Anti-apoptotic effects of arachidonic acid and prostaglandin E2 in pancreatic beta-cells.

BACKGROUND/AIMS: The polyunsaturated fatty acid arachidonic acid (AA) has been implicated in beta-cell defence mechanisms and prostaglandin (PG) products of cyclooxygenase (COX) 2 action confer resistance to alloxan-induced apoptosis in insulin-secreting RIN cells. We have now investigated the anti-apoptotic effects of AA and its metabolite, PGE(2), in the MIN6 mouse insulin-secreting beta-cell line and mouse islets. METHODS: Apoptosis was determined in MIN6 beta-cell and mouse islet extracts by measurement of capase-3 activity, and COX2 mRNA levels were quantified by real-time RT-PCR. RESULTS: Exposure of MIN6 cells to AA (3.1-12.5 microM) caused concentration-dependent reductions in apoptosis, and similar results were obtained when endogenous AA levels were elevated in cytosolic phospholipase A(2)-overexpressing MIN6 cells. 25mM glucose caused both a significant up-regulation of MIN6 cell COX2 mRNA levels and a decrease in apoptosis. Inhibition of MIN6 cell COX2 activity with a selective inhibitor, NS-398 (10-100 microM), increased apoptosis and exogenous PGE(2) (0.2-5 microM) reduced NS-398-induced apoptosis in a concentration-dependent manner. The protective effects of AA and PGE(2) were also observed in primary mouse islets. CONCLUSION: These data show that AA and its COX2-generated metabolite, PGE(2), can protect beta-cells from apoptosis.     

Piroxicam and NS-398 rescue neurones from hypoxia/reoxygenation damage by a mechanism independent of cyclo-oxygenase inhibition

We studied whether NS-398, a selective cyclo-oxygenase-2 (COX-2) enzyme inhibitor, and piroxicam, an inhibitor of COX-2 and the constitutively expressed COX-1, protect neurones against hypoxia/reoxygenation injury. Rat spinal cord cultures were exposed to hypoxia for 20 h followed by reoxygenation. Hypoxia/reoxygenation increased lactate dehydrogenase (LDH) release, which was inhibited by piroxicam (180-270 microM) and NS-398 (30 microM). Cell counts confirmed the neuroprotection. Western blotting revealed no COX-1 or COX-2 proteins even after hypoxia/reoxygenation. Production of prostaglandin E2 (PGE2), a marker of COX activity, was barely measurable and piroxicam and NS-398 had no effect on the negligible PGE2 production. Hypoxia/reoxygenation increased nuclear factor-kappa B (NF-kappaB) binding activity, which was inhibited by piroxicam but not by NS-398. AP-1 binding activity after hypoxia/reoxygenation was inhibited by piroxicam but strongly enhanced by NS-398. However, both COX inhibitors induced activation of extracellular signal-regulated kinase (ERK) in neurones and phosphorylation of heavy molecular weight neurofilaments, cytoskeletal substrates of ERK. It is concluded that piroxicam and NS-398 protect neurones against hypoxia/reperfusion. The protection is independent of COX activity and not solely explained by modulation of NF-kappaB and AP-1 binding activity. Instead, piroxicam and NS-398-induced phosphorylation through ERK pathway may contribute to the increased neuronal survival.     

Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA phospholipase A2 in COS-1 cells

Phospholipases A2 (PLA2) and cyclooxygenases (COX) are important enzymes responsible for production of potent lipid mediators, including prostaglandins (PG) and thromboxane A2. We investigated coupling between PLA2 and COX isoforms by using transient transfection in COS-1 cells. Untransfected cells, incubated with or without phorbol ester + the Ca2+ ionophore ionomycin, generated trivial amounts of PGE2. In cells co-transfected with cytosolic PLA2 (cPLA2) and COX-1 or COX-2, phorbol ester + ionomycin markedly stimulated PGE2 production. There was no preferential coupling of cPLA2 to either of the COX isoforms. In contrast, group IIA secretory PLA2 (sPLA2) co-transfected with COX-1 or COX-2 did not lead to an increase in PGE2 production, despite high levels of sPLA2 enzymatic activity. Transfection of cPLA2 did not affect basal free arachidonic acid (AA) levels. Phorbol ester + ionomycin stimulated release of AA in cPLA2-transfected COS-1 cells, but not in untransfected cells, whereas sPLA2 transfection (without stimulation) led to high basal free AA. Thus, AA released by cPLA2 is accessible to both COX isoforms for metabolism to PG, whereas AA released by sPLA2 is not metabolized by COX.     

Regulation of bone morphogenetic protein-2 expression by endogenous prostaglandin E2 in human mesenchymal stem cells

Cyclooxygenase (COX)-2 is generally known as an inducible enzyme, and it produces arachidonic acid to prostaglandin E2 (PGE2), which modulates bone metabolism. Here, we investigated the expression and role of COX isomers in human mesenchymal stem cells. Human mesenchymal stem cells constitutively expressed COX-2 as well as COX-1, and secretion of PGE2 was completely inhibited by NS-398, a specific inhibitor of COX-2. Levels of secreted PGE2 were strikingly higher in human mesenchymal stem cells than in osteoblastic cells differentiated from the mesenchymal cells. This higher production of PGE2 in mesenchymal stem cells was due to higher expression of membrane-associated PGE synthase (mPGES) regulated by early growth response factor-1 (Egr-1). Treatment of human mesenchymal stem cells with NS-398 suppressed expression of bone morphogenetic protein-2 (BMP-2). The suppression of BMP-2 by NS-398 was abrogated by an EP4 receptor agonist as well as by PGE2. Moreover, BMP-2 expression was suppressed by an EP4 receptor antagonist. These data indicate that PGE2 produced by COX-2 increases BMP-2 expression via binding the EP4 receptor.     

In vivo activation of N-methyl-D-aspartate receptors in the rat hippocampus increases prostaglandin E(2) extracellular levels and triggers lipid peroxidation through cyclooxygenase-mediated mechanisms

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE(2) and 8-epi-PGF(2alpha) following infusion of N-methyl-D-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE(2) and 8-epi-PGF(2alpha) basal levels. These effects were largely prevented by the specific cytosolic phospholipase A(2) (cPLA(2) ) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.     

Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.     

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.     

Type IIA secretory phospholipase A2 up-regulates cyclooxygenase-2 and amplifies cytokine-mediated prostaglandin production in human rheumatoid synoviocytes

Human type IIA secretory phospholipase A2 (sPLA2-IIA) is induced in association with several immune-mediated inflammatory conditions. We have evaluated the effect of sPLA2-IIA on PG production in primary synovial fibroblasts from patients with rheumatoid arthritis (RA). At concentrations found in the synovial fluid of RA patients, exogenously added sPLA2-IIA dose-dependently amplified TNF-alpha-stimulated PGE2 production by cultured synovial fibroblasts. Enhancement of TNF-alpha-stimulated PGE2 production in synovial cells was accompanied by increased expression of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2)-alpha. Blockade of COX-2 enzyme activity with the selective inhibitor NS-398 prevented both TNF-alpha-stimulated and sPLA2-IIA-amplified PGE2 production without affecting COX-2 protein induction. However, both sPLA2-IIA-amplified PGE2 production and enhanced COX-2 expression were blocked by the sPLA2 inhibitor LY311727. Colocalization studies using triple-labeling immunofluorescence microscopy showed that sPLA2-IIA and cPLA2-alpha are coexpressed with COX-2 in discrete populations of CD14-positive synovial macrophages and synovial tissue fibroblasts from RA patients. Based on these findings, we propose a model whereby the enhanced expression of sPLA2-IIA by RA synovial cells up-regulates TNF-alpha-mediated PG production via superinduction of COX-2. Therefore, sPLA2-IIA may be a critical modulator of cytokine-mediated synovial inflammation in RA.     

The COX-2 inhibitor NS-398 causes T-cell developmental disruptions independent of COX-2 enzyme inhibition

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the function of cyclooxygenases, COX-1 and COX-2, which catalyze the first step in the synthesis of inflammatory mediators (PGE2). We sought to understand the roles of cyclooxygenases and NSAIDs in T-cell development. Our data show no significant defects in T-cell development in fetal thymic organ cultures of mice disrupted in both or either COX genes or in mice disrupted in either EP-1 or EP-2 receptor genes. On the other hand, NSAIDs reproducibly caused thymocyte developmental defects. However, the specific effects of the COX-2 inhibitors were not correlated with their potency for inhibition of COX-2 activity. We focused on the NS-398 COX-2 inhibitor and showed that its effects could not be reversed by exogenous PGE2. Furthermore, NS-398 was inhibitory even when its target, COX-2, was absent. These data show that the T-cell developmental effects of NS-398 are COX-2 and PGE2 independent.     

A role for cyclooxygenase-2 in lipopolysaccharide-induced anorexia in rats

Because nonselective cycloooxygenase (COX) inhibition attenuated anorexia after lipopolysaccharide (LPS) administration, we tested the ability of resveratrol (2.5, 10, and 40 mg/kg) and NS-398 (2.5, 10, and 40 mg/kg), selective inhibitors of the two COX isoforms COX-1 and -2, respectively, to attenuate LPS (100 microg/kg ip)-induced anorexia. NS-398 (10 and 40 mg/kg) administered with LPS at lights out attenuated LPS-induced anorexia, whereas resveratrol at all doses tested did not. Because prostaglandin (PG) E(2) is considered the major metabolite synthesized by COX, we measured plasma and cerebrospinal fluid (CSF) PGE(2) levels after LPS administration. LPS induced a time-dependent increase of PGE(2) in CSF but not in plasma. NS-398 (5, 10, and 40 mg/kg) blocked the LPS-induced increase in CSF PGE(2), whereas resveratrol (10 mg/kg) did not. These results support a role of COX-2 in mediating the anorectic response to peripheral LPS and point at PGE(2) as a potential neuromodulator involved in this response.     

Role of cyclooxygenase-2 in Helicobacter pylori- induced gastritis in Mongolian gerbils

Cyclooxygenase (COX)-2 expression is induced in the gastric mucosa of Helicobacter pylori-infected patients, but its role remains unclear. We examined the effects of NS-398 and indomethacin on gastric pathology in H. pylori-infected Mongolian gerbils. COX-1 was detected in both normal and H. pylori-infected mucosa, whereas COX-2 was expressed only in the infected mucosa. PGE(2) production was elevated by H. pylori infection, and the increased production was reduced by NS-398, which did not affect PGE(2) production in normal mucosa. Indomethacin inhibited PGE(2) production in both normal and infected mucosa. Hemorrhagic erosions, neutrophil infiltration, lymphoid follicles, and epithelium damage were induced by H. pylori infection. NS-398 and indomethacin aggravated these pathological changes but did not increase viable H. pylori number. H. pylori-increased production of neutrophil chemokine and interferon-gamma was potentiated by NS-398 and indomethacin. Neither NS-398 nor indomethacin caused any pathological changes or cytokine production in normal animals. These results indicate that COX-2 as well as COX-1 might play anti-inflammatory roles in H. pylori-induced gastritis.     

Endothelins Stimulate Expression of Cyclooxygenase 2 in Rat Cultured Astrocytes

Endothelin (ET) is one of the active endogenous substances regulating the functions of astrocytes. In the present study, we examined effects of ET on cyclooxygenase (COX) expression in cultured astrocytes. ET-3 (100 nM) caused transient increases in the expression of both COX2 mRNA and protein, but not those of COX1, in cultured astrocytes. ET-induced COX2 mRNA expression was suppressed by 5 microg/ml actinomycin D, 30 microM BAPTA/AM, inhibitors of protein kinase C (1-100 nM staurosporin and 100 microM H-7), 2 microM dexamethasone, and prolonged treatment with 100 nM phorbol 12-myristate 13-acetate. ET-3 stimulated production of prostaglandin (PG) E2 in cultured astrocytes. The effect of ET-3 on the PGE2 production was diminished by actinomycin D. Indomethacin and NS398, a selective COX2 inhibitor, comparably decreased both the basal and the ET-stimulated PGE2 production. Proliferation of cultured astrocytes was stimulated by 100 nM ET-3, and the increased proliferation was reduced by co-addition of 1 microM PGE2. Treatment with 1 microM PGE2 caused astrocytic morphological changes accompanied by disappearance of stress fibers, a prominent structure of organized cytoskeletal actin in cultured astrocytes. In the presence of 10 nM ET-3, PGE2 did not show an effect on astrocytic actin organization. The present study shows that ET is an inducer of astrocytic COX2 and suggests that ET-induced PGE2 production through COX2 may be involved in the regulation of astrocytic functions.     

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18?days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4?h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.     

NS-398: cyclooxygenase-2 independent inhibition of leukocyte priming for lipid body formation and enhanced leukotriene generation

Because the induction of new lipid body formation in leukocytes correlates with and likely contributes to their enhanced 'primed' prostaglandin and leukotriene formation, we evaluated two selective cyclooxygenase (COX)-2 inhibitors. Three types of stimuli, cis -unsaturated fatty acids, platelet activating factor and protein kinase C activators, stimulate lipid body formation. NS-398 (0.1-10 microM), but not another COX-2 inhibitor, SC58125 (0.1- 10 microM), blocked leukocyte lipid body formation elicited by all three types of stimuli and also blocked priming for enhanced LTB(4) production and PGE(2) production. The effect of NS-398 on lipid body formation was independent of its inhibitory effects on COX-2 since arachidonate-induced lipid body formation in COX-2-deficient mouse leukocytes was also inhibited by NS-398. By means of its ability to inhibit leukocyte lipid body formation, NS-398 may exert actions independent of its COX-2 inhibition and more broadly contribute to the suppression of formation of COX-1 and lipoxygenase-derived eicosanoids.     

Differential effects of COX inhibitors against beta-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimer's disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-β protein (Aβ, fragments 25–35 and 1–42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from Aβ and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that Aβ toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of Aβ neurotoxicity.     

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.     

Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1alpha, and PGE2 were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with L-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2 and PGE2 by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.     

Alcohol is a potent stimulant of immature neuronal networks: implications for fetal alcohol spectrum disorder

Ethanol consumption during development affects the maturation of hippocampal circuits by mechanisms that are not fully understood. Ethanol acts as a depressant in the mature CNS and it has been assumed that this also applies to immature neurons. We investigated whether ethanol targets the neuronal network activity that is involved in the refinement of developing hippocampal synapses. This activity appears during the growth spurt period in the form of giant depolarizing potentials (GDPs). GDPs are generated by the excitatory actions of GABA and glutamate via a positive feedback circuit involving pyramidal neurons and interneurons. We found that ethanol potently increases GDP frequency in the CA3 hippocampal region of slices from neonatal rats. It also increased the frequency of GDP-driven Ca2+ transients in pyramidal neurons and increased the frequency of GABA(A) receptor-mediated spontaneous postsynaptic currents in CA3 pyramidal cells and interneurons. The ethanol-induced potentiation of GABAergic activity is probably the result of increased quantal GABA release at interneuronal synapses but not enhanced neuronal excitability. These findings demonstrate that ethanol is a potent stimulant of developing neuronal circuits, which might contribute to the abnormal hippocampal development associated with fetal alcohol syndrome and alcohol-related neurodevelopmental disorders.     

Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.