PGE2 reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors,but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E₂ (PGE₂) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3′5′-monophosphate (cAMP). PGE₂ reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G_s-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE₂. Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE₂ were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G_s-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE₂ reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE₂ is mediated via the EP3 receptor. In contrast, PGE₂ added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE₂ is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE₂ may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.     

Molecular cloning and characterization of bovine prostaglandin E2 receptors EP2 and EP4: expression and regulation in endometrium and myometrium during the estrous cycle and early pregnancy

Prostaglandins (PGs) play important functions in the reproductive system, and PGE(2) appears necessary for recognition of pregnancy. We have found that PGE(2) is able to increase cAMP generation in the bovine endometrium. There are two PGE(2) receptors (EP), EP2 and EP4, that are coupled to adenylate cyclase to generate cAMP, but these receptors have not been studied in the bovine. We have cloned and characterized bovine EP2 and EP4 receptors and studied their expression in the uterus. The amino acid sequences of bovine EP2 and EP4 possess a high degree (>80%) of identity with the other mammalian homologs. EP2 is expressed in most tissues, and EP4 is expressed only in intestine and testis. EP2 mRNA and protein are expressed in endometrium and myometrium during the estrous cycle, whereas EP4 is undetectable. The Western analysis indicates that EP2 is maximally expressed in both endometrium and myometrium between d 10 and 18 of the estrous cycle. Immunohistochemical localization reveals that EP2 protein is expressed in all cell types of endometrium and myometrium. On d 18, pregnancy up-regulates EP2 protein, primarily in endometrial stroma and myometrial smooth muscle cells. In conclusion, EP2 is the major cAMP-generating PGE(2) receptor expressed and regulated in the bovine uterus during the estrous cycle and early pregnancy.     

Expression, localization, and signaling of PGE(2) and EP2/EP4 receptors in human nonpregnant endometrium across the menstrual cycle

This study was designed to elucidate the sites of synthesis and action of PGE(2) in the nonpregnant human uterus across the menstrual cycle. The sites of expression of PGE synthase and synthesis of PGE(2) were investigated by immunohistochemistry using full thickness uterine biopsies. Expression of PGE synthase and synthesis of PGE(2) were localized to glandular epithelial and endothelial cells in both basalis and functionalis regions of the human endometrium. By contrast, stromal staining was predominantly localized in the functionalis layer. Some cyclical variation in expression of PGE synthase and PGE(2) synthesis was observed, with reduced expression/synthesis detected in the stromal compartment of the functionalis during the late secretory phase of the menstrual cycle. Subsequently, we assessed the site of action of PGE(2) by investigating the expression of two PGE(2) receptor isoforms, namely EP2 and EP4. Cyclical variation in endometrial EP2 and EP4 receptor mRNA expression was quantified by TaqMan quantitative RT-PCR using RNA isolated from endometrial tissue collected across the menstrual cycle. No differences in EP2 receptor mRNA expression were detected; however, EP4 receptor mRNA expression was significantly higher in late proliferative stage (P < 0.05) than in early, mid, and late secretory stage endometrium. Expression patterns of EP2 and EP4 receptors were localized by nonradioactive in situ hybridization using fluorescein isothiocyanate end- labeled oligonucleotide probes. Expression of both receptors was observed in endometrial glandular epithelial and vascular cells, with no notable spatial or temporal variation. Finally, signaling of EP2/EP4 receptors was assessed by investigating cAMP generation in vitro after stimulation with PGE(2). Endometrial cAMP generation in response to PGE(2) was significantly greater in proliferative tissue compared with early and midsecretory stage tissue (3.77 +/- 0.85 vs. 1.96 +/- 0.28 and 1.38 +/- 0.23, respectively; P < 0.05). In conclusion, this study demonstrates glandular and vascular coexpression of PGE synthase, PGE(2), EP2, and EP4 receptors and suggests an autocrine/paracrine role for PGE(2) in epithelial/endothelial cell function in the human endometrium.     

16,16-Dimethyl Prostaglandin E2 Inhibits Indomethacin-Induced Small Intestinal Lesions Through EP3 and EP4 Receptors

We evaluated the effect of various PGE analogs specific to EP receptor subtypes on indomethacin-induced small intestinal lesions in rats and investigated the relationship of EP receptor subtype with the PGE action using EP receptor knockout mice. Animals were administered indomethacin subcutaneously, and they were killed 24 hr later. 16,16-dimethyl prostaglandin E₂ (dmPGE₂) or various EP agonists were administered intravenously 10 min before indomethacin. Indomethacin caused hemorrhagic lesions in the rat small intestine, accompanied with an increase in intestinal motility and the number of enteric bacteria as well as iNOS and MPO activities. Prior administration of dmPGE₂ dose-dependently prevented intestinal lesions, together with inhibition of those functional changes. These effects of dmPGE₂ were mimicked by prostanoids (ONO-NT-012 and ONO-AE1-329), only specific to EP3 or EP4 receptors, although the intestinal motility was inhibited only by ONO-AE1-329. Intestinal mucus secretion and fluid accumulation were decreased by indomethacin but enhanced by dmPGE₂, ONO-NT-012, and ONO-AE1-329 at the doses that prevented intestinal lesions. Indomethacin also caused intestinal lesions in both wild-type and knockout mice lacking EP1 or EP3 receptors, yet the protective action of dmPGE₂ was observed in wild-type and EP1 receptor knockout mice but not the mice lacking EP3 receptors. These results suggest that the intestinal cytoprotective action of PGE₂ against indomethacin is mediated by EP3/EP4 receptors and that this effect is functionally associated with an increase of mucus secretion and enteropooling as well as inhibition of intestinal hypermotility, the former two processes mediated by both EP3 and EP4 receptors, and the latter by EP4 receptors.     

Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2

Recognition and establishment of pregnancy involve several molecular and cellular interactions among the conceptus, uterus, and corpus luteum (CL). In ruminants, interferon-tau (IFNtau) of embryonic origin is recognized as the pregnancy recognition signal. Endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolysin, whereas PGE(2) is considered a luteoprotective or luteotrophic mediator at the time of establishment of pregnancy. The interplay between IFNtau and endometrial PGs production, transport, and signaling at the time of maternal recognition of pregnancy (MRP) is not well understood. We have studied the expression of enzymes involved in metabolism of PGE(2) and PGF(2alpha), cyclooxygenase-1 (COX-1) and COX-2, PG synthases (PGES and PGFS), PG 15-dehydrogenase, and PG transporter as well as PGE(2) (EP2 and EP3) and PGF(2alpha) receptors. IFNtau influences cell-specific expression of COX-2, PGFS, EP2, and EP3 in endometrium, myometrium, and CL in a spatio-temporal and tissue-specific manner, whereas it does not alter COX-1, PGES, PG 15-dehydrogenase, PG transporter, or PGF(2alpha) receptor expression in any of these tissues. In endometrium, IFNtau decreases PGFS in epithelial cells and increases EP2 in stroma. In myometrium, IFNtau decreases PGFS and increases EP2 in smooth muscle cells. In CL, IFNtau increases PGES and decreases EP3. Together, our results show that IFNtau directly or indirectly increases PGE(2) biosynthesis and EP2-associated signaling in endometrium, myometrium, and CL during MRP. Thus, PGE(2) may play pivotal roles in endometrial receptivity, myometrial quiescence, and luteal maintenance, indicating polycrine (endocrine, exocrine, paracrine, and autocrine) actions of PGE(2) at the time of MRP. Therefore, the establishment of pregnancy may depend not only on inhibition of endometrial PGF(2alpha), but also on increased PGE(2) production in cattle.     

Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4)

Prostaglandin E₂ (PGE₂) is an important chemical mediator responsible for regulation of many vital physiological processes. Four receptor subtypes have been identified to mediate its biological actions. Among these subtypes, prostaglandin E receptor subtypes 2 and 4 (EP₂ and EP₄), both coupled to cAMP-protein kinase A (cAMP-PKA) signaling pathway, are proposed to play crucial roles under both physiological and pathological conditions. Though both receptors were extensively studied in mammals, little is known about their functionality and expression in non-mammalian species including chicken. In present study, the full-length cDNAs for chicken EP₂ and EP₄ receptors were first cloned from adult chicken ovary and testis, respectively. Chicken EP₂ is 356 amino acids in length and shows high amino acid identity to that of human (61%), mouse (63%), and rat (61%). On the other hand, the full-length cDNA of EP₄ gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cattle (83%), and a comparatively lower sequence identity to zebrafish (52%). RT-PCR assays revealed that EP₂ mRNA was expressed in all tissues examined including the oviduct, while EP₄ expression was detected only in a few tissues. Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE₂ could induce luciferase activity in DF-1 cells expressing EP₂ and EP₄ in dose-dependent manners (EC₅₀: <1 nM), confirming that both receptors could be activated by PGE₂ and functionally coupled to the cAMP-PKA signaling pathway. Together, our study establishes a molecular basis to understand the physiological roles of PGE₂ in target tissues of chicken.     

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y₁₂ and EP3 receptors, act as antagonists at G_i-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G_i-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y₁₂ antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA₂ mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI₂, iloprost, PGD₂, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE₁ and PGE₂ as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y₁₂ antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.     

Upregulated Cyclooxygenase-2 Inhibits Apoptosis of Human Gastric Epithelial Cells Infected with Helicobacter pylori

Helicobacter pylori induces apoptosis and alters the proliferation of gastric mucosal epithelial cells. Cyclooxygenase-2 (COX-2), the inducible form of prostaglandin (PG) synthesis, is known to cause alteration in epithelial cell growth. The goal of this study was to determine whether COX-2 gene expression by H. pylori infection could influence gastric epithelial cell apoptosis. Expression of COX-2 mRNA and proteins was up-regulated in Hs746T gastric epithelial cell lines infected with H. pylori, when assessed by quantitative RT-PCR and western blot. Inhibition of COX-2 expression using NS-398, a specific COX-2 inhibitor, showed a significant increase of gastric epithelial cell apoptosis and caspase-3 activation in Hs746T cells infected with H. pylori. Moreover, the effect of NS-398 on H. pylori-induced apoptosis was reversed by the addition of PGE₂. These results suggest that up-regulated COX-2 expression by H. pylori infection can inhibit apoptosis of gastric epithelial cells.     

Fibroblast growth factor-2 is expressed by the bovine uterus and stimulates interferon-tau production in bovine trophectoderm

Uterine-derived factors are essential for conceptus development and secretion of the maternal recognition-of-pregnancy factor, interferon-tau (IFNT), in ruminant species. The objectives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus during early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine trophectoderm. FGF-2 mRNA was present in endometrium throughout the estrous cycle and was localized to the luminal and glandular endometrial epithelium at d 17-18 after estrus in pregnant and nonpregnant cows. Immunoreactive FGF-2 protein was detected within the endometrium and in the uterine lumen at d 17-18 after estrus, and concentrations did not differ based on pregnancy status. In a bovine trophectoderm cell line, CT-1, supplementation of medium with at least 1 ng/ml FGF-2 increased the incorporation of [(3)H]thymidine into DNA. Similarly, IFNT secretion from CT-1 cells increased after FGF-2 supplementation (1-100 ng/ml) for 72 h. Abundance of IFNT mRNA in CT-1 cells increased after 24 h exposure to 1, 10, or 100 ng/ml FGF-2. In bovine blastocysts, FGF-2 supplementation did not affect cell number after 72 h of culture but did stimulate IFNT protein concentrations in conditioned medium. In summary, FGF-2 is present in the uterine lumen during early pregnancy and increases IFNT mRNA and protein abundance in trophectoderm. The magnitude by which FGF-2 stimulates IFNT expression suggests that this uterine-derived factor plays an active role in regulating the establishment and maintenance of pregnancy in ruminants.     

Gastric Cytoprotection in Man by Prostaglandin E2

In healthy volunteers, the effects of topical prostaglandin E₂ (PGE₂), 1 mg, on transmucosal potential difference (PD), mucus secretion, and epithelial cell turnover were investigated. PGE₂ increased gastric PD by 10 mV on an average and stimulated mucus secretion of the stomach by 50%. In contrast, epithelial cell turnover remained unchanged. Gastric output of H⁺ decreased, whereas the outputs of volume, Na⁺, and CI^- rose in response to PGE₂, which effects would be compatible with increased secretion of bicarbonate. Topical administration of ethanol 40% (vol/vol) reduced PD by 25 mV (ΔPD_E) and enhanced epithelial cell shedding by 350% with concomitant discharge of mucus from stomach epithelium. Pretreatment of the stomach with 1 mg PGE₂ prevented the ethanol-dependent epithelial cell exfoliation, indicating gastric mucosal cytoprotection. ΔPD_E and discharge of mucus were not significantly altered by PGE₂. We conclude that gastric cytoprotection by PGE₂ in man might be mediated by stimulation of mucus and/or bicarbonate production.     

Progesterone and interferon tau regulate hypoxia-inducible factors in the endometrium of the ovine uterus

In ruminants, progesterone (P4) from the ovary and interferon tau (IFNT) from the elongating blastocyst regulate expression of genes in the endometrium that are hypothesized to be important for uterine receptivity and blastocyst development. These studies determined effects of the estrous cycle, pregnancy, P4, and IFNT on hypoxia-inducible factor (HIF) expression in the ovine uterus. HIF1A mRNA, HIF2A mRNA, and HIF2A protein were most abundant in the endometrial luminal and superficial glandular epithelia (LE and sGE, respectively) of the uterus and conceptus trophectoderm. During the estrous cycle, HIF1A and HIF2A mRNA levels were low to undetectable on d 10 in the endometrial LE/sGE, increased between d 10 and 14, and then declined on d 16. Both HIF1A and HIF2A mRNA were more abundant in the endometrial LE/sGE of pregnant ewes. However, HIF3A, HIF1B, HIF2B, and HIF3B mRNA abundance was low in most cell types of the endometria and conceptus. Treatment of ovariectomized ewes with P4 induced HIF1A and HIF2A in the endometrial LE/sGE, and intrauterine infusion of ovine IFNT further increased HIF2A in P4-treated ewes, but not in ewes treated with P4 and the antiprogestin ZK 136,317. HIF3A, HIF1B, HIF2B, and HIF3B mRNA abundance was not regulated by either P4 or IFNT. Two HIF-responsive genes, carboxy-terminal domain 2 and vascular endothelial growth factor A, were detected in both the endometrium and conceptus. These studies identified new P4-induced (HIF1A and HIF2A) and IFNT-stimulated (HIF2A) genes in the uterine LE/sGE, and implicate the HIF pathway in regulation of endometrial epithelial functions and angiogenesis, as well as peri-implantation blastocyst development.     

Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy

Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2alpha receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days <50 to >250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP>EP2>P3, intercaruncle EP2>EP3> or =FP, and fetal membranes EP3> or =EP2 >FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2alpha receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2alpha in uterine activities during pregnancy in bovine.     

Neuronal prostaglandin E2 receptor subtype EP3 mediates antinociception during inflammation

The pain mediator prostaglandin E₂ (PGE₂) sensitizes nociceptive pathways through EP2 and EP4 receptors, which are coupled to G_s proteins and increase cAMP. However, PGE₂ also activates EP3 receptors, and the major signaling pathway of the EP3 receptor splice variants uses inhibition of cAMP synthesis via G_i proteins. This opposite effect raises the intriguing question of whether the G_i-protein–coupled EP3 receptor may counteract the EP2 and EP4 receptor-mediated pronociceptive effects of PGE₂. We found extensive localization of the EP3 receptor in primary sensory neurons and the spinal cord. The selective activation of the EP3 receptor at these sites did not sensitize nociceptive neurons in healthy animals. In contrast, it produced profound analgesia and reduced responses of peripheral and spinal nociceptive neurons to noxious stimuli but only when the joint was inflamed. In isolated dorsal root ganglion neurons, EP3 receptor activation counteracted the sensitizing effect of PGE₂, and stimulation of excitatory EP receptors promoted the expression of membrane-associated inhibitory EP3 receptor. We propose, therefore, that the EP3 receptor provides endogenous pain control and that selective activation of EP3 receptors may be a unique approach to reverse inflammatory pain. Importantly, we identified the EP3 receptor in the joint nerves of patients with painful osteoarthritis.     

Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor

Activation of the Gαs-coupled EP2 receptor for prostaglandin E2 (PGE₂) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 μM (i) potentiated the cAMP response to a low concentration of PGE₂ by > 50%; (ii) had no effect on EP4 or β2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE₂ on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE₂ on EP2 receptors 4- to 5-fold at 10 to 20 μM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE₂ receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE₂ released in a cell-injury setting is neuroprotective.     

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E₂ (PGE₂) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3?µM) used alone or in combination with the P2Y₁₂ antagonist cangrelor (1?µM), both without and with aspirin (100?µM). Platelet aggregation and P-selectin expression were measured in whole blood (n?=?10) following stimulation with the thromboxane A₂ (TXA₂) mimetic U46619 (0.3 or 1?µM) in combination with either the EP3 agonist sulprostone (0.1?µM), or PGE₂ (1?µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n?=?42) were randomly assigned to receive no background treatment, clopidogrel (300?mg loading dose plus 75?mg daily) or clopidogrel and aspirin (75?mg daily) for 10 days alongside DG-041 (200?mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y₁₂ antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y₁₂ blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.     

Tumor cells induce COX-2 and mPGES-1 expression in microvascular endothelial cells mainly by means of IL-1 receptor activation

Prostaglandin (PG) E₂ plays a key role in immune response, tumor progression and metastasis. We previously showed that macrovessel-derived endothelial cells do not produce PGE₂ enzymatically because they do not express the inducible microsomal PGE-synthase-1 (mPGES-1). Nevertheless, differences between macro- and micro-vessel-derived endothelial cells regarding arachidonic acid (AAc) metabolism profile have been reported. The present work was conducted to evaluate the expression of PGE₂-pathway-related enzymes in human microvascular endothelial cells (HMVEC) in culture and to test the hypothesis that the tumor cell-HMVEC cross talk could increase mPGES-1 expression in HMVEC. We treated HMVEC in culture with human recombinant IL-1β. IL-1β induced PGE₂ release and COX-2 and mPGES-1 expression in terms of mRNA and protein, determined by real-time PCR and immunoblotting, respectively. HMVEC constitutively expressed mPGES-2 and cytosolic PGES (cPGES) and the IL-1β treatment did not modify their expression. PGE₂ synthesized by HMVEC from exogenous AAc was linked to mPGES-1 expression. Immunohistochemistry analysis confirmed mPGES-1 expression in microvessels in vivo. COX-2 and mPGES-1 were also induced in HMVEC by the conditioned medium from two squamous head and neck carcinoma cell lines. Conditioned medium from tumor cell cultures contained several cytokines including the IL-1β and IL-1α. Tumor cell-induced COX-2 and mPGES-1 in HMVEC was strongly inhibited by the IL-1-receptor antagonist, indicating the important implication of IL-1 in this effect. HMVEC could therefore contribute directly to PGE₂ formed in the tumor. Our findings support the concept that mPGES-1 could be a target for therapeutic intervention in patients with cancer.     

Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors

Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.     

PGE2 induces MUC2 and MUC5AC expression in human intrahepatic biliary epithelial cells via EP4/p38MAPK activation

Background. MUC2 and MUC5AC overproduction is considered to be associated with hepatolithiasis and related to inflammation. However, mechanisms underlying MUC upregulation under inflammatory stimulation in human intrahepatic biliary epithelial cells (HIBECs) are not completely understood.Material and Methods. Expression of MUC2 and MUC5AC mRNA in HIBECs was detected by real-time PCR. Expression of COX-2, EP4, and phosphorylated ERK, JNK and p38MAPK protein was detected by Western blot. Concentrations of PGE₂, IL-1ß and TNF-α in cell culture supernatants were measured using the Quantikine Elisa kit.Results. COX-2 expression as well as PGE₂ production in HIBECs was upregulated significantly by LPS, which was completely blocked by either TLR4 antagonist or NFkB inhibitor. Selective COX-2 inhibitor suppressed LPS-induced MUC2 and MUC5AC mRNA expression remarkably. Exogenous PGE₂ increased MUC2 and MUC5AC mRNA expression in a dosage-dependent manner independent of IL-1ß and TNF-α. PGE₂ receptor EP4 agonist elevated MUC2 and MUC5AC expression, whereas EP4 antagonist had the opposite effect. Expression of phosphorylated p38MAPK was upregulated by exogenous PGE₂, and p38MAPK inhibitor reduced MUC2 and MUC5AC expression in HIBECs. In addition, it was found that levels of PGE₂, MUC2 and MUC5AC in bile samples from the hepatic ducts affected by intrahepatic stones were significantly higher than those from the unaffected hepatic ducts of patients with hepatolithiasis.Conclusions. Our findings indicate that PGE₂ induces MUC2 and MUC5AC expression in HIBECs via EP4-p38MAPK signaling.     

Cyclooxygenase-2 mediates mucin secretion from epithelial cells of lipopolysaccharide-treated canine gallbladder

Biliary mucin was regarded as a major contributing factor in formation of pigment stones as well as cholesterol ones. The aim of this study was to elucidate the mechanism of biliary mucin secretion in canine gallbladder epithelial (CGBE) cells treated by lipopolysaccharides (LPS) with special reference to cyclooxygenase (COX) -2. Confluent CGBE cells were incubated with following compounds for 8, 12, and 24 hr: (1) serum-free medium, (2) serum-free medium containing LPS (100 m/ml), (3) serum-free medium containing LPS (100 m/ml) with NS-398 (10 M), and (4) serum-free medium containing LPS (100 m/ml) with indomethacin (10 M). Mucin assay and western blots for COX-1 and COX-2 were performed. Production of PGE₂, and cAMP was also measured. Mucin secretion increased with time. At 12 hr, mucin secretion increased to 200% of control (from 100 ± 5 to 200±45%, P < 0.05). LPS treatment significantly stimulated the COX-2 expression (P < 0.05). The productions of PGE₂ and cAMP were increased from 299±68 to 524±163 pg/mg (P < 0.05) and from 0.2±0.1 to 0.92±0.4 pmol/ml (P < 0.05), respectively. NS-398, which completely inhibited COX-2 expression, significantly suppressed the level of PGE₂ and cAMP as well as mucin secretion (P < 0.05). Indomethacin, which partially inhibited COX-2 expression, suppressed the production of PGE₂, but not cAMP and mucin secretion. In conclusion, our results suggested that the PGE₂ induced by COX-2 might play a role in mucin secretion from the gallbladder epithelium through the increment of cAMP.