BW A868C Research Articles

Extracellular acidification attenuates bronchial contraction via an autocrine activation of EP2 receptor: Its diminishment in murine experimental asthma

PurposeExtracellular acidification is a major component of tissue inflammation, including airway inflammation in asthmatics. However, its physiological/pathophysiological significance in bronchial function is not fully understood. Currently, the functional role of extracellular acidification on bronchial contraction was explored. MethodsLeft main bronchi were isolated from male BALB/c mice. Epithelium-removed tissues were exposed to acidic pH under submaximal contraction induced by 10−5 M acetylcholine in the presence or absence of a COX inhibitor indomethacin (10−6 M). Effects of AH6809 (10−6 M, an EP2 receptor antagonist), BW A868C (10−7 M, a DP receptor antagonist) and CAY10441 (3×10−6 M, an IP receptor antagonist) on the acidification-induced change in tension were determined. The release of prostaglandin E2 (PGE2) from epithelium-denuded tissues in response to acidic pH was assessed using an ELISA. ResultsIn the bronchi stimulated with acetylcholine, change in the extracellular pH from 7.4 to 6.8 caused a transient augmentation of contraction followed by a sustained relaxing response. The latter inhibitory response was abolished by indomethacin and AH6809 but not by BW A868C or CAY10441. Both indomethacin and AH6809 significantly increased potency and efficacy of acetylcholine at pH 6.8. Stimulation with low pH caused an increase in PGE2 release from epithelium-denuded bronchi. Interestingly, the acidic pH-induced bronchial relaxation was significantly reduced in a murine asthma model that had a bronchial hyperresponsiveness to acetylcholine. ConclusionTaken together, extracellular acidification could inhibit the bronchial contraction via autocrine activation of EP2 receptors. The diminished acidic pH-mediated inhibition of bronchial tone may contribute to excessive bronchoconstriction in inflamed airways such as asthma.

Prostanoid receptor subtypes involved in treprostinil-mediated vasodilation of rat pulmonary arteries and in treprostinil-mediated inhibition of collagen gene expression of human lung fibroblasts

Treprostinil (TRE) is a potent pulmonary vasodilator with effects on other pathological aspects of pulmonary arterial hypertension. In this study, the prostanoid receptors involved in TRE-induced relaxation of isolated rat pulmonary arteries and TRE-induced inhibition of increased gene expression in collagen synthesis and contractility of human lung fibroblasts were determined. TRE (0.01−100 μM) relaxed prostaglandin F2α-precontracted rat pulmonary arteries which was attenuated by denudation of the vascular endothelium. TRE-induced relaxation was predominantly blocked by the IP receptor antagonist RO3244194 (1 μM), with slightly greater inhibition in endothelium-denuded tissue. At higher TRE concentrations (> 1 μM), the DP1 receptor antagonist BW A868C (1 μM) also inhibited relaxation reaching significance above 10 μM. In contrast, the EP3 receptor antagonist L798106 (1 μM) accentuated TRE-induced relaxation of pulmonary arteries with intact endothelium. In human lung fibroblasts, the EP2 receptor antagonist PF-04418948 (1 μM) blocked transforming growth factor β1 (TGF-β1)-increased expression of collagen synthesis (COL1A1 and COL1A2) and fibroblast contractility (ACTG2) genes in presence of TRE (0.1 μM). In conclusion, the IP receptor located on rat pulmonary vascular smooth muscle and endothelium is the primary receptor mediating vasorelaxation, while the DP1 receptor present on the rat endothelium is involved only at higher TRE concentrations. In human lung fibroblasts, the EP2 receptor is the dominant receptor subtype involved in suppression of increased collagen synthesis and fibroblast contractility gene expression induced by TGF-β1 in the presence of TRE.

Rubimetide, humanin, and MMK1 exert anxiolytic-like activities via the formyl peptide receptor 2 in mice followed by the successive activation of DP1, A2A, and GABAA receptors

Rubimetide (Met-Arg-Trp), which had been isolated as an antihypertensive peptide from an enzymatic digest of spinach ribulose-bisphosphate carboxylase/oxygenase (Rubisco), showed anxiolytic-like activity prostaglandin (PG) D2-dependent manner in the elevated plus-maze test after administration at a dose of 0.1mg/kg (ip.) or 1mg/kg (p.o.) in male mice of ddY strain. In this study, we found that rubimetide has weak affinities for the FPR1 and FPR2, subtypes of formyl peptide receptor (FPR). The anxiolytic-like activity of rubimetide (0.1mg/kg, ip.) was blocked by WRW4, an antagonist of FPR2, but not by Boc-FLFLF, an antagonist of FPR1, suggesting that the anxiolytic-like activity was mediated by the FPR2. Humanin, an endogenous agonist peptide of the FPR2, exerted an anxiolytic-like activity after intracerebroventricular (icv) administration, which was also blocked by WRW4. MMK1, a synthetic agonist peptide of the FPR2, also exerted anxiolytic-like activity. Thus, FPR2 proved to mediate anxiolytic-like effect as the first example of central effect exerted by FPR agonists. As well as the anxiolytic-like activity of rubimetide, that of MMK1 was blocked by BW A868C, an antagonist of the DP1-receptor. Furthermore, anxiolytic-like activity of rubimetide was blocked by SCH58251 and bicuculline, antagonists for adenosine A2A and GABAA receptors, respectively. From these results, it is concluded that the anxiolytic-like activities of rubimetide and typical agonist peptides of the FPR2 were mediated successively by the PGD2-DP1 receptor, adenosine-A2A receptor, and GABA-GABAA receptor systems downstream of the FPR2.

Prostaglandin D2‐DP1 receptor signaling inhibits sympathetic nerve activity and development of angiotensin II‐salt hypertension (874.11)

Cerebrospinal fluid levels of lipocalin‐prostaglandin D synthase protein (L‐PGDS) and the lipid mediator prostaglandin D2 (PGD2) are increased in early stages of AngII‐salt hypertension (AngII‐HS‐HTN) in rats. Because L‐PGDS inhibition reduces neurogenic AngII‐HS‐HTN development and its product PGD2 acts on central DP‐1 receptors (DP‐1Rs), we hypothesized that PGD2 acting through DP1‐Rs mediated AngII‐HS‐HTN development.Method: Two sets of studies were performed. First, anesthetized rats received an injection of PGD2 (50µg/5µl) intracerebroventricularly (icv). PGD2 caused a maximum 22±6 mmHg increase in BP. In other anesthetized rats the DP1‐R antagonist BW A868C (78ng/5µl) was injected icv followed by PGD2 injection; BW A868C blocked PGD2 mediated increases in BP. In the second study we tested the effect of chronic DP1‐R blockade on AngII‐salt HTN. Continuous radio‐telemetric recording of BP was used. After control BP recording, rats on 2% NaCl diet were pretreated with vehicle (n=4) or BW A868C (425nM/h, sc, n=5). All rats then received AngII (150ng/kg/min, sc) for 14 days. Ganglionic blockade with hexamethonium (30mg/kg, ip) was performed on day 10 of AngII administration to assess neurogenic pressor activity. Finally, PGD2 (50µg/5µl) was injected acutely into lateral ventricles to test if central DP‐1Rs were inhibited.Result: Compared to the control period, AngII infusion increased mean arterial pressure (MAP, mmHg) from 95±2 to143±10 (31±10 increase) in vehicle, and from 109±7 to140±1 (56±3 increase) in BW A868C treated rats (p<0.05). Neurogenic pressor activity (mmHg) was significantly higher in BW A868 (86±10) than vehicle (46±11) treated rats. Finally, central PGD2 injection failed to elicit a BP response in BW A868C treated rats confirming central DP1‐R inhibition.Conclusion: Acute icv PGD2 induced increases in BP are mediated via DP‐1R. However, chronic PGD2 signaling through central DP1‐R inhibits AngII‐HS‐HTN development and causes sympathoinhibition.Grant Funding Source: “Supported by” RO1 HL 076312 and RCO HL 060363

Role of prostaglandin D2 in mast cell activation-induced sensitization of esophageal vagal afferents

Sensitization of esophageal afferents plays an important role in esophageal nociception, but the mechanism is less clear. Our previous studies demonstrated that mast cell (MC) activation releases the preformed mediators histamine and tryptase, which play important roles in sensitization of esophageal vagal nociceptive C fibers. PGD2 is a lipid mediator released by activated MCs. Whether PGD2 plays a role in this sensitization process has yet to be determined. Expression of the PGD2 DP1 and DP2 receptors in nodose ganglion neurons was determined by immunofluorescence staining, Western blotting, and RT-PCR. Extracellular recordings were performed in ex vivo esophageal-vagal preparations. Action potentials evoked by esophageal distension were compared before and after perfusion of PGD2, DP1 and DP2 receptor agonists, and MC activation, with or without pretreatment with antagonists. The effect of PGD2 on 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal nodose neurons was determined by patch-clamp recording. Our results demonstrate that DP1 and DP2 receptor mRNA and protein were expressed mainly in small- and medium-diameter neurons in nodose ganglia. PGD2 significantly increased esophageal distension-evoked action potential discharges in esophageal nodose C fibers. The DP1 receptor agonist BW 245C mimicked this effect. PGD2 directly sensitized DiI-labeled esophageal nodose neurons by decreasing the action potential threshold. Pretreatment with the DP1 receptor antagonist BW A868C significantly inhibited PGD2 perfusion- or MC activation-induced increases in esophageal distension-evoked action potential discharges in esophageal nodose C fibers. In conclusion, PGD2 plays an important role in MC activation-induced sensitization of esophageal nodose C fibers. This adds a novel mechanism of visceral afferent sensitization.

Colonic mucosal mediators from patients with irritable bowel syndrome excite enteric cholinergic motor neurons

Mediators released in the mucosal milieu have been suggested to be involved in visceral hypersensitivity and abdominal pain in patients with irritable bowel syndrome (IBS). However, their impact on myenteric neurons remains unsettled. Mucosal biopsies were obtained from the descending colon of patients with IBS and controls. Mucosal mast cells were identified immunohistochemically. The impact of spontaneously released mucosal mediators on guinea pig electrically stimulated longitudinal muscle myenteric plexus (LMMP) preparations was assessed in vitro by means of selective receptor antagonists and inhibitors. Patients with IBS showed an increased mast cell count compared with controls. Application of mucosal mediators of IBS to LMMPs potentiated cholinergic twitch contractions, an effect directly correlated with mast cell counts. Enhanced contractions were inhibited by 50.3% with the prostaglandin D2 antagonist BW A868C, by 31.3% and 39% with the TRPV1 antagonists capsazepine and HC-030031, respectively, and by 60.5% with purinergic P2X antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Conversely, the serotonin1-4, histamine1-3, tachykinin1-3 receptor blockade, and serine protease inhibition had no significant effect. Colonic mucosal mediators from patients with IBS excite myenteric cholinergic motor neurons. These effects were correlated with mast cell counts and mediated by activation of prostanoid receptors, TRPV1, and P2X receptors. These results support the role of mucosal inflammatory mediators and mast cell activation in altered motor function of IBS.

Prostaglandin D2 induces apoptosis of human osteoclasts by activating the CRTH2 receptor and the intrinsic apoptosis pathway

Prostaglandin D2 (PGD2) is a lipid mediator synthesized from arachidonic acid that directly activates two specific receptors, the D-type prostanoid (DP) receptor and chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). PGD2 can affect bone metabolism by influencing both osteoblast and osteoclast (OC) functions, both cells involved in bone remodeling and in in vivo fracture repair as well. The objective of the present study was to determine the effects of PGD2, acting through its two specific receptors, on human OC apoptosis. Human OCs were differentiated in vitro from peripheral blood mononuclear cells in the presence of receptor activator for nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and treated with PGD2, its specific agonists and antagonists. Treatment with PGD2 for 24hours in the presence of naproxen (10μM) to inhibit endogenous prostaglandin production increased the percentage of apoptotic OCs in a dose-dependent manner, as did the specific CRTH2 agonist compound DK-PGD2 but not the DP agonist compound BW 245C. In the absence of naproxen, the CRTH2 antagonist compound CAY 10471 reduced OC apoptosis rate but the DP antagonist BW A868C had no effect. The induction of PGD2-CRTH2 dependent apoptosis was associated with the activation of caspase-9, but not caspase-8, leading to caspase-3 cleavage. These data show that PGD2 induces human OC apoptosis through activation of CRTH2 and the apoptosis intrinsic pathway.

896 Protease-Activated Receptor 2 Activates Transient Receptor Potential Vanilloid 4 by SRC Kinase-Mediated Tyrosine Phosphorylation

Introduction: PGD2 is an important mediator released after mast cell activation. Our previous studies demonstrated PGD2 sensitizes esophageal nodose C-fibers to increase their response to esophageal distension. This study aims to determine the underlying mechanism of the sensitization process. Methods: PGD2 DP1 receptor expressions in nodose neurons were determined by immunofluorescent-staining, Western blot, and RT-PCR. Extracellular single fiber recordings were performed from vagal nodose neurons using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. We recorded action potentials evoked by esophageal distensions before and after perfusion of PGD2 with/without pretreatment of DP1 receptor antagonist BWA 868C. Whole-cell patch-clamp recording was performed to investigate the action of PGD2 on isolated nodose neurons. Viscermotor responses (VMR) to esophageal distension were recorded by electromyography from acromiotrapezius muscles in animals receiving PGD2 injection (20 μl, 100 nM) into the wall of the esophagus before and after truncal vagatomy. Results: DP1 receptor expressions were identified in nodose neurons by immunofluorescent-labeling, Western blot, and RT-PCR. 98% of DP1positive neurons were of smalland medium-diameters (φ 0.05, n=4). Whole-cell patch-clamp recording from isolated nodose neurons demonstrated PGD2 (1 μM) depolarized recorded neurons (13±2 mV, n=5) accompanied by an increase in the neuronal input resistance from 360±20 to 420±25 MΩ. IV relationship studies showed current reversed at 105 mV suggesting the effect was mediated by the closure of K+ channel(s). Compared to controls, PGD2 injection into the esophagus significantly enhanced esophageal distension evoked-VMRs at distension pressures 20, 40, 60, and 80 mmHg respectively (p <0.05, n=3). Left truncal vagatomy attenuated PGD2induced enhancements of VMR in response to esophageal distension (p <0.05, n=3) indicating the participation of vagal afferent pathway in this sensitization process. Conclusion: These data demonstrate mast cell mediator PGD2 plays a crucial role in sensitization of esophageal nodose C-fiber neurons to induce esophageal mechanical hypersensitivity. This sensitization effect is mediated via DP1 receptor and involves downstream K+ channel.

898 Chronic Stress-Induced Visceral Hyperalgesia: Evidence That the Formation of CB1 and TRPV1 Receptor Complexes Modulates Pain in Dorsal Root Ganglion (DRG) Neurons

Introduction: PGD2 is an important mediator released after mast cell activation. Our previous studies demonstrated PGD2 sensitizes esophageal nodose C-fibers to increase their response to esophageal distension. This study aims to determine the underlying mechanism of the sensitization process. Methods: PGD2 DP1 receptor expressions in nodose neurons were determined by immunofluorescent-staining, Western blot, and RT-PCR. Extracellular single fiber recordings were performed from vagal nodose neurons using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. We recorded action potentials evoked by esophageal distensions before and after perfusion of PGD2 with/without pretreatment of DP1 receptor antagonist BWA 868C. Whole-cell patch-clamp recording was performed to investigate the action of PGD2 on isolated nodose neurons. Viscermotor responses (VMR) to esophageal distension were recorded by electromyography from acromiotrapezius muscles in animals receiving PGD2 injection (20 μl, 100 nM) into the wall of the esophagus before and after truncal vagatomy. Results: DP1 receptor expressions were identified in nodose neurons by immunofluorescent-labeling, Western blot, and RT-PCR. 98% of DP1positive neurons were of smalland medium-diameters (φ 0.05, n=4). Whole-cell patch-clamp recording from isolated nodose neurons demonstrated PGD2 (1 μM) depolarized recorded neurons (13±2 mV, n=5) accompanied by an increase in the neuronal input resistance from 360±20 to 420±25 MΩ. IV relationship studies showed current reversed at 105 mV suggesting the effect was mediated by the closure of K+ channel(s). Compared to controls, PGD2 injection into the esophagus significantly enhanced esophageal distension evoked-VMRs at distension pressures 20, 40, 60, and 80 mmHg respectively (p <0.05, n=3). Left truncal vagatomy attenuated PGD2induced enhancements of VMR in response to esophageal distension (p <0.05, n=3) indicating the participation of vagal afferent pathway in this sensitization process. Conclusion: These data demonstrate mast cell mediator PGD2 plays a crucial role in sensitization of esophageal nodose C-fiber neurons to induce esophageal mechanical hypersensitivity. This sensitization effect is mediated via DP1 receptor and involves downstream K+ channel.

Rubimetide (Met-Arg-Trp) derived from Rubisco exhibits anxiolytic-like activity via the DP 1 receptor in male ddY mice

In this study, we found that Met-Arg-Trp (rubimetide), which had been isolated as a hypotensive peptide from a pepsin–pancreatin digest of spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), has anxiolytic-like activity in the elevated plus-maze test at a dose of 0.1 mg/kg (i.p.) or 1.0 mg/kg (p.o.) in mice with p < 0.01 and p < 0.05, respectively. The anxiolytic-like activity of rubimetide (0.1 mg/kg, i.p.) was blocked by BW A868C (60 μg/kg, i.p.), an antagonist for the DP 1 receptor, suggesting the anxiolytic-like activity of rubimetide is mediated by prostaglandin D 2 and the DP 1 receptor.

Met-Arg-Trp derived from Rubisco lowers blood pressure via prostaglandin D 2-dependent vasorelaxation in spontaneously hypertensive rats

Met-Arg-Trp (MRW) has been isolated as an inhibitor for angiotensin I-converting enzyme (ACE) from a pepsin–pancreatin digest of spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco) (IC 50 = 0.6 μM). It has been reported that hypotensive activity of ACE-inhibitory peptides derived from food proteins are weakened in spontaneously hypertensive rats older than 25 weeks (old SHR). However, MRW reduced blood pressure after oral administration at a dose of 5 mg/kg in old SHR as well as in younger SHR. MRW exhibited vasorelaxing activity above 1 μM in isolated mesenteric artery from adult and old SHR. The vasorelaxing activity of MRW was blocked by indomethacin and BW A868C, a cyclooxygenase inhibitor and an antagonist for DP 1 receptor, respectively. However, N G-nitro- l-arginine methyl ester, an inhibitor for nitric oxide synthase, had no effect on the relaxation. The hypotensive activity of MRW was also blocked by indomethacin and BW A868C, respectively, in adult and old SHR. Taken together, the vasorelaxing and hypotensive activities of MRW may be mediated by prostaglandin D 2 and the DP 1 receptor. These findings suggest that the hypotensive activity of MRW is mainly caused by vasorelaxation rather than by ACE-inhibition.

Involvement of chemical mediators in nasal allergic responses of HDC-KO mice

The present study was undertaken to investigate the involvement of chemical mediators in nasal allergic responses using histidine decarboxylase knockout (HDC-KO) mice. An allergic rhinitis model was developed in HDC-KO and wild-type mice by the intraperitoneal injection of ovalbumin, aluminum hydroxide gel and pertussis toxin. Five days later, they were boosted by a subcutaneous injection of ovalbumin into the back. From day 18 after the first immunization to day 39, intranasal sensitization with ovalbumin was performed every day and the severity of allergic rhinitis was observed by measuring nasal allergic responses and total IgE levels. It was found that the intranasal administration of antigen caused a significant increase of nasal sneezing and rubbing from day 25 to day 39 both in sensitized HDC-KO and wild-type mice. In addition, a significant elevation of total IgE levels in serum was also found both in sensitized HDC-KO and wild-type mice from day 18 to day 39 after the first immunization. L-733,060, a tachykinin NK 1 receptor antagonist at a dose of 10 mg/kg (s.c.), resulted in the dose-dependent inhibition of nasal allergic responses induced by antigen in both HDC-KO and wild-type mice. In addition, both chlorpheniramine at doses of 3 and 10 mg/kg (p.o.) and BW A868C at doses of 0.3 and 1 mg/kg (i.v.) also showed a dose-related reduction of the nasal allergic responses induced by antigen in sensitized wild-type mice. On the other hand, they had no effects on the nasal signs induced by antigen in HDC-KO mice. From these results, it was revealed that substance P induces nasal allergic responses in the mouse model of chronic allergic rhinitis through the activation of tachykinin NK 1 receptors. Therefore, it can be concluded that not only histamine, but also substance P and prostaglandin D 2, participated in the nasal allergic responses induced by antigen in mice.

Effects of TS-022, a newly developed prostanoid DP 1 receptor agonist, on experimental pruritus, cutaneous barrier disruptions and atopic dermatitis in mice

TS-022, {4-[(1R, 2S, 3R, 5R)-5-Chloro-2-(( S)-3-cyclohexyl-3-hydroxyprop-1-ynyl)-3-hydroxycyclopentyl] butylthio} acetic acid monohydrate, inhibits ADP-induced platelet aggregation, an effect significantly antagonized, as in the case of prostaglandin D 2 by the prostanoid DP 1 receptor antagonist (BW A868C). TS-022 is a prostanoid DP 1 receptor agonist, originally developed as a novel anti-pruritic drug for patients with atopic dermatitis. We examined the effects of TS-022 on experimental pruritus, cutaneous barrier disruption, and atopic dermatitis and in in vitro immune function tests. Topically applied TS-022 significantly suppressed scratching in skin-lesioned NC/Nga mice from a concentration of 2.5 nM, and this scratch-suppressive activity was significantly antagonized by BW A868C. Tacrolimus (FK-506) and dexamethasone, used as reference drugs for atopic dermatitis, also exhibited suppressive effects against scratching, but only at concentrations of 125 and 25,000 μM. TS-022 applied topically, once a day for 2 days, significantly accelerated repair of the cutaneous barrier disruption caused by mechanical scratching, from concentrations of 2.5 nM. This acceleration of repair of the disrupted cutaneous barrier by this drug was also significantly antagonized by BW A868C. FK-506 and dexamethasone showed no beneficial effects on the repair of the disrupted cutaneous barrier. Repeated topical application of 2.5 μM of TS-022 and 12.5 μM of FK-506 once a day for 6 weeks significantly improved the skin inflammation scores in the NC/Nga mice. In regard to the effects of TS-022 in vitro, the inhibitory activity of TS-022 against concanavalin A-induced cytokine production by splenocytes was marginal as compared with that of FK-506 or dexamethasone. These results suggest that the beneficial therapeutic effects of TS-022 in NC/Nga mice with atopic dermatitis are mediated by its suppressive effect on scratching and its effect of accelerating repair of the disrupted cutaneous barrier, both effects being attributable to its prostanoid DP 1 receptor agonistic activity.

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.

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.

Piglet saphenous vein contains multiple relaxatory prostanoid receptors: evidence for EP4, EP2, DP and IP receptor subtypes

Prostaglandin E(2) produced endothelium-independent relaxation of phenylephrine- and 5-HT-contracted piglet saphenous vein (PSV; pEC(50)=8.6+/-0.2; n=6). The prostanoid EP(4) receptor antagonist GW627368X (30-300 nM) produced parallel rightward displacement of PGE(2) concentration-effect (E/[A]) curves (pK(b)=9.2+/-0.2; slope=1). Higher concentrations of GW627368X did not produce further rightward shifts, revealing the presence of non-EP(4) prostanoid receptors. In all, 18 other prostanoid receptor agonists relaxed PSV in a concentration-related manner. Relative potencies of agonists most sensitive to 10 muM GW627368X (and therefore predominantly activating EP(4) receptors) correlated well with those at human recombinant EP(4) receptors in human embryonic kidney (HEK-293) cells (r(2)=0.74). In the presence of 10 microM GW627368X, the rank order of agonist relative potency matched that of the human recombinant EP(2) receptor in Chinese hamster ovary cells (r(2)=0.72). Iloprost, cicaprost and PGI(2) relaxed PSV maximally and were antagonised by 10 microM GW627368X, demonstrating that they were full EP(4) receptor agonists. Residual responses to these compounds in the presence of GW627368X suggested the presence of IP receptors.BW245C relaxed PSV maximally (pEC(50)=6.8+/-0.1). In the presence of 10 microM GW627368X, BW245C produced biphasic E/[A] curves (phase one pEC(50)=6.6; alpha=24%; phase two pEC(50)=5.1; alpha=112%). Phase two was antagonised by the DP receptor antagonist BW A868C (1 microM), demonstrating that BW245C is an agonist at DP and EP4 receptors. We conclude that PSV contains EP(4), EP(2), DP and IP receptors; IP receptor agonists are also porcine EP(4) receptor agonists.

Prostaglandin D2-Induced Eosinophilic Airway Inflammation Is Mediated by CRTH2 Receptor

Mast cell-derived prostaglandin D(2) (PGD(2)) is one of the essential modulators of eosinophilic airway inflammation in asthma and allergic rhinitis. Two G protein-coupled receptors for PGD(2), prostaglandin D(2) receptor (DP) and chemoattractant receptor-homologous molecule expressed on Th(2) cells (CRTH2), are both expressed on the surface of eosinophils, and CRTH2 has been demonstrated to mediate PGD(2)-induced eosinophil mobilization in vitro. However, it has not yet been determined whether PGD(2) and its receptors mediate in vivo eosinophil trafficking into the airways or other organs. We demonstrated that intratracheal administration of PGD(2) in rats pretreated with systemic interleukin-5 (IL-5) injection induced marked airway eosinophilia, determined by the differential counts of cells in bronchoalveolar lavage (BAL) fluid and lung histology, within 2 h. Systemic IL-5 alone significantly increased the number of eosinophils in the peripheral blood but showed no effect on airway eosinophilia. Three CRTH2-specific agonists (13,14-dihydro-15-keto-PGD(2), 11-deoxy-11-methylene-15-keto-PGD(2), and indomethacin) demonstrated equivalent induction of BAL eosinophilia to that of PGD(2), but a DP agonist (BW 245C [5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin]) or a thromboxane A(2) receptor (TP) agonist ([1S-1alpha,2beta(5Z), 3alpha(1E,3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid) showed no effect. PGD(2) or CRTH2 agonist-induced BAL eosinophilia was almost completely inhibited by pretreatment with a CRTH2/TP antagonist, ramatroban [BAY-u3405; (+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,2,3,4-tetra-hydrocarbazole-9-propionic acid], whereas a TP-specific antagonist, SQ29,548 (5-heptenoic, 7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]-hept-2-yl]-[1S-[1alpha,2alpha(Z),3alpha,4alpha]]), or a DP-specific antagonist, BW A868C [3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexy-2-hydroxyethylamino)-hydantoin], did not inhibit the effects of PGD(2). These results suggest that CRTH2 plays a significant role in the eosinophil trafficking from the bloodstream into the airways in PGD(2)-related airway inflammation.

A Functional Study on Prostanoid Receptors Involved in Cultured Human Iridal Melanocyte Stimulation

The effects of various prostanoids on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (bFGF) (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium or various deleted media with or without various prostanoids at different concentrations. After 6 days, the numbers of cells and dendrites were counted and melanin content was measured and compared with controls. Prostaglandin E2, an EP2receptor agonist (AH 13205) and AGN 192093 (thromboxane mimetic) stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. A mixed EP1and EP3receptor agonist (sulprostone), a EP4receptor agonist (ONO-AE1-329), IP receptor agonists (cicaprost or iloprost) and a TP receptor agonist (U-46619) showed no effect. Prostaglandin D2showed stimulating effects. However, these stimulating effects could not be blocked by the addition of a DP receptor antagonist (BW A868C). Furthermore, a DP receptor agonist (BW 245C) showed no effects, indicating that the effect of prostaglandin D2may involve receptors other than the DP receptor subtype. The present study indicates that: (1) among various EP receptor agonists, only an EP2receptor agonist has stimulating effects on iridal melanocytes; (2) DP, IP and TP receptor agonists do not have stimulating effects; and (3) the mechanisms of action of prostaglandin D2and AGN 192093 need further study.

Role of the parasite-derived prostaglandin D2 in the inhibition of epidermal Langerhans cell migration during schistosomiasis infection.

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-alpha, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-alpha-triggered migration of LCs through the adenylate cyclase-coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

The human prostanoid DP receptor stimulates mucin secretion in LS174T cells.

This study demonstrates the localization of the prostaglandin (PG)D(2) receptor (DP) within the mucous-secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells. The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD(2) and the highly selective DP-specific agonist L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE(2) competed for [(3)H]-PGD(2)-specific binding to LS174T cell membranes (K:(i) values of 0.4 nM and 7 nM, respectively). The DP-specific agonists PGD(2), PGJ(2), BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)), and L-644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC(50) values: 45 - 90 nM) and demonstrated the expected rank order of potency. PGE(2) also elicited cyclic AMP production in this cell line (EC(50) value: 162 nM). The activation of cyclic AMP production by PGD(2) and L-644,698, but not PGE(2), was inhibited by the selective DP antagonist BW A868C. Thus, PGD(2) and L-644,698 act through hDP in LS174T cells. PGD(2), L-644,698 and PGE(2) (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration-dependent manner (EC(50)<50 nM). However, BW A868C effectively antagonized only the mucin secretion mediated by PGD(2) and L-644,698 and not PGE(2). These data support a role for the DP receptor in the regulation of mucous secretion.