CRTH2 Receptors Research Articles

PGD 2 metabolism in plasma: Kinetics and relationship with bioactivity on DP1 and CRTH2 receptors

Prostaglandin (PG)D 2, an important mediator in allergic diseases, is rapidly transformed in plasma to active metabolites that bind and activate two distinct receptors, DP1 and CRTH2. Since the rate of PGD 2 degradation and the bioactivity of the resulting metabolites are still unclear, the aim of our study was to analyze the kinetics and biological effects of PGD 2 metabolites formed in plasma. Eosinophil shape change was taken as a parameter of chemotactic activation mediated by CRTH2 whereas inhibition of platelet aggregation served as a measure of DP1 activity. PGD 2 was degraded in plasma with an apparent half-life of approximately 30 min, accompanied by a loss of potency in inhibiting platelet aggregation as well as inducing eosinophil stimulation. Incubation of PGD 2 in plasma for 120 min caused an increase in the IC 50 for platelet aggregation by a factor of 6.5 and an increase of the EC 50 for eosinophil shape change by a factor of 7.2. However, tandem mass spectrometry analysis showed that incubation of PGD 2 in plasma for 120 min resulted in clearance of PGD 2 of more than 92%, which was mirrored by a continuous formation of Δ 12-PGD 2 and Δ 12-PGJ 2, whereas only small amounts of 15d-PGD 2 and 15d-PGJ 2 were detected. Interestingly, a rapid degradation of PGD 2 was also observed in serum, which was not prevented by pepsin digestion of serum preceding the addition of PGD 2. Therefore, despite extensive non-enzymatic metabolization of PGD 2 in plasma, its biological activity with respect to DP1 and CRTH2 is maintained through the formation of bioactive metabolites.

Interactive effect of histamine and prostaglandin D 2 on nasal allergic symptoms in rats

This study was undertaken to investigate the interactive effect of histamine and prostaglandin D 2 in nasal allergic symptoms in rats. The intranasal application of histamine at doses lower than 10 μmol/site caused no sneezing or nasal rubbing. In addition, prostaglandin D 2 also showed no significant increase in these responses, even at a dose of 10 nmol/site. On the other hand, the simultaneous instillation of histamine and prostaglandin D 2 resulted in a 1000 times more potent effect in inducing nasal symptoms than the administration of histamine alone. Thus, prostaglandin D 2 enhanced the actions of histamine in inducing sneezing and nasal rubbing in a dose-dependent manner, and significant effects were observed at doses higher than 1 nmol/site. The responses induced by the simultaneous application of histamine and prostaglandin D 2 were inhibited by chlorpheniramine, cyproheptadine, BW A868C and ramatroban. Chlorpheniramine and cyproheptadine showed the dose-related inhibition of nasal symptoms induced by the combined administration of histamine (10 nmol) and prostaglandin D 2 (10 nmol), but the effect of cyproheptadine was relatively weak compared with chlorpheniramine. Moreover, BW A868C and ramatroban also showed the inhibition of nasal symptoms induced by the simultaneous administration of histamine and prostaglandin D 2 in a dose-dependent manner. BW A868C was more potent in inhibiting the nasal symptoms than ramatroban. These results clearly indicate that prostaglandin D 2 showed a synergistic effect on sneezing and nasal rubbing induced by histamine in rats, and its effect occurred through both prostaglandin D 2 and CRTH2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) receptors.

GW627368X ((N‐{2‐[4‐(4,9‐diethoxy‐1‐oxo‐1,3‐dihydro‐2H‐benzo[f]isoindol‐2‐yl)phenyl]acetyl} benzene sulphonamide): a novel, potent and selective prostanoid EP4 receptor antagonist

1. N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulphonamide (GW627368X) is a novel, potent and selective competitive antagonist of prostanoid EP4 receptors with additional human TP receptor affinity. 2. At recombinant human prostanoid EP4 receptors expressed in HEK293 cells, GW627368X produced parallel rightward shifts of PGE2 concentration-effect (E/[A]) curves resulting in an affinity (pKb) estimate of 7.9 +/- 0.4 and a Schild slpoe not significantly different from unity. The affinity was independent of the agonist used. 4. In rings of phenylephrine precontracted piglet saphenous vein, GW627368X (30-300 nM) produced parallel rightward displacement of PGE2 E/[A] curves (pKb = 9.2 +/- 0.2; slope = 1). 4. GW627368X appears to bind to human prostanoid TP receptors but not the TP receptors of other species. In human washed platelets, GW627368X (10 microM) produced 100% inhibition of U-46619 (EC100)-induced aggregation (approximate pA2 approximately 7.0). However, in rings of rabbit and piglet saphenous vein and of guinea-pig aorta GW627368X (10 microM) did not displace U-46619 E/[A] curves indicating an affinity of < 5.0 for rabbit and guinea-pig prostanoid TP receptors. 5. In functional assays GW627368X is devoid of both agonism and antagonist affinity for prostanoid CRTH2, EP2, EP3, IP and FP receptors. At prostanoid EP1 receptors, GW627368X was an antagonist with a pA2 of 6.0, and at prostanoid IP receptors the compound increased the maximum effect of iloprost by 55%. At rabbit prostanoid EP2 receptors the pA2 of GW627368X was < 5.0. 6. In competition radioligand bioassays, GW627368X had affinity for human prostanoid EP4 and TP receptors (pKi = 7.0 +/- 0.2 (n = 10) and 6.8 (n = 2), respectively). Affinity for all other human prostanoid receptors was < 5.3. 7. GW627368X will be a valuable tool to explore the role of the prostanoid EP4 receptor in many physiological and pathological settings.

Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.

Identification of Indole Derivatives Exclusively Interfering with a G Protein-Independent Signaling Pathway of the Prostaglandin D2 Receptor CRTH2

The anti-inflammatory drugs indomethacin and ramatroban, the latter showing clinical efficacy in treating allergic asthma, have been shown to act as a classic agonist and antagonist, respectively, of the G protein-coupled chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 receptor). Here, we report the identification of two indole derivatives 1-(4-ethoxyphenyl)-5-methoxy-2-methylindole-3-carboxylic acid and N(alpha)-tosyltryptophan (hereafter referred to as 1 and 2, respectively), which are structurally related to indomethacin and ramatroban but which selectively interfere with a specific G protein-independent signaling pathway of CRTH2. In whole-cell saturation-binding assays, 1 and 2 both increase the number of [(3)H]prostaglandin D2 (PGD2)-recognizing CRTH2 sites and the affinity of PGD2 for CRTH2. Enzyme-linked immunosorbent assays show that they do not alter the total number of CRTH2 receptors on the cell surface. Analysis of their binding mode indicates that unlike indomethacin or ramatroban, 1 and 2 can occupy CRTH2 simultaneously with PGD2. On a functional level, however, 1 and 2 do not interfere with PGD2-mediated activation of heterotrimeric G proteins by CRTH2. In contrast, both compounds inhibit PGD2-mediated arrestin translocation via a G protein-independent mechanism. In human eosinophils endogenously expressing CRTH2, 1 selectively decreases the efficacy but not the potency of PGD2-induced shape change, unlike ramatroban, which displays competitive antagonistic behavior. These data show for the first time that "antagonists" can cause markedly dissimilar degrees of inhibition for different effector pathways and suggest that it may be possible to develop novel classes of specific signal-inhibiting drugs distinct from conventional antagonists.

Production of Prostaglandin D2 by Human Osteoblasts and Modulation of Osteoprotegerin, RANKL, and Cellular Migration by DP and CRTH2 Receptors

Human osteoblasts produce PGD(2), which acts on the DP receptor to decrease osteoprotegerin production and on the CRTH2 receptor to decrease RANKL expression and to induce osteoblast chemotaxis. These results indicate that activation of CRTH2 may lead to an anabolic response in bone. Whereas the actions of prostaglandin (PG)E(2) as a modulator of bone and osteoblast function are relatively well characterized, little is known about PGD(2) and bone metabolism. The objectives of this study were to determine if human osteoblasts can produce PGD(2), which prostaglandin D(2) synthases are implicated in this synthesis, to identify the PGD(2) receptors (DP and CRTH2) on these cells and to characterize the biological effects resulting from their activation. RT-PCR analysis and immunohistochemistry were used to detect PGD(2) receptor and synthases in cultured human osteoblasts. Immunohistochemistry was used to identify the synthases and receptors in human bone tissue. Intracellular cAMP and calcium levels were determined to verify receptor activation. The cells were stimulated with PGD(2) or the specific agonists BW 245C (DP) and DK-PGD(2) (CRTH2), and the resulting effects on osteoprotegerin (OPG) secretion, RANKL expression, and chemotaxis were determined. Osteoblast production of PGD(2) was evaluated by measuring PGD(2) in the culture supernatants after stimulation with interleukin (IL)-1, TNF-alpha, PTH, vascular endothelial growth factor (VEGF), and insulin-like growth factor I (IGF-I). Human osteoblasts in culture generated PGD(2) when stimulated. Both osteoblasts in culture and in situ present the lipocalin-type PGD(2) synthase only. Both DP and CRTH2 receptors were present in human osteoblasts in culture and in situ. Stimulation of DP resulted in an increase in cAMP, whereas CRTH2 increased the intracellular calcium level. OPG production was reduced by 60% after DP receptor stimulation, whereas CRTH2 receptor stimulation decreased RANKL expression on human osteoblasts. As reported for other cell types, CRTH2 was a potent inducer of chemotaxis for human osteoblasts in culture. Human osteoblasts in culture produce PGD(2) under biologically relevant stimuli through the lipocalin-type PGD(2) synthase (L-PGDS) pathway. As an autacoid, PGD(2) can act on DP and CRTH2 receptors, both present on these cells. Specific activation of CRTH2 could lead directly and indirectly to an anabolic response in bone.

Recent Progress in Work on PGD2 Antagonists for Drugs Targeting Allergic Diseases

Prostaglandin (PG) D(2), the major cyclooxygenase metabolite generated from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases due to its various inflammatory effects. However, the lack of PGD(2) (DP) receptor antagonists has limited the study of its essential roles in the disease state. Recent discoveries of several DP receptor antagonists, the development of the mutant mouse and the discovery of a second PGD(2) receptor, CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) receptor, have revealed the crucial roles of PGD(2) and CRTH2 receptors in allergic disorders. This review presents biological evidence that PGD(2) plays an essential role in allergy disorders and discusses the therapeutic possibilities of recently reported PGD(2) antagonists.

Lipocalin-type and hematopoietic prostaglandin D synthases as a novel example of functional convergence

Prostaglandin (PG) D 2 is a major PG produced in the central nervous system and is involved in the regulation of sleep and pain responses through DP receptors. It is also actively produced by mast cells, basophils, and Th2 cells, acting as an allergic mediator through DP and CRTH2 receptors. PGD 2 is further dehydrated to produce PGJ 2, Δ 12-PGJ 2, and 15-deoxy-Δ 12,14-PGJ 2, the last being a ligand for the nuclear receptor PPARγ. PGD synthase (PGDS) catalyzes the isomerization of PGH 2 to PGD 2 in the presence of sulfhydryl compounds. Two distinct types of PGDS have been identified: one is the lipocalin-type PGDS (L-PGDS); and the other, the hematopoietic PGDS (H-PGDS). We isolated the human and mouse cDNAs and genes for L-PGDS and H-PGDS, determined their X-ray crystallographic structures, examined their tissue distribution profiles and cellular localization, and generated gene-knockout mice and human enzyme-overexpressing transgenic mice. L-PGDS and H-PGDS are quite different from each other, in terms of their amino acid sequence, tertiary structure, evolutional origin, chromosomal and cellular localization, tissue distribution, and also functional relevance. Therefore, L-PGDS and H-PGDS are considered to be a novel example of functional convergence.