Low prevalence of secondary endosymbionts in aphids sampled from rapeseed crops in Germany.

Peach-potato aphids, Myzus persicae Sulzer (Hemiptera:Aphididae), and cabbage aphids, Brevicoryne brassicae Linnaeus (Hemiptera:Aphididae), are herbivorous insects of significant agricultural importance. Aphids can harbour a range of non-essential (facultative) endosymbiotic bacteria that confer multiple costs and benefits to the host aphid. A key endosymbiont-derived phenotype is protection against parasitoid wasps, and this protective phenotype has been associated with several defensive enodsymbionts. In recent years greater emphasis has been placed on developing alternative pest management strategies, including the increased use of natural enemies such as parasitoids wasps. For the success of aphid control strategies to be estimated the presence of defensive endosymbionts that can potentially disrupt the success of biocontrol agents needs to be determined in natural aphid populations. Here, we sampled aphids and mummies (parasitised aphids) from an important rapeseed production region in Germany and used multiplex PCR assays to characterise the endosymbiont communities. We found that aphids rarely harboured facultative endosymbionts, with 3.6% of M. persicae and 0% of B. brassicae populations forming facultative endosymbiont associations. This is comparable with endosymbiont prevalence described for M. persicae populations surveyed in Australia, Europe, Chile, and USA where endosymbiont infection frequencies range form 0-2%, but is in contrast with observations from China where M. persicae populations have more abundant and diverse endosymbiotic communities (endosymbionts present in over 50% of aphid populations).

Can corticosteroids be beaten in future asthma therapy?

Despite the enormous therapeutic advance, there is a general trend towards increasing morbidity and mortality due to asthma, which suggests that there is a need for new and improved treatments. The past decade was determined by the so-called "new biology" that identified and cloned almost all receptors and ion channels. This scientific revolution should lead to a more rapid identification of novel targets for major diseases and processes like high throughput screening and combinatorial chemistry should have improved and fastened the development of new drugs. Interestingly, exactly the opposite has happened. With the exception of leukotriene receptor antagonists and some monoclonal antibodies, no new developments have been introduced into asthma therapy during the last decade. The most promising approach is still to find drugs like corticosteroids with multiple functions. However, there is no evidence at the very moment that corticosteroids can be beaten in the next ten years. Therefore, our task is to improve the corticosteroids and make therapy with them even safer. The so-called soft-steroids such as loteprednol and etiprednol belong to the future promising therapeutically effective and safe treatments of allergic disorders.

Possibilities in improvement of glucocorticoid treatments in asthma with special reference to loteprednol etabonate.

Allergic conditions contribute significantly to the burden of chronic disease in the industrialized world. The increasing prevalence has lead research into the discovery and development of various new therapeutic strategies. Despite considerable efforts of the pharmaceutical industry, the leukotriene antagonists were the only new class of asthma treatments to be licensed in the past 30 years. Topical glucocorticoids (GCs) are the most potent and effective therapy for treating allergic diseases. However, their use is limited by diverse undesired effects. Changes in pharmacokinetic parameters of GCs may be an interesting and promising approach to improve efficacy and safety of inhaled GCs. Loteprednol etabonate has been developed on the basis of the retrometabolic drug design. In animal studies, it has been demonstrated to have long-lasting anti-allergic (anti-asthmatic) effects without influencing the hypothalamic-pituitary axis (HPA). This soft steroid is now in phase III of the clinical development. Recently, loteprednol has been proven to be effective in the management of allergic rhinitis (400 microg once daily). No suppression of HPA was observed at clinically effective and higher doses. In conclusion, loteprednol as the first representative of soft steroids elicits marked anti-inflammatory effects, but has no impact on endocrine responses. It may represent a promising new therapy in the treatment of allergic rhinitis and asthma.

Asthma therapy in the new millennium.

Bronchial asthma is one of the most common chronic diseases in modern society and yet, despite the availability of highly effective drugs, there is increasing evidence to suggest that its incidence is increasing. It is a general health problem in several industrialised countries and will remain one for the next decades. With regard to asthma pathogenesis, our understanding has increased tremendously over the last two decades. Therefore, the potential for specific targeted and constructed therapies has become evident. Monoclonal antibodies to IgE, soluble receptors or antibodies to certain cytokines such as IL-4 and IL-5 are being investigated as possible treatments for asthma. Besides the already known receptor antagonists, new compounds directed to novel receptor types (e.g. cytokine, adenosine, adhesion molecules, etc.) are now under development. New targets in the cytosol will come into focus. Preliminary studies of selective phosphodiesterase (PDE) inhibitors in asthmatic patients have been encouraging. It is also very likely that the use of glucocorticoids cannot be excluded from therapy. However, we should generate new glucocorticoids with less side-effects, probably by using the so-called retrometabolic drug design. The first representative of this new steroid class, loteprednol is already approved for the therapy of certain allergic disorders. Because asthma is a disease of many different gene polymorphisms, gene therapy seems to be of low success at present. Alternatively, antisense oligonucleotides could be used. Future developments may also include strategies targeting the remodeling of structural elements of the airways. Today's intensive search for new treatments should ensure a greater diversity of therapeutic possibilities for the management of asthma in the next millennium.

Drug therapy in asthma bronchiale in the new millennium.

Asthma bronchiale represents a major health issue in industrialized countries and will likely remain so for decades. The drug treatment of asthma demonstrates certain peculiarities: revolutionary new drug introductions happen almost each quarter century. With improved understanding of asthma pathogenesis and drug metabolism, the potential for specific targeted and constructed therapies has become evident. Monoclonal antibodies to IgE and certain cytokines such IL-4 and IL-5 are being investigated as possible treatments for asthma. Similarly, preliminary studies of selective phosphodiesterase inhibitors in asthmatic patients have been encouraging. Other potential therapies include for example inhibitors of cytokine synthesis, promoters of Th2-Th1 switch, adenosine receptor agonists or antagonists, etc.. A new way is represented by a modified retrometabolic drug design resulting in so-called soft drugs. The first representative of this new drug class is loteprednol etabote (LE), a non-fluorinated glucocorticoid approved for the allergic ophthalmological indications and now in clinical trial for the treatment of allergic airway diseases. Today's intensive search for new treatments should ensure a greater diversity of therapeutic possibilities for the management of asthma in the new millennium.

Expression of cyclooxygenase-1 and -2 in normal and glaucomatous human eyes.

PURPOSE: Primary open-angle glaucoma (POAG) is the predominant form of chronic glaucoma, but the underlying pathologic mechanisms are largely unknown. Because prostaglandins (PGs) have been introduced into POAG treatment with remarkable success, this study was undertaken to investigate whether a change in the expression of the PG-synthesizing enzymes cyclooxygenase (COX)-1 and -2 might be involved in the pathogenesis of POAG. METHODS: Expression of COX-1 and -2 was assessed by confocal laser microscopy, immunohistochemistry, Western blot analysis, and real-time RT-PCR in human eyes with different forms of glaucoma (primary open-angle, angle-closure, congenital juvenile, and steroid-induced), as well as in age-matched control eyes. Additionally, PGE2 was measured in aqueous humor by means of an enzyme-linked immunoassay as a product of COX activity. RESULTS: In normal eyes, ocular COX-1 and -2 expression were largely confined to the nonpigmented secretory epithelium of the ciliary body. By immunohistochemistry and real-time RT-PCR, COX-2 expression was completely lost in the nonpigmented secretory epithelium of the ciliary body of eyes with end-stage POAG, whereas COX-1 expression was unchanged. By immunohistochemistry, in the ciliary bodies of eyes in five patients with diagnosis of early POAG, eyes in two had complete loss of COX-2 expression and in three showed only a few remaining scattered COX-2-expressing cells. COX-2 expression in the ciliary body was also lost in patients with steroid-induced glaucoma and was reduced in patients receiving topical steroid treatment. Eyes of patients with either congenital juvenile or angle-closure glaucoma showed COX-2 expression indistinguishable from control eyes. Aqueous humor of eyes with POAG contained significantly less PGE2 than control eyes. CONCLUSIONS: Both cyclooxygenase isoforms are constitutively expressed in the normal human eye. Specific loss of COX-2 expression in the nonpigmented secretory epithelium of the ciliary body appears to be linked to the occurrence of POAG and steroid-induced glaucoma.

R(+)-methanandamide induces cyclooxygenase-2 expression in human neuroglioma cells via a non-cannabinoid receptor-mediated mechanism.

Cannabinoids affect prostaglandin (PG) formation in the central nervous system through as yet unidentified mechanisms. Using H4 human neuroglioma cells, the present study investigates the effect of R(+)-methanandamide (metabolically stable analogue of the endocannabinoid anandamide) on the expression of the cyclooxygenase-2 (COX-2) enzyme. Incubation of cells with R(+)-methanandamide was accompanied by concentration-dependent increases in COX-2 mRNA, COX-2 protein, and COX-2-dependent PGE(2) synthesis. Moreover, treatment of cells with R(+)-methanandamide in the presence of interleukin-1beta led to an overadditive induction of COX-2 expression. The stimulatory effect of R(+)-methanandamide on COX-2 expression was mimicked by the structurally unrelated cannabinoid Delta(9)-tetrahydrocannabinol. Stimulation of both COX-2 mRNA expression and subsequent PGE(2) synthesis by R(+)-methanandamide was not affected by the selective CB(1) receptor antagonist AM-251 or the G(i/o) protein inactivator pertussis toxin. Enhancement of COX-2 expression by R(+)-methanandamide was paralleled by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK. Consistent with the activation of both kinases, R(+)-methanandamide-induced COX-2 mRNA expression and PGE(2) formation were abrogated in the presence of specific inhibitors of p38 MAPK (SB203580) and p42/44 MAPK activation (PD98059). Together, our results demonstrate that R(+)-methanandamide induces COX-2 expression in human neuroglioma cells via a cannabinoid receptor-independent mechanism involving activation of the MAPK pathway. In conclusion, induction of COX-2 expression may represent a novel mechanism by which cannabinoids mediate PG-dependent effects within the central nervous system.

Constitutive expression and localization of COX-1 and COX-2 in rabbit iris and ciliary body.

Prostaglandins are involved in the regulation of intraocular pressure and the blood-aqueous barrier of the eye, and are used for the treatment of glaucoma. The decrease of the constitutively expressed PG-synthesizing enzyme cyclooxygenase-2 (COX-2) has been demonstrated in the ciliary non-pigmented epithelial layer of patients with primary open angle glaucoma. Little is known about the distribution of COX-1 and COX-2 in animals. We investigated this in the iris and ciliary body of the normal rabbit eye. The presence of COX-1 and COX-2 in freshly excised iris and ciliary body tissue from adult New Zealand White albino rabbits was demonstrated by real-time RT-PCR, and Western blot analysis. The localization of both isoforms and of the neuron-specific protein gene product 9.5 was determined by indirect immunofluorescence. Both enzymes are expressed in the iris and the ciliary body. Immunofluorescence studies including double staining techniques localized COX-1 and COX-2 to about 50% of cells in the stromal tissue of iris and ciliary body, mainly on the corneal side. They were co-localized in about 75% of these cells. Whereas all stained cells were positive for COX-1, COX-2 showed a gradient-like distribution in the stroma, with some restriction of expression near the epithelial layers, which we clearly showed to be completely negative for both COX-1 and COX-2. Also, neuronal elements did not show COX-1 or COX-2 immunoreactivity. These results establish the presence of COX-1 and COX-2 on the RNA and protein levels in normal, unstimulated rabbit iris and ciliary body. The pattern of distribution suggests a role for both enzymes in maintaining the physiology of the eye. In contrast to our results in man, non-pigmented epithelial cells of the ciliary body did not express immunoreactivity. This could account for differences in the regulation of intraocular pressure and/or blood-aqueous barrier between human and rabbit eyes.

Flurbiprofen enantiomers inhibit inducible nitric oxide synthase expression in RAW 264.7 macrophages.

PURPOSE: Using RAW 264.7 macrophages, the present study investigates the influence of optically pure enantiomers of the nonsteroidal anti-inflammatory drug flurbiprofen on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression. METHODS: iNOS and cyclooxygenase-2 (COX-2) mRNA levels were measured by quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR). Concentrations of nitrite (index of cellular NO production) and prostaglandin E2 (index of COX-2 activity) in cell culture supernatants were determined by Griess assay and enzyme immunoassay, respectively. RESULTS: R(-)- and S(+)-flurbiprofen decreased LPS-induced iNOS mRNA and nitrite levels in an equipotent and concentration-dependent manner. Suppression of iNOS mRNA expression by R(-)- and S(+)-flurbiprofen was gene-specific in that both substances failed to inhibit LPS-induced COX-2 mRNA expression. By contrast, flurbiprofen enantiomers suppressed LPS-induced prostaglandin E2 formation enantioselectively with S(+)-flurbiprofen being considerably more potent than its R(-)-antipode. CONCLUSIONS: Our results show that R(-)- and S(+)-flurbiprofen, albeit differing in their potency as inhibitors of COX-2 activity, equipotently suppress iNOS expression. Because sustained high NO levels are associated with pain and tissue injury under various pathological conditions, a suppression of the inducible NO pathway may contribute to the pharmacological action of both R(-)- and S(+)-flurbiprofen.

Role of nitric oxide in zymosan induced paw inflammation and thermal hyperalgesia.

OBJECTIVE: To assess the involvement of spinal inducible nitric oxide synthase (iNOS) in inflammation and nociception. MATERIALS AND METHODS: The time course of iNOS mRNA expression in rat spinal cord and inflamed paw was assessed by means of quantitative real time RT-PCR. In addition, the effects of the iNOS inhibitor L-NIL on inflammatory paw edema and thermal hyperalgesia were studied in comparison to those of the NO-donor RE-2047. L-NIL (3, 9, 27 and 81 mg/kg) and RE-2047 (3, 9 and 27 mg/kg) or vehicle were administered orally 15 min prior to the intraplantar injection of 0.625 mg zymosan. RESULTS: Following zymosan injection, mRNA expression of iNOS increased in the inflamed paw and spinal cord with a maximum at 2.5 and 4 h, respectively. In the spinal cord iNOS mRNA started to decline at 10 h whereas it remained at maximum in the inflamed paw up to the end of the observation period of 24 h. As expected, RE-2047 had significant pronociceptive and proinflammatory effects. L-NIL significantly reduced paw inflammation at 27 and 81 mg/kg but failed to reduce hyperalgesia at the doses tested. CONCLUSIONS: The results show that iNOS is upregulated in the inflamed tissue and spinal cord with a similar time course. The effects obtained with L-NIL suggest that iNOS differently contributes to the inflammatory and nociceptive response induced by zymosan.

Modulation of synaptic transmission by nociceptin/orphanin FQ and nocistatin in the spinal cord dorsal horn of mutant mice lacking the nociceptin/orphanin FQ receptor.

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are two neuropeptides derived from the same precursor protein that exhibit opposing effects on spinal neurotransmission and nociception. Here, we have used whole-cell, patch-clamp recordings from visually identified neurons in spinal cord dorsal horn slices of genetically modified mice to investigate the role of the N/OFQ receptor (N/OFQ-R) in the modulatory action of both peptides on excitatory glutamatergic and inhibitory glycinergic and gamma-aminobutyric acid (GABA)-ergic synaptic transmission. In wild-type mice, N/OFQ selectively suppressed excitatory transmission in a concentration-dependent manner but left inhibitory synaptic transmission unaffected. In contrast, NST reduced only inhibitory but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated excitatory synaptic transmission. N/OFQ-mediated inhibition of excitatory transmission was completely absent in N/OFQ-R receptor-deficient (N/OFQ-R(-/-)) mice and significantly reduced in heterozygous (N/OFQ-R(+/-)) mice, whereas the action of NST on inhibitory neurotransmission was completely retained. To test for the relevance of these results for spinal nociception, we investigated the effects of intrathecally injected N/OFQ in the mouse formalin test, an animal model of tonic pain. N/OFQ (3 nmol/mouse) induced significant antinociception in wild-type mice, but had no antinociceptive effects in N/OFQ-R(-/-) mice. These results indicate that the inhibitory action of N/OFQ on excitatory glutamatergic synaptic transmission and its spinal antinociceptive action are mediated via the N/OFQ receptor, whereas the action of NST is independent of this receptor.

Importance of Kupffer cells for T-cell-dependent liver injury in mice.

T cells seem to be responsible for liver damage in any type of acute hepatitis. Nevertheless, the importance of Kupffer cells (KCs) for T-cell-dependent liver failure is unclear. Here we focus on the role of KCs and tumor necrosis factor (TNF) production after T cell stimulation in mice. T-cell- and TNF-dependent liver injury were induced either by Pseudomonas exotoxin A (PEA), by concanavalin A (Con A), or by the combination of subtoxic doses of PEA and the superantigen Staphylococcus enterotoxin B (SEB). KCs were depleted by clodronate liposomes. Although livers of PEA-treated mice contained foci of confluent necrosis and numerous apoptotic cells, hardly any apoptotic cells were observed in the livers of Con A-treated mice. Instead, large bridging necroses were visible. Elimination of KCs protected mice from PEA-, Con A-, or PEA/SEB-induced liver injury. In the absence of KCs, liver damage was restricted to a few small necrotic areas. KCs were the main source of TNF. Hepatic TNF mRNA and protein production were strongly attenuated because of KC-depletion whereas plasma TNF levels were unaltered. Our results suggest that KCs play an important role in T cell activation-induced liver injury by contributing TNF. Plasma TNF levels are poor diagnostic markers for the severity of TNF-dependent liver inflammation.

Cyclooxygenase-2 expression in lipopolysaccharide-stimulated human monocytes is modulated by cyclic AMP, prostaglandin E(2), and nonsteroidal anti-inflammatory drugs.

Using human blood monocytes (for determination of cyclooxygenase-2 (COX-2) mRNA by RT-PCR) and human whole blood (for prostanoid determination), the present study investigates the influence of the second messenger cAMP on lipopolysaccharide (LPS)-induced COX-2 expression with particular emphasis on the role of prostaglandin E(2) (PGE(2)) in this process. Elevation of intracellular cAMP with a cell-permeable cAMP analogue (dibutyryl cAMP), an adenylyl cyclase activator (cholera toxin), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) substantially enhanced LPS-induced PGE(2) formation and COX-2 mRNA expression, but did not modify COX-2 enzyme activity. Moreover, up-regulation of LPS-induced COX-2 expression was caused by PGE(2), butaprost (selective agonist of the adenylyl cyclase-coupled EP(2) receptor) and 11-deoxy PGE(1) (EP(2)/EP(4) agonist), whereas sulprostone (EP(3)/EP(1) agonist) left COX-2 expression unaltered. Abrogation of LPS-induced PGE(2) synthesis with the selective COX-2 inhibitor NS-398 caused a decrease in COX-2 mRNA levels that was restored by exogenous PGE(2) and mimicked by S(+)-flurbiprofen and ketoprofen. Overall, these results indicate a modulatory role of cAMP in the regulation of COX-2 expression. PGE(2), a cAMP-elevating final product of the COX-2 pathway, may autoregulate COX-2 expression in human monocytes via a positive feedback mechanism.

Suppressed injury-induced rise in spinal prostaglandin E2 production and reduced early thermal hyperalgesia in iNOS-deficient mice.

It is widely accepted that peripheral injury increases spinal inducible cyclooxygenase (COX-2) expression and prostaglandin E(2) (PGE(2)) formation as key mediators of nociceptive sensitization. Here, we used inducible nitric oxide synthase (iNOS) gene-deficient (iNOS-/-) mice to determine the contribution of iNOS-derived nitric oxide (NO) to this process. iNOS-/- mice exhibited reduced thermal hyperalgesia after zymosan injection. Spinal NO and PGE(2) formation both remained at baseline levels, in contrast to wild-type (wt) mice. In wt mice reduced hyperalgesia similar to that seen in iNOS-/- mice was induced by local spinal, but not by systemic treatment with the iNOS inhibitor l-NIL, suggesting that the reduced heat sensitization in iNOS-/- mice was attributable to the lack of spinal rather than peripheral iNOS. Two additional observations indicate that the antinociceptive effects of iNOS inhibition are dependent on a loss of stimulation of PG synthesis. First, intrathecal injection of the COX inhibitor indomethacin, which exerted pronounced antinociceptive effects in wt mice, was completely ineffective in iNOS-/- mice. Second, treatment with the NO donor RE-2047 not only completely restored spinal PG production and thermal sensitization in iNOS-/- mice but also its sensitivity to indomethacin. In both types of mice induction of thermal hyperalgesia was accompanied by similar increases in COX-1 and COX-2 mRNA expression. The stimulation of PG production by NO therefore involves an increase in enzymatic activity, rather than an alteration of COX gene expression. These results indicate that NO derived from spinal iNOS acts as a fast inductor of spinal thermal hyperalgesia.

Upregulation of cyclooxygenase-1 and the PGE2 receptor EP2 in rat and human mesangioproliferative glomerulonephritis.

OBJECTIVE AND DESIGN: Glomerular expression and localization of the two cyclooxygenase isoforms, Cox-1 and Cox-2, and the prostaglandin E2 receptor EP2 were investigated in a rat model of transient mesangioproliferative glomerulonephritis. Cox expression was also studied in biopsies from patients with IgA nephropathy. MATERIALS AND TREATMENT: After induction of glomerulonephritis by i.v. injection of a monoclonal anti-Thy1.1 antibody, rats were sacrificed at day 2, 6, 12 and 56. Changes in protein expression were detected by immunohistochemistry. Glomerular mRNA levels were analyzed by real time polymerase chain reaction (PCR). RESULTS: In normal rat kidney, immunoreactivity of Cox-1 was detected predominantly in collecting duct cells and that of Cox-2 in the macula densa. Cox-1 staining showed a massive transient increase in diseased glomeruli at day 6, localized mainly to mesangial cells coinciding with cell proliferation, which also peaked at day 6. Upregulation of Cox-1 was also evident at the mRNA level (4 fold). Cox-2 expression in the macula densa region transiently increased at day 6, but no significant upregulation of Cox-2 was observed in glomerular cells at any time point. Prostaglandin E2 receptor EP2 mRNA and protein were detected in rat glomeruli. EP2 immunoreactivity was prominent on podocytes in normal rats while at day 6 of the disease also mesangial cells stained positive. In biopsies of patients with IgA nephritis, predominant expression of Cox-1, but not Cox-2, was found in glomeruli, whereas Cox-2 was strongly expressed in infiltrating interstitial cells. CONCLUSIONS: The upregulation of glomerular Cox-1 but not Cox-2 and the parallel induction of the EP-2 receptor, which was shown to mediate cAMP accumulation in mesangial cells, suggest that induction of prostaglandin formation may contribute to the resolution rather than to the progression of anti-Thy1.1 nephritis. The expression pattern of Cox-1 and Cox-2 in human IgA nephritis points to a role for both Cox isoforms in human glomerular inflammation.

Deficiency in the transcription factor interferon regulatory factor (IRF)-2 leads to severely compromised development of natural killer and T helper type 1 cells.

Interferon (IFN) regulatory factor (IRF)-2 was originally described as an antagonist of IRF-1-mediated transcriptional regulation of IFN-inducible genes. IRF-1(-/)- mice exhibit defective T helper type 1 (Th1) cell differentiation. We have used experimental leishmaniasis to show that, like IRF-1(-/)- mice, IRF-2(-/)- mice are susceptible to Leishmania major infection due to a defect in Th1 differentiation. Natural killer (NK) cell development is compromised in both IRF-1(-/)- and IRF-2(-/)- mice, but the underlying mechanism differs. NK (but not NK(+) T) cell numbers are decreased in IRF-2(-/)- mice, and the NK cells that are present are immature in phenotype. Therefore, like IRF-1, IRF-2 is required for normal generation of Th1 responses and for NK cell development in vivo. In this particular circumstance the absence of IRF-2 cannot be compensated for by the presence of IRF-1 alone. Mechanistically, IRF-2 may act as a functional agonist rather than antagonist of IRF-1 for some, but not all, IFN-stimulated regulatory element (ISRE)-responsive genes.

Salicylate metabolites inhibit cyclooxygenase-2-dependent prostaglandin E(2) synthesis in murine macrophages.

The poor cyclooxygenase (COX) inhibitor and major aspirin metabolite salicylic acid is known to exert analgesic and anti-inflammatory effects by still unidentified mechanisms. In RAW 264.7 macrophages, lipopolysaccharide (LPS)-induced COX-2-dependent synthesis of prostaglandin E(2) (PGE(2)) was suppressed by aspirin (IC(50) of 5. 35 microM), whereas no significant inhibition was observed in the presence of sodium salicylate and the salicylate metabolite salicyluric acid at concentrations up to 100 microM. However, the salicylate metabolite gentisic acid (2,5-dihydroxybenzoic acid; 10-100 microM) and salicyl-coenzyme A (100 microM), the intermediate product in the formation of salicyluric acid from salicylic acid, significantly suppressed LPS-induced PGE(2) production. In contrast, gamma-resorcylic acid (2,6-dihydroxybenzoic acid) as well as unconjugated coenzyme A failed to affect prostanoid synthesis, implying that the para-substitution of hydroxy groups and the activated coenzyme A thioester are important for COX-2 inhibition. Using real-time RT-PCR, none of the salicylate derivatives tested were found to interfere with COX-2 expression. Overall, our results suggest that certain metabolites of salicylic acid may contribute to the pharmacological action of its parent compound by inhibiting COX-2-dependent PGE(2) formation at sites of inflammation.

Prostaglandin E(2) upregulates cyclooxygenase-2 expression in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Prostaglandin E(2) (PGE(2)) has been implicated in the regulation of inflammatory and immunological events. Using RAW 264.7 macrophages, the present study investigates the influence of PGE(2) on the expression of cyclooxygenase-2 (COX-2). Incubation of cells with PGE(2) increased lipopolysaccharide (LPS)-induced COX-2 mRNA levels in a concentration-dependent manner. Upregulation of COX-2 expression by PGE(2) was completely abolished by the specific adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and mimicked by butaprost, a selective agonist of the adenylyl cyclase-coupled PGE(2) receptor subtype 2 (EP(2)), or 11-deoxy PGE(1), an EP(2)/EP(4) receptor agonist. By contrast, the EP(3)/EP(1) receptor agonists 17-phenyl-omega-trinor PGE(2) and sulprostone left LPS-induced COX-2 expression virtually unaltered. Upregulation of LPS-induced COX-2 expression and subsequent PGE(2) synthesis was also observed in the presence of the cell-permeable cAMP analogue dibutyryl cAMP and the adenylyl cyclase activator cholera toxin. Together, our data demonstrate that PGE(2) potentiates COX-2 mRNA expression via an adenylyl cyclase/cAMP-dependent pathway. In conclusion, upregulation of COX-2 expression via an autocrine feed-forward loop may in part contribute to the well-known capacity of PGE(2)/cAMP to modulate inflammatory processes.

Nitric oxide inhibits inducible nitric oxide synthase mRNA expression in RAW 264.7 macrophages.

Using cultured murine RAW 264.7 macrophages, the present study investigates the influence of nitric oxide (NO) on the expression of the inducible NO synthase (iNOS) enzyme at the transcriptional level. Incubation of cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) led to a marked increase in iNOS mRNA levels. Inhibition of LPS/IFN-gamma-induced NO synthesis with the L-arginine analogue N(G)-monomethyl-L-arginine (L-NMMA) was accompanied by a significant up-regulation of iNOS mRNA that was reversed in the presence of the NO donor sodium nitroprusside (SNP). Treatment of cells with SNP alone decreased LPS/IFN-gamma-induced iNOS mRNA levels in a concentration-dependent manner. The inhibitory effect of SNP on iNOS mRNA expression was not prevented by 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase. In agreement with this finding, incubation of cells with the membrane-permeable cyclic GMP analogue 8-bromo cyclic GMP left LPS/IFN-gamma-induced iNOS mRNA expression virtually unaltered. Together, our results demonstrate that both iNOS-derived and exogenous NO exert an inhibitory effect on the expression of iNOS by a mechanism independent of the soluble guanylyl cyclase/cyclic GMP pathway. In conclusion, NO may control the extent of iNOS mRNA expression by a negative autoregulatory feedback.