Transcription factor p53 regulates healthy human ovarian cells function.

The aim of our study was to understand the role of transcription factor p53 in the control of healthy human ovarian cell functions. Ovarian granulosa cells were transfected with a cDNA construct encoding p53. The intracellular accumulation of p53, of the apoptosis marker bax, and of the proliferation marker PCNA, as well as the release of progesterone (P4), insulin-like growth factor I (IGF-I), oxytocin (OT), and prostaglandin F (PGF) and E2 (PGE) were evaluated by quantitative immunocytochemistry and RIA/IRMA. Transfection with the p53 cDNA construct resulted in the accumulation of p53 and bax, in a reduced level of released PCNA and PGF, and in an increased PGE output. No changes in P4, IGF-I, and OT secretion were found. These observations are the first demonstration of the involvement of p53 in the control of healthy human ovarian cell functions, namely, in the downregulation of proliferation, in the upregulation of apoptosis, and in the alteration of PGF and PGE release, but not of P4, IGF-I, or OT.

Increased eicosanoid levels in the Sugen/chronic hypoxia model of severe pulmonary hypertension.

Inflammation and altered immunity are recognized components of severe pulmonary arterial hypertension in human patients and in animal models of PAH. While eicosanoid metabolites of cyclooxygenase and lipoxygenase pathways have been identified in the lungs from pulmonary hypertensive animals their role in the pathogenesis of severe angioobliterative PAH has not been examined. Here we investigated whether a cyclooxygenase-2 (COX-2) inhibitor or diethylcarbamazine (DEC), that is known for its 5-lipoxygenase inhibiting and antioxidant actions, modify the development of PAH in the Sugen 5416/hypoxia (SuHx) rat model. The COX-2 inhibitor SC-58125 had little effect on the right ventricular pressure and did not prevent the development of pulmonary angioobliteration. In contrast, DEC blunted the muscularization of pulmonary arterioles and reduced the number of fully obliterated lung vessels. DEC treatment of SuHx rats, after the lung vascular disease had been established, reduced the degree of PAH, the number of obliterated arterioles and the degree of perivascular inflammation. We conclude that the non-specific anti-inflammatory drug DEC affects developing PAH and is partially effective once angioobliterative PAH has been established.

A heterogeneous mixture of F-series prostaglandins promotes sperm guidance in the Caenorhabditis elegans reproductive tract.

The mechanisms that guide motile sperm through the female reproductive tract to oocytes are not well understood. We have shown that Caenorhabditis elegans oocytes synthesize sperm guiding F-series prostaglandins from polyunsaturated fatty acid (PUFA) precursors provided in yolk lipoprotein complexes. Here we use genetics and electrospray ionization tandem mass spectrometry to partially delineate F-series prostaglandin metabolism pathways. We show that omega-6 and omega-3 PUFAs, including arachidonic and eicosapentaenoic acids, are converted into more than 10 structurally related F-series prostaglandins, which function collectively and largely redundantly to promote sperm guidance. Disruption of omega-3 PUFA synthesis triggers compensatory up-regulation of prostaglandins derived from omega-6 PUFAs. C. elegans F-series prostaglandin synthesis involves biochemical mechanisms distinct from those in mammalian cyclooxygenase-dependent pathways, yet PGF(2α) stereoisomers are still synthesized. A comparison of F-series prostaglandins in C. elegans and mouse tissues reveals shared features. Finally, we show that a conserved cytochrome P450 enzyme, whose human homolog is implicated in Bietti's Crystalline Dystrophy, negatively regulates prostaglandin synthesis. These results support the model that multiple cyclooxygenase-independent prostaglandins function together to promote sperm motility important for fertilization. This cyclooxygenase-independent pathway for F-series synthesis may be conserved.

Prostaglandin (F and E, 2- and 3-series) production and cyclooxygenase (COX-2) gene expression of wild and cultured broodstock of Senegalese sole (Solea senegalensis).

Prostaglandin levels in different tissues and cyclooxygenase (COX-2) gene expression were compared between wild and cultured Senegalese sole (Solea senegalensis) broodstock in which a significantly different fatty acid profile, particularly lower tissue levels of arachidonic acid (ARA, 20:4n-6) and higher levels of eicosapentaenoic acid (EPA, 20:5n-3) in the cultured fish compared to wild had already been described. This is the first report of the COX-2 mRNA expression in Senegalese sole. Cyclooxygenase (COX-2) mRNA expression and prostaglandin (2- and 3-series) levels were determined in tissues from 32 broodstock fish, 16 (8 males and 8 females) from each origin wild and cultured (G1). Transcripts of COX-2 were highly expressed in gills, sperm-duct (s-duct), testis, oviduct and spleen compared to liver, kidney and muscle. Differences in COX-2 transcripts expression were found in response to the origin of the fish and expression was significantly higher in s-duct and gills from wild fish compared to cultured. Wild fish showed significantly higher levels of total 2-series PGs and lower levels of 3-series compared to cultured fish. The significance of the lower COX-2 expression and lower PG 2-series production in some of the tissues of cultured fish was discussed in relation to the previously described differences in fatty acid profile (lower tissue levels of ARA and higher levels of EPA and EPA/ARA ratio in cultured fish) and the reproductive failure to spawn viable eggs from G1 cultured Senegalese sole compared to successful spawning from captive wild broodstock.

The effect of the pro-inflammatory cytokine tumor necrosis factor-alpha on human joint capsule myofibroblasts.

INTRODUCTION: Previous studies have shown that the number of myoblastically differentiated fibroblasts known as myofibroblasts (MFs) is significantly increased in stiff joint capsules, indicating their crucial role in the pathogenesis of post-traumatic joint stiffness. Although the mode of MFs' function has been well defined for different diseases associated with tissue fibrosis, the underlying mechanisms of their regulation in the pathogenesis of post-traumatic joint capsule contracture are largely unknown. METHODS: In this study, we examined the impact of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on cellular functions of human joint capsule MFs. MFs were challenged with different concentrations of TNF-alpha with or without both its specifically inactivating antibody infliximab (IFX) and cyclooxygenase-2 (COX2) inhibitor diclofenac. Cell proliferation, gene expression of both alpha-smooth muscle actin (alpha-SMA) and collagen type I, the synthesis of prostaglandin derivates E2, F1A, and F2A, as well as the ability to contract the extracellular matrix were assayed in monolayers and in a three-dimensional collagen gel contraction model. The alpha-SMA and COX2 protein expressions were evaluated by immunofluorescence staining and Western blot analysis. RESULTS: The results indicate that TNF-alpha promotes cell viability and proliferation of MFs, but significantly inhibits the contraction of the extracellular matrix in a dose-dependent manner. This effect was associated with downregulation of alpha-SMA and collagen type I by TNF-alpha application. Furthermore, we found a significant time-dependent upregulation of prostaglandin E2 synthesis upon TNF-alpha treatment. The effect of TNF-alpha on COX2-positive MFs could be specifically prevented by IFX and partially reduced by the COX2 inhibitor diclofenac. CONCLUSIONS: Our results provide evidence that TNF-alpha specifically modulates the function of MFs through regulation of prostaglandin E2 synthesis and therefore may play a crucial role in the pathogenesis of joint capsule contractures.

[Marine fungus Stilbella aciculosa as a potential producer of prostaglandins].

The amount and composition of fatty acids in the fungus Stilbella aciculosa associated with the marine macroorganism Apostichopus japonica (trepang) were determined by gas-liquid chromatography and gas chromatography-mass spectrometry. In the culture liquid of S. aciculosa, prostaglandins (PG) of groups E and F were revealed by UV spectroscopy. This finding was confirmed by the presence of direct precursors of PG, polyunsaturated eicosapentaenoic and docosahexaenoic acids, in the culture liquid. The biomass of this fungus contained PG of group B.

Regulation of luteal prostaglandin F(2 alpha) production and its relevance to cell death: an in vitro study using rat dispersed luteal cells.

We investigated the mechanism by which rat luteal cells produce prostaglandin F(2 alpha) (PGF(2 alpha)) and its relevance to cell death in vitro. Treatment with progesterone (P4) of dispersed luteal cells prepared from rats on day 9 of pseudopregnancy caused dose-dependent inhibition of PGF(2 alpha) secretion. Cytokines, tumor necrosis factor alpha (TNFalpha) or interferon gamma (IFN gamma) alone had no or modest regulatory effects. Arachidonyl trifluoromethyl ketone (AACOCF(3)), a specific group IVA phospholipase A(2) inhibitor, depressed both basal and cytokine-regulated PGF(2 alpha) production. A combination of TNFalpha and IFN gamma stimulated PGF(2 alpha) synthesis and cytotoxicity (both, P<0.05). Agonistic anti-Fas antibody challenge caused a significant cytotoxic effect but without affecting PGF(2 alpha) production. The present data suggest that P4 inhibits and TNFalpha and IFN gamma cooperatively stimulate PGF(2 alpha) release by rat luteal cells. They also suggest that luteal cell death induced by TNFalpha/IFN gamma and Fas stimulation seems to occur via distinct signaling pathways involving PGF(2 alpha) production.

Labor-associated regulation of prostaglandin E and F synthesis and action in the ovine amnion and cervix.

OBJECTIVE: Prostaglandins (PGs) are key regulators of cervical dilatation and membrane breakdown at the onset of labor. PG synthase and receptor expression has been previously documented in uterine tissues; however, mechanisms governing the changes occurring in the cervix and amnion are less well established. The aim of the current study was to determine the level of expression of PG synthetic enzymes and receptors in these tissues in association with induced labor in sheep. METHODS: Labor was induced in sheep at 135 days of gestation by continuous fetal dexamethasone infusion. Amnion and cervical tissue was obtained before and after labor for measurement of mRNA encoding enzymes (cytosolic phospholipase A2 [cPLA2], PGH synthase-2 [PGHS-2], PGF synthase [PGFS], and PGE synthase [PGES]) and receptors (FP and EP1-4) by real-time polymerase chain reaction (PCR). RESULTS: cPLA2 expression increased significantly in cervical tissue at labor onset, whereas expression of the other enzymes measured did not change. There was a marked rise in EP3 expression in the cervix, but abundance of this receptor was lower than EP2 and FP expression, which did not change. The amnion exhibited a labor-associated decrease in PGHS-2, PGFS, and FP mRNA expression. CONCLUSION: The regulation of PG synthesis and action occurring in the amnion and cervix in association with labor appear to differ markedly between the two tissues, indicating tissue-specific roles for PGs. The data support a role for increased PG synthesis and action in the cervix and suggest a decrease in PG production and action in the amnion, in sharp contrast to the pattern reported in human amnion.

The role of oxytocin, protein kinase A, and ERK-related MAP-kinase in the control of porcine ovarian follicle functions.

The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A (PKA), and mitogen-activated protein kinase (ERKs MAP-kinase) in the control of ovarian cell functions as well as the role of PKA and MAPK in mediating OT effects on these processes. The whole porcine ovarian follicles were cultured in the presence or absence of OT (1, 10, 100 ng/ml), PKA inhibitor Rp-cAMPS (10 nM), MAP-kinase inhibitor PD98059 (1 microg/ml), or their combination. The release of prostaglandins F (PGF) and E (PGE) were determined by RIA, PKA (alpha-cat subunit), the proliferation-associated peptide PCNA and ERK-1, -2 expression in cell lyzates were analysed by Western-blotting. OT stimulated the release of PGF and PGE, and accumulation of PKA, ERK-1/-2, and PCNA in cell lysate. PD98059 decreased the basal PGF and PGE output, as well as reduced both ERK-1 and ERK-2 accumulation in cell lysates. Rp-cAMPS decreased PKA accumulation in cell lysates. Rp-cAMPS prevented the OT-induced stimulation of PKA, ERK-1, ERK-2, PGF, and PGE, PD98059 did so for PKA, PGF, and PGE. However, PD98059 reduced either basal or OT-induced p-ERK level. OT-stimulated PCNA accumulation was only slightly modified by these blockers. These observations suggest that OT, PKA, and ERKs MAPK can be involved in the control of PGs release and proliferation of ovarian cells. The influence of OT on both PKA and MAPK, and the ability of PKA and MAPK blockers to prevent completely or partially OT effects suggest, that effects of OT on PGF and PGE can be mediated by both PKA and MAPK. The role of MAPK and PKA in mediating the proliferative effects of OT seems to be minor assuming the involvement of other intracellular messengers.

C-reactive protein decreases prostacyclin release from human aortic endothelial cells.

BACKGROUND: In addition to being a risk marker for cardiovascular disease, much recent data suggest that C-reactive protein (CRP) promotes atherogenesis. Decreased endothelial NO and prostacyclin (PGI2) contribute to a proatherogenic and prothrombotic state. We have shown that CRP decreases endothelial NO synthase expression and bioactivity in human aortic endothelial cells (HAECs). PGI2 is a potent vasodilator and inhibitor of platelet aggregation. Hence, the aim of this study was to examine the effect of CRP on PGI2 release from HAECs and human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: HAECs and HCAECs were incubated with human CRP (0 to 50 microg/mL for 24 hours). The release of PGF-1alpha, a stable product of PGI2, was also assayed in the absence and presence of a potent agonist, A23187. CRP significantly decreased PGF-1alpha release from HAECs under basal (48% decrease, P<0.001; n=5) and stimulated (26% decrease, P<0.01; n=5) conditions. CRP had no effect on PGI2 synthase (PGIS) mass. By increasing both superoxide and inducible NO synthase, CRP resulted in increased nitration of PGIS by peroxynitrite. The increased nitration and decreased activity of PGIS by CRP was reversed with peroxynitrite scavengers. CONCLUSIONS: Thus, CRP decreases PGI2 release from HAECs by inactivating PGIS via nitration, additionally contributing to its atherogenicity.

Prostaglandin F synthase.

Prostaglandin (PG) F2 is synthesized via three pathways from PGE2, PGD2, or PGH2 by PGE 9-ketoreductase, PGD 11-ketoreductase, or PGH 9-, 11-endoperoxide reductase, respectively. The enzymological and molecular biological properties of these enzymes have been reported in work over the last 30 years. Here, these three pathways of PGF synthesis by these enzymes are reviewed, and the physiological roles of the enzymes are discussed.

Investigations into the mechanisms controlling prostaglandin production by the guinea-pig placenta: roles of calcium and gonadotrophin-releasing hormone.

The outputs of PGF(2 alpha), PGE(2) and 6-keto-PGF(1 alpha) were higher from the day 29 guinea-pig placenta than from the sub-placenta in culture, with PGF(2 alpha)being the major prostaglandin produced by the placenta. Lack of extracellular calcium reduced the production of all three prostaglandins by the sub-placenta and 6-keto-PGF(1 alpha) production by the placenta, but had no effect on the production of PGF(2 alpha) and PGE(2) by the placenta. EGTA (a calcium chelator) and a low concentration (30 microM) of TMB-8 (an intracellular calcium antagonist) generally inhibited prostaglandin output from the placenta and sub-placenta at various time points during culture, although EGTA had no effect on PGE(2) output from the placenta. Trifluoperazine and W-7 (calmodulin inhibitors) had no inhibitory effect on the outputs of PGF(2 alpha) and PGE(2) from the placenta, nor on the outputs of any prostaglandin from the sub-placenta. However, these two compounds inhibited the output of 6-keto-PGF(1 alpha) from the placenta. Nifedipine and verapamil (calcium channel blocking drugs) generally reduced the outputs of prostaglandins from the placenta and sub-placenta, except verapamil had no inhibitory effect on PGF(2 alpha) output from the sub-placenta. Gonadotrophin-releasing hormone (GnRH) did not stimulate the output of prostaglandins from the placenta, and tended to have a weak inhibitory action on this tissue. On the sub-placenta, GnRH had an initial inhibitory action on the outputs of PGF(2alpha) and 6-keto-PGF(1 alpha), which was then followed by a stimulation of the outputs of PGF(2 alpha) and, to a lesser extent, of PGE(2).

Role of prostaglandins in the suppression of apoptosis in hen granulosa cells by transforming growth factor alpha.

Although transforming growth factor alpha (TGF-alpha) is known to be an important survival factor for granulosa cells, the cellular and molecular mechanisms involved are uncertain. The purpose of the present study was to investigate the possible involvement of prostaglandins in the anti-apoptotic action of TGF-alpha. Hen granulosa cells from healthy prehierarchical follicles (2-6 mm) cultured in serum-free medium underwent spontaneous apoptosis as demonstrated by DNA fragmentation and nuclear chromatin condensation. TGF-alpha (20 ng ml(-1)) stimulated maximum synthesis of prostaglandins (PGE and PGF) in granulosa cells and completely inhibited serum deprivation-induced apoptosis. The addition of an inhibitor of cyclooxygenase (COX; N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) or ibuprofen) or phospholipase A(2) (PLA(2); aristolochic acid, 2-p-amylcinnamoyl amino-4-chlorobenzoic acid (ONO-RS-82) or arachidonyl triflouro methyl ketone (TFMK)), to the culture medium markedly suppressed the TGF-alpha-induced prostaglandin synthesis and significantly increased granulosa cell apoptosis. The apoptotic effect of NS398 and aristolochic acid was completely inhibited by exogenous prostaglandins (PGF(2 alpha), PGE(1), PGE(2)) and arachidonic acid, respectively. However, exogenous prostaglandins failed to inhibit the PLA(2) inhibitor-induced apoptotic DNA fragmentation, implying that in addition to prostaglandins, arachidonic acid or leukotrienes may be important in transducing the anti-apoptotic action of TGF-alpha. In the absence of exogenous TGF-alpha, prostaglandins had no significant influence on granulosa cell apoptosis induced by serum withdrawal. These findings indicate that prostaglandin synthesis is a necessary, but not sufficient, event in the suppression of granulosa cell apoptosis by TGF-alpha. Whether arachidonic acid or leukotrienes are important in the anti-apoptotic action of TGF-alpha in hen granulosa cells remains to be determined.

The presence and role of the dopamine DA-2 receptor in the human decidua.

OBJECTIVES: Our objectives were to identify the presence of the dopamine DA-2 receptor in human decidua and to study its function in human parturition. METHODS: Human term decidual tissues were obtained during vaginal delivery and then homogenized. The P3 fraction was prepared for a radiolabeled receptor assay with [3H] spiperone as the ligand. Human decidual tissues obtained at cesarean section before the onset of labor were incubated in Krebs-Ringer buffer at 37 degrees C for 30 minutes in the presence of dopamine with or without (-)-sulpiride. The level of prostaglandin (PG) F in the medium was measured with a RIA kit. Differences were assessed with the Wilcoxon non-parametric test. RESULTS: Scatchard analysis showed a single class of binding sites having an equilibrium dissociation constant (Kd) of 2.25 +/- 0.59 nM (mean +/- SD) and a maximum binding capacity (Bmax) value of 166.5 +/- 77.7 fmol/mg protein (n = 3). Dopamine significantly increased the production of PGF. This stimulatory effect of dopamine was suppressed by (-)-sulpiride (p < 0.05; n = 7). CONCLUSION: The DA-2 receptor was demonstrated in the human decidua. Dopamine can stimulate PGF production via this receptor.

Allergen-induced synthesis of F(2)-isoprostanes in atopic asthmatics. Evidence for oxidant stress.

It is thought that reactive oxygen species (ROS) participate in the inflammation which characterizes asthma, but the evidence supporting this contention is incomplete. F(2)-isoprostanes (F(2)-IsoPs) are arachidonate products formed on membrane phospholipids by the action of ROS and thereby represent a quantitative measure of oxidant stress in vivo. Using a mass spectrometric assay we measured urinary release of F(2)-IsoPs in 11 patients with mild atopic asthma after inhaled allergen challenge. The excretion of F(2)-IsoPs increased at 2 h after allergen (1.5 +/- 0.2 versus 2.6 +/- 0.3 ng/mg creatinine) and remained significantly elevated in all urine collections for the 8-h period of the study (analysis of variance [ANOVA]). The measured compounds were of noncyclooxygenase origin because neither aspirin nor indomethacin given before challenge suppressed them. Urinary F(2)-IsoPs remained unchanged after inhaled methacholine challenge. In nine atopic asthmatics, F(2)-IsoPs were quantified in bronchoalveolar lavage fluid (BALF) at baseline values and in a separate segment 24 h after allergen instillation. F(2)-IsoPs were elevated late in the BALF (0.9 +/- 0.2 versus 11.4 +/- 3.0 pg /ml, baseline versus allergen, respectively, p = 0.007). The increase was inhibited by pretreatment of the subjects with inhaled corticosteroids. These findings provide a new evidence for a role for ROS and lipid peroxidation in allergen-induced airway inflammation.

Benzydamine reduces prostaglandin production in human gingival fibroblasts challenged with interleukin-1 beta or tumor necrosis factor alpha.

Benzydamine [1-benzyl-3-(3-dimethylamino)propoxy-1H-indazole] is a drug with analgesic, anesthetic, antimicrobial and anti-inflammatory activity. The purpose of the present study was to investigate the effect of benzydamine on prostaglandin production in human gingival fibroblasts. Benzydamine significantly reduced the basal production of both prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable breakdown product of prostaglandin I2 (PGI2), in unstimulated human gingival fibroblasts. When the cells were treated simultaneously with benzydamine and the cytokines IL-1 beta or TNF alpha, the agent benzydamine reduced (P < 0.05) the stimulatory effect of IL-1 beta and TNF alpha respectively, on PGE2 and PGI2 production in human gingival fibroblasts. Furthermore, benzydamine reduced (P < 0.05) both the basal level and the cytokine-induced 3H-arachidonic acid release 3H-(AA) in gingival fibroblasts. The addition of exogenous arachidonic acid to the cells resulted in enhanced PGE2 production, which was reduced (P < 0.05) in the presence of benzydamine. The study indicates that benzydamine reduces the prostaglandin synthesis in gingival fibroblasts, partly at the level of phospholipase A2, by diminishing the liberation of arachidonic acid (AA) from phospholipids, and partly at the level of cyclooxygenase. The inhibitory effect of benzydamine on prostaglandin production may explain the anti-inflammatory effect of the drug in the management of patients with oral inflammatory conditions.

Establishment and characterization of an osteocyte-like cell line, MLO-Y4.

Much attention has focused on osteoblast and osteoclast biology, but little research has been performed on the cell that composes 90% of all bone cells--the osteocyte. Osteocyte function has been difficult to study because these cells are embedded in mineralized tissue and are difficult to obtain in reasonable numbers and purity. Establishment of an osteocyte cell line makes it possible to study osteocyte function more readily and easily by the application of protein chemistry and molecular biology. We have established a cell line that appears to have the properties of primary osteocytes.

Thromboxane and prostacyclin in maternal and fetal circulation in pre-eclampsia.

OBJECTIVES: A major pathophysiologic change of pre-eclampsia has been attributed to the overproduction of thromboxane A2 (TXA2) mainly from activated platelets. On the other hand, increased biosynthesis of TXA2 has also been reported from preeclamptic placentas. The systemic role of these different sources of TXA2 has not been clarified. The purpose of this study is to define the changes of TXA2 and the antagonizing prostacyclin (PC) in maternal and fetal circulations. METHODS: The stable metabolites of TXA2 and PC [Thromboxine B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), respectively] in the cord and maternal blood of nine patients with pre-eclampsia and nine normal parturients were measured by radioimmunoassay. RESULT: In normal pregnancy, the cord blood contained much higher TXB2 (1697+/-898 vs. 267+/-128 ng/ml, P < 0.01) and 6-keto-PGF1alpha (266+/-263 vs. 12.5+/-3.9 ng/ml, P < 0.05) levels than the maternal blood. In the preeclamptic state, a marked increase of TXB2 was noted in both maternal and cord blood, reaching levels which were significantly higher than during normal pregnancy (2995+/-1103 vs. 267+/-128 ng/ml in maternal blood, P < 0.0001, and 3197+/-1288 vs. 1697+/-898 ng/ml in cord blood, P < 0.005). A less significant increase in 6-keto-PGF1alpha (134+/-10.8 vs. 12.5+/-3.9 ng/ml, P < 0.05) was also noted in the maternal blood. Moreover, the level of TXB2 correlated with the diastolic blood pressure of preeclamptic patients before and after delivery. CONCLUSION: The results suggest an abundant source of eicosanoids in the feto-placental circulation, which does not readily cross the placental barrier. In pregnancy complicated with pre-eclampsia, thromboxane level of both fetal and maternal circulations are markedly increased, which may be responsible for the pathophysiologic changes. The lack of adverse systemic effects on the fetus highlights a placental source of TXA2 of transient bioactivity which is rapidly hydrolyzed to non-active TXB2. Federation of Gynecology and Obstetrics

The role of cyclooxygenase-1 and cyclooxygenase-2 in lipopolysaccharide and interleukin-1 stimulated enterocyte prostanoid formation.

Lipopolysaccharide is an inflammatory agent and interleukin-1 is a cytokine. Their pro-inflammatory effects may be mediated by prostanoids produced by inducible cyclooxygenase-2. The aim of this study was to determine the prostanoids produced by lipopolysaccharide and interleukin-1 stimulated enterocytes through the cyclooxygenase-1 and 2 pathways. Cultured enterocytes were stimulated with lipopolysaccharide or interleukin-1beta with and without cyclooxygenase inhibitors. Low concentrations of indomethacin and valerylsalicylic acid (VSA) were evaluated as cyclooxygenase-1 inhibitors and their effects compared with the effects of a specific cyclooxygenase-2 inhibitor, SC-58125. Prostaglandin E2, 6-keto prostaglandin F1alpha, prostaglandin D2 and leukotriene B4 levels were determined by radioimmunoassay. Immunoblot analysis using isoform-specific antibodies showed that the inducible cyclooxygenase enzyme (COX-2) was expressed by 4 h in LPS and IL-1beta treated cells while the constitutive COX-1 remained unaltered in its expression. Interleukin-1beta and lipopolysaccharide stimulated the formation of all prostanoids compared with untreated cells, but failed to stimulate leukotriene B4. Indomethacin at 20 microM concentration, and VSA inhibited lipopolysaccharide and interleukin 1beta stimulated prostaglandin E2, but not 6-keto prostaglandin F1alpha formation. SC-58125 inhibited lipopolysaccharide and interleukin-1beta stimulated 6-keto prostaglandin F1alpha but not prostaglandin E2 release. The specific cyclooxygenase-2 inhibitor also inhibited lipopolysaccharide produced prostaglandin D2 but not interleukin-1beta stimulated prostaglandin D2. While SC-58125 inhibited basal 6-keto prostaglandin-F1alpha formation it significantly increased basal prostaglandin E2 and prostaglandin D2 formation. As SC-58125 inhibited lipopolysaccharide and interleukin-1beta induced 6-keto prostaglandin F1alpha production but not prostaglandin E2 production, it suggests that these agents stimulate prostacyclin production through a cyclooxygenase-2 mediated mechanism and prostaglandin E2 production occurs through a cyclooxygenase-1 mediated mechanism. Prostaglandin D2 production appeared to be variably produced by cyclooxygenase-1 or cyclooxygenase-2, depending on the stimulus.