COX-1 and COX-2 activities to microglial PGE(2) secretion. COX-2 and in part COX-1-selective inhibitors inhibited LPS-induced PGE(2) secretion, whereas the AA-induced PGE(2) secretion was reduced by COX-1-selective inhibitors only. Apparently, adult human microglia in vitro (1) constitutively express COX-1, and (2) do not express COX-2 upon exposure to either Abeta or plaque associated cytokines. In the light of microglial COX activity as a potential therapeutical target in AD, the data presented in this study suggest that classical NSAIDs, rather than selective COX-2 inhibitors, are more potent in reducing microglial prostaglandin secretion.     

Characterization of a delayed rectifier potassium channel in the slowly adapting stretch receptor neuron of crayfish

Prostaglandins, which are cyclooxygenase (COX) products, are pathologically up-regulated, and have been proven to be closely associated with neuronal death. In this study, we investigated a role of COX isoforms (COX-1 and COX-2) in kainic acid-induced neuronal death in cultured murine cortical or hippocampal neurons. In primary cortical neurons, both indomethacin (COX-1/-2 nonselective inhibitor) and aspirin (COX-1 preferential inhibitor) reduced basal and kainic acid-induced PGE(2) production significantly and prevented neuronal cell death after kainic acid treatment. In contrast, NS398 (COX-2 selective inhibitor) had no effect on kainic acid-induced neuronal cell death. In hippocampal neurons, however, COX-2 inhibitors prevented both kainic acid-induced neuronal death and PGE(2) production. COX-2 expression was remarkably up-regulated by kainic acid in hippocampal neurons; whereas in cortical neurons, COX-2 expression was comparatively less significant. Astrocytes were unresponsive to kainic acid in terms of PGE(2) production and cell death. In conclusion, we suggest that the release of PGE(2) induced by kainic acid occurred through COX-1 activity rather than COX-2 in cortical neurons. The inhibition of PGE(2) release by COX-1 inhibitors prevented kainic acid-induced cortical neuronal death, while in the hippocampal neurons, COX-2 inhibitors prevented kainic acid-induced PGE(2) release and hippocampal neuronal death.     

Reactive astrocyte COX2‐PGE2 production inhibits oligodendrocyte maturation in neonatal white matter injury

Inflammation is a major risk factor for neonatal white matter injury (NWMI), which is associated with later development of cerebral palsy. Although recent studies have demonstrated maturation arrest of oligodendrocyte progenitor cells (OPCs) in NWMI, the identity of inflammatory mediators with direct effects on OPCs has been unclear. Here, we investigated downstream effects of pro‐inflammatory IL‐1β to induce cyclooxygenase‐2 (COX2) and prostaglandin E2 (PGE2) production in white matter. First, we assessed COX2 expression in human fetal brain and term neonatal brain affected by hypoxic‐ischemic encephalopathy (HIE). In the developing human brain, COX2 was expressed in radial glia, microglia, and endothelial cells. In human term neonatal HIE cases with subcortical WMI, COX2 was strongly induced in reactive astrocytes with “A2” reactivity. Next, we show that OPCs express the EP1 receptor for PGE2, and PGE2 acts directly on OPCs to block maturation in vitro. Pharmacologic blockade with EP1‐specific inhibitors (ONO‐8711, SC‐51089), or genetic deficiency of EP1 attenuated effects of PGE2. In an IL‐1β‐induced model of NWMI, astrocytes also exhibit “A2” reactivity and induce COX2. Furthermore, in vivo inhibition of COX2 with Nimesulide rescues hypomyelination and behavioral impairment. These findings suggest that neonatal white matter astrocytes can develop “A2” reactivity that contributes to OPC maturation arrest in NWMI through induction of COX2‐PGE2 signaling, a pathway that can be targeted for neonatal neuroprotection.     

Cytosolic phospholipase A alpha modulates NMDA neurotoxicity in mouse hippocampal cultures

The arachidonic acid-specific cytosolic phospholipase A(2) alpha (cPLA(2)alpha) has been implicated in the generation of neurological injuries. cPLA(2)alpha-dependent neurological injury has been postulated to be mediated through inflammatory and eicosanoid pathways. We determined if cPLA(2)alpha amplifies the injury of a non-inflammatory, excitotoxic stimulus by modifying a well-described toxicity assay to measure the toxicity of N-methyl-d-aspartate (NMDA) in the CA1 region of organotypic, mouse hippocampal cultures. Hippocampal cultures from wild-type and cPLA(2)alpha knockout mice were exposed to 5, 7.5 or 10 microm NMDA for 1 h. Toxicity was measured 23 h later. Cultures derived from cPLA(2)alpha(-/-) mice and cultures treated with the selective inhibitor AACOCF(3) were significantly protected from NMDA toxicity, as compared with wild-type cultures. To determine if cPLA(2)alpha-dependent toxicity is cyclooxygenase (COX)-2 dependent, COX-2 and PGE(2) levels were measured 7 and 25 h after NMDA treatment. NMDA treatment failed to induce COX-2 protein or increase PGE(2) in the culture media in either genotype at either time. In contrast, phorbol 12-myristate 13-acetate and ionophore treatment caused robust induction of COX-2 and PGE(2) in both genotypes. We conclude that cPLA(2)alpha may have a hitherto unrecognized direct effect on excitatory neurotoxicity, suggesting that cPLA(2)alpha inhibition is a therapeutic candidate for treatment of the early, excitotoxic injury observed in stroke.     

Exposure of glia to pro-oxidant agents revealed selective Stat1 activation by H2O2 and Jak2-independent antioxidant features of the Jak2 inhibitor AG490

The JAK/STAT pathway is activated in response to cytokines and growth factors. In addition, oxidative stress can activate this pathway, but the causative pro-oxidant forms are not well identified. We exposed cultures of rat glia to H2O2, FeSO4, nitroprussiate, or paraquat. We assessed oxidative stress by measuring reactive oxygen species (ROS) and oxidated proteins, we determined phosphorylated Stat1 (pStat1), and we evaluated the effect of antioxidants (trolox, propyl gallate, and N-acetylcysteine) and of Jak2 (Janus tyrosine kinases) inhibitors (AG490 and Jak2-Inhibitor-II). Pro-oxidant agents induced ROS and protein oxidation, excluding nitroprussiate that induced protein nitrosylation. H2O2, and to a lesser extent FeSO4, increased the level of pStat1, whereas nitroprussiate and paraquat did not. Trolox and propyl gallate strongly prevented ROS formation but they did not abolish H2O2-induced pStat1. In contrast, NAC did not reduce the level of ROS but it prevented the increase of pStat1 induced by H2O2, evidencing a differential effect on ROS formation and on Stat1 phosphorylation. H2O2 induced pStat1 in mixed glia cultures and, to a lesser extent, in purified astroglia, but not in microglia. Jak2 inhibitors reduced H2O2-induced pStat1, suggesting the involvement of this kinase in the increased phosphorylation of Stat1 by peroxide. Unexpectedly, AG490, but not Jak2-Inhibitor-II, reduced ROS formation, and it abrogated lipid peroxidation in microsomal preparations. Furthermore, AG490 reduced ROS in glial cells that were transfected with siRNA to silence Jak2 expression. These findings reveal previously unrecognized Jak2-independent antioxidant properties of AG490, and show that Jak2-dependent Stat1 activation by peroxide is dissociated from ROS generation.     

Co-localization of cytosolic phospholipase A2 and cyclooxygenase-2 in Rhesus monkey cerebellum

Cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX)-1 and COX-2 play important and integrated roles in the release and subsequent metabolism of arachidonic acid, an important second messenger, in brain and other tissues. Antibodies to each of these enzymes were used to examine their cellular localization and expression in the cerebellum of the adult macaque, using Western blotting and immunohistochemical methods. COX-2 and cPLA2 immunoreactivities co-localized on the plasma membrane of Purkinje cells, and within punctate intracellular regions. In contrast, COX-1 immunoreactivity was relatively uniform in Purkinje cell cytoplasm, and was more homogeneous in cells of the granular cell layer and occasionally of the molecular layer. COX-1 immunoreactivity was not found on the cell surface. Labeling of Purkinje cell dendrites was not marked for any of the enzymes. cPLA2 and COX-2 have been shown to be functionally coupled in a number of cell systems, and in brain following lithium chloride administration to rats. The co-localization of cPLA2 and COX-2 is consistent with evidence of their functional coupling at brain synapses, and of the presence of an unesterified brain arachidonate pool released by cPLA2 which is the precursor for prostaglandin formation via COX-2.     

Thrombin-induced oxidative stress contributes to the death of hippocampal neurons: role of neuronal NADPH oxidase

The present study investigated whether thrombin can induce the production of reactive oxygen species (ROS) through activation of neuronal NADPH oxidase and whether this contributes to oxidative damage and consequently to neurodegeneration. Immunocytochemical and biochemical evidence demonstrated that, in neuron-enriched hippocampal cultures, thrombin induces neurodegeneration in a dose-dependent manner. In parallel, ROS production was evident as assessed by analyzing DCF and hydroethidine. Real-time PCR analysis, at various time points after thrombin treatment, also demonstrated that expression of NADPH oxidase subunits (p47(phox) and p67(phox)) occurs. In addition, Western blot analysis and double-label immunocytochemistry showed an up-regulation in the expression of cytosolic components (Rac 1 and p67(phox)), the translocation of cytosolic proteins (p47(phox) and p67(phox)) to the membrane, and the localization of gp91(phox) or p47(phox) expression in hippocampal neurons of cultures and CA1 layer. The thrombin-induced ROS production, protein oxidation, and loss of cultured hippocampal neurons were partially attenuated by an NADPH oxidase inhibitor and/or by several antioxidants. Collectively, the present study is the first to demonstrate that, in cultured hippocampal neurons, thrombin-induced neurotoxicity is, at least in part, caused by neuronal NADPH oxidase-mediated oxidative stress. This strongly suggests that thrombin can act as an endogenous neurotoxin, and inhibitors of thrombin and/or antioxidants can be useful agents for treating oxidative stress-mediated hippocampal neurodegenerative diseases, such as Alzheimer's disease.     

特异性抑制JAK2/STAT3信号通路对大鼠急性脑缺血再灌注损伤后COX-2和VEGF表达水平的影响

目的 探讨特异性抑制蛋白酪氨酸激酶2/信号转导与激活子3(JAK2/STAT3)信号通路对急性脑缺血再灌注损伤大鼠脑组织环氧合酶-2(COX-2)和血管内皮生长因子(VEGF)表达水平的影响。方法 采用线栓法制备大鼠急性大脑中动脉闭塞再灌注模型; 大鼠随机分为假手术组、模型组和给药组; 给药组大鼠于再灌注前5 min腹腔注射JAK2/STAT3信号通路抑制剂AG490(1 mg/kg),其余2组给予等量生理盐水; 再灌注24 h后各组行神经功能缺损评分,用氯化三苯基四氮唑(TTC)染色法检测大鼠脑梗死体积,用实时荧光定量PCR(RT-qPCR)检测脑组织中JAK2、STAT3、环氧合酶2(COX-2)和血管内皮生长因子(VEGF)的mRNA相对表达水平,用蛋白印迹(Western blot)检测脑组织磷酸化JAK2(p-JAK2)、磷酸化STAT3(p-STAT3)、COX-2和VEGF的蛋白相对表达水平。结果 与假手术组比较,模型组大鼠神经功能缺损评分、脑梗死体积、JAK2和STAT3的mRNA,p-JAK2和p-STAT3蛋白、COX-2的mRNA和蛋白相对表达水平均显著升高(P<0.05),VEGF的mRNA和蛋白相对表达水平显著降低(P<0.05); 与模型组比较,给药组大鼠神经功能缺损评分、脑梗死体积、JAK2和STAT3的mRNA、p-JAK2和p-STAT3蛋白、COX-2的mRNA和蛋白相对表达水平均显著降低(P<0.05),VEGF的mRNA和蛋白相对表达水平显著升高(P<0.05)。结论 特异性抑制JAK2/STAT3信号通路可能通过降低脑组织中COX-2表达和促进VEGF表达来保护大鼠急性脑缺血再灌注损伤。     

The action mechanism of cyclooxygenase-2 inhibitor for treatment of experimental allergic neuritis

We previously reported the effect of a cyclooxygenase (COX)-2 inhibitor, nimesulide, on experimental allergic neuritis (EAN) in both the induction and effector phases, in contrast to the usual COX inhibitor, which was effective only when administered in the induction phase. To assess the mechanism of action of a COX-2 inhibitor, we studied the expression of COX-2 and assayed plasma levels of prostaglandins, and also compared the clinical effect of a COX-2 inhibitor with a 5-lipoxygenase (LO) inhibitor, which is responsible for another pathway of arachidonic acid metabolism. Nerves of EAN rats showed distinct expression of COX-2, which is derived mostly from endoneurial macrophages. Treatment with a COX-2 inhibitor had no effect on its expression. However, prostaglandin estradiol (E(2)) concentration of plasma was significantly lower compared with the control group. The LO inhibitor showed no clinical effect. These results suggest that a selective COX-2 inhibitor is effective in the effector phase by its influence on macrophages that are responsible for nerve degeneration.     

Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells.

Activation of vascular smooth muscle cells (VMSC) by thrombin induces the expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1). We investigated in cultured human and rat VSMC whether reactive oxygen species (ROS) derived from the vascular NADPH oxidase contribute to this effect. Exposure of cultured VSMC to thrombin rapidly increased ROS formation, phosphorylation of p38 MAP kinase as well as the expression of MCP-1. Specific inhibition of the p22phox subunit of the vascular NADPH oxidase using either p22phox neutralizing antibody or p22phox antisense oligonucleotides attenuated thrombin-induced ROS generation. Furthermore, thrombin-induced p38 MAP kinase activation as well as MCP-1 expression were impaired by antioxidants as well as by p22phox antisense oligonucleotides. Inhibition of p38 MAP kinase diminished the thrombin-induced expression of MCP-1. CONCLUSION: Thrombin, by activating a p22phox-containing NADPH oxidase, elicits ROS generation and activation of p38 MAP kinase in VSMC. The subsequent induction of MCP-1 expression highligts the crucial role of the p22phox-containing NADPH oxidase in thrombin-induced signal transduction in VSMC.     

Intraparenchymal administration of interleukin-1beta induces cyclooxygenase-2-mediated expression of membrane- and cytosolic-associated prostaglandin E synthases in mouse brain

Interleukin (IL)-1beta is a proinflammatory cytokine expressed in neural tissue following injury and in neurodegenerative states. To understand the consequences of its presence in brain, we carried out intraparenchymal IL-1beta injections and found significant increases in prostaglandin (PG)E2, a critical factor in inflammatory and physiological processes. Elevated mRNA and protein expression of the PGE2-synthetic-related enzymes cyclooxygenase (COX)-2 and membrane-associated PGE synthase (PGES) accompanied local PGE2 production. In addition, IL-1beta stimulated protein expression of cytosolic PGES. Finally, we showed attenuation of these IL-1beta-inductions by COX-2 inhibition, suggesting in vivo regulation of both PGE synthase isoforms in the brain.