Inhibited Prostaglandin E2 Release Research Articles

A Combination of Polymethoxyflavones from Citrus sinensis and Prenylflavonoids from Humulus lupulus Counteracts IL-1β-Induced Differentiated Caco-2 Cells Dysfunction via a Modulation of NF-κB/Nrf2 Activation.

We here investigated the anti-inflammatory activity of a polymethoxylated flavone-containing fraction (PMFF) from Citrus sinensis and of a prenylflavonoid-containing one (PFF) from Humulus lupulus, either alone or in combination (MIX). To this end, an in vitro model of inflammatory bowel disease (IBD), consisting of differentiated, interleukin (IL)-1β-stimulated Caco-2 cells, was employed. We demonstrated that non-cytotoxic concentrations of either PMFF or PFF or MIX reduced nitric oxide (NO) production while PFF and MIX, but not PMFF, also inhibited prostaglandin E2 release. Coherently, MIX suppressed both inducible NO synthase and cyclooxygenase-2 over-expression besides NF-κB activation. Moreover, MIX increased nuclear factor erythroid 2-related factor 2 (Nrf2) activation, heme oxygenase-1 expression, restoring GSH and reactive oxygen and nitrogen species (RONs) levels. Remarkably, these effects with MIX were stronger than those produced by PMFF or PFF alone. Noteworthy, nobiletin (NOB) and xanthohumol (XTM), two of the most represented phytochemicals in PMFF and PFF, respectively, synergistically inhibited RONs production. Overall, our results demonstrate that MIX enhances the anti-inflammatory and anti-oxidative effects of the individual fractions in a model of IBD, via a mechanism involving modulation of NF-κB and Nrf2 signalling. Synergistic interactions between NOB and XTM emerge as a relevant aspect underlying this evidence.

Parainfluenza virus infection enhances NSAIDs–induced inhibition of PGE2 generation and COX-2 expression in human airway epithelial cells

PurposeRespiratory viral infection and nonsteroidal anti-inflammatory drugs (NSAIDs) may affect arachidonic acid (AA) metabolism in the airway epithelium, however their joint effect has not been studied. We hypothesized, that alternations of AA metabolism in human airway epithelial cells (ECs) – induced by Parainfluenza virus type 3 (PIV3) – may be modified by concomitant treatment with NSAIDs. Materials and methodsNasal (RPMI 2650) and bronchial (BEAS-2B) epithelial cells were cultured into confluence and then infected with PIV3. Prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) levels in cell supernatants were measured by ELISA and expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) mRNA in cells was evaluated after reverse transcription with real-time polymerase chain reactions. ResultsPGE2 generation was decreased by PIV3 infection in the upper airway epithelial cells, and increased in the lower airway epithelial cells. Both naproxen and celecoxib induced significant reduction in PGE2 release in both infected and non-infected upper and lower airway epithelial cells. However, in PIV3-infected epithelial cells celecoxib inhibited PGE2 release and COX-2 expression to significantly higher degree as compared to non-infected cells. 15-HETE generation or COX-1, 5-LO and 15-LO expression were not affected by the virus infection or by NSAIDs. ConclusionVirus infection in airway epithelial cells enhances inhibitory effect of NSAIDs on prostaglandin E2 generation.

Expression and Molecular Regulation of the Cox2 Gene in Gastroenteropancreatic Neuroendocrine Tumors and Antiproliferation of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs).

BackgroundGastroenteropancreatic neuroendocrine tumors (GEP-NETs) has had a significant increase over the past 4 decades. The pathophysiological role of the cyclooxygenase-2 (cox-2) gene and factors responsible for the expression in GEP-NETs is of clinical value. Current study determined the expression of cox-2 gene in human GEP-NET tissues and corresponding cell lines, investigated the molecular mechanisms underlying the regulation of cox-2 gene expression and assessed the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on both anchorage-dependent and independent growth of GEP-NET cells.Material/MethodsGEP-NET tissues and QGP-1, BON, and LCC-18 GEP-NET cell lines were used. The expression of cox-2 gene was analyzed by immunohistochemistry, western blot, RT-PCR, and enzyme immunoassay. Transient transfection and luciferase assays along with electrophoretic mobility shift assays were conducted to explore the regulation of cox-2 gene expression. The effect of COX-inhibitors on GEP-NET cell growth was determined by proliferation assays and colony growth assessment.ResultsWe found 87.8% of GEP-NET tissues stained positive for COX-2. QGP-1 and LCC-18 cells expressed cox-2 gene. PGE2 (prostaglandin E2) amounts quantified in the supernatants of NET cells matched to cox-2 expression level. The CRE-E-box element (−56 to −48 bp) and binding of USF1, USF2, and CREB transcription factors to this proximal promoter element were essential for cox-2 promoter activity in GEP-NET cells. COX-2-specific inhibitor NS-398 potently and dose-dependently inhibited PGE2 release from QGP-1 cells. Interestingly, both NS-398 and acetylic salicylic acid effectively suppressed proliferation of QGP-1 and BON cells in a dose-dependent manner.ConclusionsThe majority of GEP-NETs over express cox-2 gene. The binding of CREB and USF-1/-2 transcription factors to a proximal, overlapping CRE-Ebox element is the underlying mechanism for cox-2 gene expression. NSAIDs potently suppressed the proliferations and may offer a novel approach for chemoprevention and therapy of GEP-NETs.

Compounds from Geijera parviflora with prostaglandin E2 inhibitory activity may explain its traditional use for pain relief

Ethnopharmacological relevanceAustralian Aboriginal people used crushed leaves of Geijera parviflora Lindl. both internally and externally for pain relief, including for toothache (Cribb and Cribb, 1981). This study tested the hypothesis that this traditional use might be at least in part explained by the presence of compounds with anti-inflammatory activity. Materials and methodsA crude extract (95% EtOH) was prepared from powdered dried leaves. From the CH3Cl fraction of this extract compounds were isolated by bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine leukemic monocyte–macrophages, (2) prostaglandin E2 (PGE2) inhibitory activity in 3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO) and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5) antibacterial activity against a panel of Gram-positive (Staphylococcus aureus ATCC 29213 and ATCC 25923, Staphylococcus epidermidis ATCC 35984, biofilm-forming) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains by broth microdilution. ResultsEleven compounds were isolated, including one new flavone and one new natural product, with a further four compounds reported from this species for the first time. Some of the compounds showed good anti-inflammatory activity in vitro. In particular, flindersine (1) and N-(acetoxymethyl) flindersine (3) inhibited PGE2 release with IC50 values of 5.0μM and 4.9μM, respectively, without any significant cytotoxicity. Several other compounds showed moderate inhibition of NO (5, 6, 7) and TNF-α (6), with IC50 in the low micromolar range; however much of this apparent activity could be accounted for by the cytotoxicity of these compounds. None of the compounds showed anti-bacterial activity. ConclusionsThe inhibition of PGE2, an important mediator of inflammation and pain, by flindersine and a derivative thereof, along with the moderate anti-inflammatory activity shown by several other compounds isolated from Geijera parviflora leaf extract, support the traditional use of this plant for pain relief by Australian Aboriginal people.

Ethanol extract of Poria cocos reduces the production of inflammatory mediators by suppressing the NF-kappaB signaling pathway in lipopolysaccharide-stimulated RAW 264.7 macrophages

BackgroundPoria cocos Wolf, a medicinal fungus, is widely used in traditional medicines in East Asian countries owing to its various therapeutic potentials. Although several studies have demonstrated the anti-inflammatory activity of this fungus, its underlying mechanisms have not yet been clearly defined.MethodsIn the present study, we have demonstrated the anti-inflammatory effects of ethanol extract of P. cocos (EEPC) in lipopolysaccaride (LPS)-stimulated RAW 264.7 macrophages. As inflammatory parameters, the productions of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-1β and tumor necrosis factor (TNF)-α were evaluated. We also examined the EEPC’s effect on the nuclear factor-kappaB (NF-κB) signaling pathway.ResultsOur results indicated that EEPC exhibits a potent inhibitory effect on NO production and inhibits PGE2 release in LPS-induced macrophages without affecting cell viability. EEPC also significantly attenuated LPS-induced secretion of inflammatory cytokines IL-1β and TNF-α. Additionally, LPS-induced expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, IL-1β, and TNF-α was decreased by pre-treatment with EEPC at the transcriptional level. Moreover, EEPC clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits, which correlated with EEPC’s inhibitory effects on inhibitor kappaB (IκB) degradation. Moreover, EEPC clearly suppressed the LPS-induced DNA-binding activity of NF-κB, as well as the nuclear translocation of the NF-κB p65, which correlated with EEPC’s inhibitory effects on inhibitor kappaB (IκB) degradation.ConclusionsTaken together, our data indicates that EEPC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2, IL-1β, and TNF-α through inactivation of the NF-κB signaling pathway, supporting the pharmacological basis of P. cocos as a traditional herbal medicine for treatment of inflammation and its associated disorders.

Regulation of Nerve Growth Factor by Anti-Inflammatory Drugs, a Steroid, and a Selective Cyclooxygenase 2 Inhibitor in Human Intervertebral Disc Cells Stimulated With Interleukin-1

Regulation of nerve growth factor (NGF) by 2 different anti-inflammatory drugs was investigated in vitro using isolated human intervertebral disc (IVD) cells stimulated with the proinflammatory cytokine interleukin-1 (IL-1). To investigate the regulation of NGF by a synthetic steroid and a selective cyclooxygenase-2 (COX-2) inhibitor and to clarify the biological role of prostaglandin E2 (PGE2) in this process. NGF is known to play an important role in pain, including low back pain, and to be induced by proinflammatory cytokines in IVD cells. However, the effect of clinically used drugs for managing low back pain on the regulation of NGF is unclear. Isolated human IVD cells were stimulated with interleukin-1 (IL-1) in the presence or absence of dexamethasone or a selective COX-2 inhibitor (NS-398). NGF expression and release were determined by real-time polymerase chain reaction and enzyme-linked immuno sorbent assay, respectively. Inhibition of PGE2 release was determined by enzyme-linked immuno sorbent assay. The effects of exogenous PGE2 and its receptor (E-series prostanoid receptors [EPs] 1-4) agonists were also tested for NGF regulation. IL-1 transiently induced, in a dose-dependent manner, the induction of NGF in human IVD cells. Pretreatment with dexamethasone strongly inhibited the NGF expression, whereas NS-398 significantly enhanced it at the concentration at which PGE2 release was substantially inhibited. Exogenous PGE2 inhibited IL-1 induction of NGF and this effect was mimicked when EP2 and EP4, but not EP1 and EP3, agonists were supplemented to the culture. Although selective COX-2 inhibitors have been shown to be effective for acute low back pain by inhibiting PGE2 release, our findings suggest that it may have a limited efficacy because it exaggerated NGF expression, whereas dexamethasone inhibited it. On the other hand, PGE2 had an inhibitory function for NGF induction by mediating EP2/4 in human IVD cells. Further studies are needed to clarify whether these observations could take place in vivo. N/A.

Inhibition of prostaglandin E2 release by salicylates, benzoates and phenols: a quantitative structure-activity study.

Concentrations inhibiting 50% of the prostaglandin E2 release from phorbol ester-stimulated mouse peritoneal macrophages in-vitro were determined for 59 monosubstituted congeners of salicylic acid, benzoic acid and phenol. Twenty-seven further compounds, mainly benzoic acids, were found to be inactive. An attempt was made to establish a quantitative structure-activity relationship (QSAR) form our experimental data using literature or calculated values for the logarithmic n-octanol/water partition coefficients of the compounds, molar refractivity and sigma values of substituents as well as structural indicator variables. The equations found have moderate predictive power and must be considered as a first step in the investigation of factors determining the biological activity of salicylates, benzoates and phenols.

A selective cyclooxygenase‐2 inhibitor decreases transforming growth factor‐β1 synthesis and matrix production in human peritoneal mesothelial cells

High glucose concentration, which provides the chief driving force in peritoneal dialysis, has been considered to be an important initial factor that contributes to peritoneal thickening and fibrosis. Human peritoneal mesothelial cells (HPMCs) and the expansion of extracellular matrix (ECM) play important roles in the pathological process of peritoneal fibrosis. Peritoneal matrix accumulation is a characteristic of peritoneal fibrosis (PF). Continuous ambulatory peritoneal dialysis (CAPD) patients with upregulation of transforming growth factor-beta1 (TGF-beta1) in their drained effluent show an increased risk of PF. Inhibition of TGF-beta1 expression in human peritoneal mesothelial cells (HPMCs) may provide a potential treatment for PF. sc58236, a highly selective cyclooxygenase-2 (COX-2) inhibitor, reduces proteinuria and mRNA expression of TGF-beta1 and collagen III and IV in the remnant kidney, but their effects on ECM turnover in HPMCs are unknown. The aims of this study were to investigate the effects of sc58236 on TGF-beta1 expression and matrix production in HPMCs. HPMCs were cultured from human omentum by an enzyme digestion method. To examine the effect of sc58236 on TGF-beta1 and ECM secretion, HPMC were incubated in medium F12 with high concentration of D-glucose (4.25%) in the presence and absence of 20 microM sc58236. The mRNA expression of COX-2, TGF-beta1 and collagen I (Col I) were determined by semi-quantification reverse transcription PCR (RT-PCR). Prostaglandin E2 (PGE2) concentration in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA). The protein of TGF-beta1 was determined by ELISA and the activity of TGF-beta1 present in the conditioned media was measured using the mink lung epithelial cell (MLEC) growth inhibition assay. Proteins of COX-2 and Col were determined by Western blot. The results showed that primary cultures of HPMCs do express the mRNA for COX-2 and stimulation of these cells with 4.25% D-glucose induced a marked increase in COX-2 mRNA expression and protein. PGE2 expression was obviously up-regulated with stimulation by 4.25% D-glucose. Addition of 20 microM sc58236 significantly inhibited PGE2 release into the culture medium. mRNA expression and bioactive and total protein of TGF-beta1 and Col I were significantly increased in HPMCs stimulated with 4.25% D-glucose compared to the control group with F12 media, which was reversed in the presence of sc58236 (20 microM). An obvious decrease in TGF-beta1 mRNA expression and bioactive and total protein were found in sc58236 treated groups compared to the group stimulated with 4.25% D-glucose. Exposure of HPMCs to sc58236 reduced Col I secretion. Sc58236 reduces the expression of TGF-beta1 in HPMCs stimulated by 4.25% D-glucose and reduces ECM production through PGE2 production. These studies suggest that sc58236, a highly selective cyclooxygenase-2 (COX-2) inhibitor, may have a specific role in ameliorating the course of progressive peritoneal fibrosis under long-term peritoneal dialysis states.

Prostaglandin E2 receptors EP1 and EP4 are up-regulated in rabbit chondrocytes by IL-1β, but not by TNFα

Prostaglandin E2 (PGE2) exerts its actions through the binding of the high affinity EP receptors. We wanted to evaluate the regulation of EP1 and EP4, and the expression of cyclooxygenase (COX)-2, main enzyme responsible for PGE2 synthesis in inflammatory situations, in healthy rabbit chondrocytes stimulated with inflammatory mediators locally increased during osteoarthritis. Articular chondrocytes obtained from healthy rabbits were stimulated with interleukin (IL)-1beta (0.1-100 u/ml) or tumour necrosis factor (TNF)alpha (100 ng/ml). Where indicated, cells were preincubated with non-steroidal antiinflammatory drugs (NSAIDs) (10(-6) M) to inhibit PGE2 synthesis. IL-1beta induced a dose and time-dependent increase in EP1, EP4 and COX-2 expression. However, TNFalpha presence did not induce a significant modification in EP1, EP4 or COX-2 gene expression at any time of study. NSAID presence significantly inhibited PGE2 release but did not modify the EP receptors or COX-2 expression induced by IL-1beta. Our results indicate that EP1 and EP4 receptors, and COX-2 are up-regulated in IL-1beta-stimulated chondrocytes, while no significant modifications are observed in TNFalpha-stimulated cells. NSAIDs were unable to modify the expression of these mediators induced by IL-1beta. Therefore, the increase in PGE2 synthesis, induced by IL-1beta, does not seem to mediate the increase in EP receptor expression, in rabbit chondrocytes.

Glucocorticoids and TGF-beta1 synergize in augmenting fibroblast mediated contraction of collagen gels.

TGF-beta plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF-beta and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF-beta in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF-beta for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E2 (PGE2) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE2 release by HFL-1 fibroblasts. TGF-beta significantly augmented gel contraction but also induced a 30% increase in PGE2 release and increased the expression of COX-1. Glucocorticoids inhibited TGF-beta1 induced-PGE2 release, and enhanced TGF-beta augmented gel contraction without significantly affecting TGF-beta augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF-beta augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF-beta in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE2 production. Such interactions between glucocorticoids and TGF-beta may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.

Fluoxetine and amitriptyline inhibit nitric oxide, prostaglandin E2, and hyaluronic acid production in human synovial cells and synovial tissue cultures.

To evaluate the effects of fluoxetine and amitriptyline on nitric oxide (NO), prostaglandin E2 (PGE2), and hyaluronic acid (HA) production in human synovial cells and synovial tissue cultures. Human synovial cells, synovial tissue, and cartilage were cultured in the presence or absence of cytokines, lipopolysaccharides (LPS), fluoxetine, or amitriptyline. Production of NO, PGE2, and HA was determined in culture media. Sulfated glycosaminoglycan (S-GAG) synthesis was evaluated in cartilage by 35S incorporation. Fluoxetine (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) inhibited NO release by 56%, 62%, and 71%, respectively, in the media of synovial cells stimulated by interleukin-1alpha (IL-1alpha; 1 ng/ml) plus tumor necrosis factor alpha (TNFalpha; 30 ng/ml). Amitriptyline (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) caused a 16%, 27.3%, and 51.4% inhibition of NO release. Fluoxetine and amitriptyline (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) significantly (P<0.05) inhibited PGE2 release in the media of human synovial cells in the presence of IL-1alpha plus TNFalpha, in a dose-dependent manner (up to 88% inhibition). Fluoxetine (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) and amitriptyline (1 microg/ml and 3 microg/ml) significantly (P<0.05) inhibited PGE2 release in the media of human synovial tissue in the presence of LPS. Fluoxetine and amitriptyline (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) also significantly (P<0.05) inhibited HA production by human synovial cells in the presence of IL-1beta plus TNFalpha. Fluoxetine and amitriptyline (1 microg/ml) partially reversed IL-1beta-induced inhibition of 35S-GAG synthesis by human cartilage cultures (P<0.05). Neither fluoxetine nor amitriptyline had a toxic effect on cells in the concentrations used. Inhibition of NO and PGE2 production by connective tissue cells is a mechanism by which some antidepressant medications may affect pain, articular inflammation, and joint damage.

Inhibitory effects of shimotsu-to, a traditional Chinese herbal prescription, on ultraviolet radiation-induced cell damage and prostaglandin E2 release in cultured Swiss 3T3 cells

We have investigated the effects of Shimotsu-to, a traditional Chinese herbal prescription (Kampo medicine), on ultraviolet (UV) radiation-induced cell damage and prostaglandin E2 (PGE2) release in cultured Swiss 3T3 cells to examine the anti-inflammatory mechanism of Shimotsu-to. Short-term UV irradiation significantly induced cell damage and stimulated PGE2 release in Swiss 3T3 cells cultured for 4 h. The UV-radiation-induced cell damage and the stimulation of PGE2 release were significantly suppressed by the treatment with Shimotsu-to. Among the single crude drug components of Shimotsu-to, Rehmanniae Radix showed significant protective effects against UV-radiation-induced cell damage and PGE2 release. Other synthetic anti-inflammatory agents, such as dexamethasone, dipotassium glycyrrhizinate and allantoin also protected against UV-induced cell damage and inhibited PGE2 release in cultured Swiss 3T3 cells. These results suggest that the anti-inflammatory mechanism of Shimotsu-to against UV-irradiated erythema (acute skin inflammation) in guinea pigs in vivo may be mediated by the inhibition of PGE2 release from cutaneous target cells.

Sodium salicylate inhibits cyclo-oxygenase-2 activity independently of transcription factor (nuclear factor kappaB) activation: role of arachidonic acid.

Acetylsalicylic acid (aspirin) is the drug most commonly self-administered to reduce inflammation, swelling, and pain. The established mechanism of action of aspirin is inhibition of the enzyme cyclo-oxygenase (COX). Once taken, aspirin is rapidly deacetylated to form salicylic acid, which may account, at least in part, for the therapeutic actions of aspirin. However, where tested, salicylic acid has been found to be a relatively inactive inhibitor of COX activity in vitro, despite being an effective inhibitor of prostanoids formed at the site of inflammation in vivo. Recently, the identification of a cytokine-inducible isoform of COX, COX-2, has led to the suggestion that salicylate produces its anti-inflammatory actions by inhibiting COX-2 induction through actions on nuclear factor kappaB (NF-kappaB). We have used interleukin 1beta-induced COX-2 in human A549 cells to investigate the mechanism of action of salicylate on COX-2 activity. Sodium salicylate inhibited prostaglandin E2 release when added together with interleukin 1beta for 24 hr with an IC50 value of 5 microg/ml, an effect that was independent of NF-kappaB activation or COX-2 transcription or translation. Sodium salicylate acutely (30 min) also caused a concentration-dependent inhibition of COX-2 activity measured in the presence of 0, 1, or 10 microM exogenous arachidonic acid. In contrast, when exogenous arachidonic acid was increased to 30 microM, sodium salicylate was a very weak inhibitor of COX-2 activity with an IC50 of >100 microg/ml. Thus, sodium salicylate is an effective inhibitor of COX-2 activity at concentrations far below those required to inhibit NF-kappaB (20 mg/ml) activation and is easily displaced by arachidonic acid.

The co-ordinate regulation of lipocortin 1, COX 2 and cPLA2 by IL-1 beta in A549 cells.

In the A549 human lung adenocarcinoma cell line, generation of prostaglandin E2 (PGE2) is an important regulator of cell proliferation in vitro (1). Both epidermal growth factor (EGF) and the pro-inflammatory cytokine IL-1β up regulate cell proliferation by inducing PGE2 release. The PGE2 mediated increase in cell proliferation can be inhibited by dexamethasone. The inhibition of PGE2 release by the dexamethasone is mediated via the induction of lipocortin 1 on the surface of A549 cells (1). Furthermore, we have shown the EGF simulated arachidonic acid release, PGE2 release and subsequent cell proliferation can all be repressed by the addition of fragments of lipocortin-1 to the cells (2). These results imply that cell surface lipocortin 1 can regulate arachidonic acid release, and when EGF simulated cells are treated with dexamethasone the increase in cell surface lipocortin 1 is responsible for down-regulating arachidonic acid release, PGE2 release and cell proliferation.

T-614, a novel antirheumatic drug, inhibits both the activity and induction of cyclooxygenase-2 (COX-2) in cultured fibroblasts.

To elucidate the mechanism for the selective inhibition of prostaglandin E2 (PGE2) production in inflammatory tissue by T-614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne), its effects on both the activity and the induction of cyclooxygenase (COX)-2 were investigated in vitro. T-614 inhibited the activity of purified COX-2 enzyme (IC50: 7.7 micrograms/ml), but was inactive against both COX-1 activities of microsomal and purified enzymes (IC50: > 300 micrograms/ml). On the other hand, when the inhibition of PGE2 production by T-614 was examined in the cultured fibroblasts stimulated with bradykinin, T-614 at 1 microgram/ml or less inhibited PGE2 release more effectively than that in the above cell-free system. Therefore, we examined which of the COX enzymes was expressed in bradykinin-stimulated fibroblasts by using both the reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses. As a result, COX-1 mRNA was constitutively expressed in the cells, whereas COX-2 mRNA was not detected without stimulation with bradykinin, but was expressed within 30 min when stimulated. Furthermore, it was found that the addition of T-614 reduced the COX-2 mRNA levels in 30 min after stimulation. These studies suggest that at least some of inhibitory effects of T-614 on prostanoids production are mediated by the synergy of the inhibition of COX-2 activity and the inhibition of induction, and such an action of T-614 may explain the pharmacological properties of this drug.

Effects of butylated hydroxyanisole on arachidonic acid and linoleic acid metabolism in relation to gastrointestinal cell proliferation in the rat.

In order to determine the effect of oral administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA; dose-level: 1.5% BHA of the diet) on arachidonic acid (AA) and linoleic acid (LA) metabolism in correlation with changes in gastrointestinal cell kinetics, we coadministered two inhibitors of prostaglandin H synthase, acetylsalicylic acid (ASA) and indomethacin (IM), to rats. Coadministration of ASA (0.2%) and IM (0.002%) in the drinking water, resulted in a significant reduction of the BHA-induced enhancement of cell proliferation in forestomach and glandular stomach. ASA completely counteracted the effect of BHA on labeling indices in colon/rectum whereas IM exhibited no effect in this organ. Both inhibitors had no direct effect on cell kinetics in the control groups. ASA, and to a lesser degree IM, inhibited prostaglandin E2 release in all tissues examined. Whereas ASA did inhibit lipoxygenase-mediated metabolism of AA in forestomach tissue, ASA did not affect the release of AA- and LA-derived hydroxy fatty acids in glandular stomach and colon/rectum. IM did not affect lipoxygenase production. BHA, however, appeared to be a strong inhibitor of both routes of AA metabolism. While ASA nor IM affected LA metabolism, BHA inhibited both prostaglandin H synthase-mediated and lipoxygenase-mediated metabolism of AA and LA. A causal role of AA or LA metabolites in the process of cell proliferation enhancement induced by BHA, can therefore be excluded. Prostaglandin H synthase may, however, be involved in BHA activation by converting the hydroquinone metabolite of BHA to the corresponding quinone by redox cycling, which is probably accompanied by reactive intermediate production.

Interleukin 1 alpha inhibits prostaglandin E2 release to suppress pulsatile release of luteinizing hormone but not follicle-stimulating hormone.

Interleukin 1 alpha (IL-1 alpha), a powerful endogenous pyrogen released from monocytes and macrophages by bacterial endotoxin, stimulates corticotropin, prolactin, and somatotropin release and inhibits thyrotropin release by hypothalamic action. We injected recombinant human IL-1 alpha into the third cerebral ventricle, to study its effect on the pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in conscious, freely moving, ovariectomized rats. Intraventricular injection of 0.25 pmol of IL-1 alpha caused an almost immediate reduction of plasma LH concentration; this decrease was statistically significant 20 min after injection and occurred through a highly significant reduction in the number of LH pulses, with no effect on pulse amplitude. In contrast, there was no change in pulse frequency but a small significant elevation in amplitude of FSH pulses. Intraventricular injection of the diluent had no effect on gonadotropin release. The results provide further evidence for separate hypothalamic control mechanisms for FSH and LH release. To determine the mechanism of the suppression of LH release, mediobasal hypothalamic fragments were incubated in vitro with IL-1 alpha (10 pM) and the release of LH-releasing hormone (LHRH) and prostaglandin E2 into the medium was measured by RIA in the presence or absence of norepinephrine (50 microM). IL-1 alpha reduced basal LHRH release and blocked LHRH release induced by norepinephrine. It had no effect on the basal release of prostaglandin E2; however, it completely inhibited the release of PGE2 evoked by norepinephrine. To evaluate the possibility that IL-1 alpha might also interfere with the epoxygenase pathway of arachidonic acid metabolism, epoxyeicosatrienoic acids were also measured. IL-1 alpha had no effect on the content of epoxyeicosatrienoic acids in the hypothalamic fragments as measured by gas chromatography and mass spectrometry. In conclusion, IL-1 alpha suppresses LH but not FSH release by an almost complete cessation of pulsatile release of LH in the castrated rat. The mechanism of this effect appears to be by inhibition of prostaglandin E2-mediated release of LHRH.

Inhibition of prostaglandin E 2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.

Down-regulation of natural killer cell activity by autologous polymorphonuclear leucocytes. Role of indomethacin.

It has been recently reported that neutrophils are involved in the regulation of NK cell activity. However, the mechanism of such regulation is unclear. The present study was designed to investigate the mechanisms involved in the regulation of NK cytotoxicity by human neutrophils. The role of indomethacin, an anti-inflammatory drug, in this interaction was studied. NK cells were purified from peripheral blood obtained from normal individuals. NK cell cytotoxicity was tested on K 562 cell line by Cr release assay. Autologous neutrophils obtained from peripheral blood were stimulated by opsonized zymosan either in the presence or absence of indomethacin. The role of neutrophil supernatant containing oxygen radicals and prostaglandins on NK cytotoxicity was examined. It was shown that supernatants from stimulated neutrophils significantly inhibited (P less than 0.05) the autologous NK cell cytotoxicity. The presence of indomethacin in the in vitro reaction mixture, or given orally to donors, partially or completely abolished the inhibitory effect of neutrophil supernatant. Indomethacin inhibited prostaglandin E2 release, and luminol-enhanced, myeloperoxidase-mediated chemiluminescence of activated PMN. Diafiltration of neutrophil supernatant showed that the inhibitory activity was present in the fraction containing molecules lower than 5,000 daltons. In conclusion, our findings indicate that down-regulation of NK cytotoxicity is mediated by prostaglandins produced by stimulated neutrophils and possibly by oxygen radicals.

Prostaglandin production by calvariae from sham operated and oophorectomized rats: effect of 17 beta-estradiol in vivo.

The mechanism by which estrogen inhibits bone resorption in vivo is not known. Since prostaglandin E2 (PGE2) is a potent stimulator of bone resorption in vitro, we tested the hypothesis that estrogens might affect bone indirectly by inhibiting endogenous PGE2 synthesis. Cultured parietal bones from 7- to 9-week-old rats showed a gradual release of immunoreactive PGE2 in vitro. Oophorectomy resulted in a two-fold increase in PGE2 release. Treatment in vivo with low doses of 17 beta-estradiol or high doses of 17 alpha-estradiol 26 h and 2 h prior to sacrifice inhibited bone PGE2 release in vitro. Addition of 17 beta-estradiol to calvariae in vitro did not affect PGE2 release, whereas cortisol inhibited PGE2 release.