The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

Tryptase activates calcium-independent phospholipase A2 and releases PGE2 in airway epithelial cells

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.     

5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B₄ (LTB₄) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E₂ (PGE₂) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0–120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB₄ signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB₄. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE₂ reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro. leukotriene B₄; prostaglandin E₂; spreading; migration; bridges     

Bradykinin increases IL-8 generation in airway epithelial cells via COX-2-derived prostanoids

Interleukin (IL)-8, the C-X-C chemokine, is a potent neutrophil chemoattractant that has been implicated in a number of inflammatory airway diseases such as cystic fibrosis. Here we tested the hypothesis that bradykinin, an inflammatory mediator and chloride secretagogue, would increase IL-8 generation in airway epithelial cells through autocrine generation of endogenous prostanoids. Bradykinin increased IL-8 generation in both a non-cystic fibrosis (A549) and cystic fibrosis epithelial cell line (CFTE29) that was inhibited by the nonselective cyclooxygenase (COX) inhibitor indomethacin and the COX-2 selective inhibitor NS-398. COX-2 was the only isoform of COX expressed in both cell lines. Furthermore, the COX substrate arachidonic acid and exogenous prostaglandin E(2) both increased IL-8 release in A549 cells. These results suggest that bradykinin may contribute to neutrophilic inflammation in the airway by generation of IL-8 from airway epithelial cells. The dependence of this response on endogenous production of prostanoids by COX-2 suggests that selective COX-2 inhibitors may have a role in the treatment of airway diseases characterized by neutrophilic inflammation such as cystic fibrosis or chronic obstructive pulmonary disease.     

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.     

Cyclooxygenase and cytochrome P-450 pathways induced by fetal calf serum regulate wound closure in 3T6 fibroblast cultures through the effect of prostaglandin E2 and 12 and 20 hydroxyeicosatetraenoic acids

Wound-induced injury of 3T6 fibroblast cultures initiated a repair process stimulated by fetal calf serum (FCS) that restored the integrity of cell cultures. In these experimental conditions, FCS induced arachidonic acid (AA) release and eicosanoid production. Our results show that the inhibition of the cyclooxygenase (COX) and/or cytochrome P-450 pathways significantly decreases the wound closure, whereas that of the lipoxygenase pathway does not modify the wound repair process. Both EP(1) and EP(4) receptors of prostaglandin E(2) (PGE(2)) mediate PGE(2) stimulated 3T6 fibroblast wound closure. Our data suggest that calcium and cAMP are involved in the signaling event induced by PGE(2) during the 3T6 fibroblast wound repair process. On the other hand, we show that ketoconazole, a cytochrome P-450 inhibitor, hinders the wound closure induced by FCS in wounded 3T6 fibroblast cultures. 12 and 20 Hydroxyeicosatetraenoic acids (HETEs), which are key AA metabolites synthesized by cytochrome P-450, partially revert the effects of ketoconazole on the wound repair process. Thus, the COX and cytochrome P-450 pathways of the arachidonate cascade are involved in 3T6 fibroblast wound closure.     

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.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

Colon carcinoma cell growth is associated with prostaglandin E2/EP4 receptor-evoked ERK activation

Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E(2) (PGE(2)) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE(2.) Treatment with indomethacin, or COX-selective inhibitors, prevents PGE(2) biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE(2) or the EP4 receptor-selective agonist PGE(1)-OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE(2)-induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE(2), suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers.     

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.     

Thiyl radicals in biosystems: inhibition of the prostaglandin metabolism by the cis-trans-isomerization of arachidonic acid double bonds

This paper describes parallel and comparative experiments on the enzymatic cyclooxygenase (COX) driven conversion of arachidonic acid (AA, all-cis-5,8,11,14-eicosatetraenoic acid) into prostaglandins by using pure arachidonic acid and AA samples containing relatively small amounts of thiyl radical induced trans-isomers. The experiments were performed in a liquid aqueous model system using COX-1 as well as by the in vitro feeding of VD(3)-differentiated and LPS-stimulated promyelocytic HL-60 cells using the cell's own COX-2. In the model solution, all the different test methods used (oxygen consumption, ROS induced luminescence, and TMPD oxidation) indicated the greatly disproportionate, non-stoichiometric inhibition of the prostaglandin metabolism by the trans-isomers. Accordingly, measurements performed in the cell system gave comparable results: both luminescence ROS detection and the ELISA test on PGE(2) expression resulted in the strong inhibition of the prostaglandin metabolism. We interpret these findings as enzyme blocking caused by just one mono-trans-isomerized double bond of AA.     

Inhibition of both COX-1 and COX-2 is required for development of gastric damage in response to nonsteroidal antiinflammatory drugs.

We examined the gastric ulcerogenic property of selective COX-1 and/or COX-2 inhibitors in rats, and investigated whether COX-1 inhibition is by itself sufficient for induction of gastric damage. Animals fasted for 18 h were given various COX inhibitors p.o., either alone or in combination, and they were killed 8 h later. The nonselective COX inhibitors such as indomethacin, naproxen and dicrofenac inhibited PG production, increased gastric motility, and provoked severe gastric lesions. In contrast, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach, with no effect on the mucosal PGE(2) contents and gastric motility. Likewise, the selective COX-1 inhibitor SC-560 also did not cause gastric damage, despite causing a significant decrease in PGE(2) contents. The combined administration of SC-560 and rofecoxib, however, provoked gross damage in the gastric mucosa, in a dose-dependent manner. SC-560 also caused a marked gastric hypermotility, whereas rofecoxib had no effect on basal gastric motor activity. On the other hand, the COX-2 mRNA was expressed in the stomach after administration of SC-560, while the normal gastric mucosa expressed only COX-1 mRNA but not COX-2 mRNA. These results suggest that the gastric ulcerogenic property of conventional NSAIDs is not accounted for solely by COX-1 inhibition and requires the inhibition of both COX-1 and COX-2. The inhibition of COX-1 up-regulates the COX-2 expression, and this may counteract the deleterious influences, such as gastric hypermotility and the subsequent events, due to a PG deficiency caused by COX-1 inhibition.     

Expression and activity of cyclooxygenase isoforms in skeletal muscles and myocardium of humans and rodents.

Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity in striated muscles, including skeletal muscles and myocardium, in particular it is still unclear whether muscle cells are able to produce prostaglandins (PGs). We characterized the expression and enzymatic activity of COX-1 and COX-2 in the skeletal muscles and in the myocardium of mice, rats and humans. By RT-PCR, COX-1 and COX-2 mRNAs were observed in homogenates of mouse and rat hearts, and in different types of skeletal muscles from all different species. By Western blotting, COX-1 and -2 proteins were detected in skeletal muscles and hearts from rodents, as well as in skeletal muscles from humans. Immunoperoxidase stains showed that COX-1 and -2 were diffusely expressed in the myocytes of different muscles and in the myocardiocytes from all different species. In the presence of arachidonic acid, which is the COX enzymatic substrate, isolated skeletal muscle and heart samples from rodents released predominantly PGE(2). The biosynthesis of PGE(2) was reduced between 50 and 80% (P < 0.05 vs. vehicle) in the presence of either COX-1- or COX-2-selective blockers, demonstrating that both isoforms are enzymatically active. Exogenous PGE(2) added to isolated skeletal muscle preparations from rodents did not affect contraction, whereas it significantly fastened relaxation of a slow type muscle, such as soleus. In conclusion, COX-1 and COX-2 are expressed and enzymatically active in myocytes of skeletal muscles and hearts of rodents and humans. PGE(2) appears to be the main product of COX activity in striated muscles.     

Tumor cell-derived prostaglandin E2 inhibits monocyte function by interfering with CCR5 and Mac-1.

The cyclooxygenases (COX)-1 and COX-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Whereas COX-1 is expressed ubiquitously, COX-2 is an immediate-early gene often associated with malignant transformation, and a role for the COX enzymes in tumor initiation and promotion is discussed. Nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin and indomethacin that block COX-1 and -2 have been shown to have beneficial effects for tumor patients. Therefore, these compounds have gained interest also among oncologists. However, the molecular mechanism by which NSAIDs inhibit carcinogenesis is not clearly understood. The prostaglandin-dependent and -independent effect may both account for their antineoplastic action. We show here that tumor cells derived from different tumors regularly produce prostaglandin E(2) (PGE(2)) interfering with the function of monocytes. In particular, PGE(2) inhibits the potential of monocytes to migrate in the direction of a chemotactic stimulus and to adhere to endothelial cell. This inhibition is most probably due to a modulation of the chemokine receptor CCR5 and the beta2-integrin Mac-1. Both down-regulation of CCR5 and reduced expression of Mac-1 may diminish the potential of peripheral blood monocytes to leave blood vessels and invade target tissues. Since both dysfunctions can be restored with NSAIDs, our findings help to explain the molecular chemopreventive action of NSAIDs on tumor formation and progression.     

ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E(2) (PGE(2)) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 +/- 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 +/- 1.8 and 14.3 +/- 1.7% compared with K(+) (72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP(1) antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE(2) induced only a slight phasic contraction in guinea pig trachea, but PGE(2) induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE(2) generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE(2) production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP(1) receptor.     

Cyclooxygenase-2 stimulates production of amyloid beta-peptide in neuroblastoma x glioma hybrid NG108-15 cells

Cyclooxygenase (COX) synthesizes bioactive prostaglandins from arachidonic acid, and there are COX-1 and COX-2 isoforms with distinct pathophysiological functions. Recent studies demonstrated that COX-2 expression was up-regulated in the brain of patients with Alzheimer's disease. We established mouse neuroblastoma x rat glioma hybrid NG108-15 cells stably expressing human COX-2. The COX-2-expressing cells showed 3- to 4-fold increases in both COX activity and prostaglandin E(2) production. The mRNA level of amyloid precursor protein (APP) was elevated by approximately 2-fold in the COX-2-expressing cells compared with mock-transfected cells. Amyloid beta-peptide and a secreted form of APP, both derived from APP by proteolysis was also increased. Interestingly, neurite outgrowth was stimulated in the COX-2-expressing cells with concomitant reduction of the cell proliferation rate. A selective COX-2 inhibitor (JTE-522) and a nonselective COX inhibitor (indomethacin) suppressed production of amyloid beta-peptide and a secreted form of APP by inhibition of APP mRNA level, suggesting that COX-2 plays important roles in the neurodegenerative processes of Alzheimer's disease.     

Prostaglandins mediate tonic contraction of the guinea pig and human gallbladder

The gallbladder (GB) maintains tonic contraction modulated by neurohormonal inputs but generated by myogenic mechanisms. The aim of these studies was to examine the role of prostaglandins in the genesis of GB myogenic tension. Muscle strips and cells were treated with prostaglandin agonists, antagonists, cyclooxygenase (COX) inhibitors, and small interference RNA (siRNA). The results show that PGE2, thromboxane A2 (TxA2), and PGF(2alpha) cause a dose-dependent contraction of muscle strips and cells. However, only TxA2 and PGE2 (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and TxB2 (TxA2 stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PGE2 levels. Both inhibitors and the nonselective COX inhibitor indomethacin abolished the tonic contraction. Transfection of human GB muscle strips with COX-1 siRNA partially lowered the tonic contraction and reduced COX-1 protein expression and TxB2 levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PGE2. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, TxB2, and PGE2 levels; a COX-1 inhibitor prevented the increase in tension and TxB2; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PGE2. Indomethacin blocked the rise in tension and TxB2 and PGE2 levels. We conclude that PGE2 generated by COX-2 and TxA2 generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PGE2 and TxA2 levels.