17-phenyl Trinor PGE2 Research Articles

Identification of novel 1,2,3,6-tetrahydropyridyl-substituted benzo[d]thiazoles: Lead generation and optimization toward potent and orally active EP1 receptor antagonists

Herein we described the design, synthesis and evaluation of a novel series of benzo[d]thiazole derivatives toward an orally active EP1 antagonist. Lead generation studies provided benzo[d]thiazole core from the four designed scaffolds. Optimization of this scaffold in terms of EP1 antagonist potency and ligand-lipophilicity efficiency (LLE; pIC50-clogP) led to a 1,2,3,6-tetrahydropyridyl-substituted benzo[d]thiazole derivative, 7r (IC50 1.1nM; LLE 4.7), which showed a good pharmacological effect when administered intraduodenally in a 17-phenyl trinor-PGE2 (17-PTP)-induced overactive bladder model in rats.

Abstract TMP51: Activation of PGE2 EP1 Receptor Promotes Microglial Phagocytosis Though CD36 Receptor Recycling

Background and Purpose: We have shown that the deletion of prostaglandin E2 (PGE2) EP1 receptor undermined microglial phagocytosis in vivo. However, the related mechanism still remains unknown. CD36 is a type II scavenger receptor that mediates microglial phagocytosis. It was reported that microglial CD36 contributes to hematoma clearance after intracerebral hemorrhage (ICH). In this study, we sought to identify the effects of two EP1 agonists on microglial phagocytosis and CD36 receptor recycling in primary microglial cultures. Method: The EP1 agonists: 17-phenyl trinor-PGE2 (17-pt-PGE2) and ONO-DI-004 were used to treat primary microglial cultures, respectively. Fluorescent latex beads or CFSE-labeled red blood cells (CFSE-RBCs) were added to microglial cells at a concentration of ten beads/RBCs per cell for live-cell imaging. Immunocytochemistry was also performed with antibody against ionized calcium-binding adapter molecule 1 (Iba1, a marker to microglia). Furthermore, we analyzed CD36 receptor recycling by using an established receptor recycling assay. Result: The primary microglial cells were incubated with fluorescent latex beads and treated with the EP1 agonist 17-pt-PGE2 for 120 min. We found that 17-pt-PGE2 significantly increased the number of attached beads to microglial cells at the concentration of 10 μM from 40 min to 120 min of incubation. Furthermore, we analyzed CD36 receptor recycling using an established receptor recycling assay and the quantitative results showed that 10 μM of 17-pt-PGE2 treatment markedly increased the area of recycled CD36 receptor in microglial cells. We then treated primary microglial cells with another selective EP1 agonist ONO-DI-004 for 120 min at the concentration of 5 μM and 10 μM, respectively. The area of CFSE-RBCs after ice-cold PBS washing was markedly increased with 10 μM of ONO-DI-004 treatment. In the live-cell imaging experiment, 10 μM of ONO-DI-004 treatment also significantly increased the number of attached CFSE-RBCs to microglial cells from 25 min to 60 min of incubation. Conclusions: This study showed that the EP1 agonists: 17-pt-PGE2 and ONO-DI-004 can promote microglial phagocytosis in vitro and enhanced CD36 receptor recycling could mediate some of these effects.

Prostaglandin E2 inhibits mast cell–dependent bronchoconstriction in human small airways through the E prostanoid subtype 2 receptor

Inhaled prostaglandin (PG) E2 might inhibit asthmatic responses, but the mechanisms involved remain undefined. We sought to characterize the direct and indirect effects of PGE2 on human small airways with particular reference to the receptors mediating the responses. Contraction and relaxation were studied in isolated human bronchi with an inner diameter of 1 mm or less. Low concentrations of PGE2 (0.01-1 μmol/L) relaxed the bronchi precontracted by histamine. The bronchodilator response was inhibited by the E prostanoid (EP) subtype 4 receptor antagonist ONO-AE3-208 but unaffected by the EP2 receptor antagonist PF-04418948. Higher concentrations of PGE2 (10-100 μmol/L) contracted the small airways. However, the TP receptor agonists U-46,619, PGF2α, and PGD2 were more potent than PGE2. Moreover, the bronchoconstrictor responses to PGE2 and all other tested prostanoids, including the EP1/EP3 receptor agonist 17-phenyl trinor PGE2 and the partial FP receptor agonist AL-8810, were uniformly abolished by the TP receptor antagonist SQ-29,548. In the presence of TP and EP4 antagonists, PGE2 inhibited the mast cell-mediated bronchoconstriction resulting from anti-IgE challenge. Measurement of the release of histamine and cysteinyl leukotrienes documented that this bronchoprotective action of PGE2 was mediated by the EP2 receptor, unrelated to bronchodilation, and increased with time of exposure. The pharmacology of PGE2 in isolated human small airways was different from its profile in animal models. This first demonstration of powerful EP2 receptor-mediated inhibition of IgE-dependent contractions in human airways introduces a new selective target for the treatment of asthma. This EP2 control of mast cell-mediated bronchoconstriction is presumably exaggerated in patients with aspirin-exacerbated respiratory disease.

Prostaglandin E2 upregulates β1 integrin expression via the E prostanoid 1 receptor/nuclear factor κ-light-chain-enhancer of activated B cells pathway in non-small-cell lung cancer cells

The prostaglandin E2 (PGE2) E prostanoid (EP)1 receptor shown to be associated with lung cancer cell invasion. However, the mechanism of EP1 receptor-mediated cell migration remains to be elucidated. β1 integrin is an essential regulator of the tumorigenic properties of non-small-cell lung carcinoma (NSCLC) cells. To date, little is known regarding the association between the EP1 receptor and β1 integrin expression. The present study investigated the effect of EP1 receptor activation on β1 integrin expression and cell migration in NSCLC cells. A total of 34 patients with clinical diagnosis of NSCLC and 10 patients with benign disease were recruited for the present study. The expression levels of the EP1 receptor and β1 integrin expression were studied in resected lung tissue using immunohistochemistry. A statistical analysis was performed using Stata se12.0 software. The effects of PGE2, EP1 agonist 17-phenyl trinor-PGE2 (17-PT-PGE2) and the nuclear factor κ-B (NF-κB) inhibitor on β1 integrin expression were investigated on A549 cells. The expression of β1 integrin and the phosphorylation of NF-κB‑p65 Ser536 was investigated by western blot analysis. Cell migration was assessed by a transwell assay. The results demonstrated that β1 integrin and EP1 receptor expression exhibited a positive correlation of evident significance in the 44 samples. The in vitro migration assay revealed that cell migration was increased by 30% when the cells were treated with 5 µM 17-PT-PGE2 and that the pre-treatment of β1 integrin monoclonal antibody inhibited 17-PT-PGE2‑mediated cell migration completely. PGE2 and 17-PT-PGE2 treatment increased β1 integrin expression. RNA interference against the EP1 receptor blocked the PGE2-mediated β1 integrin expression in A549 cells. Treatment with 17-PT-PGE2 induced NF-κB activation, and the selective NF-κB inhibitor pyrrolidinedithiocarbamate inhibited 17-PT-PGE2-mediated β1 integrin expression. In conclusion, the present study indicated that the PGE2 EP1 receptor regulates β1 integrin expression and cell migration in NSCLC cells by activating the NF-κB signaling pathway. Targeting the PGE2/EP1/β1 integrin signaling pathway may aid in the development of new therapeutic strategies for the prevention and treatment of this type of cancer.

Prostaglandin E2 receptor EP1-mediated phosphorylation of focal adhesion kinase enhances cell adhesion and migration in hepatocellular carcinoma cells

The prostaglandin E₂ (PGE₂) EP1 receptor has been implicated in hepatocellular carcinoma (HCC) cell invasion. However, little is known about the mechanisms of EP1 receptor-mediated cell adhesion and migration. We previously showed that PGE₂ promotes cell adhesion and migration by activating focal adhesion kinase (FAK). The present study was designed to elucidate the association between the EP1 receptor and FAK activation in HCC cells and to investigate the related signaling pathways. The effects of PGE₂, EP1 agonist 17-phenyl trinor-PGE₂ (17-PT-PGE₂), PKC and EGFR inhibitors on FAK activation were investigated by treatment of Huh-7 cells. Phosphorylation of FAK Y397 and c-Src Y416 was investigated by western blotting. Cell adhesion and migration were analyzed by WST and transwell assays, respectively. Protein kinase C (PKC) activity was measured with a PKC assay kit. The results showed that 17-PT-PGE₂ (3 µM) increased FAK Y397 phosphorylation by more than 2-fold and promoted cell adhesion and migration in Huh-7 cells. In transfected 293 cells, expression of the EP1 receptor was confirmed to upregulate FAK phosphorylation, while the EP1 receptor antagonist sc-19220 decreased PGE₂-mediated FAK activation. PKC activity and c-Src Y416 phosphorylation were enhanced after 17-PT-PGE₂ treatment. Both PKC and c-Src inhibitor suppressed the 17-PT-PGE₂-upregulated FAK phosphorylation, as well as 17-PT-PGE₂-induced cell adhesion and migration. In addition, exogenous epidermal growth factor (EGF) treatment increased FAK phosphorylation. The EGF receptor (EGFR) inhibitor also suppressed 17-PT-PGE₂-upregulated FAK phosphorylation. Our study suggests that the PGE₂ EP1 receptor regulates FAK phosphorylation by activating the PKC/c-Src and EGFR signal pathways, which may coordinately regulate adhesion and migration in HCC.

Prostaglandin E2 receptor 1 activity regulates cell survival following hypoxia in cultured rat cortical neurons

The clinical side-effects of increased cyclooxygenase (COX) activity induced by pathologic conditions have raised concerns recently. However, a better understanding of the mechanisms underlying the subsequent neurotoxicity requires knowledge of pathways downstream of COX, especially prostaglandin E2 (PGE2) and its receptors. Therefore, this study was performed to investigate the effects of PGE2 receptor 1 (EP1) activity on neuronal cell death resulting from hypoxia/reoxygenation (Hyp). As cyclinD1 activity has been shown to regulate neuronal apoptosis as well, the role of cyclinD1 was investigated, as well. Cortical neural cells isolated from fetal Wistar rats were cultured for 12 d and exposed to Hyp conditions to establish an in vitro Hyp model. To determine the effects of EP1 activity on Hyp-induced neurotoxicity, cells were treated with 17-phenyl trinor-PGE2 (17-pt), a synthetic EP1 agonist, or sc-51089, an EP1 antagonist, then exposed to hypoxic conditions for 3 h and reoxygenated for 21 h. Following Hyp, cell viability was quantified by MTT assays, and apoptosis was assessed by flow cytometry. Protein expression levels of caspase-3 and cyclinD1 were examined by Western blot analysis. Treatment of cultured cortical neurons with 17-pt significantly decreased the survival rate of Hyp-treated neurons ( p < 0.05), while treatment with sc-51089 increased the survival rate. Treatment with 17-pt also led to increased expression levels of caspase-3, further supporting a role for EP1 in the observed neurotoxicity. However, cyclinD1 expression levels were unchanged following treatment with either 17-pt or sc-51089. Therefore, EP1 may play an important role in Hyp-induced neuronal apoptosis, but this neurotoxic activity is unlikely to involve cyclinD1.

Prostaglandin E2Differentially Modulates Human Platelet Function through the Prostanoid EP2 and EP3 Receptors

Activated human platelets synthesize prostaglandin (PG) E(2), although at lower rate than thromboxane A(2). PGE(2) acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized compared with thromboxane. We studied the effect of PGE(2) and its analogs on in vitro human platelet function and platelet and megakaryocyte EP expression. Platelets preincubated with PGE(2) or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method; platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin, and microaggregates were investigated by flow cytometry. PGE(2) at nanomolar concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC(50), 25.6 ± 6 nM; E(max) of 100 ± 19% increase versus vehicle-treated), without affecting final maximal aggregation. PGE(2) stabilized reversible aggregation induced by low ADP concentrations (EC(50), 37.7 ± 9 nM). The EP3 agonists, 11-deoxy-16,16-dimethyl PGE(2) (11d-16dm PGE(2)) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC(50) of 48.6 ± 10 nM (E(max), 252 ± 51%) and 5 ± 2 nM (E(max), 300 ± 35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC(50), 40 ± 20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE(2) alone raised intraplatelet Ca(2+) and enhanced ADP-induced Ca(2+) increase. 11d-16dm PGE(2) and 17-phenyltrinor PGE(2) (EP3 > EP1 agonist) at nanomolar concentrations counteracted PGE(1)-induced VASP phosphorylation and induced platelet microaggregates and P-selectin expression. EP1, EP2, EP3, and EP4 were expressed on human platelets and megakaryocytes. PGE(2) through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.

Prostaglandin E 2 reduces amyloid β-induced phagocytosis in cultured rat microglia

Treatment with amyloid β 1–42 (Aβ 1–42) at 1 µM for 60 min increased phagocytosis of latex beads by cultured rat microglia. This increase was reduced dose-dependently by prostaglandin E 2 (PGE 2), but PGD 2, PGF 2α, iloprost, or U-46619 had no effects. PGE 2 also reduced the phagocytosis of fluorescent-labeled Aβ 1–42. Aβ 1–42-induced phagocytosis was reduced by butaprost but not by 17-phenyl trinor PGE 2, sulprostone, or PGE 1 alcohol. The reduction effect of PGE 2 on phagocytosis was reversed by AH6809, an E-prostanoid receptor 2 (EP2) antagonist, which inhibited cyclic adenosine monophosphate (AMP) accumulation induced by PGE 2. Butaprost, but not 17-phenyl trinor PGE 2, sulprostone, or PGE 1 alcohol increased intracellular cyclic AMP accumulation. In western blotting analysis, EP2-like immunoreactivity was detected in the crude membrane fraction of microglia. On the other hand, Aβ 1–42-induced phagocytosis was not affected by SC-560, a cyclooxygenase-1 (COX-1) inhibitor, NS-398, a COX-2 inhibitor, or ibuprofen, a non-specific COX inhibitor. Aβ 1–42 or PGE 2 had little effect on the expression levels of COX-1 or COX-2. These results indicate that Aβ 1–42-induced microglial phagocytosis is reduced by PGE 2 through EP2.

Cyclooxygenase-2 Derived Prostaglandin E2 Is Required for Dendritic Cell Differentiation From Hematopoietic Progenitor Cells.

Abstract 1499Poster Board I-522Dendritic cells (DCs) are an attractive target for therapeutic manipulation of the immune system due to their potent antigen presentation capacity and ability to induce effective immune response. In steady-state conditions different DC subsets including myeloid DCs (CD11c+CD11b+B220neg) and plasmacytoid DCs (CD11c+CD11bnegB220+) are generated in bone-marrow (BM) from hematopoietic stem cells through a series of differentiation steps. We recently demonstrated that prostaglandin (PGE2), the predominant metabolite of arachidonic acid metabolism by cyclooxygenase (COX) enzymes, enhances the homing, survival and proliferation of hematopoietic stem cells (Hoggatt et. al., Blood 2009). In this study, we examined the requirement of prostaglandins in development of DCs from hematopoietic progenitor cells. In vivo treatment of mice for 4 days with the non-steroidal anti-inflammatory drug (NSAID) indomethacin (2.5 mg/kg/bid), a dual COX1/COX2 inhibitor, produced a 59.5±17.8 % (p<0.02) reduction in total bone-marrow DC number compared to control mice treated with vehicle alone. Interestingly, indomethacin selectively decreased marrow CD11c+CD11b+B220neg myeloid DCs without affecting CD11c+CD11bnegB220+ plasmacytoid DCs. To determine whether fewer DCs in the bone marrow of indomethacin treated mice was due to the impairment of DC differentiation from their hematopoietic progenitor cells, we stimulated differentiation of DCs from lineage depleted (CD5−,CD45R−, CD11b−, Anti-Gr-1−, 7-4− and Ter-119−) bone marrow cells using Flt-3 ligand for 9 days in vitro and treated cultures with indomethacin (1 microM), SC-560 (10 microM), a selective COX-1 inhibitor or NS-398 (10 microM), a selective COX-2 inhibitor. Indomethacin produced a 1.98±0.38 fold, (p<0.02) reduction and the COX-2 inhibitor NS-398 produced a 1.52±0.04 fold, (p<0.05) reduction in CD11c+CD11b+B220neg myeloid DC generation compared to control, while the COX-1 specific inhibitor SC-560 was without effect. As expected, Flt-3-ligand induced plasmacytoid DC (CD11c+CD11bnegB220+) differentiation was not affected by selective COX inhibitors. Indomethacin also impaired generation of CD11a+CD14neg Langerhans DC from human umbilical cord blood CD34+ cells. Measurement of PGE2 production in culture supernatants from DC-producing cultures demonstrated detectable PGE2 after 6 days of culture and DC generation from BM progenitors in these cultures was impaired when PGE2 synthesis was blocked on day 6 by indomethacin administration. Indomethacin treatment during the first 5 days of Flt3-ligand stimulated DC differentiation cultures did not decrease DC production. To identify mechanisms responsible for this impairment in Flt-3 ligand-induced DC generation from hematopoietic progenitor cells, we analyzed the effect of indomethacin on DC-committed precursor cell proliferation and survival. Survival of DC-committed precursors defined as CD11clow CD11bbrightMHCIIlow was reduced 35±2.5% (p<0.05) in indomethacin treated cultures compared to control. However, indomethacin did not affect DC precursor proliferation as measured by BrdU incorporation assay. To elucidate the signaling mechanisms by which indomethacin impaired the survival of DC precursors, we added selective receptor agonists to each of known PGE receptors, EP1-4, during Flt3-ligand induced DC differentiation. The EP1/EP3 agonist 17-phenyl trinor PGE2 rescued the DC precursors from indomethacin mediated death, whereas Butaprost, a specific EP2 agonist and L902688, a selective EP4 agonist, failed to rescue DC precursor death. DCs developed in the presence of prostaglandin inhibitors did not show any defect in LPS-induced activation and expressed CD40, CD80, CD86 and MHCII levels similar to control as measured by flow cytometry. In addition, DC developed in the absence of endogenous PGE2 production successfully induced T-cell activation as measured by mix lymphocyte reaction assay (MLR). In conclusion, COX-2 mediated prostaglandin production by DC-committed hematopoietic precursors confers resistance to cell death via signaling through EP1/EP3 receptors and promotes dendritic cell development. Disclosures:No relevant conflicts of interest to declare.

Intermolecular cross‐talk between the prostaglandin E2 receptor (EP)3 subtype and thromboxane A2 receptor signalling in human erythroleukaemic cells

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E(2) receptor (EP) and the TPalpha and TPbeta isoforms of the human thromboxane (TX)A(2) receptor (TP), 17-phenyl trinor PGE(2)-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP(1) subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells. Specificity of 17-phenyl trinor PGE(2) signalling and its possible cross-talk with signalling by TPalpha/TPbeta receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)(3) generation and intracellular calcium ([Ca(2+)](i)) mobilization. While 17-Phenyl trinor PGE(2) led to activation of phospholipase (PL)Cbeta to yield increases in IP(3) generation and [Ca(2+)](i), it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA(2) mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE(2) was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G(i)/G(betagamma)-dependent activation of PLCbeta. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE(2)-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP(3), rather than the EP(1), EP(2) or EP(4) receptor subtype in HEL cells. The EP(1) and EP(3) subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP(1) receptors impaired or desensitized, while that of EP(3) receptors augmented signalling through TPalpha/TPbeta receptors, in a cell type-specific manner.

Prostaglandin E 2 reduces extracellular ATP-induced migration in cultured rat microglia

Treatment with 100 μM adenosine triphosphate (ATP) for 120 min augmented migration of cultured rat microglia by about 4-fold. This augmentation was effectively reduced by 0.1–10 μM prostaglandin E 2 (PGE 2). PGE 2-mediated reduction was reversed by the EP2 antagonist AH6809 at 10 μM. The EP2 agonist butaprost also reduced ATP-induced migration at 10 μM, whereas the EP1 agonist 17-phenyl trinor PGE 2, the EP3 agonist sulprostone, and the EP4 agonist PGE 1 alcohol all had no effect at 10 μM. In addition, ATP-induced migration was reduced by the adenylate cyclase activator forskolin at 100 μM, whereas the adenylate cyclase inhibitor SQ22536 reversed the effect of PGE 2 on ATP-induced migration at 100 μM. Over the same experimental duration, PGE 2, butaprost, and forskolin had little effect on cell viability. These findings indicate that ATP-induced microglial migration is reduced by PGE 2 through EP2 and adenylate cyclase.

Prostaglandin E2-Mediated Migration of Human Trophoblast Requires RAC1 and CDC421

The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E(2) produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE(2)-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE(2) or 17-phenyl trinor PGE(2) (PGE receptor PTGER1 agonist) or PGE(1) Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE(2)-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE(2), PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE(2)-mediated migration of human EVTs.

Effects of gestational age on prostaglandin EP receptor expression and functional involvement during in vitro contraction of the guinea pig uterus

Prostaglandin E 2 (PGE 2) exerts diverse biological effects through four G-protein-coupled cell surface receptor subtypes, EP1–4. This study's objective was to characterize EP1–4 receptor mRNA expression within pregnant guinea pig myometrium during early implantation stage (gestation day [GD] 6) and late stage gestation (GD 50) and evaluate in vitro contractile activity of receptor subtype selective agonists. Using RT-PCR, qualitative gene expression patterns of EP2, EP3, and EP4 mRNA were detected in the myometrium and remained unchanged between the gestational ages. EP1 mRNA remained undetected in pregnant tissue. In vitro contractile activity was evaluated in GD 6 and GD 50 myometrium using vehicle and EP agonists PGE 2, 17-phenyl trinor PGE 2, sulprostone, misoprostol, and CP-533,536. All spasmogens in pregnant myometrium were EP1/EP3 selective agonists, though likely acting via EP3 receptors in this test model. CP-533,536—a highly selective EP2 receptor agonist—and the vehicle failed to induce myometrial contraction at both gestational ages.

PGE 2 receptors and their intracellular mechanisms in rabbit small intestine

The effects of PGE 2 on longitudinal smooth muscle, the intracellular mechanisms involved, and the localization of EP receptors were investigated in rabbit small intestine. PGE 2 evoked contractions in small intestine that were reduced by tetrodotoxin and hexamethonium. 17-Phenyl trinor PGE 2, sulprostone, misoprostol and 16,16-dimethyl PGE 2 evoked contractions. Butaprost did not modify spontaneous motility. AH 6809 reduced PGE 2 and 17-phenyl trinor PGE 2-induced contractions. Verapamil, Ca 2+ free medium, staurosporine, forskolin, theophylline, and rolipram diminished, while IP-20 and H-89 increased PGE 2-induced contractions. Western blot analysis showed protein bands of 41 kDa for EP 1, 71 kDa for EP 2 and 62 kDa for EP 3 receptors. EP 1, EP 2 and EP 3 receptors were detected in neurons of the myenteric and submucosal ganglia, but only EP 3 receptors were found in smooth muscle layers. This study did not detect EP 4 receptor. PGE 2-induced contractions would be mediated through EP 1 and EP 3 receptors, and voltage-dependent Ca 2+ channels, protein kinase C, and cAMP would be implicated in these responses.

Involvement of EP1 and EP2 receptors in the regulation of the Na,K-ATPase by prostaglandins in MDCK cells

Prostaglandins are key regulators of ion transport in the kidney. In MDCK cells, which model distal tubule cells, the transcription of the Na,K-ATPase β1 subunit is regulated by PGE 1 and PGE 2. To identify the EP receptors that mediate transcriptional regulation, transient transfection studies are conducted using the human β1 promoter/luciferase construct, pHβ1-1141 Luc. The involvement of EP1 and EP2 receptors is indicated by studies with the EP1 selective agonist 17-phenyl trinor PGE 2, and the EP2 selective agonist butaprost (which stimulate), as well as by studies with the antagonists SC-51089 (EP1 specific) and AH 6809 (EP1 and EP2 specific). Consistent with the involvement of Gs coupled EP2 receptors, is that the PGE 1 stimulation is inhibited by the PKAI expression vector (encoding the protein kinase A (PKA) inhibitory protein), as well as by the myristolated PKA inhibitory peptide PKI. In addition to this evidence (for the involvement of EP2 receptors), evidence for the involvement of EP1 receptors in the PGE 1 mediated stimulation of Na,K-ATPase β subunit gene transcription includes the stimulatory effect of 17-phenyl trinor PGE 2, as well as the inhibitory effects of SC-51089. Also consistent with the involvement of Gq coupled EP1 receptors, the PGE 1 stimulation is inhibited by the PKCI vector (encoding the PKC inhibitory domain), the PKC inhibitor Go 6976, thapsigargin, as well as the calmodulin antagonists W7 and W13.

Prostaglandin E 2 deteriorates N-methyl- d-aspartate receptor-mediated cytotoxicity possibly by activating EP2 receptors in cultured cortical neurons

The activation of glutamate receptors, particularly N-methyl- d-aspartate (NMDA) receptors, initiates ischemic cascade in the early stages of cerebral ischemia. Postischemia, cerebral ischemia is also associated with an inflammatory reaction that contributes to tissue damage. The up-regulation of neuronal cyclooxygenase-2 (COX-2) and elevation of prostaglandin E 2 (PGE 2) have been reported to occur after cerebral ischemic insult. We therefore studied whether the COX-2 reaction product PGE 2 affects glutamate receptor-mediated cell death in cultured rat cortical cells. PGE 2 was found to augment NMDA-mediated cell death. The transcription of EP1, EP2, EP3 and EP4 PGE 2 receptor genes was investigated using reverse transcriptase–polymerase chain reaction (RT-PCR). EP1, EP2 and EP3 receptor genes were found in cortical cells. Butaprost (an EP2 agonist) markedly enhanced NMDA-mediated cell death, whereas 17-phenyl trinor-PGE 2 (an EP1 agonist) and sulprostone (an EP3 agonist) had little effect. Both PGE 2 and butaprost elevated cAMP intracellular levels in the cortical cells; moreover, forskolin, an activator of adenylate cyclase, enhanced NMDA-mediated cell death. These results suggest that PGE 2, acting via EP2 receptors, aggravates excitotoxic neurodegeneration by a cAMP-dependent mechanism.

Prostaglandin E2 Stimulates Fibronectin Expression through EP1 Receptor, Phospholipase C, Protein Kinase Cα, and c-Src Pathway in Primary Cultured Rat Osteoblasts

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE(2) enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE(2) also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP(1) receptor but not other PGE receptors is involved in PGE(2)-mediated up-regulation of Fn. At the mechanistic level, Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE(2)-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE(2). Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including alpha, beta, epsilon, and delta, demonstrated that alpha isozyme plays an important role in the enhancement action of PGE(2) on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE(2) and 17-phenyl trinor PGE(2) (EP(1)/EP(3) agonist) increased the surface expression and mRNA level of alpha5 or beta1 integrins. Fn promoter activity was enhanced by PGE(2) and 17-phenyl trinor PGE(2) in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited the potentiating action of PGE(2) on Fn promoter activity. Local administration of PGE(2) or 17-phenyl trinor PGE(2) into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and alpha5beta1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE(2) increased Fn and promoted bone formation in rat osteoblasts via the EP(1)/phospholipase C/PKCalpha/c-Src signaling pathway.

Metabotropic signal transduction for bradykinin in submucosal neurons of guinea pig small intestine.

Intracellular recording methods with "sharp" microelectrodes were used to study signal transduction mechanisms underlying the excitatory action of bradykinin (BK) in morphologically identified neurons in the small intestinal submucosal plexus. Exposure to BK evoked slowly activating membrane depolarization and enhanced excitability associated with increased input resistance in AH-type and decreased input resistance in S-type neurons. Preincubation with pertussis toxin did not affect the BK-evoked responses. Pretreatment with the cyclooxygenase inhibitors indomethacin or piroxicam suppressed or abolished the BK-evoked responses. Application of prostaglandin (PG) E(2) or PG analogs evoked BK-like depolarizing responses in the submucosal plexus with a potency order of PGE(2) > PGE(1) > 17-phenyl trinor-PGE(2) > PGI(2) > sulprostone > PGF(2alpha). Depolarizing responses to bradykinin or PGE(2) in S-type neurons were suppressed in the presence of the phospholipase C inhibitor U73122 [(1-6-[([17beta]-3-methoxyestra-1,3,5[10]-tren-17-71)amino]hexyl)-1H-pyrrole-2,5-dione)], but not the inactive analog U73343 [(1-6-[([17beta]-3-methoxyestra-1,3,5[10]trien-17yl)amino]hexyl)-2,5-pyrrolidinedione)]. The inositol-1,4,5-trisphosphate receptor antagonist 2-aminoethoxy-diphenylborane and the calmodulin inhibitor W-7, but not ryanodine, suppressed both bradykinin- and PGE(2)-evoked responses. KN-62, an inhibitor of calmodulin kinases, or GF109203X, a specific protein kinase C inhibitor, suppressed both BK- and PGE(2)-evoked depolarizing responses. Selective protein kinase A inhibitors did not alter BK- or PGE(2)-evoked depolarizing responses in S neurons. The results suggest that BK stimulates synthesis and release of PGE(2), which acts at EP(1) receptors to evoke depolarizing responses in submucosal neurons. The postreceptor transduction cascade includes activation of phospholipase C, inositol-1,4,5-trisphosphate production, intraneuronal Ca2+ mobilization, activation of protein kinase C and/or calmodulin kinases, and phosphorylation of cationic channels.

Spinal EP receptors mediating prostaglandin E 2–induced mechanical hyperalgesia, thermal hyperalgesia, and touch-evoked allodynia in rats

Intrathecal administration of prostaglandin E 2 (PGE 2) produces mechanical hyperalgesia, thermal hyperalgesia, and touch-evoked allodynia in rats. Experiments were conducted to examine the effects of intrathecal administration of relatively selective PGE 2 receptor (EP receptor) agonists to establish which spinal EP receptors mediate these behavioral effects of spinally administered PGE 2. Administration of either sulprostone (EP 3 receptor agonist) or PGE 1 alcohol (EP 4 receptor agonist) produced marked mechanical and thermal hyperalgesia and touch-evoked allodynia. Neither 17-phenyl trinor PGE 2 (EP 1 receptor agonist) nor butaprost (EP 2 receptor agonist) produced any significant changes in behavioral response thresholds to mechanical or thermal stimuli. However, 17-phenyl trinor PGE 2 (EP 1 receptor agonist) did produce marked touch-evoked allodynia. These data suggest that in rats activation of spinal EP 3 and EP 4 receptors by PGE 2 is important for development of both mechanical and thermal hyperalgesia as well as for touch-evoked allodynia. PGE 2-induced allodynia also appears to involve activation of spinal EP 1 receptors.

Prostaglandin (PG) FP and EP1 receptors mediate PGF2alpha and PGE2 regulation of interleukin-1beta expression in Leydig cell progenitors.

Prostaglandins (PG) mediate IL-1beta regulation of several interleukin mRNAs in progenitor Leydig cells. PGE(2) and PGF(2alpha) potently reverse indomethacin (INDO; a cyclooxygenase inhibitor) inhibition of IL-1beta autoinduction. IL-1beta increases PGE(2) and PGF(2alpha) production. To determine the PG receptors involved in this regulation, this study established by RT-PCR and Western analyses which specific receptors for PGE(2) (EP receptors) and PGF(2alpha) (FP receptors) are expressed in progenitors. Pharmacological characterization of receptors involved in PGE(2) and PGF(2alpha) regulation of IL-1beta mRNA levels was ascertained using real-time PCR analyses. FP, EP(1), EP(2), and EP(4) receptor mRNAs and proteins, and an EP(3) receptor subtype were detected. IL-1beta treatment (24-h) significantly decreased EP(1) receptor levels; INDO abrogated this down-regulation. FP, EP(2), and EP(4) receptor levels increased after IL-1beta and IL-1beta + INDO. A selective FP agonist, cloprostenol (0.1 micro M), and PGF(2alpha) (10 micro M) had similar effects on IL-1beta mRNA levels in progenitors treated with IL-1beta + INDO. None of the EP(2)/EP(4) agonists [butaprost, misoprostol, or 11-deoxy PGE(1) (10 micro M)] affected IL-1beta mRNA levels. In contrast, EP(1)/EP(3) agonists (17-phenyl trinor PGE(2) and sulprostone) increased IL-1beta mRNAs in a dose-dependent manner. EP(1) receptor subtype-selective antagonist, SC-51322, blocked IL-1beta-induced and [IL-1beta + INDO + 17-phenyl trinor PGE(2)]-induced increases in IL-1beta mRNAs. Taken together, our data demonstrate that FP and EP(1) receptors mediate PGF(2alpha) and PGE(2) induction of progenitor IL-1beta expression.