The EP3 and EP4 Receptor Subtypes both Mediate the Fever-producing Effects of Prostaglandin E2 in the Rostral Ventromedial Preoptic Area of the Hypothalamus in Rats.

This study aimed to re-examine the receptor subtype that mediates the fever-producing effects of prostaglandin E2 (PGE2) in the rostral ventromedial preoptic area (rvmPOA) of the hypothalamus. Among the four subtypes of PGE2 receptors (EP1, EP2, EP3, and EP4), EP3 receptor is crucially involved in the febrile effects of PGE2. However, it is possible for other subtypes of PGE2 receptor to contribute in the central mechanism of fever generation. Accordingly, effects of microinjection of PGE2 receptor subtype-specific agonists or antagonists were examined at the locus where a microinjection of a small amount (420 fmol) of PGE2 elicited prompt increases in the O2 consumption rate (VO2), heart rate, and colonic temperature (Tc) in the rvmPOA of urethane-chloralose-anesthetized rats. The EP3 agonist sulprostone mimicked, whereas its antagonist L-798,106 reduced, the febrile effects of PGE2 microinjected into the same site. Similarly, the EP4 agonist rivenprost mimicked, whereas its antagonist ONO-AE3-208 reduced, the effects of PGE2 microinjected into the same site. In contrast, microinjection of the EP1 agonist iloprost induced a very small increase in VO2 but did not have significant influences on the heart rate and Tc, whereas its antagonist, AH6809, did not affect the PGE2-induced responses. Microinjection of the EP2 agonist butaprost had no effects on the VO2, heart rate, and Tc. The results suggest that the EP3 and EP4 receptor subtypes are both involved in the fever generated by PGE2 in the rvmPOA.

PGE2 receptor agonist misoprostol protects brain against intracerebral hemorrhage in mice.

Intracerebral hemorrhage (ICH) is a devastating form of stroke. Misoprostol, a synthetic prostaglandin E1 (PGE1) analog and PGE2 receptor agonist, has shown protection against cerebral ischemia. In this study, we tested the efficacy of misoprostol in the 12-month-old mice subjected to 1 of 2 complementary ICH models, the collagenase model (primary study) and blood model (secondary study, performed in an independent laboratory). We also investigated its potential mechanism of action. Misoprostol posttreatment decreased brain lesion volume, edema, and brain atrophy and improved long-term functional outcomes. In the collagenase-induced ICH model, misoprostol decreased cellular inflammatory response; attenuated oxidative brain damage and gelatinolytic activity; and decreased high-mobility group box 1 (HMGB1) expression, Src kinase activity, and interleukin-1ß expression without affecting cyclooxygenase-2 expression. Furthermore, HMGB1 inhibition with glycyrrhizin decreased Src kinase activity, gelatinolytic activity, neuronal death, and brain lesion volume. Src kinase inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) decreased gelatinolytic activity and brain edema and improved neurologic function but did not decrease HMGB1 protein level. These results indicate that misoprostol protects brain against ICH injury through mechanisms that may involve the HMGB1, Src kinase, and matrix metalloproteinase-2/9 pathways.

An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening.

BACKGROUND: Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E(1) (PGE(1)) and E(2) (PGE(2)) can be identified in the water-soluble non-purified fraction. PGE(1) is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE(2) is an inflammatory molecule. METHODS: We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP(1) receptor stably expressed in HEK293 cells (human embryonic kidney). PGE(1) and PGE(2) were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened. RESULTS: After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 µM concentration) using fluorescence microscopy. CONCLUSIONS: Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, PGE(1) and PGE(2).

Neuroprotection by selective allosteric potentiators of the EP2 prostaglandin receptor.

Activation of the Galphas-coupled EP2 receptor for prostaglandin E2 (PGE(2)) 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 microM (i) potentiated the cAMP response to a low concentration of PGE(2) by > 50%; (ii) had no effect on EP4 or beta2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE(2) 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 PGE(2) on EP2 receptors 4- to 5-fold at 10 to 20 microM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE(2) receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE(2) released in a cell-injury setting is neuroprotective.

Prostaglandin E receptor subtype EP3 in conjunctival epithelium regulates late-phase reaction of experimental allergic conjunctivitis.

BACKGROUND: We previously demonstrated that the prostaglandin E(2) (PGE(2))-EP3 pathway negatively regulates allergic reactions in a murine allergic asthma model. OBJECTIVES: We investigated whether the PGE(2)-EP3 pathway also regulates the development of murine experimental allergic conjunctivitis (EAC). METHODS: The expression of EP3 was examined by means of RT-PCR and immunohistochemistry in wild-type mice, as well as by means of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining in mice deficient in EP3 (Ptger3(-/-) mice) carrying the beta-galactosidase gene at the EP3 gene locus. EAC was induced by immunization of mice with short ragweed pollen (RW), followed by challenge with eye drops of RW, and eosinophil infiltration and eotaxin-1 mRNA expression in the conjunctiva were examined. Mice were also treated with a topical application of an EP3-selective agonist during the elicitation phase. Quantitative RT-PCR was used to detect expression of COXs and prostaglandin E synthases, and ELISA was used to measure PGE(2) production in the eyelid. RESULTS: EP3 was constitutively expressed in conjunctival epithelium on the ocular surface. Ptger3(-/-) mice demonstrated significantly increased eosinophil infiltration in conjunctiva after RW challenge compared with wild-type mice. Consistently, significantly higher expression of eotaxin-1 mRNA was observed in Ptger3(-/-) mice. Conversely, treatment of wild-type mice with an EP3-selective agonist resulted in a significant decrease in eosinophil infiltration, which was blunted in Ptger3(-/-) mice. Expression of COX-2 and prostaglandin E synthases was upregulated and PGE(2) content was increased in the eyelids after RW challenge. CONCLUSION: These data suggest that PGE(2) acts on EP3 in conjunctival epithelium and downregulates the progression of EAC.

PGE2 signal via EP2 receptors evoked by a selective agonist enhances regeneration of injured articular cartilage.

OBJECTIVE: The effect of the prostaglandin E2 (PGE2) signal through prostaglandin E receptor 2 (EP2) receptors on the repair of injured articular cartilage was investigated using a selective agonist for EP2. METHODS: Chondral and osteochondral defects were prepared on the rabbit femoral concave in both knee joints, and gelatin containing polylactic-co-glycolic acid microspheres conjugated with or without the EP2 agonist was placed nearby. Animals were sacrificed at 4 or 12 weeks post-operation, and regenerated cartilage tissues and subchondral structure remodeling were evaluated by histological scoring. The quality of regenerated tissues was also evaluated by the immunohistochemical staining of EP2, type II collagen, and proliferating cell nuclear antigen (PCNA). As an evaluation of side effects, the inflammatory reaction of the synovial membrane was analyzed based on histology and the mRNA expression of matrix metalloproteinase3 (MMP3), tissue inhibitor of metalloproteinase 3 (TIMP3), and interleukin-1 beta (IL-1 beta). Also, the activity of MMP3 and the amount of tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein in joint fluid were measured. RESULTS: In both models, the EP2 agonist enhanced the regeneration of the type II collagen-positive tissues containing EP2- and PCNA-positive chondrocytes, and the histological scale of regenerated tissue and subchondral bone was better than that of on the control side, particularly at 12 weeks post-operation. No inflammatory reaction in the synovial membrane was observed, and no induction of pro-inflammatory cytokines was found in joint fluid. CONCLUSION: Selective stimulation of the PGE2 signal through EP2 receptors by a specific agonist promoted regeneration of cartilage tissues with a physiological osteochondral boundary, suggesting the potential usefulness of this small molecule for the treatment of injured articular cartilages.

Synthesis and evaluation of novel pyrazolidinone analogs of PGE2 as EP2 and EP4 receptors agonists.

Replacement of the hydroxy cyclopentanone ring in PGE(2) with chemically more stable heterocyclic rings and substitution of the unsaturated alpha-alkenyl chain with a metabolically more stable phenethyl chain led to the development of potent and selective analogs of PGE(2). Compound 10f showed the highest potency and selectivity for EP(4) the receptor.

A comparative study of the bone-restorative efficacy of anabolic agents in aged ovariectomized rats.

INTRODUCTION: The study was designed to compare the bone anabolic effects of basic fibroblast growth factor (bFGF), a selective agonist for prostaglandin E receptor subtype EP4, and parathyroid hormone (PTH) in aged ovariectomized (OVX) rats with severe cancellous osteopenia. METHODS: Groups of aged OVX rats were maintained untreated for 1 year postovariectomy (15 months of age) to develop severe tibial cancellous osteopenia. These animals were then treated with bFGF or the EP4 agonist (EP4) for 3 weeks. Other groups of aged OVX rats were treated with EP4 or PTH alone for 11 weeks, or sequentially with bFGF or EP4 for 3 weeks followed by PTH for 8 weeks. Cancellous and cortical bone histomorphometry were performed in the right proximal tibial metaphysis and tibial diaphysis respectively. RESULTS: Treatment with bFGF for 3 weeks markedly increased serum osteocalcin, osteoid volume, and osteoblast and osteoid surfaces to a greater extent than EP4. Basic FGF, but not EP4 or PTH, induced formation of osteoid islands within bone marrow. EP4 stimulated cancellous bone turnover, but failed to restore lost cancellous bone in the severely osteopenic proximal tibia after 11 weeks of treatment. In contrast, EP4, much like PTH, increased cortical bone mass in the tibial diaphysis by stimulating both periosteal and endocortical bone formation. Treatment of aged OVX rats with PTH alone tended to partially reverse the severe tibial cancellous osteopenia, whereas sequential treatment with bFGF and PTH increased tibial cancellous bone mass to near the level of vehicle-treated control rats. These findings indicate that bFGF had the strongest stimulatory effect on cancellous bone formation, and was the only anabolic agent to induce formation of osteoid islands within the bone marrow of the severely osteopenic proximal tibia. Therefore, bFGF may be more effective for the reversal of severe cancellous osteopenia. PTH and EP4 increased cortical bone mass to nearly the same extent, but cancellous bone mass was greater by two-fold in PTH-treated OVX rats than in EP4-treated OVX rats. CONCLUSION: These findings in aged OVX rats suggest that PTH is more efficacious than EP4 for augmentation of cancellous bone in the severely osteopenic, estrogen-deplete skeleton.

[Effect of guizhi decoction on PKA and PKC activities of hypothalamus in fever rats].

OBJECTIVE: To investigate the changes of the activity of both protein kinase A and C and the mechanisms of antipyretic action of Guizhi decoction. METHOD: The fever responses were observed after combination injection of H-89 (a selective inhibitor of PKA) and calphostin C (a selective inhibitor of PKC), and oral pretreatment of Guizhi decoction in fever rats induced by an intra-cerebroventricular (icv) injection of an EP3 agonist, and both PKA and PKC activity in hypothalamus were measured in rats pretreated with Guizhi decoction and vehicle using isotopic tracing assay. RESULT: The rise in rat body temperature was inhibited by H-89, Calphostin C, and Guizhi decoction, moreover, pretreatment with Guizhi decoction reduced PKA activity obviously. PKC activity in model rats exhibited a tendency to drop compared with that of control group, Oral administration of Guizhi decoction in large dose inhibited the response significantly, while the low dose of Guzhi decoction has no effect on PKC. CONCLUSION: Both PKA and PKC may participate in the mechanism of fever induction by EP3 agonist. The decrease of PKA and PKC may contribute to the antipyretic action of Guizhi decoction, some isoenzyme of PKC may play a role in the fever production.

Evaluation of selective prostaglandin E2 (PGE2) receptor agonists as therapeutic agents for the treatment of asthma.

Prostaglandin E2 (PGE2) released in asthmatic airways has bronchodilator properties and inhibits allergen-induced bronchoconstriction and release of inflammatory mediators. Although considered as a potential treatment for asthma, PGE2 also has some proinflammatory properties. PGE2 acts through four different receptor subtypes (EP1, EP2, EP3, and EP4) that may explain some of PGE2's diverse effects. In a mouse model of allergic inflammation in which the four receptors were individually deleted, only EP3(-/-) mice showed an enhancement of inflammation, whereas an EP3 agonist was inhibitory, with PGE2 being inactive. Thus, EP3 agonists may lead to a new approach for the treatment of asthma. However, other PGE2 receptor subtypes may also have beneficial effects, and a greater understanding of the signaling pathways of these receptor subtypes will help to clarify the role of these receptors in asthma.

Effect of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the cortical bone response to mechanical loading.

The influence of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the bone response to mechanical loading was evaluated. Six-month-old female Wistar rats were used and assigned to three groups (n = 12/group): Vehicle administration (EP4-V), low-dose ONO-4819 administration (EP4-L, 3 microg/kg BW), and high-dose ONO-4819 administration (EP4-H, 30 microg/kg BW). ONO-4819 was subcutaneously injected in the back twice a day for 3 weeks. Loads on the right tibia at 39.4 N for 36 cycles at 2 Hz were applied in vivo by 4-point bending every other day for 3 weeks. Whole-body bone mineral content showed a significant difference between EP4-V and EP4-H (P < 0.05). Bone mineral density (BMD) of the total and regional tibia (the region with maximal bending at the central diaphysis) was higher in EP4-H than EP4-V, showing a significant effect of loading (P < 0.001) and ONO-4819 (P < 0.05). BMD of the total femur was higher in EP4-H than EP4-V (P < 0.01) and that of the distal femur was higher in EP4-H than EP4-V (P < 0.001). Histomorphometry of the cortical bone showed that loading increased formation surface (FS/BS), mineral appositional rate (MAR), and bone formation rate (BFR/BS) significantly at the lateral periosteal surface (P < 0.001); however, the effect of ONO-4819 was not significant. At the medial periosteal surface, loading increased the three parameters (P < 0.001) and ONO-4819 increased FS/BS (P < 0.001) and MAR (P < 0.05) significantly. At the endocortical surface, the effects of both loading and ONO-4819 were significant on all three parameters (for loading; FS/BS P < 0.01, MAR P < 0.05, BFR/BS P < 0.03, for ONO-4819 all P < 0.001). It was concluded that ONO-4819 increased cortical bone formation in rats and there was an additive effect on the bone response to external loading by 4-point bending.

Prostaglandin E2 as a mediator of fever: the role of prostaglandin E (EP) receptors.

Prostaglandin E2 (PGE2) is a principal fever mediator that induces hyperthermia when injected into the organum vasculosum lamina terminalis (OVLT) and the adjacent preoptic area of the hypothalamus (POA). PGE (EP) receptors have four subtypes, i.e. EP1, EP2, EP3, and EP4. In the rat OVLT/POA region, at least three of these receptors, i.e. EP1, EP3, and EP4 receptors, have distinctively different distribution patterns. In rats, intracerebroventricular injection of EP1 receptor agonists and EP3 receptor agonists increased core temperature (Tc) and that of an EP4 receptor agonist decreased it. IntraPOA injection of an EP1 receptor agonist increased Tc. IntraPOA injection of an EP3 receptor agonist, however, induced hyperalgesia but not hyperthermia. Studies using mice with EP receptor gene deletions have indicated that EP3 receptors play a crucial role in febrile response. Therefore, the involvement of EP3 receptors at other levels of the nervous system should be considered. Such nuclei include the raphe pallidus nucleus, intermediolateral cell column in the spinal cord, or the nucleus of the solitary tract.

Prostaglandin E2 receptor (EP4) selective agonist (ONO-4819.CD) accelerates bone repair of femoral cortex after drill-hole injury associated with local upregulation of bone turnover in mature rats.

Prostaglandin E(2) (PGE(2)) is essential for fracture healing. Systemic administration of EP4 ligands such as PGE(2) and other synthetic EP4 agonists appears to transduce anabolic signals by binding to receptor EP4. Therefore, the present study was designed to test whether administration of EP4 agonist accelerates the healing of drill-hole injury in the femoral diaphysis. After surgery, a total of 128 Wistar rats, at the age of 12 weeks, were assigned to basal control (n = 8), and three groups with respective doses of 0 (vehicle control), 10 (low-dose), and 30 (high-dose) microg/kg body weight of the agent were subcutaneously injected twice a day. Femoral specimens were obtained at 0, 5, 7, 14, 21, and 28 days. In EP4 agonist-treated groups, the total bone volume of the regenerating bone in the defect did not significantly differ, but the regenerated cortical bone volume measured by histomorphometry and cortical bone mineral content (Ct. BMC) by pQCT dose-dependently increased at 14 and 21 days compared to the control. In the high-dose group, the value of osteoclast surface significantly increased compared with that in the control at 14 days. Expression levels of osteocalcin and TRAP mRNAs in the injured tissue increased at 14 days. Expression levels of EP4, BMP-2, and RANKL mRNAs increased at 7 days in the high-dose group. The bone mineral values of the lumbar bone at 28 days, measured by DXA, did not differ in the three groups. These data indicated that systemic administration of EP4 agonist ONO-4819.CD accelerated cortical bone healing after drill-hole injury by upregulating the local turnover of the regenerating bone.

Prostaglandin E2 activates the histaminergic system via the EP4 receptor to induce wakefulness in rats.

Prostaglandin (PG)E2 promotes the wakeful state when administered into the posterior hypothalamus, in which the histaminergic tuberomammillary nucleus (TMN) is located. To explore the neurotransmitter mechanisms responsible for PGE2-induced wakefulness in rats, we examined the effect of PGE2 on the activity of the histaminergic system and the involvement of PGE2 receptor subtypes in the response. PGE2 perfusion in the TMN at doses of 100, 200, and 400 pmol/min for 2 hr significantly increased histamine release from the medial preoptic area and frontal cortex in a dose-dependent manner, as measured by in vivo microdialysis. Among the agonists of the four distinct subtypes of PGE2 receptors (EP1-4) tested, only the EP4 receptor agonist (ONO-AE1-329) mimicked the excitatory effect of PGE2 on histamine release from both the medial preoptic area and frontal cortex. Perfusion of either PGE2 or the EP4 agonist into the TMN at a dose of 200 pmol/min for 1 hr increased histidine decarboxylase activity, histidine decarboxylase mRNA level, and histamine content in the hypothalamus. In situ hybridization revealed that EP4 receptor mRNA was expressed in histidine decarboxylase-immunoreactive neurons of the TMN region. Furthermore, EP4 agonist perfusion into the TMN induced wakefulness. These findings indicate that PGE2 induces wakefulness through activation of the histaminergic system via EP4 receptors.

Contrasting effects of E type prostaglandin (EP) receptor agonists on core body temperature in rats.

Prostaglandin E2 (PGE2) is thought to be a principal fever mediator. There are four subtypes of PGE (EP) receptors, EP1-EP4. We investigated which EP receptors mediate PGE2-induced hyperthermia by injecting selective EP receptor agonists into the rat lateral cerebral ventricle under unrestrained condition. ONO-DI-004, an EP1 receptor agonist, increased the core temperature (T(c)) in a dose-dependent manner (1.6+/-0.1 degrees C at 20 nmol, with the peak 30 min after injection) with a time course similar to PGE2-induced hyperthermia. ONO-AE1-259-01 (20 nmol), an EP2 receptor agonist, did not change the T(c). ONO-AE-248 (20 nmol), an EP3 receptor agonist, also increased the T(c). However, the peak effect was delayed (1.2+/-0.2 degrees C, 50 min after injection) compared to PGE2. In contrast, ONO-AE1-329, an EP4 receptor agonist, decreased the T(c). These findings suggest that the EP1, EP3, and EP4 receptors all may contribute to the thermoregulatory response to PGE2, but each may have a different role.

[Effect of guizhi tang and its active components on the fever induced by EP3 agonist].

OBJECTIVE: To investigate the effect of Guizhi Tang and its active components on the fever induced by EP3 receptor agonist sulprostone in rats. METHOD: The rise in body temperature evoked by a LCV(lateral cerebroventricle)-injection of sulprostone was compared with that of sulprostone induced-fever rats pretreated with Guizgi Tang and its active compounds, cinnamaldehyde, cinnamic acid and total glucosides of paeony. RESULT: Pretreatments with Guizhi Tang and cinnamaldehyde inhibited the rise in body temperature induced by sulprostone, while cinnamic acid tended to augment the fever. The sulprostone-induced fever was blocked by an ip pretreatment of total glucosides of paeony even below the basement. CONCLUSION: Present data suggest that interruption with the down-stream events of EP3 receptor may contribute to the antipyretic action of Guizhi Tang, cinnamaldehyde and the total glucosides of paeony, while cinnamic acid may have no such effect.