2-Methoxyestradiol Ameliorates Angiotensin II-Induced Hypertension by Inhibiting Cytosolic Phospholipase A2α Activity in Female Mice.

[Figure: see text].

Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances ß-cell proliferation and identity and relieves oxidative damage.

OBJECTIVE: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes ß-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance ß-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). METHODS: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and ß-cell proliferation and ß-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. RESULTS: EP3 blockade increased ß-cell mass in db/db mice through enhanced ß-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. CONCLUSIONS: The current study suggests that EP3 blockade promotes ß-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

Optimization of physicochemical properties of pyridone-based EP3 receptor antagonists.

A novel series of pyridone-based EP3 receptor antagonists was optimized for good physical properties and oral bioavailability in rodents. The lead compounds 3h, 3l and 4d displayed good in vitro profiles, moderate to good metabolic stability and good rodent PK profiles with low clearance, high oral exposure and acceptable half-life.

Rat prostaglandin EP3 receptor is highly promiscuous and is the sole prostanoid receptor family member that regulates INS-1 (832/3) cell glucose-stimulated insulin secretion.

Chronic elevations in fatty acid metabolites termed prostaglandins can be found in circulation and in pancreatic islets from mice or humans with diabetes and have been suggested as contributing to the ß-cell dysfunction of the disease. Two-series prostaglandins bind to a family of G-protein-coupled receptors, each with different biochemical and pharmacological properties. Prostaglandin E receptor (EP) subfamily agonists and antagonists have been shown to influence ß-cell insulin secretion, replication, and/or survival. Here, we define EP3 as the sole prostanoid receptor family member expressed in a rat ß-cell-derived line that regulates glucose-stimulated insulin secretion. Several other agonists classically understood as selective for other prostanoid receptor family members also reduce glucose-stimulated insulin secretion, but these effects are only observed at relatively high concentrations, and, using a well-characterized EP3-specific antagonist, are mediated solely by cross-reactivity with rat EP3. Our findings confirm the critical role of EP3 in regulating ß-cell function, but are also of general interest, as many agonists supposedly selective for other prostanoid receptor family members are also full and efficacious agonists of EP3. Therefore, care must be taken when interpreting experimental results from cells or cell lines that also express EP3.

Cross-effect of TRPV1 and EP3 receptor on coughs and bronchopulmonary C-neural activities.

Prostaglandin E2 (PGE2)-induced coughs in vivo and vagal nerve depolarization in vitro are inhibited by systemic and local administration of prostaglandin EP3 receptor (L-798106) and TRPV1 antagonists (JNJ 17203212). These results indicate a modulating effect of TRPV1 on the EP3 receptor-mediated cough responses to PGE2 likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA, mainly stimulating TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically are co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) there was a cross-effect of TRPV1 and EP3 receptor on these neural excitations. To this end, aerosolized CA or PGE2 was inhaled by unanesthetized guinea pigs pretreated without or with each antagonist given in aerosol form. Immunofluorescence was applied to identify the co-expression of TRPV1 and EP3 receptor in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell voltage patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in individual vagal pulmonary C-neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the evoked currents. We found that PGE2-induced cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. Approximately 1/4 of vagal pulmonary C-neurons co-expressed EP3 with a cell size < 20 µm. Both CAP- and PGE2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the cross-effect of both antagonists on cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the cough response to PGE2.

Larger endothelium-dependent contractions in iliac arteries of adult SHRs are attributed to differential downregulation of TP and EP3 receptors in the vessels of WKYs and SHRs during the transition from adolescence to adulthood.

This study was to determine how endothelium-dependent contractions (EDCs) change in iliac arteries of Wistar-Kyoto (WKYs) and spontaneously hypertensive rats (SHRs) during the transition from adolescence to adulthood and the underlying mechanism(s). We also aimed to elucidate effects of L-798106, an EP3 receptor antagonist, on EDCs and the blood pressure increase in adolescent SHRs. Blood vessels were isolated for functional and biochemical analyses. EDCs were comparable in adolescent iliac arteries of both strains, and contractions to ACh, prostacyclin (PGI2), the EP3 receptor agonist sulprostone and the TP receptor agonist U46619 in adult vessels were less prominent compared with those in the adolescents, while the attenuation of vasoconstrictions to ACh, PGI2 or U46619 with age was to a lesser extent in SHRs. PGI2 production was decreased to a similar level in adult arteries. TP and EP3 expressions were downregulated in adult vessels, whereas the extent of TP downregulation was less in SHRs. L-798106 partially suppressed the vasoconstrictions to U46619 and attenuated EDCs to a greater extent than SQ29548, and administration of L-798106 blunted the blood pressure increase with age in prehypertensive SHRs. These results demonstrate the comparable EDCs in iliac arteries of the adolescents are decreased in the adults, but relatively larger EDCs in adult SHRs can be a reflection of differential downregulation of TP and EP3 receptors during the transition from adolescence to adulthood. Also, our data suggest that blockade of both TP and EP3 receptors starting from the prehypertensive stage suppresses EDCs and the development of hypertension in SHRs.

Central Blockade of E-Prostanoid 3 Receptor Ameliorated Hypertension Partially by Attenuating Oxidative Stress and Inflammation in the Hypothalamic Paraventricular Nucleus of Spontaneously Hypertensive Rats.

Hypertension, as one of the major risk factors for cardiovascular disease, significantly affects human health. Prostaglandin E2 (PGE2) and the E3-class prostanoid (EP3) receptor have previously been demonstrated to modulate blood pressure and hemodynamics in various animal models of hypertension. The PGE2-evoked pressor and biochemical responses can be blocked with the EP3 receptor antagonist, L-798106 (N-[(5-bromo-2methoxyphenyl)sulfonyl]-3-[2-(2-naphthalenylmethyl) phenyl]-2-propenamide). In the hypothalamic paraventricular nucleus (PVN), sympathetic excitation can be introduced by PGE2, which can activate EP3 receptors located in the PVN. In such a case, the central knockdown of EP3 receptor can be considered as a potential therapeutic modality for hypertension management. The present study examined the efficacy of the PVN infusion of L-798106, by performing experiments on spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). The rats were administered with chronic bilateral PVN infusion of L-798106 (10 µg/day) or the vehicle for 28 days. The results indicated that the SHRs had a higher mean arterial pressure (MAP), an increased Fra-like (Fra-LI) activity in the PVN, as well as a higher expression of gp91phox, mitogen-activated protein kinase (MAPK), and proinflammatory cytokines in the PVN compared with the WKYs. Additionally, the expression of Cu/Zn-SOD in the PVN of the SHRs was reduced compared with the WKYs. The bilateral PVN infusion of L-798106 significantly reduced MAP, as well as plasma norepinephrine (NE) levels in the SHRs. It also inhibited Fra-LI activity and reduced the expression of gp91phox, proinflammatory cytokines, and MAPK, whereas it increased the expression of Cu/Zn-SOD in the PVN of SHRs. In addition, L-798106 restored the balance of the neurotransmitters in the PVN. On the whole, the findings of the present study demonstrate that the PVN blockade of EP3 receptor can ameliorate hypertension and cardiac hypertrophy partially by attenuating ROS and proinflammatory cytokines, and modulating neurotransmitters in the PVN.

The deleterious role of the prostaglandin E2 EP3 receptor in angiotensin II hypertension.

Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E2 (PGE2) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP3 and EP4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP3-transgenic mice, which overexpress EP3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN.NEW & NOTEWORTHY This study is the first to show that systemic treatment with an EP3 receptor antagonist (L798,106) attenuates the angiotensin II-induced increase in blood pressure in mice. The results from this project could complement existing hypertension therapies by combining blockade of the EP3 receptor with antihypertensive drugs.

Prostaglandin E2 receptor EP3 subtype in the paraventricular hypothalamic nucleus mediates corticotropin-releasing factor-induced elevation of plasma noradrenaline levels in rats.

Corticotropin-releasing factor (CRF) plays an important role in sympathetic regulation. Centrally administered CRF elevates plasma catecholamine levels, resulting in CRF-dependent hypertension and tachycardia. We previously reported that brain thromboxane A2 mediates CRF-induced elevation of plasma adrenaline levels, whereas prostanoids other than thromboxane A2 mediate elevations in plasma noradrenaline levels. However, the mechanism by which CRF induces elevations in plasma noradrenaline levels remains unknown. Previous studies have revealed that brain prostaglandin (PG) E2, but not other PGs, causes sympathetic activation. In this study, we examined the roles of brain PGE2 and its receptors in CRF-induced elevation of plasma noradrenaline levels in rats. Our results showed that intracerebroventricular pretreatment with an antagonist of the PGE2 receptor EP3 subtype, but not other subtypes, suppressed CRF-induced elevations in plasma noradrenaline levels. We also examined the role of PGE2 and EP3 receptors in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation, in CRF-induced elevation of plasma noradrenaline levels. Centrally administered CRF increased PGE2 levels in PVN microdialysates, and microinjection of an EP3 receptor agonist into the PVN elevated plasma noradrenaline levels. Bilateral blockade of EP3 receptors in the PVN suppressed the elevation of plasma noradrenaline levels evoked by intracerebroventricular administration and PVN-microinjection of CRF. Our results suggest that CRF stimulates PGE2 release into the PVN that activates EP3 receptors in the PVN, resulting in the elevation of plasma noradrenaline levels.

The effect of the EP3 antagonist DG-041 on male mice with diet-induced obesity.

BACKGROUND/AIMS: The prostaglandin E2 (PGE2) EP3 receptor has a multifaceted role in metabolism. Drugs targeting EP3 have been proposed as therapeutics for diabetes; however, studies utilizing global EP3 knockout mice suggest that EP3 blockade increases obesity and insulin resistance. The present studies attempt to determine the effect of acute EP3 antagonist treatment on the diabetic phenotype. METHODS: DG-041 was confirmed to be a high affinity antagonist at the mouse EP3 receptor by competition radioligand binding and by blockade of EP3-mediated responses. DG-041 pharmacokinetic studies were performed to determine the most efficacious route of administration. Male C57BL/6 × BALB/c (CB6F1) mice were fed diets containing 10%, 45%, or 60% calories from fat to induce obesity. Changes to the metabolic phenotype in these mice were evaluated after one week treatment with DG-041. RESULTS: Subcutaneous injections of DG-041 at 20 mg/kg blocked the sulprostone-evoked rise in mean arterial pressure confirming the efficacy of this administration regime. Seven day treatment with DG-041 had minimal effect on body composition or glycemic control. DG-041 administration caused a reduction in skeletal muscle triglyceride content while showing a trend toward increased hepatic triglycerides. CONCLUSION: Short term EP3 administration of DG-041 produced effective blockade of the EP3 receptor and decreased skeletal muscle triglyceride content but had no significant effects on the diabetic phenotype.

Effects of an EP2 and EP3 Receptor Dual Agonist, ONO-8055, on a Radical Hysterectomy-Induced Underactive Bladder Model in Monkeys.

OBJECTIVES: The objective was to develop an underactive bladder (UAB) model in primates and to evaluate the potential of prostanoid EP2 and EP3 receptor dual agonist ONO-8055 to become a therapeutic agent for UAB. METHODS: A surgical procedure resembling radical hysterectomy was performed on female cynomolgus monkeys. Subsequently, in vitro muscle strip studies were performed using bladder muscle strips from normal monkeys and monkeys that underwent surgery. In addition, uroflowmetric data were obtained at specified days after the surgery. To evaluate the effects of ONO-8055 and distigmine (DIS) on voiding function in the UAB monkey model, uroflowmetry was performed approximately 1 week after the surgery, before and after the cumulative intravenous administration of the compounds at 2 h intervals. RESULTS: In the bladder muscle strip studies, the responses to potassium chloride at 2 months, and carbachol and electrical field stimulation from 2 weeks decreased significantly. Voided volume (VV), maximum flow rate (Qmax), and average flow rate (Qave) decreased after surgery, while voiding time (VT) increased. In this model, ONO-8055 and DIS significantly increased VV and Qmax. DIS prolonged VT, while ONO-8055 had no effect. The results also showed that ONO-8055 increased Qave. CONCLUSIONS: We developed a neurogenic UAB model in primates. As ONO-8055 improved voiding function in this model to at least the same degree as DIS, this EP2 and EP3 receptor dual agonist has the potential to be a candidate for neurogenic UAB pharmacotherapy.

Increased role of E prostanoid receptor-3 in prostacyclin-evoked contractile activity of spontaneously hypertensive rat mesenteric resistance arteries.

This study aimed to determine whether E prostanoid receptor-3 (EP3) is involved in prostacyclin (PGI2)-evoked vasoconstrictor activity of resistance arteries and if so, how it changes under hypertensive conditions. Mesenteric resistance arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were isolated for functional and biochemical studies. Here we show that in vessels from WKYs, PGI2 or the endothelial muscarinic agonist ACh (which stimulates in vitro PGI2 synthesis) evoked vasoconstrictor activity, which increased in SHRs. The thromboxane-prostanoid receptor (TP) antagonist SQ29548 partially removed the vasoconstrictor activity, and an increased contractile activity of PGI2 resistant to SQ29548 was observed in SHRs. Interestingly, L798106, an antagonist of EP3 (whose expression was higher in SHRs than in WKYs), not only added to the effect of SQ29548 but also caused relaxation to PGI2 more than that obtained with SQ29548. In accordance, EP3 deletion, which reduced PGI2-evoked contraction, together with SQ29548 resulted in relaxation evoked by the agonist in mouse aortas. These results thus demonstrate an explicit involvement of EP3 in PGI2-evoked vasoconstrictor activity in rat mesenteric resistance arteries and suggest that up-regulation of the receptor contributes significantly to the increased contractile activity evoked by PGI2 under hypertensive conditions.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin promotes endothelial cell apoptosis through activation of EP3/p38MAPK/Bcl-2 pathway.

Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose-dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)-2 expression and its downstream prostaglandin (PG) production (mainly PGE2 and 6-keto-PGF1α ) in HUVECs. Interestingly, inhibition of COX-2, but not COX-1, markedly attenuated TCDD-triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE2 receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD-treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl-2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl-2 overexpression attenuated the apoptosis in TCDD-treated HUVECs. TCDD increased EP3-dependent Rho activity and subsequently promoted p38MAPK/Bcl-2 pathway-mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re-endothelialization after femoral artery injury in wild-type (WT) mice, but not in EP3-/- mice. In summary, TCDD promotes endothelial apoptosis through the COX-2/PGE2 /EP3/p38MAPK/Bcl-2 pathway. Given the cardiovascular hazard of a COX-2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD-associated cardiovascular diseases.

The EP3 Receptor/Gz Signaling Axis as a Therapeutic Target for Diabetes and Cardiovascular Disease.

Cardiovascular disease is a common co-morbidity found with obesity-linked type 2 diabetes. Current pharmaceuticals for these two diseases treat each of them separately. Yet, diabetes and cardiovascular disease share molecular signaling pathways that are increasingly being understood to contribute to disease pathophysiology, particularly in pre-clinical models. This review will focus on one such signaling pathway: that mediated by the G protein-coupled receptor, Prostaglandin E2 Receptor 3 (EP3), and its associated G protein in the insulin-secreting beta-cell and potentially the platelet, Gz. The EP3/Gz signaling axis may hold promise as a dual target for type 2 diabetes and cardiovascular disease.

Opposing effects of prostaglandin E2 receptors EP3 and EP4 on mouse and human ß-cell survival and proliferation.

OBJECTIVE: Hyperglycemia and systemic inflammation, hallmarks of Type 2 Diabetes (T2D), can induce the production of the inflammatory signaling molecule Prostaglandin E2 (PGE2) in islets. The effects of PGE2 are mediated by its four receptors, E-Prostanoid Receptors 1-4 (EP1-4). EP3 and EP4 play opposing roles in many cell types due to signaling through different G proteins, Gi and GS, respectively. We previously found that EP3 and EP4 expression are reciprocally regulated by activation of the FoxM1 transcription factor, which promotes ß-cell proliferation and survival. Our goal was to determine if EP3 and EP4 regulate ß-cell proliferation and survival and, if so, to elucidate the downstream signaling mechanisms. METHODS: ß-cell proliferation was assessed in mouse and human islets ex vivo treated with selective agonists and antagonists for EP3 (sulprostone and DG-041, respectively) and EP4 (CAY10598 and L-161,982, respectively). ß-cell survival was measured in mouse and human islets treated with the EP3- and EP4-selective ligands in conjunction with a cytokine cocktail to induce cell death. Changes in gene expression and protein phosphorylation were analyzed in response to modulation of EP3 and EP4 activity in mouse islets. RESULTS: Blockade of EP3 enhanced ß-cell proliferation in young, but not old, mouse islets in part through phospholipase C (PLC)-γ1 activity. Blocking EP3 also increased human ß-cell proliferation. EP4 modulation had no effect on ex vivo proliferation alone. However, blockade of EP3 in combination with activation of EP4 enhanced human, but not mouse, ß-cell proliferation. In both mouse and human islets, EP3 blockade or EP4 activation enhanced ß-cell survival in the presence of cytokines. EP4 acts in a protein kinase A (PKA)-dependent manner to increase mouse ß-cell survival. In addition, the positive effects of FoxM1 activation on ß-cell survival are inhibited by EP3 and dependent on EP4 signaling. CONCLUSIONS: Our results identify EP3 and EP4 as novel regulators of ß-cell proliferation and survival in mouse and human islets ex vivo.

Platelets as crucial partners for tumor metastasis: from mechanistic aspects to pharmacological targeting.

Platelets are anucleated cells that circulate in the blood as sentinels of tissue integrity. In fact, they are rich in a plethora of proteins and other factors stored in different granules which they selectively release upon stimulation. Moreover, platelets synthesize a vast number of lipids and release various types of vesicles, including exosomes which are rich in genetic material. Platelets possess a central function to interact with other cell types, including inflammatory cells and cancer cells. Recent findings have enlightened the capacity of platelets to induce changes in the phenotype of cancer cells which acquire invasiveness thus enhancing their metastatic potential. Thus, it has been hypothesized that targeting the platelet may represent a novel strategy to prevent the development and progression of cancer. This is supported by the efficacy of the antiplatelet agent low-dose aspirin. Studies are ongoing to verify whether other antiplatelet agents share the anticancer effectiveness of aspirin.

Structural features of subtype-selective EP receptor modulators.

Prostaglandin E2 is a potent endogenous molecule that binds to four different G-protein-coupled receptors: EP1-4. Each of these receptors is a valuable drug target, with distinct tissue localisation and signalling pathways. We review the structural features of EP modulators required for subtype-selective activity, as well as the structural requirements for improved pharmacokinetic parameters. Novel EP receptor subtype selective agonists and antagonists appear to be valuable drug candidates in the therapy of many pathophysiological states, including ulcerative colitis, glaucoma, bone healing, B cell lymphoma, neurological diseases, among others, which have been studied in vitro, in vivo and in early phase clinical trials.

Deorphaning the Macromolecular Targets of the Natural Anticancer Compound Doliculide.

The cyclodepsipeptide doliculide is a marine natural product with strong actin-polymerizing and anticancer activities. Evidence for doliculide acting as a potent and subtype-selective antagonist of prostanoid E receptor 3 (EP3) is presented. Computational target prediction suggested that this membrane receptor is a likely macromolecular target and enabled immediate in vitro validation. This proof-of-concept study demonstrates the in silico deorphanization of phenotypic screening hits as a viable concept for future natural-product-inspired chemical biology and drug discovery efforts.

Periaqueductal Grey EP3 Receptors Facilitate Spinal Nociception in Arthritic Secondary Hypersensitivity.

UNLABELLED: Descending controls on spinal nociceptive processing play a pivotal role in shaping the pain experience after tissue injury. Secondary hypersensitivity develops within undamaged tissue adjacent and distant to damaged sites. Spinal neuronal pools innervating regions of secondary hypersensitivity are dominated by descending facilitation that amplifies spinal inputs from unsensitized peripheral nociceptors. Cyclooxygenase-prostaglandin (PG) E2 signaling within the ventrolateral periaqueductal gray (vlPAG) is pronociceptive in naive and acutely inflamed animals, but its contributions in more prolonged inflammation and, importantly, secondary hypersensitivity remain unknown. In naive rats, PG EP3 receptor (EP3R) antagonism in vlPAG modulated noxious withdrawal reflex (EMG) thresholds to preferential C-nociceptor, but not A-nociceptor, activation and raised thermal withdrawal thresholds in awake animals. In rats with inflammatory arthritis, secondary mechanical and thermal hypersensitivity of the hindpaw developed and was associated with spinal sensitization to A-nociceptor inputs alone. In arthritic rats, blockade of vlPAG EP3R raised EMG thresholds to C-nociceptor activation in the area of secondary hypersensitivity to a degree equivalent to that evoked by the same manipulation in naive rats. Importantly, vlPAG EP3R blockade also affected responses to A-nociceptor activation, but only in arthritic animals. We conclude that vlPAG EP3R activity exerts an equivalent facilitation on the spinal processing of C-nociceptor inputs in naive and arthritic animals, but gains in effects on spinal A-nociceptor processing from a region of secondary hypersensitivity. Therefore, the spinal sensitization to A-nociceptor inputs associated with secondary hypersensitivity is likely to be at least partly dependent on descending prostanergic facilitation from the vlPAG. SIGNIFICANCE STATEMENT: After tissue damage, sensitivity to painful stimulation develops in undamaged areas (secondary hypersensitivity). This is found in many painful conditions, particularly arthritis. The periaqueductal gray (PAG) is an important center that controls spinal nociceptive processing, on which secondary hypersensitivity depends. Prostaglandins (PGs) are mediators of inflammation with pronociceptive actions within the PAG under normal conditions. We find that secondary hindpaw hypersensitivity in arthritic rats results from spinal sensitization to peripheral A-nociceptor inputs. In the PAG of arthritic, but not naive, rats, there is enhanced control of spinal A-nociceptor processing through PG EP3 receptors. The descending facilitatory actions of intra-PAG PGs play a direct and central role in the maintenance of inflammatory secondary hypersensitivity, particularly relating to the processing of A-fiber nociceptive information.

Optimization of amide-based EP3 receptor antagonists.

Prostaglandin E receptor subtype 3 (EP3) antagonism may treat a variety of symptoms from inflammation to cardiovascular and metabolic diseases. Previously, most EP3 antagonists were large acidic ligands that mimic the substrate, prostaglandin E2 (PGE2). This manuscript describes the optimization of a neutral small molecule amide series with improved lipophilic efficiency (LipE) also known as lipophilic ligand efficiency (LLE) ((a) Nat. Rev. Drug Disc.2007, 6, 881; (b) Annu. Rep. Med. Chem.2010, 45, 380).