Genetic evidence for distinct roles of COX-1 and COX-2 in the immediate and delayed phases of prostaglandin synthesis in mast cells.

Activation of mast cells by aggregation of their high-affinity IgE receptors stimulates prostaglandin (PG) D(2) synthesis and secretion. An immediate phase of PGD(2) synthesis, complete within 30 min, is followed by a delayed, second phase of PGD(2) production that reaches a maximum 4 to 8 h after activation. Activation of mast cells from COX-2 (-/-) mice stimulates the release of PGD(2) during the first 30 min, whereas activation of mast cells from COX-1 (-/-) mice does not generate any PGD(2) in the first 2 h. On the other hand, COX-2 (-/-) cells do not participate in delayed phase of PGD(2) synthesis, while COX-1 (-/-) cells secrete low levels of PGD(2) between 2 and 4 h after activation. These data demonstrate that (i) the first phase of PG synthesis is COX-1 dependent and (ii) the second, delayed phase of PG synthesis is dependent on activation-induced synthesis and activity of COX-2.     

Group V secretory phospholipase A2 amplifies the induction of cyclooxygenase 2 and delayed prostaglandin D2 generation in mouse bone marrow culture-derived mast cells in a strain-dependent manner

Activation of mouse bone marrow-derived mast cells (BMMC) with stem cell factor (SCF) or IgE and antigen elicits exocytosis and an immediate phase of prostaglandin (PG) D(2) and leukotriene (LT) C(4) generation. Activation of BMMC by SCF, IL-1beta and IL-10 elicits a delayed phase of PGD(2) generation dependent on cyclooxygenase (COX) 2 induction. Cytosolic phospholipase A(2) alpha provides arachidonic acid in both phases and amplifies COX-2 induction. Pharmacological experiments implicate an amplifying role for secretory (s) PLA(2). We used mice lacking the gene encoding group V sPLA(2) (Pla2g5-/-) to definitively test its role in eicosanoid generation by BMMC. Pla2g5-/- BMMC on a C57BL/6 genetic background showed a modest reduction in exocytosis and immediate PGD(2) generation after activation with SCF or with IgE and antigen, while LTC(4) generation was not modified. Delayed-phase PGD(2) generation and COX-2 induction were reduced approximately 35% in C57BL/6 Pla2g5-/- BMMC and were restored by exogenous PGE(2). There was no deficit in either phase of eicosanoid generation by Pla2g5-/- BMMC on a BALB/c background. Thus, group V sPLA(2) amplifies COX-2 expression and delayed phase PGD(2) generation in a strain-dependent manner; it has at best a limited role in immediate eicosanoid generation by BMMC.     

Sequential induction of pro- and anti-inflammatory prostaglandins and peroxisome proliferators-activated receptor-gamma during normal wound healing: a time course study

Lipid mediators generated from metabolism of arachidonic acid play a crucial role in the initiating and resolution of acute inflammation by shifting from pro-inflammatory prostaglandin (PG) E2 to anti-inflammatory PGD2 and its metabolites. The changes in PG levels over time during the normal wound-repair process have not, however, been reported. We determined the temporal expression of PG and their biosynthetic enzymes using the full thickness incisional model of normal wound healing in mice. We demonstrate that during normal wound repair, there is a shift in the metabolism of arachidonate from PGE2 during the acute inflammatory phase to PGD2 during the repair phase. This shift is mediated by temporal changes in the expression of cyclooxygenases (COX) and microsomal PGES (mPGES)-1. Inducible COX (COX-2) expression is sustained throughout the initiation and repair process, but mPGES-1 is increased only during the acute inflammatory phase and its disappearance coincides with increased PGD2. PGD2 and its degradation products are known to mediate their anti-inflammatory effects by binding to peroxisome proliferators-activated receptor gamma (PPARgamma). In this study, we show that PPARgamma is upregulated during the resolution phase of wound repair concomitant with the shift to PGD2, and may be responsible for initiating endogenous mechanism resulting in healing/resolution.     

Role of COX-1 and COX-2 on skin PGs biosynthesis by mechanical scratching in mice

We examined the involvement of cyclooxygenase (COX)-1 and COX-2 on mechanical scratching-induced prostaglandins (PGs) production in the skin of mice. The dorsal regions of mice were scratched using a stainless brush. COXs expressions in the skin were analyzed using real-time PCR and Western blotting. The effect of acetylsalicylic acid (ASA) on the ability of PGs production were determined based on skin PGs level induced by arachidonic acid (AA) application. Mechanical scratching increased PGD2, PGE2, PGI2 and PGF(2 alpha). COX-1 was constitutively expressed and COX-2 expression was enhanced by scratching. Intravenous administration of ASA inhibited PGs biosynthesis in the normal skin. PGs levels of the skin 6h after ASA administration (ASA 6 h) were almost equal to those of the skin 10 min after ASA administration (ASA 10 min). In the scratched skin, AA-induced PGE2 and PGI2 of ASA 6 h were significantly higher than those of ASA 10 min. The skin PGD2 and PGF(2 alpha) of ASA 10 min were almost same to those of ASA 6 h. In the normal skin of COX-1-deficient mice, skin PGD2 level was lower than that of wild-type mice, although PGE2, PGI2 and PGF(2 alpha) levels were almost equal to those of wild type. In the scratched skin of COX-1-deficient mice, PGD2, PGE2, PGI2 and PGF(2 alpha) levels were lower than those of wild-type mice. These results suggested that cutaneous PGD2 could be mainly produced by COX-1, and PGE2 and PGI2 could be produced by COX-1 and COX-2, respectively, in mice.     

Mechanotransduction of stretch-induced prostanoid release by fetal lung epithelial cells

Mechanical ventilation is the primary supportive treatment for infants and adults suffering from severe respiratory failure. Adverse mechanical ventilation (overdistension of the lung) triggers a proinflammatory response. Along with cytokines, inflammatory mediators such as bioactive lipids are involved in the regulation of the inflammatory response. The arachidonic acid pathway is a key source of bioactive lipid mediators, including prostanoids. Although ventilation has been shown to influence the production of prostanoids in the lung, the mechanotransduction pathways are unknown. Herein, we established that cyclic stretch of fetal lung epithelial cells, but not fibroblasts, can evoke an extremely sensitive, rapid alteration in eicosanoid metabolism through a cyclooxygenase (COX)-2 dependent mechanism. Cyclic stretch significantly increased PGI(2), PGF(2alpha), PGD(2), PGE(2), and thromboxane B(2) levels in the media of epithelial cells, but did not alter leukotriene B(4) or 12-hydroxyeicosatetraenoic acid levels. Inhibition of COX-2, but not COX-1, attenuated the cyclic stretch-induced PG increase in the media, suggesting that cyclic stretch primarily affected PG synthesis. Substrate (free arachidonic acid) availability for PG generation was increased because of a cyclic stretch-induced activation of cytosolic phospholipase A(2) (cPLA(2)) via an influx of extracellular calcium and phosphorylation by mitogen-activated protein kinase, p44/42MAPK. The data are compatible with cPLA(2) and COX-2 being intimately involved in regulating the injury response to adverse mechanical ventilation.     

Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice

Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/-)) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/-) mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/-) mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/-) mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/-) compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/-) mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/-) mice. After KA exposure, COX-2(-/-) mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/-) mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the glutamatergic system.     

Oxygen free radicals in rat limbic structures after kainate-induced seizures

Several indices of free radical generation were determined in limbic structures after kainate (KA)-induced seizure activity in adult and postnatal day (PND) 12 and 17 rats. Superoxide dismutase, catalase, and glutathione peroxidase activities were measured in piriform cortex and hippocampal subfields at 8, 16, 48 h, and 5 days after KA injection in adults and pups, and also at 3 weeks postinjection in adults. KA-induced seizure activity had no significant effect on enzyme activities in PND 12 and 17 rats. In adults, superoxide dismutase and catalase activities were significantly increased at 5 days after KA administration, and returned to preinjection levels by 3 weeks. Glutathione peroxidase activity was also increased significantly at 5 days postinjection, but remained elevated at 3 weeks. Lipid peroxidation, as indicated by malondialdehyde (MDA) concentration, exhibited an early significant increase at 8 and 16 h, followed at 48 h and 5 days by a significant decrease. At 3 weeks postinjection, MDA levels were still significantly decreased in CA3 and dentate gyrus. KA administration in PND 12 and 17 rats had no significant effect on MDA content. KA-induced seizure activity in adults also resulted in a large and sustained increase in protein oxidation in piriform cortex and hippocampus. The early increase in MDA and protein oxidation in adult rats strongly suggests the involvement of oxygen free radicals in the initial phases of KA-induced pathology, whereas the changes in scavenging enzyme activities and MDA content at 5 days and 3 weeks post KA injection possibly reflect glial proliferation subsequent to neuronal death.     

Kainate-induced mitochondrial oxidative stress contributes to hippocampal degeneration in senescence-accelerated mice

We have demonstrated that kainate (KA) induces a reduction in mitochondrial Mn-superoxide dismutase (Mn-SOD) expression in the rat hippocampus and that KA-induced oxidative damage is more prominent in senile-prone (SAM-P8) than senile-resistant (SAM-R1) mice. To extend this, we examined whether KA seizure sensitivity contributed to mitochondrial degeneration in these mouse strains. KA-induced seizure susceptibility in SAM-P8 mice paralleled prominent increases in lipid peroxidation and protein oxidation and was accompanied by significant impairment in glutathione homeostasis in the hippocampus. These findings were more pronounced in the mitochondrial fraction than in the hippocampal homogenate. Consistently, KA-induced decreases in Mn-SOD protein expression, mitochondrial transmembrane potential, and uncoupling protein (UCP)-2 expression were more prominent in SAM-P8 than SAM-R1 mice. Marked release of cytochrome c from mitochondria into the cytosol and a higher level of caspase-3 cleavage were observed in KA-treated SAM-P8 mice. Additionally, electron microscopic evaluation indicated that KA-induced increases in mitochondrial damage and lipofuscin-like substances were more pronounced in SAM-P8 than SAM-R1 animals. These results suggest that KA-mediated mitochondrial oxidative stress contributed to hippocampal degeneration in the senile-prone mouse.     

Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A₂-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.     

NO介质在大鼠红藻氨酸诱导癫痫发作中的作用

目的：进一步探讨脑内一氧化氮(NO)介质(NO或NO衍生物)在复杂部分性及全身强直阵挛性癫痫发作中的作用。方法：采用红藻氨酸(KA)诱导大鼠癫痫发作,以NO合酶抑制剂L-硝基精氨酸(L-NNA)或NO前体L-精氨酸(L-Arg)予以预处理,观察其癫痫发作行为及海马结构内NO含量(NO2^-/NO3^-)的变化。结果：给予大鼠惊厥剂量KA(10mg/kg),15min时出现湿狗样抖动(WDS),1～3h出现全身痉挛;经L-NNA(50mg/kg)或L-Arg(40mg/kg)预处理的大鼠,注射相同剂量的KA后,其癫痫行为发生明显变化,L-NNA预处理的大鼠癫痫发作行为明显加重,表现为全身痉挛的潜伏期缩短、时间延长、死亡率提高;L-Arg预处理的大鼠癫痫发作行为减弱,WDS和全身痉挛的潜伏期均延长,发作程度减轻、时间缩短,观察时间内无一例死亡。KA给药后30min海马结构内的NO2^-/NO3^-含量迅速增多,7d时仍持续增高;与NS预处理组相比,经L-Arg预处理的动物,KA给药后3h及3d,其NO2^-/NO3^-浓度升高明显。结论：兴奋诱导性癫痫发作过程中内源性NO介质的变化可能具有重要的抗发作作用。     

Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D

Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE(2) production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.     

Does phenobarbital protect against trimethyltin-induced neuropathology of limbic structures?

Because of the similarity in the pattern of limbic sites damaged by both compounds, it has been suggested that trimethyltin (TMT) may be an excitotoxin like kainic acid (KA). KA produces seizures which eventually result in neuronal damage similar to that found in epilepsy. Anticonvulsants reduce both the seizures and pathology associated with KA. Because TMT may also produce seizures, we undertook to determine whether or not some of the TMT-induced limbic neuropathology could result from seizure activity. To do this, a single dose of TMT chloride (either 7.5 or 15 mg/kg) was given per os to rats, and then phenobarbital (30 mg/kg) was administered subcutaneously in repeated doses. Treatment with phenobarbital did not prevent pathologic changes in the hippocampus, dentate gyrus, and pyriform or prepyriform cortex. Since phenobarbital did not protect against TMT-induced neuronal damage, as it has been reported by others to protect against KA-induced damage, the present findings suggest that these two toxicants probably produce hippocampal pathology via different mechanisms and that the TMT-induced pathologic changes do not require sustained electrical seizure activity.     

Do human platelets express COX-2?

The rate-limiting enzyme in prostaglandin (PG)- and thromboxane (TX)-synthesis is known as cyclooxygenase (COX). The COX-enzyme family consists of the classical COX-1 and the inducible COX-2-enzyme. To investigate whether platelets contain COX-2, we measured thiobarbituric acid reactive substances (TBARS) after either blocking COX-1 or COX-2 or adding compounds known to affect COX-expression. To stimulate platelets' different reagents such as collagen, thrombin and arachidonic acid (AA) were used. The inhibitors used in this study were acetylsalicylic acid (ASA), indomethacin and NS-398. Using the western-blot technique, we failed to detect COX-2 in platelets while COX-1 was detectable. We were not able to discover COX-2 in platelets using the methods we applied. As the amount of COX-2 in platelets might be below the detection limit of the methods used, the biological relevance COX-2 in platelets, if even existing at low amounts, remains to be established.     

Yin-yang: balancing act of prostaglandins with opposing functions to regulate inflammation

For many years, cyclooxygenase-2 (COX-2), a critical enzyme for PG production, has been the favorite target for anti-inflammatory drug development. However, recent revelations regarding the adverse effects of selective COX-2 inhibitors have stimulated intense debate. Interestingly, in the early phase of inflammation, COX-2 facilitates inflammatory PG production while in the late phase it has anti-inflammatory effects. Moreover, although some PGs are proinflammatory, others have anti-inflammatory effects. Thus, it is likely that PGs with opposing effects maintain homeostasis, although the molecular mechanism(s) remains unclear. We report here that an inflammatory PG, PGD2, via its receptor, mediates the activation of NF-kappaB stimulating COX-2 gene expression. Most interestingly, an anti-inflammatory PG (PGA1) suppresses NF-kappaB activation and inhibits COX-2 gene expression. We propose that while pro- and anti-inflammatory PGs counteract each other to maintain homeostasis, selective COX-2 inhibitors may disrupt this balance, thereby resulting in reported adverse effects.     

Cytosolic phospholipase A2 translocates to forming phagosomes during phagocytosis of zymosan in macrophages

Resident tissue macrophages mediate early innate immune responses to microbial infection. Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is activated in macrophages during phagocytosis of non-opsonized yeast (zymosan) triggering arachidonic acid release and eicosanoid production. cPLA(2)alpha translocates from cytosol to membrane in response to intracellular calcium concentration ([Ca(2+)](i)) increases. Enhanced green fluorescent protein (EGFP)-cPLA(2)alpha translocated to forming phagosomes, surrounding the zymosan particle by 5 min and completely overlapping with early endosome (Rab5) and plasma membrane (F4/80) markers but only partially overlapping with resident endoplasmic reticulum proteins (GRP78 and cyclooxygenase 2). EGFP-cPLA(2)alpha also localized to membrane ruffles during phagocytosis. Zymosan induced an initial high amplitude calcium transient that preceded particle uptake followed by a low amplitude sustained calcium increase. Both phases were required for optimal phagocytosis. Extracellular calcium chelation prevented only the sustained phase but allowed a limited number of phagocytic events, which were accompanied by translocation of cPLA(2)alpha to the phagosome although [Ca(2+)](i) remained at resting levels. The results demonstrate that cPLA(2)alpha targets the phagosome membrane, which may serve as a source of arachidonic acid for eicosanoid production.     

Prolonged exposure to cigarette smoke blocks the neurotoxicity induced by kainic acid in rats

We examined the effects of cigarette smoke (CS) on three parameters associated with kainic acid (KA)-induced neurotoxicity: seizure activity, cell loss in the hippocampus, and increased Fos-related antigen (FRA) expression. Animals were exposed to the main stream of CS from 15 Kentucky 2R1F research cigarettes containing 28.6 mg tar and 1.74 mg nicotine per cigarette, for 10 min a day, 6 days per week, for 4 weeks, using an automatic smoking machine. KA administration (10 mg/kg, i.p.) produced robust behavioral convulsions lasting 4-5 h. Pre-exposure to CS significantly reduced the seizures, mortality, and severe loss of cells in regions CA1 and CA3 of the hippocampus after KA administration. Consistently, pre-exposure to CS significantly attenuated the KA-induced increased FRA immunoreactivity in the hippocampus. In contrast, pretreatment with central nicotinic antagonist, mecamylamine (2 or 10 mg/kg, i.p.) blocked the neuroprotective effects mediated by CS in a dose-dependent manner. These results indicate that CS exposure provides neuroprotection against the KA insult via nicotinic receptor activation.     

Aldosterone augments endothelin-1-induced cardiac myocyte hypertrophy with the reinforcement of the JNK pathway

Monocyte-derived dendritic cells (moDCs) are increasingly used in clinical settings to stimulate tumor immunity. Prostaglandin E2 (PGE2), which is a member of the eicosanoid family of oxygenated arachidonic acid derivatives generated through the action of cyclooxygenases (COXs), is frequently used to enhance the tumor necrosis factor-alpha-induced terminal maturation of moDCs. We show here that one effect of interleukin (IL)-4, which is used together with GM-CSF to generate moDCs, is the suppression of endogenous PGE2 production in moDCs. IL-4 inhibits the cytoplasmic form of phospholipase A2, the enzyme that specifically liberates arachidonic acid from membrane phospholipids. Although moDCs failed to mobilize endogenous arachidonic acid, they converted exogenous arachidonic acid into PGE2 in a COX-1- and COX-2-dependent fashion. IL-4-mediated suppression of PGE2 biosynthesis in human moDCs explains the previously reported maturation-enhancing effect of exogenous PGE2. The general suppression of eicosanoid biosynthesis may, however, limit the immunological efficacy of moDCs generated with IL-4.