Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on October 13, 2006; DOI: 10.1124/jpet.106.111062
0022-3565/07/3201-173-179$20.00
JPET 320:173-179, 2007
INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Agonist and Antagonist Effects of 15R-Prostaglandin (PG) D2 and 11-Methylene-PGD2 on Human Eosinophils and Basophils
Chantal Cossette,
Sinead E. Walsh,
Seongjin Kim,
Gue-Jae Lee,
John A. Lawson,
Sophie Bellone,
Joshua Rokach, and
William S. Powell
Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada (C.C., S.E.W., W.S.P.); Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, Florida (S.K., G.-J.L., S.B., J.R.); and the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania (J.A.L.)
Received for publication
July 25, 2006
Accepted
October 11, 2006.
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Abstract
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Prostaglandin (PG) D2 acts through both the DP1 receptor, which is coupled to adenylyl cyclase, and the DP2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells), which is present on eosinophils, basophils, and Th2 cells and results in cell activation and migration. The most potent prostanoid DP2 agonist so far reported is 15R-methyl-PGD2, in which the hydroxyl group has the unnatural R configuration. In contrast, the corresponding analog possessing the natural 15S configuration is 
75 times less potent. This raised the question of whether the isoprostane 15R-PGD2 might have potent DP2 receptor-mediated biological activity. We therefore chemically synthesized 15R-PGD2 and investigated its biological activity. This compound elicited DP2 receptor-mediated CD11b expression in human basophils and eosinophils and induced actin polymerization and migration in eosinophils with a potency about the same as that of PGD2. In contrast, it had only a weak effect on DP1 receptor-mediated adenylyl cyclase activity in human platelets. We also investigated the effects of modification of the 9-hydroxyl and 11-oxo groups of PGD2. Both PGK2, in which the 9-hydroxyl group is replaced by an oxo group, and 11-deoxy-11-methylene PGD2, in which the 11-oxo group is replaced by a CH2 group, have little or no DP1 or DP2 agonist activity. However, the 11-methylene analog is a DP2 antagonist (IC50, 2 µM). We conclude that 15R-PGD2, which may be generated by oxidative stress, is a potent and selective DP2 agonist and that modification of the 11-oxo group of PGD2 can result in DP2 antagonist activity.
Prostaglandins (PGs) are formed by the action of cyclooxygenases on arachidonic acid, resulting in the addition of oxygen at positions 9, 11, and 15. The 15-hydroxyl group of enzymatically formed PGs is almost exclusively in the S configuration (Schneider et al., 2002
), an interesting exception to this being 15R-hydroxy-5,8,11,13-eicosatetraenoic acid, which is formed by aspirin-inhibited cyclooxygenase-2 (Lecomte et al., 1994). However, isoprostanes, formed by the nonenzymatic autoxidation of arachidonic acid, are a mixture of stereoisomers, including those containing 15R-hydroxyl groups (Montuschi et al., 2004). Prostanoids act through a series of highly selective receptors, including an eight-member family of receptors selective for PGD2 (DP1 receptor), PGE2 (EP1–4 receptors), PGF2
(FP receptor), PGI2 (IP receptor), and thromboxane A2 (TP receptor) (Narumiya and FitzGerald, 2001). These receptors distinguish different PGs from one another principally on the basis of the nature of the hydroxyl or keto groups at positions 9, 11, and 15. The presence of a hydroxyl group in the S configuration at C15 is critical for optimal activation of all of the above receptors (Main and Whittle, 1975; Powell et al., 1975; Miller and Sutton, 1976), and its oxidation by 15-hydroxy-PG dehydrogenase results in termination of biological activity (Tai et al., 2002).
We (Monneret et al., 2001) and others (Hirai et al., 2001) recently discovered another PG receptor (the DP2 receptor or chemoattractant receptor-homologous molecule expressed on Th2 cells) that is associated with a variety of responses in eosinophils, including calcium mobilization (Monneret et al., 2002), actin polymerization CD11b expression, L-selectin shedding, and cell migration (Monneret et al., 2001). This receptor is highly selective for PGD2 over any of the other prostanoids mentioned above. However, in contrast to other prostanoid receptors, it is activated by the PGD2 metabolite 15-keto-13,14-dihydro-PGD2, in which the 15S-hydroxyl group has been oxidized (Gervais et al., 2001; Hirai et al., 2001; Monneret et al., 2001). The sequence of the DP2 receptor is considerably different from those of the closely related family of classic prostanoid receptors and bears much more resemblance to receptors for chemoattractants such as leukotriene B4 (Hirai et al., 2001), which may explain its different pattern of selectivity. Certain other modifications of the alkyl side chain (C13–C20) of PGD2 are also well tolerated by the DP2 receptor, including complete removal of the C15 hydroxyl group and rearrangement of the 
13-double bond as in 15-deoxy-12,14-PGD2, which is equipotent with PGD2 in activating eosinophils (Monneret et al., 2002).
We recently identified 15R-methyl-PGD2 as the most potent DP2 receptor agonist yet reported (Monneret et al., 2003). This compound is about 5 times more potent than PGD2 and about 75 times more potent than 15S-methyl-PGD2, which bears the natural S configuration at C15. Although neither of these 15-methyl analogs is a potent agonist of the DP1 receptor, which is associated with activation of adenylyl cyclase, 15S-methyl-PGD2 is several times more active than the corresponding 15R isomer. The high activity of 15R-methyl-PGD2 at the DP2 receptor was unexpected and immediately raised the question as to whether 15R-PGD2, which lacks the 15-methyl group, might be a potent activator of this receptor, perhaps even considerably more potent than PGD2 itself. This is an important issue in view of the mounting evidence for a critical role for PGD2 and its receptors in asthma and other allergic diseases (Kostenis and Ulven, 2006). Furthermore, 15R-PGD2 may be formed in vivo along with other isoprostanes, as a result of oxidative stress, which is prominent in inflammatory diseases such as asthma (Caramori and Papi, 2004). To evaluate the biological activity of 15R-PGD2, we prepared this substance by total chemical synthesis and investigated its ability to activate a number of DP2 receptor-mediated responses in eosinophils and basophils as well as DP1 receptor-mediated cAMP formation in platelets. We also tested the biological effects of other PGD2 analogs in which the 9- and 11-hydroxyl groups were modified (Fig. 1).
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Fig. 1. Structures of PGD2 and PGD2 analogs investigated in the present study. 15R-PGD2 was prepared by chemical synthesis, whereas other prostaglandins were obtained commercially.
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Materials and Methods
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Materials. PGD2, PGK2, 11-deoxy-11-methylene PGD2 (11-CH2-PGD2), 15R-methyl-PGD2, and 15-deoxy-12,14-PGD2 were purchased from Cayman Chemical (Ann Arbor, MI). 15R-PGD2 was prepared by total chemical synthesis as described previously (Kim et al., 2005). Reagents used for staining cells for flow cytometry were as follows: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin (Molecular Probes, Eugene, OR), fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD11b (Bear1), and PC5-labeled anti-CD16 (Beckman-Coulter, Fullerton, CA); phycoerythrin (PE)-labeled mouse anti-human very late antigen 4 (CD49d) and PC5-labeled CD49d (Becton Dickinson, San Jose, CA); and polyclonal FITC-conjugated anti-human IgE (BioSource International, Camarillo, CA).
Analysis of CD11b Expression in Eosinophils and Basophils. CD11b expression on eosinophils and basophils was measured in whole blood from healthy human subjects, following the removal of plasma as described previously (Monneret et al., 2005). Blood from healthy volunteers, collected using citrate as the anticoagulant, was diluted with 5 volumes of phosphate-buffered saline (PBS), centrifuged, and the pellet was resuspended to the original volume in PBS containing CaCl2 (1.8 mM) and MgCl2 (1 mM). The cells were preincubated at 37°C for 5 min, followed by incubation with either vehicle (10 µl of PBS containing 0.1% BSA) or prostaglandins for 10 min. After termination of the incubations with ice-cold FACSFlow (2 ml; Becton-Dickinson), the tubes were centrifuged, and the pellets were incubated for 30 min at 4°C with a mixture of FITC-labeled anti-IgE, PE-labeled anti-CD11b, and PC5-labeled anti-CD49d in PBS. Red cells were then lysed with Optilyse C (Beckman-Coulter; 15 min at 23°C). The cells were centrifuged and resuspended in PBS containing 1% formaldehyde. The distribution of fluorescence intensities was measured by flow cytometry using a FACSCalibur instrument (Becton-Dickinson) using WinMDI software for data analysis. Eosinophils and basophils were identified on the basis of intense labeling with anti-CD49d and anti-IgE, respectively, along with side and forward scatter.
In experiments in which CD11b was measured in eosinophils alone, mixed leukocytes were prepared as described above. Aliquots (0.5 ml; 106 cells/ml) were preincubated for 5 min at 37°C and then for a further for 10 min with agonists. The incubations were terminated by addition of ice-cold FACSFlow and centrifugation. The cells were then stained with a mixture of PE-labeled anti-CD49d and FITC-labeled anti-CD11b and treated with Optilyse and formaldehyde as described previously (Monneret et al., 2002). CD11b expression was measured in eosinophils (high CD49d, high side scatter, low forward scatter) by flow cytometry.
Measurement of Actin Polymerization in Eosinophils. Actin polymerization was measured in a mixed leukocyte fraction obtained by treatment of whole blood with Dextran T-500 for 45 min at 4°C, followed by hypotonic lysis as described previously (Monneret et al., 2002). Leukocytes were prelabeled with PC5-labeled mouse antihuman CD16 for 30 min on ice. After washing by centrifugation the cells were resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4) containing Ca2+ (1.8 mM) and Mg2+ (1 mM). Aliquots of the leukocyte suspension (90 µl, 5 x 106 cells/ml) were preincubated for 5 min at 37°C followed by the addition of either vehicle (10 µl of PBS containing 0.1% BSA) or prostaglandins. After 20 s, the incubations were terminated by the addition of formaldehyde (final concentration, 8.5%), and the samples were kept on ice for 30 min. Cytosolic F-actin was stained by incubation with a mixture of lysophosphatidylcholine (30 µg in 23.8 µl of PBS) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin (49 pmol in 6.2 µl of methanol; final concentration, 0.3 µM) overnight in the dark at 4°C. The cells were then washed by centrifugation and resuspended in 300 µl of PBS containing 1% formaldehyde. F-actin levels were measured by flow cytometry in eosinophils, which were identified on the basis of high side scatter and low CD16 expression.
Evaluation of Eosinophil Migration. Eosinophil migration was measured as described previously (Monneret et al., 2001) by means of 48-well microchemotaxis chambers (Neuro Probe, Cabin John, MD) and Sartorius cellulose nitrate filters (8-mm pore size; 140-mm thickness) (Neuro Probe). PGs were added to the bottom well in a volume of 30 µl of PBS containing 1.8 mM CaCl2, 1 mM MgCl2, and 0.3% bovine serum albumin, whereas eosinophils or neutrophils (150 000 cells in 55 µl of RPMI containing 0.4% ovalbumin) were added to each of the top wells. Following incubation for 2 h at 37°C, the filters were fixed with mercuric chloride and stained with hematoxylin and chromotrope 2R (Kay, 1970). The numbers of cells on the bottom surfaces of the filters were counted in five different fields at a magnification of 400x for each incubation, each of which was performed in duplicate.
Determination of cAMP Levels in Platelets. Platelets were prepared from whole blood obtained from healthy volunteers as described previously (Monneret et al., 2003). After washing by centrifugation, they were resuspended in PBS containing Ca2+ (1.8 mM) and Mg2+ (1 mM) to give a concentration of 3 x 108 platelets/ml. Aliquots (100 µl) of this suspension were preincubated for 2 min at 37°C with isobutylmethylxanthine (1 mM) and then incubated for a further 2 min with either vehicle (10 µl of PBS containing 0.1% BSA) or prostaglandins. The incubations were terminated by the addition of ice-cold ethanol (300 µl), and the precipitated proteins were removed by centrifugation (600g for 15 min). cAMP in the supernatants was measured using a competitive protein-binding radiometric assay (Diagnostic Products, Los Angeles, CA) according to the manufacturer's instructions.
Data Analysis. The statistical significance of differences between means was determined using either analysis of variance with the Bonferroni test as a multiple comparison method or Student's t test as appropriate. EC50 and IC50 values are the means ± S.E. of values obtained from "n" individual experiments.
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Results
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15R-PGD2 Elicits CD11b Expression by Eosinophils and Basophils. We examined the effect of 15R-PGD2, PGD2, and 15R-methyl-PGD2 on CD11b expression by human eosinophils using whole blood from which the plasma had been removed (Fig. 2A). 15R-PGD2 (EC50, 0.5 ± 0.1 nM) and 15R-methyl-PGD2 (EC50, 0.5 ± 0.25 nM) appeared to be slightly more potent than PGD2 (EC50, 1.2 ± 0.5 nM) in stimulating CD11b expression, but these differences were not statistically significant. We also measured the effects of these compounds on CD11b expression in basophils in the same blood samples and obtained essentially similar results, although the EC50 values tended to be slightly higher in basophils compared with eosinophils (Fig. 2B). The potencies of the three compounds were in the order: 15R-methyl-PGD2 (EC50, 1.3 ± 0.6 nM) > 15R-PGD2 (EC50, 3.9 ± 2.8 nM) > PGD2 (EC50, 5.6 ± 3.1 nM), but these differences were not statistically significant.
15R-PGD2 Is a Potent Stimulator of Actin Polymerization in Eosinophils. The effect of 15R-PGD2 on actin polymerization in eosinophils was determined by flow cytometry using a preparation of mixed leukocytes (Fig. 3). The concentration-response curve for 15R-PGD2 (EC50, 12 ± 5 nM) was virtually identical to that for PGD2 (EC50, 19 ± 8 nM). The potencies of both substances for actin polymerization were about 10 times lower than for CD11b expression, perhaps due to the shorter time of the assay. The actin response to PGD2 in eosinophils is very rapid, peaking after 20 s and then diminishing, whereas agonist-induced CD11b expression rises much more slowly and plateaus by 10 min (Monneret et al., 2002).
15R-PGD2 Promotes Eosinophil Migration. We investigated the effect of 15R-PGD2 on eosinophil migration using eosinophils purified by positive immunomagnetic selection (Fig. 4). As with the other responses, the potency of 15R-PGD2 (EC50, 2 ± 0.5 nM) was very similar to that of PGD2 (EC50, 4 ± 2.4 nM). However, the maximal response to PGD2 (57 ± 6 cells/high-power field) was somewhat greater than that to 15R-PGD2 (44 ± 6 cells/high-power field) (P < 0.005).
Effects of 15R-PGD2 on cAMP Formation by Platelets. To determine whether 15R-PGD2 is selective for DP2 receptors, we investigated its effect on cAMP formation in human platelets (Fig. 5). Compared with PGD2 (EC50, 79 ± 11 nM), 15R-PGD2 is a very weak stimulator of DP1 receptor-mediated cAMP formation. Although the maximal response to 15R-PGD2 was not achieved at the highest concentration tested (10 µM), this concentration gave rise to a response that was only about 50% that to a 100 times lower concentration of PGD2. The highest concentration of 15R-methyl-PGD2 tested (10 µM) elicited a response that was about one quarter that due to the same concentration of 15R-PGD2.
Effects of Modification of the 9- and 11-Substituents of PGD2 on DP1 and DP2 Receptor-Mediated Responses. We also investigated the effects of oxidation of the C9 hydroxyl group of PGD2 to an oxo group (i.e., PGK2) and replacement of the C11 oxo group by a methylene group (i.e., 11-CH2-PGD2) on PGD2 receptor-mediated responses in eosinophils and platelets. PGK2 did not elicit either CD11b expression (Fig. 6A) or actin polymerization (Fig. 6B) in eosinophils, whereas 11-CH2-PGD2 appeared to have a slight stimulatory effect on CD11b expression at the highest concentration (10 µM) tested (P < 0.005). Although both 11-CH2-PGD2 and PGK2 had significant stimulatory effects on cAMP formation by platelets (P < 0.001; Fig. 6C) at the highest concentration tested (10 µM), these responses were only about 5% of the maximal response to PGD2. Thus, these two substances have very little agonist activity on both DP1 and DP2 receptors.
Antagonistic Effects of 11-CH2-PGD2 and PGK2 on DP2 Receptor-Mediated Responses. We investigated whether 11-CH2-PGD2 and PGK2 could antagonize the responses to DP2 receptor agonists in eosinophils. 11-CH2-PGD2 nearly completely blocked the effect of the selective DP2 receptor agonist 15-deoxy-12,14-PGD2 on CD11b expression in eosinophils with an IC50 value of 2.8 ± 1.3 µM (Fig. 7A). PGK2 had a similar effect but was less potent, with about 50% inhibition being achieved at the highest concentration tested (10 µM). 11-CH2-PGD2 had a similar effect on PGD2-mediated actin polymerization in eosinophils, inhibiting this response with an IC50 of 1.7 ± 0.9 µM (Fig. 7B). In contrast, 11-CH2-PGD2 had no effect on 5-oxo-6,8,11,14-eicosatetraenoic acid-induced actin polymerization in these cells (data not shown).
To determine whether 11-CH2-PGD2 also inhibits DP1-mediated responses, its effects on PGD2-induced cAMP formation in platelets were investigated. At the highest concentration tested (10 µM), 11-CH2-PGD2 inhibited the response to PGD2 (100 nM) by 44% (Fig. 7C). The same degree of inhibition of DP2-mediated responses by 11-CH2-PGD2 in eosinophils was achieved at lower concentrations, and actin polymerization and CD11b expression were inhibited by 44% by concentrations of 1 and 2 µM, respectively.
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Discussion
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The present study was prompted by our recent finding that 15R-methyl-PGD2, in which the C15 hydroxyl group is in the unnatural R configuration, is a much more potent DP2 receptor agonist than its 15S-methyl counterpart, in which the C15 hydroxyl group has the same configuration as in PGD2 (Monneret et al., 2003). This was surprising because PGs with 15R hydroxyl groups normally display little biological activity (Main and Whittle, 1975; Powell et al., 1975; Miller and Sutton, 1976) and raised the question of whether 15R-PGD2 itself, without the additional methyl group, might be a potent DP2 receptor agonist, perhaps even more potent than PGD2 itself. To investigate the ability of 15R-PGD2 to activate DP2 receptor-mediated responses, this compound was prepared by total chemical synthesis (Kim et al., 2005). We then evaluated the ability of 15R-PGD2 to stimulate DP2 receptor-mediated responses in eosinophils, including actin polymerization, which is associated with cell movement, expression of CD11b, an adhesion molecule that is required for migration of granulocytes through the endothelium, and cell migration. To determine the activity of 15R-PGD2 at the DP1 receptor, we investigated its effects on cAMP formation in platelets, which highly express this receptor. 15R-PGD2 was found to be approximately equipotent with PGD2 in stimulating all of the DP2 receptor-mediated responses in eosinophils. 15R-PGD2 also strongly stimulated CD11b expression in basophils, which highly express both DP1 and DP2 receptors (Hirai et al., 2001). In contrast, it is between 2 and 3 orders of magnitude less potent than PGD2 in stimulating DP1 receptor-mediated cAMP formation in platelets.
In addition to inversion of the stereochemistry of the 15S-hydroxyl group of PGD2, various other modifications of the substituents at C15 of PGD2 have little effect on DP2-mediated responses. For example, oxidation of the hydroxyl group to a keto group, as in 13,14-dihydro-15-keto-PGD2, only slightly reduces DP2 agonist activity but completely abolishes DP1 agonist activity (Gervais et al., 2001; Hirai et al., 2001; Monneret et al., 2001). Likewise, removal of the 15-hydroxyl group coupled with the addition of a double bond to the alkyl side chain of PGD2 (15-deoxy-12,14-PGD2) does not affect DP2 activity (Monneret et al., 2002) but dramatically reduces DP1-mediated responses (Bundy et al., 1983). As noted above, inversion of the stereochemistry at C15 coupled with addition of a methyl group increases potency at the DP2 receptor and nearly completely eliminates it at the DP1 receptor (Monneret et al., 2003). These findings might be interpreted to suggest that the alkyl side chain of PGD2 does not play a role in DP2 receptor-mediated activity. However, in contrast to the above modifications, addition of a methyl group to PGD2, without alteration of the configuration at C15 (15S-methyl-PGD2), results in an over 10-fold loss in potency at the DP2 receptor (Monneret et al., 2003). This suggests that the alkyl side chain is required for full DP2 agonist activity, perhaps due to a hydrophobic interaction with the receptor, because the hydroxyl group itself is not required for activity (Monneret et al., 2002). It is possible that addition of a methyl group to PGD2 places the 15S-hydroxyl group in a position that interferes sterically with this interaction, whereas the hydroxyl group in the 15R-methylhydroxy analog does not have this effect.
In contrast to the DP2 receptor, the 15S-hydroxyl group of PGD2 is clearly required for activation of the DP1 receptor because its removal (15-deoxy-12,14-PGD2), oxidation (13,14-dihydro-15-keto-PGD2), addition of a methyl group (Powell, 2003), or inversion (15R-PGD2) all nearly abolish agonist activity at this receptor. This suggests that unlike the DP2 receptor, there must be a strong hydrophilic interaction between the hydroxyl group of the alkyl side chain of PGD2 and the DP1 receptor.
The strong DP2 agonist activity of 15R-PGD2 may have biological implications because this compound could be formed in vivo as a result of autoxidation of arachidonic acid. This process gives rise to a large number of isoprostanes, some of which are identical to enzymatically generated prostaglandins (Rokach et al., 2004). It has been shown that in addition to enzymatically generated PGD2, large amounts of racemic PGD2, presumably synthesized by a free radical process, are formed as a result of subjection of rats to oxidative stress in vivo. Ent-PGD2 was detected in significant amounts in both rat and human urine under basal conditions and increased to about 35 times control levels in response to oxidative stress induced by administration of carbon tetrachloride to rats (Gao et al., 2003). Although no attempt to identify 15R-PGD2 was made in this study, it would seem likely that it would be formed along with other PGD2 isomers under conditions of oxidative stress. The potent DP2 agonist activity of this compound could contribute to proinflammatory responses following oxidative damage to tissues in various inflammatory diseases such as asthma.
Like the 15-hydroxyl group, the 9-hydroxyl group of PGD2 is not a requirement for activation of DP2 receptors. PGJ2, which lacks this hydroxyl group, has about one-fifth the potency of PGD2, whereas 15-deoxy-12,14-PGJ2, which lacks both the 9- and 15-hydroxyl groups, is equipotent with PGD2 (Monneret et al., 2002). Neither does activation of the DP1 receptor require the 9-hydroxyl group because PGJ2 is equipotent with PGD2 in stimulating DP1 receptor-mediated elevation of cAMP levels in platelets (Wright et al., 1998). However, it would seem that this part of the molecule does play some role in activation of the DP2 receptor by PGD2 because the present study demonstrates that oxidation of the 9-hydroxyl group to an oxo group, as in PGK2, abolishes agonist activity and results in a small degree of antagonist activity.
The 11-oxo group of PGD2 is clearly critical for activation of both the DP1 and DP2 receptors. We have shown that reduction of this group to a hydroxyl group (11
-PGF2) results in a 100-fold reduction in potency at the DP2 receptor (Monneret et al., 2002), and it has been shown by others (Giles et al., 1991) that this modification results in an approximately 50-fold reduction in DP1-mediated activity. The importance of this oxo group is confirmed by the present study because 11-CH2-PGD2 has little or no agonist activity on both DP1 and DP2 receptors. In contrast, 11-CH2-PGD2 has antagonist activity at the DP2 receptor, inhibiting both DP2-mediated CD11b expression and actin polymerization in eosinophils by about 80% at a concentration of 10 µM and having an IC50 of about 2 µM. PGK2 also has antagonist activity but is less potent, exhibiting about 50% inhibition of DP2 receptor-mediated CD11b expression at a concentration of 10 µM. 11-CH2-PGD2 also has some antagonist activity at the DP1 receptor but is less potent because it inhibited PGD2-induced cAMP formation in platelets by less than 50% at a concentration of 10 µM.
Although the present study is the first to identify a PGD2 analog with DP2 receptor antagonist activity, a number of nonprostanoids have been demonstrated to be fairly potent DP2 antagonists. The finding that indomethacin, in addition to its potent cyclooxygenase inhibitory activity, is also a DP2 receptor agonist (Hirai et al., 2002) led to a search for other indolic compounds that might have antagonist activity. The first DP2 antagonist to be identified was ramatroban (IC50 approximately 100 nM) (Sugimoto et al., 2003), which is an indolic compound that was originally developed by Bayer as a selective TP receptor antagonist (Theis et al., 1992). Minor structural modification of ramatroban resulted in a highly selective and potent DP2 antagonist (Ulven and Kostenis, 2005). Other indolic compounds with selective DP2 receptor antagonist activities have also recently been reported (Armer et al., 2005; Birkinshaw et al., 2006). A series of tetrahydroquinoline derivatives have also been identified as selective DP2 antagonists (Mimura et al., 2005).
In conclusion, 15R-PGD2 is a potent and selective DP2 receptor agonist. It is equipotent to PGD2 in stimulating a variety of responses in eosinophils and basophils but has very little DP1 receptor-mediated activity in platelets. Replacement of the 11-oxo group of PGD2 by a methylene group resulted in conversion of agonist to antagonist activity at the DP2 receptor, a finding that may be useful in the design of future DP2 receptor antagonists.
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Acknowledgements
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We are grateful to Sylvie Gravel for assistance with the chemotaxis assays.
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Footnotes
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This work was supported by the Canadian Institutes of Health Research (Grant MOP-6254 to W.S.P.), by the J.T. Costello Memorial Research Fund, and by the National Institutes of Health (Grants HL81873 and HL69835 to J.R.). J.R. acknowledges the National Science Foundation for a Bruker 400 MHz NMR instrument (Grant CHE-03 42251).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.111062.
ABBREVIATIONS: PG, prostaglandin; 11-CH2-PGD2, 11-methylene-PGD2; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
Address correspondence to: Dr. William S. Powell, Meakins-Christie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, Quebec H2X 2P2, Canada. E-mail: William.Powell{at}McGill.ca
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Abstract
Materials and Methods
), 15R-D2 (
), or 15R-methyl-PGD2 (15Me-D2; 
) for 10 min at 37°C. The cells were immunostained as described under Materials and Methods. Eosinophils were identified on the basis of high expression of CD49d, and high side scatter, whereas basophils were identified by their forward and side scatter properties and high anti-IgE staining. All values are means ± S.E. (n = 6).
). CD11b (A) and F-actin (B) were measured by flow cytometry, whereas cAMP was measured using a protein binding assay as described under Materials and Methods. All values are means ± S.E. For A and B, n = 5 (PGD2), 4 (11-CH2-PGD2), or 3 (PGK2). For C, n = 4.