Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs

Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.     

Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin 1alpha, tumor necrosis factor-alpha, glucocorticoids, and 17beta-estradiol

OBJECTIVE: To determine the effects of interleukin 1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), dexamethasone, and 17beta-estradiol on the expression of cyclooxygenase-1 (COX-1) and COX-2 in bovine chondrocytes. METHODS: Northern blot analysis was used to quantify COX-1 and COX-2 mRNA expression in primary cultures of bovine chondrocytes and prostaglandin production to evaluate COX activity. RESULTS: IL-1alpha and TNF-alpha increased the expression of COX-2. This effect was independent of de novo protein synthesis and dependent on increased mRNA stability in the case of IL-1alpha. Dexamethasone inhibited the effects of both cytokines. 17beta-estradiol inhibited COX-2 mRNA expression in basal conditions, but had no effect on COX-2 expression induced by cytokines. The specific COX-2 inhibitor compound NS 398 prevented the increase in prostaglandin E2 (PGE2) production induced by the cytokines. COX-1 levels remained stable with all treatments. CONCLUSION: Increase in mRNA stability is a mechanism implicated in the induction of COX-2 by some cytokines. The effects of IL-1alpha and TNF-alpha on PGE2 production are mainly due to an increase in COX-2 activity as shown by the effect of compound NS 398. 17beta-estradiol inhibits COX-2 mRNA expression in basal conditions, suggesting that estrogens could be implicated in the control of cartilage metabolism.     

Induction of cyclooxygenase-2 by parathyroid hormone in human osteoblasts in culture

OBJECTIVE: Parathyroid hormone (PTH) induced bone resorption by osteoclasts depends on the presence of osteoblasts. PTH induced production of prostaglandins by osteoblasts and induction of bone resorption by prostaglandins suggest that these autacoids may be implicated in the effects of PTH on bone. Our objective was to determine if the increase in prostaglandin production induced in human osteoblasts by PTH is due to an increase in cyclooxygenase-2 (COX-2) expression. METHODS: Primary cultures of human osteoblasts were obtained from specimens of trabecular bone. Confluent cells were treated with PTH, dexamethasone or compound NS-398, a specific COX-2 inhibitor. The concentration of prostaglandin E2 (PGE2) in the supernatants was determined by radioimmunoassay and COX-2 mRNA levels evaluated by Northern blot. RESULTS: PTH induced COX-2 mRNA expression and PGE2 production. These effects were time and concentration dependent and were inhibited by dexamethasone. Compound NS-398 reduced PGE2 production to the same extent as dexamethasone, and neither compound had an additive effect on this variable. CONCLUSION: These results show that PTH induces COX-2 expression in human osteoblasts in culture and suggest that this isoenzyme is the main factor in the control of prostaglandin synthesis in these experimental conditions.     

Identification of hepatitis B surface antigen variants with alterations outside the "a" determinant in immunized Singapore infants

Although histological studies have suggested that endothelial cells in bone (BDECs) are associated with some osteolytic bone diseases, it is still unclear how BDECs contribute to bone remodeling. Here we examined the response of BDECs to basic fibroblast growth factor (bFGF, FGF-2) using primary and cloned murine BDECs isolated from the femurs of BALB/c mice. Treatment of primary and cloned BDECs with bFGF induced cyclooxygenase-2 (COX-2) mRNA and protein expression. Furthermore, bFGF promotes the production of prostaglandin E2 (PGE2), which is known to be a potent stimulator of bone resorption and to induce osteoclast formation. Because the secretion of PGE2 was suppressed by COX-2 specific inhibitor NS-398 and by COX-2 antisense oligodeoxynucleotides, bFGF promotes the synthesis of PGE2 in a COX-2-dependent manner. Therefore, endothelial cells in bone are associated with bone remodeling by controlling COX-2 expression and consequently PGE2 production.     

Pharmacodynamics and pharmacokinetics of tolfenamic acid in ruminating calves: evaluation in models of acute inflammation

Injections of mild irritants intradermally (carrageenan, zymosan and dextran) and intracaveally (carrageenan) in a tissue cage model of inflammation were used in studies of the pharmacodynamics and pharmacokinetics of tolfenamic acid administered intramuscularly in calves. Inhibition of serum thromboxane (TX)B2 and inflammatory exudate prostaglandin (PG)E2 were used as indicators of the magnitude and time course of blockade of cyclo-oxygenase isoforms COX-1 and COX-2, respectively. Single doses of 2, 4 and 8 mgkg-1 tolfenamic acid partially inhibited irritant-induced rises in skin temperature (non-dose dependently) and skin oedema (dose-dependently). These doses also markedly inhibited serum TXB2 synthesis and the duration of inhibition was dose-related. A dose of 2 mgkg-1 tolfenamic acid also attenuated skin temperature rise over carrageenan-injected tissue cages, and markedly inhibited exudate PGE2 synthesis, even though drug penetration into both exudate and tissue cage transudate was limited. Tolfenamic acid pharmacokinetics were characterized by a relatively short tmax (0.94-2.04 h), a high estimated Vdarea (1.79-3.20 Lkg-1), an estimated t1/2 beta of 8.01-13.50 h and Cl beta of 0.142-0.175 Lkg-1h-1. The actions of tolfenamic acid in inhibiting PGE2 synthesis and in attenuating two of the cardinal signs of inflammation (heat and swelling) suggest that a dosage of 2 mgkg-1 administered intramuscularly should be effective clinically as an anti-inflammatory agent.     

Is there one autism or are there several kinds?]

Macrophages exudating into inflammatory sites (thioglycollate-elicited macrophages, TGM) have a diminished ability to synthesize prostaglandins (PG) as compared with resident peritoneal macrophages (RM). Constitutive expression of cyclooxygenase-1 (COX-1) was lower in TGM than in RM but the releasability of arachidonic acid was not significantly different. Thus, the differences in expression of COX-1 were primarily responsible for the abilities of TGM and RM to synthesize PGE2 upon calcium ionophore (CaI) stimulation. COX-1 expression in RM and TGM was also correlated with their ability to synthesize PGE2 from exogenously added arachidonic acid. When exposed to lipopolysaccharide (LPS), the induction of COX-2 and the enhancement of PGE2 synthesis upon CaI were much lower in TGM as compared with RM the releasability of arachidonic acid upon CaI stimulation was relatively unchanged in RM but was reduced in TGM Thus, in TGM as compared with RM, a lower level of COX-1 expression and a lower level of COX-2 induction, and the reduction of arachidonate releasability by LPS exposure, are mainly responsible for lower PGE2 synthetic ability upon CaI stimulation. However, the different COX-2 induction by LPS in RM and TGM was not reflected in their increase in the ability to synthesize PGE2 from exogenously added arachidonic acid.     

Tumor necrosis factor-alpha promotes sustained cyclooxygenase-2 expression: attenuation by dexamethasone and NSAIDs

Prostaglandin (PG) release is characteristic of most inflammatory diseases. The committed step in the formation of free arachidonic acid into PG products is catalyzed by cyclooxygenase (COX, prostaglandin H2 synthase, PGHS), which exists as two genetically distinct isoforms. COX-1 is constitutively expressed and produces PGs and thromboxane A2 during normal physiologic activities, while COX-2 is an inducible enzyme stimulated by growth factors, lipopolysaccharide, and cytokines during inflammation or cell injury. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) released into the amniotic fluid in the setting of infection have been proposed to signal amnion and decidual cells to produce PGs that may culminate in preterm labor. However, since the molecular control of this phenomenon has not been established, this study used amnion-derived WISH cells to determine if TNF-alpha promoted the formation of PGs through COX-2 activity. Treatment of WISH cells with TNF-alpha (0.1 ng/mL-100 ng/mL) caused a dose-dependent increase in COX-2 expression and the subsequent biosynthesis of PGE2 that persisted for at least 48 hrs. In contrast, COX-1 mRNA and protein levels were unaltered by TNF-alpha treatment as determined by RT-PCR and immunoblot analysis, respectively. TNF-alpha-stimulated COX-2 expression and the subsequent formation of PGE2 were inhibited by dexamethasone (0.1 microM). In addition, indomethacin (1 microM) and the novel COX-2-selective inhibitor, NS-398 (IC50 approximately 1.1 x 10(-9) M), attenuated TNF-alpha-elicited PGE2 production. Results presented here demonstrate that TNF-alpha elicits prolonged and regulatable induction of COX-2 in WISH cells, while COX-1 is constitutively expressed and unchanged in response to TNF-alpha stimulation.     

Inflammatory agonists induce cyclooxygenase type 2 expression by human neutrophils

The synthesis of prostanoids is regulated by cyclooxygenases (prostaglandin H synthases), which catalyze the conversion of arachidonic acid to PGH2. Cyclooxygenases are the target of aspirin and other nonsteroidal anti-inflammatory agents. In this study, we found that human polymorphonuclear leukocytes (PMNs) express the inducible isoform of cyclooxygenase, COX-2, when stimulated by LPS whereas the protein was not detectable in freshly isolated human PMNs. We also found by immunohistochemical analysis that COX-2 is expressed in PMNs in inflamed human tissues. COX-2 was induced in a time- and concentration-dependent fashion when isolated human PMNs were exposed to LPS; COX-2 was also induced, or its expression was increased, by TNF-alpha, IL-1, and IL-8. Expression of COX-2 in stimulated PMNs was paralleled by secretion of PGE2. The release of PGE2 was blocked by a selective nonsteroidal inhibitor of COX-2, indicating that the enzyme is responsible for the prostanoids produced, and was inhibited by dexamethasone. The time course of LPS-induced COX-2 expression and other features were different in freshly isolated PMNs, monocytes, and macrophages, indicating that COX-2 expression is differentially regulated in myeloid cells of different lineages and degrees of maturation. Consistent with this, IL-4 and IL-10, which suppressed LPS-induced COX-2 expression in monocytes, had little effect on this response by PMNs. These experiments demonstrate that PMNs express COX-2 when appropriately stimulated. Thus, they may actively influence the eicosanoid composition of the acute inflammatory milieu.     

Contribution of cyclooxygenase-1 and cyclooxygenase-2 to prostanoid formation by human enterocytes stimulated by calcium ionophore and inflammatory agents

The stimulation of intestinal epithelial cell cyclooxygenase (COX) enzymes with inflammatory agents and the inhibition of COX-1 and COX-2 enzymes has the potential to increase understanding of the role of these enzymes in intestinal inflammation. The aim of this study was to determine the contributions of COX-1 and -2 to the production of specific prostanoids by unstimulated and stimulated intestinal epithelial cells. Cultured enterocytes were stimulated with lipopolysaccharide (LPS), interleukin-1 (IL-1)beta (IL-1 beta), and calcium ionophore (Ca Ion), with and without COX inhibitors. Valerylsalicylic acid (VSA) was employed as the COX-1 inhibitor, and SC-58125 and NS398 were used as the COX-2 inhibitors. Prostanoids were quantitated by Elisa assay. Western immunoblotting demonstrated the presence of constitutive COX-1 and inducible COX-2 enzyme. Unstimulated prostanoid formation was not decreased by the COX-1 inhibitor. All of the stimulants evaluated increased prostaglandin E2 (PGE2) production. Only Ca Ion stimulated prostaglandin D2 (PGD2) production while IL-1 beta, and Ca Ion, but not LPS, increased prostaglandin F2 alpha (PGF2 alpha) formation. Ca Ion-stimulated prostanoid formation was uniformly inhibited by COX-2, but not COX-1, inhibitors. IL-1 beta-stimulated PGE2 and PGE2 alpha formation was significantly decreased by both COX-1 and COX-2 inhibitors. VSA, in a dose-dependent manner, significantly decreased IL-1 beta-stimulated PGE2 and PGF2 alpha production. Unstimulated prostanoid formation was not dependent on constitutive COX-1 activity. The stimulation of intestinal epithelial cells by Ca Ion seemed to uniformly produce prostanoids through COX-2 activity. There was no uniform COX-1 or COX-2 pathway for PGE and PGF2 alpha formation stimulated by the inflammatory agents, suggesting that employing either a COX-1 or COX-2 inhibitor therapeutically will have varying effects on intestinal epithelial cells dependent on the prostanoid species and the inflammatory stimulus involved.     

Prostaglandin E2 amplifies cytosolic phospholipase A2- and cyclooxygenase-2-dependent delayed prostaglandin E2 generation in mouse osteoblastic cells. Enhancement by secretory phospholipase A2

We used the MC3T3-E1 cell line, which originates from C57BL/6J mouse that is genetically type IIA secretory phospholipase A2 (sPLA2)-deficient, to reveal the type IIA sPLA2-independent route of the prostanglandin (PG) biosynthetic pathway. Kinetic and pharmacological studies showed that delayed PGE2 generation by this cell line in response to interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) was dependent upon cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2. Expression of these two enzymes was reduced by cPLA2 or COX-2 inhibitors and restored by adding exogenous arachidonic acid or PGE2, indicating that PGE2 produced by these cells acted as an autocrine amplifier of delayed PGE2 generation through enhanced cPLA2 and COX-2 expression. Exogenous addition or enforced expression of type IIA sPLA2 significantly increased IL-1beta/TNFalpha-initiated PGE2 generation, which was accompanied by increased expression of both cPLA2 and COX-2 and suppressed by inhibitors of these enzymes. Thus, our results revealed a particular cross-talk between the two PLA2 enzymes and COX-2 for delayed PGE2 biosynthesis by a type IIA sPLA2-deficient cell line. cPLA2 is responsible for initiating COX-2-dependent delayed PGE2 generation, and sPLA2, if introduced, enhances PGE2 generation by increasing cPLA2 and COX-2 expression via endogenous PGE2.     

No deterioration in insulin sensitivity, but impairment of both pancreatic beta-cell function and glucose sensitivity, in Japanese women with former gestational diabetes mellitus

Recent studies have demonstrated a strong correlation between infection and preterm labor. Preterm delivery is also associated with high levels of cytokines and prostaglandins in amniotic fluid. The purpose of this study was to investigate the effect of tumor necrosis factor-alpha (TNF-alpha) on the levels of cyclooxygenase, prostaglandin E2 production (PGE2), and expression of the PGE2 receptor subtype EP1 in amnion WISH cell culture. Amnion WISH cell cultures were incubated in increasing concentrations of TNF-alpha (0-50 ng/ml). Changes in cyclooxygenase and EP1 receptor proteins were evaluated by Western blot analysis. Changes in EP1 mRNA were evaluated by Northern blot, and culture fluid concentrations of PGE2 were estimated by enzyme immunoassay (EIA). EP1 protein (p<0.01), EP1 mRNA (p<0.05), cyclooxygenase-2 (COX-2) protein (p<0.001), and PGE2 concentrations (p<0.01) all increased with increasing concentrations of TNF-alpha. Changes in COX-1 protein were not observed following TNF-alpha-incubation. The results suggest that TNF-alpha may play a role in infection-induced preterm labor by its pleiotropic ability to simultaneously stimulate COX-2 activity, PGE2 concentrations, and PGE2 EP1 receptor levels in human amnion.     

Enhancement by cyclo-oxygenase inhibitors of platelet-activating factor production in thapsigargin-stimulated macrophages

1. Thapsigargin stimulated the accumulation of cell-associated platelet-activating factor (PAF) and extracellular prostaglandin E2 (PGE2) in rat peritoneal macrophages. PAF in the conditioned medium was less than the detectable amount. To obtain further insight into the mechanism of PAF accumulation, the role of PGE2 in PAF accumulation was investigated. 2. When macrophages were incubated in medium containing thapsigargin (30 ng ml(-1), 46.1 nM) and cyclo-oxygenase inhibitors such as indomethacin, naproxen or ibuprofen, the PAF content of the cells at 10 min was increased in a concentration-dependent manner in accordance with inhibition of PGE2 production. The stimulation by thapsigargin, cyclo-oxygenase inhibitors did not increase PAF accumulation. 3. In thapsigargin-stimulated macrophages, when PGE2(10(-7) M) was added to the medium, the cyclo-oxygenase inhibitor-induced stimulation of PAF accumulation at 10 min was markedly inhibited. 4. The accumulation of PAF induced by thapsigargin alone at 10 min was inhibited by exogenous PGE2 (10(-8) and 10(-7) M), or arachidonic acid (10(-6) and 10(-5) M) in accordance with the increase in PGE2 production. 5. The accumulation of PAF induced by thapsigargin alone or by thapsigargin and indomethacin (10(-6) M) was inhibited by dibutyryl cyclic AMP. 6. These results indicate that the concurrently produced PGE2 in thapsigargin-stimulated macrophages down-regulates PAF accumulation by increasing intracellular cyclic AMP levels, and that cyclo-oxygenase inhibitors increase PAF accumulation by inhibiting PGE2 production.     

Pathogenesis of Woronoff ring in psoriasis

As a result of ultraviolet light and coal tar therapy, a white ring (Woronoff) may develop in the normal skin adjacent to psoriatic plaques. Injection of prostaglandin E2 (PGE2) 1 cm outside of Woronoff ring produced redness in the ring, demonstrating that vessels within the ring were not unresponsive to PGE2. Whole skin homogenates from Woronoff ring contained an inhibitor of prostaglandin synthesis that was not found in uninvolved skin that was obtained from either psoriatics or normal controls. Prostaglandin E2 levels in the ring were one third of those in uninvolved skin from either psoriatics or normal controls. These findings suggest that the white ring that surrounds ultraviolet-light-treated psoriatic plaques is produced by a local inability to synthesize PGE2 in response to an ultraviolet light stimulus, resulting from the presence of an inhibitor of prostaglandin synthesis.     

The foot in hereditary motor and sensory neuropathies in children

We investigated the regulation of COX-2 expression and activity by adenosine receptors in rat microglial cells. The selective adenosine A2a-receptor agonist CGS21680 and the non-selective adenosine A1- and A2-receptor agonist 5'-N-ethylcarboxiamidoadenosine (NECA) induced an increase in COX-2 mRNA levels and the synthesis of prostaglandin E2 (PGE2). The adenosine A1-receptor agonist cyclopentyladenosine (CPA) was less potent, and the adenosine A1-receptor-specific agonist N6-2-(-aminophenylo)ethyladenosine (APNEA) showed only marginal effects. Microglia expressed adenosine A1-, A2a-, and A3-, but not A2b-receptor mRNAs, whereas astroglial cells expressed adenosine A2b- but not A2a-receptor mRNA. The adenosine A2a-receptor selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) inhibited both CGS21680-induced COX-2 expression and PGE2 release. CGS21680-increased PGE2 levels were inhibited by dexamethasone, by the nonsteroidal antiinflammatory drug meloxicam, and by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CGS21680 and NECA both increased intracellular cAMP levels in microglial cells. Dibutyryl cAMP as well as forskolin induced the release of PGE2. The results strongly suggest that adenosine A2a-receptor-induced intracellular signaling events cause an up-regulation of the COX-2 gene and the release of PGE2. Apparently, the cAMP second messenger system plays a crucial role in COX-2 gene regulation in rat microglial cells. The results are discussed with respect to neurodegenerative disorders of the CNS such as Alzheimer's disease, in which activated microglia are critically involved and COX inhibitors may be of therapeutic benefit.     

Suppression of CYP1A1 transcription by H2O2 is mediated by xenobiotic-response element

OBJECTIVE: To investigate the effect of amniotic fluid on prostaglandin synthesis and metabolism in the fetal membranes. DESIGN: A cell culture study of amnion and chorion obtained at elective cesarean section incubated with amniotic fluid collected following either spontaneous labor and delivery, or elective cesarean section. SUBJECTS: Forty-eight pregnant women at 3742 weeks gestation: 24 in spontaneous labor and 24 delivered by elective cesarean section. RESULTS: Significantly more PGE2 and PGF2alpha were produced by amnion and chorion treated with amniotic fluid from spontaneous labor compared with elective cesarean section. Spontaneous labor amniotic fluid favors PGE2 and PGFM production by amnion and chorion respectively; while elective section fluid stimulates PGE2 synthesis by both tissues (reflected as PGEM in chorion). Amniotic fluid, from either spontaneous labor or elective section, had no effect on the metabolism of exogenous PGE2 or PGF2alpha by chorion cells. CONCLUSION: Spontaneous labor is associated with the presence of a substance in amniotic fluid which facilitates prostaglandin synthesis in the fetal membranes, but which is without effect on prostaglandin metabolism.