Targeted Gene Deletion or Antagonism of the Prostaglandin E2 EP3 Receptor Protects Against Cardiac Injury Postmyocardial Infarction.

BACKGROUND: Prostaglandin E2 acts through 4 G-protein-coupled receptors (EP1-EP4). We previously reported that activation of the EP3 receptor reduces cardiac contractility, and its expression increases after a myocardial infarction (MI), mediating the reduction in cardiac function. In contrast, cardiac overexpression of the EP4 receptor in MI substantially improves cardiac function. Moreover, we recently reported that mice overexpressing EP3 have heart failure under basal conditions and worsened cardiac function after MI. Thus, the deleterious effects of the prostaglandin E2 EP receptors in the heart are mediated via its EP3 receptor. We, therefore, hypothesized that cardiomyocyte-specific knockout (CM-EP3 KO) or antagonism of the EP3 receptor protects the heart after MI. METHODS: To test our hypothesis, we made the novel CM-EP3 KO mouse and subjected CM-EP3 KO or controls to sham or MI surgery for 2 weeks. In separate experiments, C57BL/6 mice were subjected to 2 weeks of MI and treated with either the EP3 antagonist L798 106 or vehicle starting 3 days post-MI. RESULTS: CM-EP3 KO significantly prevented a decline in cardiac function after MI compared with WT animals and prevented an increase in hypertrophy and fibrosis. Excitingly, mice treated with L798 106 3 days after MI had significantly better cardiac function compared with vehicle-treated mice. CONCLUSIONS: Altogether, these data suggest that EP3 may play a direct role in regulating cardiac function, and pharmaceutical targeting of the EP3 receptor may be a therapeutic option in the treatment of heart failure.

The vasoconstrictor activities of prostaglandin D2 via the thromboxane prostanoid receptor and E prostanoid receptor-3 outweigh its concurrent vasodepressor effect mainly through D prostanoid receptor-1 ex vivo and in vivo.

Prostaglandin (PG) D2, a commonly considered vasodilator through D prostanoid receptor-1 (DP1), might also evoke vasoconstriction via acting on the thromboxane (Tx)-prostanoid receptor (the original receptor of TxA2; TP) and/or E prostanoid receptor-3 (one of the vasoconstrictor receptors of PGE2; EP3). This study aimed to test the above hypothesis in the mouse renal vascular bed (main renal arteries and perfused kidneys) and/or mesenteric resistance arteries and determine how the vasoconstrictor mechanism influences the overall PGD2 effect on systemic blood pressure under in vivo conditions. Experiments were performed on control wild-type (WT) mice and mice with deficiencies in TP (TP-/-) and/or EP3 (EP3-/-). Here we show that PGD2 indeed evoked vasoconstrictor responses in the above-mentioned tissues of WT mice, which were however not only reduced by TP-/- or EP3-/-, but also reversed by TP-/-/EP3-/- in some of the above tissues (mesenteric resistance arteries or perfused kidneys) to dilator reactions that were reduced by non-selective DP antagonism. A slight or mild pressor response was also observed with PGD2 under in vivo conditions, and this was again reversed to a depressor response in TP-/- or TP-/-/EP3-/- mice. Non-selective DP antagonism reduced the PGD2-evoked depressor response in TP-/-/EP3-/- mice as well. These results thus demonstrate that like other PGs, PGD2 activates TP and/or EP3 to evoke vasoconstrictor activities, which can outweigh its concurrent vasodepressor activity mediated mainly through DP1, and hence result in a pressor response, although the response might only be of a slight or mild extent.

Single hormone or synthetic agonist induces Gs/Gi coupling selectivity of EP receptors via distinct binding modes and propagating paths.

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.

Correlation of Reduced PTGER3 Expression with Prognosis and Immune Infiltration in Clear Cell Renal Carcinoma.

BACKGROUND: Prostaglandin E2 receptor 3 (PTGER3, EP3) is essential for many malignancies growth and metastasis. The role of PTGER3 in kidney renal clear cell carcinoma (KIRC) was assessed in terms of its prognosis and its association with immune infiltration. METHODS: Transcriptomic expression profiles of PTGER3 were acquired from The Cancer Genome Atlas (TCGA) database. Comparative analysis was performed to evaluate the disparity in PTGER3 expression between KIRC and normal tissues. The discriminative potential of PTGER3 as a distinguishing determinant was assessed through receiver operating characteristic (ROC) curves. Prognostic factors were evaluated employing COX regression and logistic models. Furthermore, the impact of PTGER3 on survival was ascertained utilizing the Kaplan-Meier method. A protein-protein interaction (PPI) network was constructed utilizing the STRING database. To investigate the correlation between immune infiltration levels and PTGER3 expression, a single-sample Gene Set Enrichment Analysis (GSEA) method was employed, employing the Gene Set Variation Analysis (GSVA) package and the Tumor Immune Estimation Resource (TIMER) database. RESULTS: Bioinformatics analysis unveiled a significant downregulation of PTGER3 expression in KIRC tissues compared to paraneoplastic tissues (p < 0.001). Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) experiments demonstrated a reduction in PTGER3 expression in 786-O cells in contrast to paraneoplastic tissues (p < 0.01). The ROC curve, employing PTGER3 as a potential diagnostic biomarker, exhibited a substantial area under the curve (AUC) value of 0.929. According to the Kaplan-Meier survival analysis, reduced PTGER3 expression increased the chance of negative overall survival (OS) (p = 0.019). A PPI network was constructed, elucidating the interaction patterns between PTGER3 and the top 10 co-expressed genes. An examination of gene enrichment and immune infiltration levels found a link between PTGER3 transcription and immune infiltration levels. Notably, high B cell counts and low Mast cell counts were connected to a poor prognosis in KIRC patients. CONCLUSIONS: The expression of PTGER3 was found to be diminished in KIRC in comparison to paracancerous tissue. This observation exhibited a correlation with both prognosis and immune cell infiltration. As a result, our findings suggest that PTGER3 could be considered a promising biomarker to forecast KIRC prognosis and as a possible target for immunotherapy.

Posttranscriptional regulation of the prostaglandin E receptor spliced-isoform EP3-γ and its implication in pancreatic ß-cell failure.

In Type 2 diabetes (T2D), elevated lipid levels have been suggested to contribute to insulin resistance and ß-cell dysfunction. We previously reported that the expression of the PGE2 receptor EP3 is elevated in islets of T2D individuals and is preferentially stimulated by palmitate, leading to ß-cell failure. The mouse EP3 receptor generates three isoforms by alternative splicing which differ in their C-terminal domain and are referred to as mEP3α, mEP3ß, and mEP3γ. We bring evidence that the expression of the mEP3γ isoform is elevated in islets of diabetic db/db mice and is selectively upregulated by palmitate. Specific knockdown of the mEP3γ isoform restores the expression of ß-cell-specific genes and rescues MIN6 cells from palmitate-induced dysfunction and apoptosis. This study indicates that palmitate stimulates the expression of the mEP3γ by a posttranscriptional mechanism, compared to the other spliced isoforms, and that the de novo synthesized ceramide plays an important role in FFA-induced mEP3γ expression in ß-cells. Moreover, induced levels of mEP3γ mRNA by palmitate or ceramide depend on p38 MAPK activation. Our findings suggest that mEP3γ gene expression is regulated at the posttranscriptional level and defines the EP3 signaling axis as an important pathway mediating ß-cell-impaired function and demise.

A Phytoprostane from Gracilaria longissima Increases Platelet Activation, Platelet Adhesion to Leukocytes and Endothelial Cell Migration by Potential Binding to EP3 Prostaglandin Receptor.

Plant phytoprostanes (PhytoPs) are lipid oxidative stress mediators that share structural similarities with mammal prostaglandins (PGs). They have been demonstrated to modulate inflammatory processes mediated by prostaglandins. The present study aims to test the effects of the most abundant oxylipin from Gracilaria longissima, ent-9-D1t-Phytoprostane (9-D1t-PhytoP), on platelet activation and vascular cells as well as clarify possible interactions with platelets and the endothelial EP3 receptor Platelet and monocyte activation was assessed by flow cytometry in the presence of purified 9-D1t-PhytoP. Cell migration was studied using the human Ea.hy926 cell line by performing a scratch wound healing assay. The RNA expression of inflammatory markers was evaluated by RT-PCR under inflammatory conditions. Blind docking consensus was applied to the study of the interactions of selected ligands against the EP3 receptor protein. The 9D1t-PhytoP exerts several pharmacological effects; these include prothrombotic and wound-healing properties. In endothelial cells, 9D1t-PhytP mimics the migration stimulus of PGE2. Computational analysis revealed that 9D1t-PhytP forms a stable complex with the hydrophobic pocket of the EP3 receptor by interaction with the same residues as misoprostol and prostaglandin E2 (PGE2), thus supporting its potential as an EP3 agonist. The potential to form procoagulant platelets and the higher endothelial migration rate of the 9-D1t-PhytoP, together with its capability to interact with PGE2 main target receptor in platelets suggest herein that this oxylipin could be a strong candidate for pharmaceutical research from a multitarget perspective.

Deleterious effects of cardiomyocyte-specific prostaglandin E2 EP3 receptor overexpression on cardiac function after myocardial infarction.

AIMS: Prostaglandin E2 (PGE2) is a lipid hormone that signals through 4 different G-protein coupled receptor subtypes which act to regulate key physiological processes. Our laboratory has previously reported that PGE2 through its EP3 receptor reduces cardiac contractility at the level of isolated cardiomyocytes and in the isolated working heart preparation. We therefore hypothesized that cardiomyocyte specific overexpression of the PGE2 EP3 receptor further decreases cardiac function in a mouse model of heart failure produced by myocardial infarction. MAIN METHODS: Our study tested this hypothesis using EP3 transgenic mice (EP3 TG), which overexpress the porcine analogue of human EP3 in the cardiomyocytes, and their wildtype (WT) littermates. Mice were analyzed 2 wks after myocardial infarction (MI) or sham operation by echocardiography, RT-PCR, immunohistochemistry, and histology. KEY FINDINGS: We found that the EP3 TG sham controls had a reduced ejection fraction, reduced fractional shortening, and an increased left ventricular dimension at systole and diastole compared to the WT sham controls. Moreover, there was a further reduction in the EP3 TG mice after myocardial infarction. Additionally, single-cell analysis of cardiomyocytes isolated from EP3 TG mice showed reduced contractility under basal conditions. Overexpression of EP3 significantly increased cardiac hypertrophy, interstitial collagen fraction, macrophage, and T-cell infiltration in the sham operated group. Interestingly, after MI, there were no changes in hypertrophy but there were changes in collagen fraction, and inflammatory cell infiltration. SIGNIFICANCE: Overexpression of EP3 reduces cardiac function under basal conditions and this is exacerbated after myocardial infarction.

Prostaglandin EP3 receptor activation is antinociceptive in sensory neurons via PI3Kγ, AMPK and GRK2.

BACKGROUND AND PURPOSE: Prostaglandin E2 is considered a major mediator of inflammatory pain, by acting on neuronal Gs protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is Gi protein-coupled and antagonizes the pronociceptive prostaglandin E2 effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons. EXPERIMENTAL APPROACH: Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)-/- , and PI3Kγkinase dead (KD)/KD mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry. KEY RESULTS: Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding. CONCLUSION AND IMPLICATIONS: Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E2 . These new insights hold the potential for a novel approach in pain therapy.

Altered Prostaglandin E Receptor Subtype 3 Expression in Lacrimal Glands of Patients with Chronic Stevens-Johnson Syndrome.

PURPOSE: To compare the prostaglandin E2 receptor subtype 3 (EP3) distribution in the lacrimal glands of normals, non-specific dacryoadenitis, and chronic Stevens-Johnson syndrome (SJS) patients. METHODS: Biopsies from lacrimal glands of four chronic SJS patients with severe dry eye disease, four dacryoadenitis patients, and five fresh body donors were assessed for EP3 expression using immunohistochemistry. RESULTS: In normal main and accessory lacrimal glands, EP3 is expressed strongly in nuclei and cytoplasm of majority (>75%) of acini with no ductular expression. In dacryoadenitis, EP3 expression was similar to normal glands. However, lacrimal glands from SJS patients (5-20/HPF mononuclear cells) showed a weak and reduced (<10% acini) EP3 expression within acinar cells. The reduction in intensity was more in glands with higher mononuclear cell infiltration (>10/HPF). CONCLUSION: There is downregulation of EP3 expression in the lacrimal glands of SJS patients, whereas EP3 expression is preserved in non-specific lacrimal gland inflammations.

Prostaglandin EP3 receptor-expressing preoptic neurons bidirectionally control body temperature via tonic GABAergic signaling.

The bidirectional controller of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here, we identify prostaglandin EP3 receptor-expressing POA neurons (POAEP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POAEP3R neurons are activated in response to elevated ambient temperature but inhibited by prostaglandin E2, a pyrogenic mediator. Chemogenetic stimulation of POAEP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POAEP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic (ceaseless) inhibitory signaling. Although many POAEP3R neuronal cell bodies express a glutamatergic messenger RNA marker, their axons in the DMH predominantly release γ-aminobutyric acid (GABA), and their GABAergic terminals are increased by chronic heat exposure. These findings demonstrate that tonic GABAergic inhibitory signaling from POAEP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.

Inhibition of Prostaglandin E2 Receptor EP3 Attenuates Oxidative Stress and Neuronal Apoptosis Partially by Modulating p38MAPK/FOXO3/Mul1/Mfn2 Pathway after Subarachnoid Hemorrhage in Rats.

Oxidative stress and neuronal apoptosis contribute to pathological processes of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies demonstrated that the inhibition of prostaglandin E2 receptor EP3 suppressed oxidative stress and apoptotic effects after Alzheimer's disease and intracerebral hemorrhage. This study is aimed at investigating the antioxidative stress and antiapoptotic effect of EP3 inhibition and the underlying mechanisms in a rat mode of SAH. A total of 263 Sprague-Dawley male rats were used. SAH was induced by endovascular perforation. Selective EP3 antagonist L798106 was administered intranasally at 1 h, 25 h, and 49 h after SAH induction. EP3 knockout CRISPR and FOXO3 activation CRISPR were administered intracerebroventricularly at 48 h prior to SAH, while selective EP3 agonist sulprostone was administered at 1 h prior to SAH. SAH grade, neurological deficits, western blots, immunofluorescence staining, Fluoro-Jade C staining, TUNEL staining, 8-OHdG staining, and Nissl staining were conducted after SAH. The expression of endogenous PGES2 increased and peaked at 12 h while the expression of EP1, EP2, EP3, EP4, and Mul1 increased and peaked at 24 h in the ipsilateral brain after SAH. EP3 was expressed mainly in neurons. The inhibition of EP3 with L798106 or EP3 KO CRISPR ameliorated the neurological impairments, brain tissue oxidative stress, and neuronal apoptosis after SAH. To examine potential downstream mediators of EP3, we examined the effect of the increased expression of activated FOXO3 following the administration of FOXO3 activation CRISPR. Mechanism studies demonstrated that L798106 treatment significantly decreased the expression of EP3, p-p38, p-FOXO3, Mul1, 4-HNE, Bax, and cleaved caspase-3 but upregulated the expression of Mfn2 and Bcl-2 in SAH rats. EP3 agonist sulprostone or FOXO3 activation CRISPR abolished the neuroprotective effects of L798106 and its regulation on expression of p38MAPK/FOXO3/Mul1/Mfn2 in the ipsilateral brain after SAH. In conclusion, the inhibition of EP3 by L798106 attenuated oxidative stress and neuronal apoptosis partly through p38MAPK/FOXO3/Mul1/Mfn2 pathway post-SAH in rats. EP3 may serve as a potential therapeutic target for SAH patients.

Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex.

Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.

PGE2 -EP3 axis promotes brown adipose tissue formation through stabilization of WTAP RNA methyltransferase.

Brown adipose tissue (BAT) functions as a thermogenic organ and is negatively associated with cardiometabolic diseases. N6 -methyladenosine (m6 A) modulation regulates the fate of stem cells. Here, we show that the prostaglandin E2 (PGE2 )-E-prostanoid receptor 3 (EP3) axis was activated during mouse interscapular BAT development. Disruption of EP3 impaired the browning process during adipocyte differentiation from pre-adipocytes. Brown adipocyte-specific depletion of EP3 compromised interscapular BAT formation and aggravated high-fat diet-induced obesity and insulin resistance in vivo. Mechanistically, activation of EP3 stabilized the Zfp410 mRNA via WTAP-mediated m6 A modification, while knockdown of Zfp410 abolished the EP3-induced enhancement of brown adipogenesis. EP3 prevented ubiquitin-mediated degradation of WTAP by eliminating PKA-mediated ERK1/2 inhibition during brown adipocyte differentiation. Ablation of WTAP in brown adipocytes abrogated the protective effect of EP3 overexpression in high-fat diet-fed mice. Inhibition of EP3 also retarded human embryonic stem cell differentiation into mature brown adipocytes by reducing the WTAP levels. Thus, a conserved PGE2 -EP3 axis promotes BAT development by stabilizing WTAP/Zfp410 signaling in a PKA/ERK1/2-dependent manner.

Pharmacological modulation of prostaglandin E2 (PGE2 ) EP receptors improves cardiomyocyte function under hyperglycemic conditions.

Type 2 diabetes (T2D) affects >30 million Americans and nearly 70% of individuals with T2D will die from cardiovascular disease (CVD). Circulating levels of the inflammatory signaling lipid, prostaglandin E2 (PGE2 ), are elevated in the setting of obesity and T2D and are associated with decreased cardiac function. The EP3 and EP4 PGE2 receptors have opposing actions in several tissues, including the heart: overexpression of EP3 in cardiomyocytes impairs function, while EP4 overexpression improves function. Here we performed complementary studies in vitro with isolated cardiomyocytes and in vivo using db/db mice, a model of T2D, to analyze the effects of EP3 inhibition or EP4 activation on cardiac function. Using echocardiography, we found that 2 weeks of systemic treatment of db/db mice with 20 mg/kg of EP3 antagonist, beginning at 6 weeks of age, improves ejection fraction and fractional shortening (with no effect on heart rate). We further show that either EP3 blockade or EP4 activation enhances contractility and calcium cycling in isolated mouse cardiomyocytes cultured in both normal and high glucose. Thus, peak [Ca2+ ]I transient amplitude was increased, while time to peak [Ca2+ ]I and [Ca2+ ]I decay were decreased. These data suggest that modulation of EP3 and EP4 activity has beneficial effects on cardiomyocyte contractility and overall heart function.

Prostaglandin E2 receptor 3 promotes M1 macrophages polarization in unexplained recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is associated with macrophage polarization, which can be modulated by prostaglandin E2 (PGE2). Our previous study demonstrated that PGE2 receptor 3 (EP3) signaling is induced in the first-trimester placentas of uRPL patients compared with its expression in healthy controls. However, whether EP3 plays a role in macrophage polarization at the maternal-fetal interface of uRPL women remains unknown. The positive expression of EP3 in decidual macrophages was confirmed by double immunofluorescence staining in the first-trimester placentas collected from uRPL patients and healthy controls. Antibodies CD68, iNOS, and CD163 were used as immunofluorescence marker for decidual macrophages, M1, and M2 macrophages. To clarify the effects of EP3 on macrophage polarization, THP-1 monocyte cells were applied as M0 macrophages after phorbol 12-myristate 13-acetate (PMA) treatment for in vitro study. The mRNA levels of representative M1 markers (interleukin-1ß and interleukin-6) and M2 markers (interleukin-10 and arginase-1) were quantified with qPCR in M0 macrophages being stimulated with sulprostone (an EP3 agonist) or L-798,106 (an EP3 antagonist). We found that EP3 expression was upregulated in the decidual macrophages of first-trimester placentas from uRPL patients compared with healthy controls. Furthermore, EP3 expression was increased in M1 macrophages compared with that in M2 macrophages in first-trimester placentas of uRPL patients. Sulprostone intensified the mRNA levels of IL-6 together with interferon-γ, whereas L-798,106 stimulated the mRNA expression of IL-10 and Arg-1 in a dose-dependent manner.

The Roles Played by FP/EP3 Receptors During Pressure-lowering in Mouse Eyes Mediated by a Dual FP/EP3 Receptor Agonist.

Purpose: We investigated the intraocular pressure (IOP)-lowering effect of topical sepetaprost (SPT), a dual agonist of the FP and EP3 receptors. We explored whether certain receptors mediated the hypotensive effect of SPT and outflow facility changes in C57BL/6 mice (wild-type [WT]) and FP and EP3 receptor-deficient mice (FPKO and EP3KO mice, respectively). Methods: IOP was measured using a microneedle. Outflow facility was measured using a two-level, constant-pressure perfusion method. Results: SPT significantly reduced IOP for 8 hours after administration to WT mice. The 2-hour IOP reductions afforded by latanoprost were 15.3 ± 2.5, 1.8 ± 2.0, and 12.3 ± 2.4% in WT, FPKO, and EP3KO mice, respectively; the SPT figures were 13.6 ± 2.1, 5.9 ± 2.7, and 6.6 ± 2.6%, respectively. Latanoprost-mediated IOP reduction was significantly decreased in FPKO mice, and SPT-mediated IOP reduction was reduced in both FPKO and EP3KO mice. At 6 hours after administration, latanoprost did not significantly reduce the IOP in any tested mouse strain. SPT-mediated IOP reduction was reduced in both FPKO and EP3KO mice. IOP reduction at 6 hours was significantly higher after simultaneous administration of selective FP and EP3 receptor agonists, but IOP did not fall on administration of (only) a selective EP3 receptor agonist. SPT significantly increased outflow facility in WT mice, but less so in FPKO and EP3KO mice. Conclusions: The IOP-lowering effect of SPT lasted longer than that of latanoprost. Our data imply that this may be attributable to augmented outflow facility mediated by the FP and EP3 receptors.

Synergistic Cytokine Production by ATP and PGE2 via P2X4 and EP3 Receptors in Mouse Bone-Marrow-Derived Mast Cells.

ATP is an important intercellular messenger in the extracellular space. In mast cells (MCs), ATP stimulates the ionotropic P2X4 receptor (P2X4R), resulting in enhanced degranulation and exacerbation of acute allergic reactions. In this study, we investigate whether ATP regulates inflammatory cytokine production in MCs. Gene expression was analyzed by quantitative RT-PCR, and cytokine production was measured using ELISA. The stimulation of mouse bone-marrow-derived MCs (BMMCs) with ATP alone had little effect on cytokine secretion. However, the co-stimulation with prostaglandin (PG) E2 resulted in a marked increase in the secretion of various cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-13, accompanied by an increase in their mRNA levels. The effects of ATP were inhibited by P2X4R antagonists and diminished in BMMCs derived from P2X4R-deficient mice, suggesting that P2X4R mediated the reaction. The effects of PGE2 were mimicked by an EP3 receptor (EP3R) agonist and blocked by an EP3R antagonist. The synergistic cytokine mRNA elevations induced by ATP and PGE2 were blocked by nuclear factor-κB and Ca2+-calcineurin signaling inhibitors. Altogether, these results suggest that combining P2X4R and EP3R signaling enhances acute degranulation and the subsequent cytokine secretion, exacerbating allergic inflammation.

Effects of DISC1 on Alzheimer's disease cell models assessed by iTRAQ proteomics analysis.

Alzheimer's disease (AD) is a form of neurodegenerative disease in the elderly with no cure at present. In a previous study, we found that the scaffold protein, disrupted in Schizophrenia 1 (DISC1) is down-regulated in the AD brains, and ectopic expression of DISC1 can delay the progression of AD by protecting synaptic plasticity and down-regulating BACE1. However, the underlying mechanisms remain not to be elucidated. In the present study, we compared the proteomes of normal and DISC1high AD cells expressing the amyloid precursor protein (APP) using isobaric tag for relative and absolute quantitation (iTRAQ) and mass spectrometry (MS). The differentially expressed proteins (DEPs) were identified, and the protein-protein interaction (PPI) network was constructed to identify the interacting partners of DISC1. Based on the interaction scores, NDE1, GRM3, PTGER3 and KATNA1 were identified as functionally or physically related to DISC1, and may therefore regulate AD development. The DEPs were functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with the DAVID software, and the Non-supervised Orthologous Groups (eggNOG) database was used to determine their evolutionary relationships. The DEPs were significantly enriched in microtubules and mitochondria-related pathways. Gene set enrichment analysis (GSEA) was performed to identify genes and pathways that are activated when DISC1 is overexpressed. Our findings provide novel insights into the regulatory mechanisms underlying DISC1 function in AD.

Prostaglandin E2 EP receptors in cardiovascular disease: An update.

This review article provides an update for the role of prostaglandin E2 receptors (EP1, EP2, EP3 and EP4) in cardiovascular disease. Where possible we have reported citations from the last decade although this was not possible for all of the topics covered due to the paucity of publications. The authors have attempted to cover the subjects of ischemia-reperfusion injury, arrhythmias, hypertension, novel protein binding partners of the EP receptors and their pathophysiological significance, and cardiac regeneration. These latter two topics bring studies of the EP receptors into new and exciting areas of research that are just beginning to be explored. Where there is peer-reviewed literature, the authors have placed particular emphasis on clinical studies although these are limited in number.

Prostaglandin E2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 channels.

BACKGROUND: Cough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin E2 (PGE2), have been implicated in both peripheral induction and sensitization of the cough reflex. In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms. METHODS: All drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS). RESULTS: We show that both PGE2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough (P ≤ 0.001, P ≤ 0.01, respectively). Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS. CONCLUSION: Collectively, our findings show that PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets.