Inhibition of the prostaglandin receptor EP2 following status epilepticus reduces delayed mortality and brain inflammation

Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood–brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.     

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as “structural kinetics,” to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.There are more than 42 kinesin genes in the human genome, representing 14 distinct classes (1). All are members of the P-loop NTPase superfamily of nucleotide triphosphate hydrolases (2–4). Like other NTPases, kinesins share a conserved Walker motif nucleotide-binding fold (2, 4) that consists of a central twisted β-sheet and three nucleotide-binding loops, which are termed switch-1, switch-2, and the P-loop. Kinesins also share a common microtubule (MT) binding interface, which isomerizes between states that either bind MTs weakly or strongly, and a mechanical element, termed the neck linker (NL). The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5–7). NL isomerization (5, 8) is hypothesized to be the force-generating transition in kinesin motors (6, 7, 9–11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).Spectroscopic and structural studies have led to a model to explain how kinesins generate force (5–7, 9, 10, 12–15) (summarized in SI Appendix, Fig. S1), which proposes that the conformations of the nucleotide binding site, the MT-binding interface, and the NL are all determined by the state of the catalytic site. It predicts that when unbound to the MT, the motor contains ADP in its catalytic site and its NL is undocked. MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5, 7–10, 14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11, 14); (ii) switch-1, which coordinates the γ-phosphate of ATP, alternates between two conformations, referred to as “open” and “closed,” and the NL alternates between docked and undocked (5, 6, 10, 13–15); and (iii) coordination between the conformations of switch-1 and the NL regulates the timing of the ATPase cycles of the two motor domains in processive kinesin dimers (11). However, the model fails to explain several features of kinesins. For example, it predicts that ATP does not bind to kinesin when the NL is docked. This prediction is inconsistent with studies of both Eg5 and kinesin-1, which suggest ATP binds more readily when the NL is docked (11, 16, 17). The model also predicts that the NL should be docked after ATP binding. However, electron paramagnetic resonance (EPR) probes attached to the NL show a significant population of both mobile and immobile NL states in the presence of both pre- and posthydrolytic ATP analogs (5). Furthermore, the model cannot explain the load dependence of stall, detachment, and back stepping, all of which require a branched pathway (11).To resolve these uncertainties, we have measured the kinetics of the structural changes that occur in switch-1 and the NL with nucleotide binding while the motor is bound to the MT in an experimental design that we refer to as “structural kinetics.” We carried out these experiments using an novel spectroscopic approach, termed transient time-resolved fluorescence resonance energy transfer, (TR)²FRET, that allows us to monitor the kinetics and thermodynamics of both the undocked/docked transition in the NL and the open/closed transition in switch-1 that accompany the process of nucleotide binding. These experiments explain differences in the enzymologies of kinesin-1 and Eg5 and suggest an interesting role for the L5 loop in controlling the timing of conformational changes in the Eg5 switch-1 and NL.     

Multidrug resistance associated protein 2 mediates transport of prostaglandin E2.

BACKGROUND/AIM: Inactivation of prostaglandin E(2) (PGE(2)) in the liver is a rapid process and occurs mainly through beta-oxidation in the peroxisome of the hepatocyte. Biliary excretion of PGE(2) is also a means of elimination from the liver. We investigated the role of multidrug resistance-associated protein 2 (MRP2) in the transport of PGE(2). METHODS: Biliary PGE(2) elimination was measured in liver perfusions in Wistar and MRP2-deficient TR(-) rats. Furthermore, transport experiments were performed in membrane vesicles from human MRP2-infected Spodoptera frugiperda 21 (Sf21) insect cells. RESULTS: The liver perfusions showed a 3.5 times higher percentage of undegraded [(3)H]PGE(2) in bile of Wistar rats in comparison with MRP2 deficient (TR(-)) rats (3.6% vs. 1.1%, respectively; P<0.05). MRP2-mediated transport of the model substrate [(3)H]DNP-SG was inhibited by PGE(2). Half maximal inhibition was achieved at a concentration of approximately 15 microM PGE(2). In addition, [(3)H]PGE(2) uptake in these vesicles was detected, and determined to be ATP dependent. CONCLUSION: MRP2 mediates the transport of PGE(2) and its breakdown products. The biliary excretion of PGE(2) via MRP2 may contribute to rapid elimination of the prostaglandin but might also serve to relay prostaglandin signalling to the biliary tree.     

Molecular basis for lipid recognition by the prostaglandin D2 receptor CRTH2

Prostaglandin D₂ (PGD₂) signals through the G protein–coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD₂ derivative, 15R-methyl-PGD₂ (15mPGD₂), by serial femtosecond crystallography. The structure revealed a “polar group in”–binding mode of 15mPGD₂ contrasting the “polar group out”–binding mode of PGE₂ in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.

Eicosanoids constitute a group of signaling lipid mediators that are derived from arachidonic acid or other polyunsaturated fatty acids (1, 2). As the name indicates, they all possess a long hydrocarbon chain with 20 carbon units, which is usually attached to a carboxylic acid head group. Prostanoids including prostaglandins D₂, E₂, and F₂ (PGD₂, PGE₂, and PGF₂); prostacyclin (PGI₂); thromboxane A₂ (TXA₂) and leukotrienes including leukotriene B₄, C₄, and D₄ (LTB₄, LTC₄, and LTD₄) are representative endogenous eicosanoids that can induce signaling through G protein–coupled receptors (GPCRs) to play critical roles in inflammation, immunity, hemostasis, and tissue repair (1, 3–5). In humans, nine prostanoid receptors have been identified: PGD₂ receptors 1 and 2 (DP1 and DP2), PGE₂ receptors 1 to 4 (EP1 to EP4), PGF₂ receptor (FP), PGI₂ receptor (IP), and TXA₂ receptor (TP) (5). All of these receptors, except for DP2, belong to the α-branch of Class A GPCRs as close phylogenetic neighbors of aminergic receptors (5, 6). DP2, chemoattractant receptor–homologous molecule expressed on type 2 helper T cells (Th2) (also named CRTH2), is more closely related to a group of chemotactic GPCRs in the γ-branch of Class A GPCRs, including chemokine receptors and receptors for LTB₄, formylpeptides, and complement peptides C3a and C5a (6, 7).Certain types of immune cells, including eosinophils, innate lymphoid cells, and Th2 cells, express high levels of CRTH2 (8–11). PGD₂ signaling through CRTH2 can induce chemotaxis of these immune cells, which is a major pathway that drives the onset of type 2 inflammation (11–15). Therefore, the roles of PGD₂–CRTH2 signaling axis in type 2 inflammation-related diseases such as asthma and allergic rhinitis have attracted outstanding research interest (14, 16, 17). CRTH2 antagonists hold the potential of being a new class of anti-inflammatory drugs (16, 18–24). Although clinical trials have generated mixed results on different CRTH2 antagonists for asthma (17–20, 25), it is likely that a certain subpopulation of patients, such as those with a high baseline of Th2 cells or eosinophils, may benefit most from anti-CRTH2 therapy (17, 24). In addition, it has been proposed that insurmountable CRTH2 antagonists with prolonged receptor residence time can provide better, therapeutic efficacy, compared to reversible and fast, dissociating CRTH2 antagonists, which needs further investigation (26).We have previously reported crystal structures of CRTH2 bound to two antagonists, CAY10471 and fevipiprant, which revealed a positively charged environment of the ligand-binding pocket and a potential ligand entry port (27). The structural analysis of antagonist-bound CRTH2 suggested that PGD₂ may enter the ligand-binding pocket through the ligand entry port by opposite charge attraction (27). Recently, crystal structures of several other prostanoid receptors, EP3, EP4, and TP, and cryogenic electron microscopy (cryo-EM) structures of the EP2 and EP4 signaling complexes with PGE₂ have also been reported (28–33). To further study the binding of lipid agonists to CRTH2 and investigate if CRTH2 differs from other prostanoid receptors in lipid recognition and receptor activation, we determined a 2.6-Å resolution, room temperature crystal structure of CRTH2 bound to a PGD₂ derivative by serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) source. We performed computational simulation studies using the CRTH2–15R-methyl-PGD₂ (15mPGD₂) structure, which helped identify the roles of specific residues surrounding the ligand entry port and provided molecular insights into the events that could facilitate ligand binding. Results from our mutational analysis of the identified residues helped us further strengthen the proposed model for ligand capture and entry in CRTH2.     

Clinical effect of E‐series of prostaglandin receptor 2 and epidermal growth factor receptor signal pathways in the development of esophageal squamous cell carcinoma

Signal pathways mediated by epidermal growth factor receptor (EGFR) and E‐series of prostaglandin receptors (EPs) are closely correlated to the pathogenesis of tumor. This experiment was designed to investigate the expression and clinical significance of EP2 and EGFR in esophageal squamous cell carcinoma (ESCC). Tissue samples were collected reterospectively from 87 patients with ESCC (first diagnosed). The patients were followed up for 5 years after radical surgery. The expression of EP‐2 and EGFR were examined by tissue chip technology and immunohistochemistry methods. Clinicopathological and prognostic impact were evaluated. Overexpression of EGFR and EP‐2 was more observed in ESCC than the control group (58.6% vs. 13.9%; 52.9% vs. 4.88%, P < 0.001, respectively); which correlated with tumor infiltration depth, lymph node metastasis, and tumor‐lymph node‐metastasis staging. Both the EP‐2 and EGFR overexpression were detected in 39 specimens and exhibited the positive correlation (P < 0.001, r = 0.404). Overexpression of EP2 and EGFR exhibited significant correlation with worse 5‐year overall survival than those with negative result (17.6% vs. 27.8%, P = 0.011; 10.9% vs. 34.1%, P < 0.001, respectively). Cox proportional hazard model showed that the T‐staging, lymph node metastasis, and EGFR overexpression were the independent risk factors of the prognosis. The present study exhibited that the overexpression of EP2 and EGFR in ESCC tissues might play an important role in carcinogenesis and the progression of ESCC.     

胃癌患者外周血单个核细胞产生前列腺素E2的研究

目的：探讨前列腺素Ｅ２（ＰＧＥ２）在胃癌中的应用价值。方法：用放免法测定３０例胃癌、３０例良性胃病患者和２３例健康人外周血单个核细胞产生ＰＧＥ２水平，同时还检测了ＴＮＦα和ＩＬ－２受体（ＩＬ－２Ｒ）表达水平，并使用消炎痛体外干预。结果：胃癌患者ＰＧＥ２和ＴＮＦα水平显著高于正常对照及良性胃病组（Ｐ＜００５），而胃癌患者ＩＬ－２Ｒ表达水平显著降低（Ｐ＜００１）；消炎痛体外干预可显著提高胃癌患者ＴＮＦα和ＩＬ－２Ｒ表达水平（Ｐ＜００１）。结论：研究ＰＧＥ２产生情况，对反映胃癌患者免疫功能状态及改善免疫治疗效果可能有一定意义     

前列腺素E2调节大鼠成骨细胞OCIL基因表达的信号通路研究

探讨前列腺素E2(PGE2)对大鼠成骨细胞破骨细胞抑制性凝集素(osteoclast inhibitory lectin OCIL)mRNA表达的调节及其信号转导机制.培养大鼠原代成骨细胞和UMRl06成骨细胞样细胞,采用不同浓度的PGE2和不同的信号通路调节剂干预细胞后,提取细胞总RNA,采用实时荧光定量PCR检测OCILmRNA的表达水平.PGE2、蛋白激酶A(PKA)激动剂福司可林、db-cAMP和钙离子载体A23187均促进OCIL mRNA表达,OCIL mRNA最大上调幅度分别为对照组的2.38倍、4.2倍、4.5倍和5.1倍(均P＜0.01).蛋白激酶C(PKC)激动剂PMA下调OCILmRNA表达约50%(P＜0.01).PKA抑制剂KT-5720、钙通道阻断剂维拉帕米、钙调蛋白抑制剂W7和丝裂原活化蛋白激酶(MAPK)阻断剂PD98059分别下调PGE2诱导的OCIL mRNA表达约56%、40%、65%和60%(均P＜0.01).PKC抑制剂白屈菜红碱促进PGE2诱导的OCIL mRNA表达约30%(P＜0.05).PKA、MAPK和Ca2+/钙调蛋白信号通路介导了PCE2诱导的大鼠成骨细胞OCIL基因表达.

Abstract：

To investigate the regulation of osteoclast inhibitory lectin (OCIL) mRNA expression by prostaglandin E2 ( PGE2 ) in rat osteoblastic cells and the involved signaling pathways. Rat primary osteoblasts and UMR106 osteoblast-like cells were cultured and treated with various doses of PGE2 or regulators of different signaling pathways for different periods of time, the cells were then harvested at indicated dates. Total RNA were isolated and OCIL mRNA expression were studied by real-time PCR. PGE2, Forskolin, db-cAMP, and A23187 increased OCIL mRNA by 2. 38 fold,4. 2 fold,4. 5 fold, and 5. 1 fold ( all P＜0. 01 ) respectively, while PMA downregulated OCIL mRNA expression by 50% ( P＜0. 01 ). KT-5720, verapamil, W7, and PD98059 downregulated PGE2 induced OCIL mRNA expression by 56%, 40%, 65%, and 60%, respectively( all P＜0. 0l ). While chelerythrine enhanced PGE2 induced OCIL mRNA expression by 30% ( P＜0. 05 ). PGE2 up-regulated the expression of OCIL in rat osteoblastic cells via PKA, MAPK, and Ca2+/Calmodulin signaling pathways.     

Enhanced levels of prostaglandin E2 and matrix metalloproteinase-2 correlate with the severity of airflow limitation in stable COPD

Background and objective: Cyclooxygenase-2 (COX-2) and its product prostaglandin E₂ (PGE₂) have been demonstrated to play critical roles in inflammation in respiratory diseases. However, the role of COX-2 in airway remodelling in COPD remains to be elucidated. Matrix metalloproteinase-2 (MMP-2) is associated with both inflammation and airway remodelling in COPD. The objective of this study was to measure the expression of COX-2 and the concentrations of PGE₂ and MMP-2, and to investigate the role of COX-2 and PGE₂ in airflow limitation mediated by MMP-2, in the pathogenesis of COPD. Methods: Forty-three patients with stable COPD, twelve smoking control subjects and ten non-smoking control subjects were enrolled. Induced sputum was obtained for measurement of the concentrations of PGE₂ and MMP-2 by ELISA. COX-2 protein expression was assessed by western blotting. Results: PGE₂ and MMP-2 concentrations were significantly higher in both smoking control subjects and patients with COPD than in non-smoking control subjects (P < 0.01). Moreover, the levels of PGE₂ and MMP-2 were inversely correlated with FEV₁% predicted in COPD patients (PGE₂: r = −0.748, P < 0.01; MMP-2: r = −0.801, P < 0.01). Levels of PGE₂ were also positively correlated with those of MMP-2 in patients with COPD (r = 0.775, P < 0.01). Expression of COX-2 protein was significantly higher in COPD patients than in non-smoking control subjects. Conclusions: COX-2 and its product PGE₂ are not only involved in airway inflammation, but may also contribute to the severity of airflow limitation mediated by MMP-2 during progression of COPD.     

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.     

Implication of the anti-inflammatory bioactive lipid prostaglandin D2-glycerol ester in the control of macrophage activation and inflammation by ABHD6

Proinflammatory macrophages are key mediators in several pathologies; thus, controlling their activation is necessary. The endocannabinoid system is implicated in various inflammatory processes. Here we show that in macrophages, the newly characterized enzyme α/β-hydrolase domain 6 (ABHD6) controls 2-arachidonoylglycerol (2-AG) levels and thus its pharmacological effects. Furthermore, we characterize a unique pathway mediating the effects of 2-AG through its oxygenation by cyclooxygenase-2 to give rise to the anti-inflammatory prostaglandin D₂-glycerol ester (PGD₂-G). Pharmacological blockade of cyclooxygenase-2 or of prostaglandin D synthase prevented the effects of increasing 2-AG levels by ABHD6 inhibition in vitro, as well as the 2-AG–induced increase in PGD₂-G levels. Together, our data demonstrate the physiological relevance of the interaction between the endocannabinoid and prostanoid systems. Moreover, we show that ABHD6 inhibition in vivo allows for fine-tuning of 2-AG levels in mice, therefore reducing lipopolysaccharide-induced inflammation, without the characteristic central side effects of strong increases in 2-AG levels obtained following monoacylglycerol lipase inhibition. In addition, administration of PGD₂-G reduces lipopolysaccharide-induced inflammation in mice, thus confirming the biological relevance of this 2-AG metabolite. This points to ABHD6 as an interesting therapeutic target that should be relevant in treating inflammation-related conditions, and proposes PGD₂-G as a bioactive lipid with potential anti-inflammatory properties in vivo.Macrophages are key players in innate and adaptive immune responses to bacterial infections or noxious agents. Their role during inflammatory processes is to eliminate the threat and protect the body (1, 2). Macrophages exhibiting an inflammatory phenotype secrete proinflammatory mediators and reactive oxygen and nitrogen species that influence the polarization of T-helper cells, further drive the inflammatory response, and activate various antimicrobial mechanisms (3). Under persistence of the proinflammatory phase, inflammation becomes chronic, thus deleterious (3). Macrophages are key mediators in the immunopathology of metabolic inflammation and autoimmune diseases such as inflammatory bowel disease (IBD) and rheumatoid arthritis (2, 4). Thus, in these pathologies, proinflammatory macrophage responses must be controlled and reduced.The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in various (patho)physiological processes and exerts numerous beneficial actions (ranging from pain modulation to reduction of anxiety) (5–11). The activity of this bioactive lipid depends on its endogenous levels, tightly controlled by the 2-AG hydrolyzing enzymes (12). Monoacylglycerol lipase (MAGL) is thought to be the primary enzyme responsible for 2-AG metabolism. Although this has been proven in the brain, where MAGL controls around 80% of 2-AG hydrolysis, it remains unclear whether this occurs in other tissues (13, 14). Because of this control of brain 2-AG levels, inhibition of MAGL leads to increased central levels of 2-AG and therefore to undesirable psychotropic side effects due to activation of the CB₁ cannabinoid receptor (15). More recently, other enzymes have been implicated in 2-AG hydrolysis, such as the newly annotated enzyme α/β-hydrolase domain 6 (ABHD6) (16–19). Here, we demonstrate the anti-inflammatory effects of inhibiting ABHD6 and thus increasing 2-AG levels, without the side effects of MAGL inhibition. Classically, 2-AG binds to and activates two G-protein–coupled receptors, termed cannabinoid receptors 1 and 2 (CB₁ and CB₂); however, it may also activate the peroxisome proliferator-activated receptors (PPARs) (20). Alternatively, this endogenous lipid, like arachidonic acid, may also be oxidized by cyclooxygenase (COX) enzymes to produce prostaglandin glycerol esters (PG-Gs) (21). So far, the biological effects of these PG-Gs are not fully elucidated. In this study, we also demonstrate that following increased 2-AG levels in macrophages, COX-2–mediated production of PGD₂-G increases, resulting in the anti-inflammatory effects observed with 2-AG (Fig. S1). We also put forth, in vivo, the anti-inflammatory properties of PGD₂-G.     

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE₂) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE₂ signaling in endometrial functions and establishment of pregnancy in ruminants.     

Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells

Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation, in particular. Here, we describe an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM). This genetically encoded high-sensitive indicator revealed that approximately 1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. The nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis.     

The role of luminal factors in prostaglandin protection against ethanol-induced gastric mucosal injury

Prostaglandins (PG) protect the gastric mucosa against damage by several irritants, but the mechanisms remain unclear. A standard rat model of gastric injury induced by 50% ethanol was used to test the hypothesis that PG protection occurs either by increasing luminal fluid volume and hence diluting the irritant, or by production of protective factors within this fluid. Quantitative histology was used to asssess microscopic mucosal damage. The increase in luminal fluid volume in prostaglandin E₂ (PGE₂)-treated animals was measured, and the ability of this increased fluid to protect saline-treated animals via dilution of the irritant was assessed. The transfer of protection by exchange of luminal fluid from PG to non-PG treated animals was also tested. Results showed that PGE₂ induced a specific increase in luminal fluid volume of 45.6%. When given together with ethanol treatment the extra fluid volume was not protective. Removal of luminal fluid after PG and before ethanol treatment did not abolish protection and no protective factor was transferred with the luminal fluid. In conclusion, this study has shown that neither dilution of ethanol by accumulation of luminal fluid nor the presence of luminal factors is responsible for PG protection.     

Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells

Many aspects of hormone receptor function that are crucial for controlling signal transduction of endocrine pathways can be monitored more accurately with the use of non-invasive, live cell resonance energy transfer (RET) techniques. Fluorescent RET (FRET), and its variation, bioluminescent RET (BRET), can be used to assess the real-time responses to specific hormonal stimuli, whilst preserving the cellular protein network, compartmentalization and spatial arrangement. Both FRET and BRET can be readily adapted to the study of membrane proteins. Here, we focus on their applications to the analysis of interactions involving the superfamily of hormone G-protein-coupled receptors. RET is also emerging as a significant tool for the determination of protein function in general. Such techniques will undoubtedly be of value in determining the functional identities of the vast array of proteins that are encoded by the human genome.     

木瓜苷对佐剂性关节炎大鼠滑膜前列腺素受体表达的影响

目的 探讨木瓜苷(GCS)对佐剂性关节炎(AA)大鼠滑膜前列腺素受体表达的影响.方法 采用弗氏完全佐剂(FCA)诱导大鼠AA模型,分离培养大鼠滑膜细胞,用四甲基噻唑蓝(MTT)法检测滑膜细胞的增殖能力,放射免疫法(RIA)测定滑膜细胞产生前列素E2(PGE2)和环磷酸腺苷(cAMP)的水平,Westem blot法检测滑膜EP2和EP4的表达.结果 GCS能显著抑制AA大鼠滑膜细胞的增殖,降低胞内的PGE,水平,升高cAMP水平,且GCS对滑膜细胞cAMP水平的提高与其抑制滑膜细胞增殖密切相关.GCS能上调滑膜EP2和EP4的表达.结论 GCS影响EP2、EP4受体的表达,恢复滑膜细胞EP-G蛋白-cAMP信号的正常转导可能是其发挥作用的主要机制之一.