New class of biphenylene dibenzazocinones as potent ligands for the human EP1 prostanoid receptor.

A new class of potent and selective ligands for the human EP1 prostanoid receptor is described. SAR studies reported herein allowed the identification of several potent dibenzazocinones bearing an acylsulfonamide side chain. The binding affinity of these compounds on all eight human prostanoid receptors is reported.     

Structure-activity relationship of triaryl propionic acid analogues on the human EP3 prostanoid receptor

Potent and selective ligands for the human EP3 prostanoid receptor are described. Triaryl compounds bearing an ortho-substituted propionic acid moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinities of key compound on all eight human prostanoid receptors is reported.     

Structure-activity relationship of biaryl acylsulfonamide analogues on the human EP(3) prostanoid receptor

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.     

Prostanoids in immunity: roles revealed by mice deficient in their receptors

Prostanoids including prostaglandins (PGs) and thromboxanes (TX) are a group of lipid mediators formed and released in response to various, often noxious, stimuli. While the roles of prostanoids in acute inflammatory responses are well known and have been extensively studied, it is generally believed that they play very little in immunity. This is partly because non-steroidal anti-inflammatory drugs that inhibit prostanoid synthesis have little effects on immune processes in vivo. Prostanoids exert their actions by acting on a family of G-protein-coupled receptors. They include PGD receptor, EP1, EP2, EP3 and EP4 subtypes of PGE receptor, PGF receptor, PGI receptor and TX receptor. We generated mice deficient in each of these prostanoid receptors individually, and examined their roles under various pathological conditions. These studies have revealed that prostanoids works at various sites or levels of immune responses and exert many, often opposing, actions. For example, using EP4-deficient mice, we found that stimulation of the PGE(2)-EP4 signaling in dendritic cells facilitates their migration and maturation, while the stimulation of the same pathway in T cells potently suppresses their activation and proliferation. The latter action is evident in PGE(2)-mediated suppression of T cell proliferation in the gut of mice subjected to dextran sodium sulfate-induced colitis, a model of inflammatory bowel disease. Here I summarize our findings obtained by these and other studies. These findings suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of certain immunological disorders.     

Suppression of prostaglandin E2 receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth

Prostaglandin E(2) (PGE(2)), a major cyclooxygenase (COX-2) metabolite, plays important roles in tumor biology and its functions are mediated through one or more of its receptors EP1, EP2, EP3, and EP4. We have shown that the matrix glycoprotein fibronectin stimulates lung carcinoma cell proliferation via induction of COX-2 expression with subsequent PGE(2) protein biosynthesis. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibited this effect and induced cellular apoptosis. Here, we explore the role of the PGE(2) receptor EP2 in this process and whether the inhibition observed with PPARgamma ligands is related to effects on this receptor. We found that human non-small cell lung carcinoma cell lines (H1838 and H2106) express EP2 receptors, and that the inhibition of cell growth by PPARgamma ligands (GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone [also known as BRL49653]) was associated with a significant decrease in EP2 mRNA and protein levels. The inhibitory effects of BRL49653 and ciglitazone, but not PGJ2, were reversed by a specific PPARgamma antagonist GW9662, suggesting the involvement of PPARgamma-dependent and -independent mechanisms. PPARgamma ligand treatment was associated with phosphorylation of extracellular regulated kinase (Erk), and inhibition of EP2 receptor expression by PPARgamma ligands was prevented by PD98095, an inhibitor of the MEK-1/Erk pathway. Butaprost, an EP2 agonist, like exogenous PGE(2) (dmPGE(2)), increased lung carcinoma cell growth, however, GW1929 and troglitazone blocked their effects. Our studies reveal a novel role for EP2 in mediating the proliferative effects of PGE(2) on lung carcinoma cells. PPARgamma ligands inhibit human lung carcinoma cell growth by decreasing the expression of EP2 receptors through Erk signaling and PPARgamma-dependent and -independent pathways.     

PGE1 and PGE2 modify platelet function through different prostanoid receptors

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.     

EP₃ receptors mediate PGE₂-induced hypothalamic paraventricular nucleus excitation and sympathetic activation

Prostaglandin E(2) (PGE(2)), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE(2) acts on at least four E-class prostanoid (EP) receptors known as EP(1), EP(2), EP(3), and EP(4). Since PGE(2) production within the brain is ubiquitous, the different functions of PGE(2) depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE(2) remain unknown. We examined this question using PGE(2), the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP(1), EP(3), and EP(4). In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE(2), sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP(3) receptor antagonist; the EP(1) and EP(4) receptor antagonists had little or no effect. ICV PGE(2) or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP(3) receptor antagonist. Real-time PCR detected EP(3) receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP(3) receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE(2) or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP(3) receptor antagonist. These data suggest that EP(3) receptors mediate the central excitatory effects of PGE(2) on PVN neurons and sympathetic discharge.     

Molecular and pharmacological characterization of zebrafish ‘contractile’ and ‘inhibitory’ prostanoid receptors

Prostanoids comprising prostaglandins (PGs) and thromboxanes (TXs) have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been established. Here, we demonstrate that there exist at least five ‘contractile’ (Ca²⁺-mobilizing) and one ‘inhibitory’ (G_i-coupled) prostanoid receptors in zebrafish; five ‘contractile’ receptors consisting of two PGE₂ receptors (EP1a and EP1b), two PGF_2α receptors (FP1 and FP2), and one TXA₂ receptor TP, and one ‘inhibitory’ receptor, the PGE₂ receptor EP3. [³H]PGE₂ specifically bound to the membranes of cells expressing zebrafish EP1a, EP1b and EP3 with a Kd of 4.8, 1.8 and 13.6 nM, respectively, and [³H]PGF_2α specifically bound to the membranes of cells expressing zebrafish FP1 and FP2, with a Kd of 6.5 and 1.6 nM, respectively. U-46619, a stable agonist for human and mouse TP receptors, significantly increased the specific binding of [³⁵S]GTPγS to membranes expressing the zebrafish TP receptor. Upon agonist stimulation, all six receptors showed an increase in intracellular Ca²⁺ levels, although the increase was very weak in EP1b, and pertussis toxin abolished only the EP3-mediated response. Zebrafish EP3 receptor also suppressed forskolin-induced cAMP formation in a pertussis toxin-sensitive manner. In association with the low structural conservation with mammalian receptors, most agonists and antagonists specific for mammalian EP1, EP3 and TP failed to work on each corresponding zebrafish receptor. This work provides further insights into the diverse prostanoid actions mediated by their receptors in zebrafish.     

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1beta of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP(2), EP(3), and EP(4) receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1beta promoted the elaboration of G-CSF, which was augmented by PGE(2). Cicaprost (IP receptor agonist) was approximately equiactive with PGE(2), whereas PGD(2), PGF(2alpha), and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP(1) receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE(2), the EP receptor agonists 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3) selective), 17-phenyl-omega-trinor PGE(2) (EP(1) selective), ONO-AE1-259, and butaprost (both EP(2) selective) were full agonists at enhancing G-CSF release. AH 6809 (10 microM) and L-161,982 (2 microM), which can be used in HASMC as selective EP(2) and EP(4) receptor antagonists, respectively, failed to displace to the right the PGE(2) concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE(2)-induced G-CSF release. Augmentation of G-CSF release by PGE(2) was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE(2) facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP(2) and EP(4) prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.     

Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.     

Mucosal prostanoid receptors and synthesis in familial adenomatous polyposis

Chronic ingestion of non-steroidal anti-inflammatory medication is reported to delay or, in part, reverse development of polyps in the colon, but the mechanism for this effect is unknown. Using mRNA and immunoglobulin probes, specific for prostanoid receptors and for prostaglandin endoperoxide synthase (COX 1 and 2), we sought to define, by in situ and in vitro techniques, changes in PGE2 receptors and synthesis in cell populations of precancerous familial adenomatous polyposis (FAP) colonic mucosa. In FAP, expression of prostanoid receptors EP3 and EP4 among colonic lamina propria mononuclear and lateral crypt epithelial cells was robust, with 53.9+/-5.3% of mononuclear cells staining EP4+. When sections of normal colonic mucosa were examined by similar techniques, prostanoid receptor EP4 was expressed on only 21.3+/-1.2% of lamina propria mononuclear cells (including CD4+ T lymphocytes), as well as on surface and lateral crypt epithelium, and this distribution was found at the mRNA level as well. When receptor expression was quantitated by densitometry, immunoreactive EP3 protein on deep basolateral (but not other) FAP crypt epithelium was enhanced 2.8-fold over normal, and the number of prostanoid receptor EP4+ mononuclear cells by 2.5-fold. On the other hand, while COX 1 expression in mononuclear cells was prominent in normal and FAP mucosa, densitometric analysis showed immunoreactive prostaglandin endoperoxide synthase levels were further increased in FAP, due to a greater than fourfold elevation of COX 2 expression among mononuclear cells and epithelia. Our data suggest enhanced cell-specific prostanoid receptor expression and increased prostanoid synthesis in precancerous FAP mucosa.     

Discovery of a potent and selective agonist of the prostaglandin EP4 receptor

Analogues of PGE(2) wherein the hydroxycyclopentanone ring has been replaced by a lactam have been prepared and evaluated as ligands for the EP(4) receptor. An optimized compound (19a) shows high potency and agonist efficacy at the EP(4) receptor and is highly selective over the other seven known prostaglandin receptors.     

An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors.

We describe in detail an automated and highly sensitive functional assay for calcium-coupled receptors (those receptors whose activation results in an increase in intracellular calcium levels) utilizing coelenterazine-charged aequorin as a probe for intracellular calcium levels ([Ca(2+)](i)). The assay was originally established to investigate Galpha(q)-coupled prostanoid receptors, which are members of the G-protein-coupled receptor (GPCR) superfamily, signaling through elevation of [Ca(2+)](i), initially focusing on the human EP(1) prostanoid receptor (hEP(1)). The parental human embryonic kidney cell line 293-AEQ17, developed by Button and Brownstein (Cell Calcium 14, 663-671, 1993), constitutively expresses apoaequorin and was used to develop a clonal cell line which stably coexpresses hEP(1). This cell line was used to optimize assay parameters in order to maximize accuracy and throughput in an automated 96-well format with the result that each 96-well plate can be completed in 70 min. Use of this flexible system will greatly simplify the functional analysis of GPCRs and other receptors which when activated result in increases in [Ca(2+)](i).     

PGE(2) receptors and synthesis in human gastric mucosa: perturbation in cancer

Recent evidence suggests that prostanoids are an important participant in the pathobiology of gastric adenocarcinoma, but the location and identity of cells in tumor-adjacent gastric mucosa able to synthesize and/or bind specific prostanoids is not clear. Using probes for cyclooxygenase 1 and 2 mRNA and protein as well as for the EP family of PGE(2) receptors, we sought to define the biology of prostanoids in adjacent human gastric mucosa at the site of tumor invasion.In mucosa adjacent to an invasive gastric adenocarcinoma, expression of cyclooxygenase was prominent, with COX 1 primarily in mucosal T lymphocytes surrounding nests of tumor cells. Densitometry showed these tumor-adjacent cells had substantial levels of COX 1 immunoreactive protein (relative intensity, 3.2). Cyclooxygenase 2 was newly expressed among these cells as well, but was limited in number (<25% of cyclooxygenase-positive T lymphocytes) in tumor-adjacent mucosa. Further, CD3(+) mononuclear cells, adjacent to tumor, strongly expressed prostanoid receptor EP(4) (relative intensity, 8.0), but cells with this receptor were not evident in the tumor itself. In contrast, normal gastric mucosa showed a consistent and structured expression of cyclooxygenase and PGE(2) receptor immunoreactive protein among mucosal cells. Cyclooxygenase 1 and PGE(2) receptor EP(4) were expressed on mucosal CD3(+) T lymphocytes in the lumenal (upper) third of gastric mucosa; and prostanoid receptors EP(2), EP(3) and EP(4), on gastric epithelia lining gastric pits. In situ hybridization with COX cDNAs confirmed these findings, and neither COX 2-specific mRNA nor protein was detected in normal gastric tissue. Our studies suggest that synthetic machinery and receptors for PGE(2), prominently expressed by T lymphocytes in gastric mucosa at the boundary of normal mucosa with tumor cells, may play a central role in prostanoid-driven tumorigenesis of this tissue.     

The constitutive activity of ghrelin receptors is decreased by co-expression with vasoactive prostanoid receptors when over-expressed in human embryonic kidney 293 cells

The functional activity of G protein-coupled receptors can be modified by their ability to form oligomeric complexes with G protein-coupled receptors from other receptor families. Emerging evidence suggests that the appetite-regulating hormone ghrelin is a directly acting vasodilator peptide with anti-inflammatory activity, therefore, we have examined the ability of ghrelin receptors to oligomerize with members of the prostanoid receptor family which are also involved in modulating vascular activity and inflammatory responses. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the ability of ghrelin receptors to hetero-oligomerize with prostaglandin E(2) receptor subtype EP(3-I,) prostacyclin receptors, and thromboxane A(2) (TPalpha) receptors, when transiently over-expressed in human embryonic kidney 293 cells. These results suggest that hetero-oligomeric interactions between ghrelin receptors and prostanoid receptors are likely to be of biological relevance. Co-transfection of cells with ghrelin receptor and prostanoid receptors significantly decreased ghrelin receptor expression and attenuated its constitutive activation of phospholipase C without changing its affinity for ghrelin. We also observed an increase in the proportion of ghrelin receptors localized intracellularly in the presence of prostanoid receptors. Taken together, these results suggest that the increased expression of prostanoid receptors in conditions of vascular inflammation, such as in atherosclerotic plaques, could influence those cellular responses dependent on the constitutive activation of ghrelin receptors.