EP4 receptor as a new target for bronchodilator therapy.

BACKGROUND: Asthma and chronic obstructive pulmonary disease are airway inflammatory diseases characterised by airflow obstruction. Currently approved bronchodilators such as long-acting ß(2) adrenoceptor agonists are the mainstay treatments but often fail to relieve symptoms of chronic obstructive pulmonary disease and severe asthma and safety concerns have been raised over long-term use. The aim of the study was to identify the receptor involved in prostaglandin E(2) (PGE(2))-induced relaxation in guinea pig, murine, monkey, rat and human airways in vitro. METHODS: Using an extensive range of pharmacological tools, the relaxant potential of PGE(2) and selective agonists for the EP(1-4) receptors in the presence and absence of selective antagonists in guinea pig, murine, monkey, rat and human isolated airways was investigated. RESULTS: In agreement with previous studies, it was found that the EP(2) receptor mediates PGE(2)-induced relaxation of guinea pig, murine and monkey trachea and that the EP(4) receptor mediates PGE(2)-induced relaxation of the rat trachea. These data have been confirmed in murine airways from EP(2) receptor-deficient mice (Ptger2). In contrast to previous publications, a role for the EP(4) receptor in relaxant responses in human airways in vitro was found. Relaxant activity of AH13205 (EP(2) agonist) was also demonstrated in guinea pig but not human airway tissue, which may explain its failure in clinical studies. CONCLUSION: Identification of the receptor mediating PGE(2)-induced relaxation represents a key step in developing a novel bronchodilator therapy. These data explain the lack of bronchodilator activity observed with selective EP(2) receptor agonists in clinical studies.

Oxidized Products of α-Linolenic Acid Negatively Regulate Cellular Survival and Motility of Breast Cancer Cells.

Despite recent advances in our understanding of the biological processes leading to the development and progression of cancer, there is still a need for new and effective agents to treat this disease. Phytoprostanes (PhytoPs) and phytofurans (PhytoFs) are non-enzymatically oxidized products of α-linolenic acid that are present in seeds and vegetable oils. They have been shown to possess anti-inflammatory and apoptosis-promoting activities in macrophages and leukemia cells, respectively. In this work, seven PhytoPs (PP1-PP7) and one PhytoFs (PF1) were evaluated for their cytotoxic, chemosensitization, and anti-migratory activities using the MCF-7 and MDA-MB-231 breast cancer cell lines. Among the tested compounds, only three PhytoPs had a significant effect on cell viability compared to the control group: Ent-9-L1-PhytoP (PP6) decreased cell viability in both cell lines, while 16-F1t-PhytoP (PP1) and 9-L1-PhytoP (PP5) decreased viability of MCF-7 and MDA-MB-231 cells, respectively. When combined with a sub-cytotoxic dose of doxorubicin, these three PhytoPs displayed significantly enhanced cytotoxic effects on MCF-7 cells while the chemotherapeutic drug alone had no effect. In cellular motility assays, Ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF could significantly inhibit cellular migration of MDA-MB-231 cells. In addition, Ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF also enhanced cellular adhesion of MDA-MB-231 cells.

Elevated thromboxane levels in the rat during endotoxic shock: protective effects of imidazole, 13-azaprostanoic acid, or essential fatty acid deficiency.

The potential deleterious role of the proaggregatory vasoconstrictor, thromboxane A(2), in endotoxic shock was investigated in rats. Plasma thromboxane A(2) was determined by radioimmunoassay of its stable metabolite thromboxane B(2). After intravenous administration of Salmonella enteritidis endotoxin (20 mg/kg), plasma thromboxane B(2) levels increased from nondetectable levels (<375 pg/ml) in normal control rats to 2,054+/-524 pg/ml (n = 8), within 30 min to 2,071+/-429 at 60 min, and decreased to 1,119+/-319 pg/ml, at 120 min. Plasma levels of prostaglandin E also increased from 146+/-33 pg/ml in normal controls (n = 5) to 2,161+/-606 pg/ml 30 min after endotoxin (n = 5). In contrast to shocked controls, rats pretreated with imidazole, a thromboxane synthetase inhibitor, or essential fatty acid-deficient rats, which are deficient in arachidonate and its metabolites, did not exhibit significant elevations in plasma levels of thromboxane B(2). Imidazole did not however inhibit endotoxin-induced elevations in plasma prostaglandin E. Essential fatty acid deficiency significantly reduced mortality to lethal endotoxic shock. This refractoriness could be duplicated in normal rats pretreated with the fatty acid cyclo-oxygenase inhibitor, indomethacin (10 mg/kg), intravenously 30 min before endotoxin injection. Imidazole (30 mg/kg) administered intraperitoneally 1 h before or intravenously 30 min before endotoxin, also significantly (P < 0.01) reduced mortality from lethal endotoxin shock to 40% compared to a control mortality of 95% at 24 h. Likewise pretreatment with 13-azaprostanoic acid (30 mg/kg), a thromboxane antagonist, reduced mortality from endotoxic shock at 24 h from 100% in control rats to only 50% (P < 0.01). The results suggest that endotoxin induces increased synthesis of thromboxane A(2) that may contribute to the pathogenesis of endotoxic shock.

Acetyl glyceryl ether phosphorylcholine-stimulated human platelets cause pulmonary hypertension and edema in isolated rabbit lungs. Role of thromboxane A2.

Macrophages, neutrophils, and platelets may play a role in acute edematous lung injury, such as that seen in the adult respiratory distress syndrome (ARDS), but their potential actions and interactions are unclear. Because stimulated human macrophages and neutrophils can release acetyl glyceryl ether phosphorylcholine (AGEPC), a potent platelet activator, we hypothesized that in ARDS, leukocyte release of AGEPC might stimulate platelets to release thromboxane A2 (TXA2), which then produces pulmonary hypertension and lung edema. In support of this premise, we found that pulmonary hypertension and edema occurred in isolated rabbit lungs perfused with human platelets and AGEPC, but not with platelets or AGEPC alone. Infusion of a vasodilator (nitroglycerin) to maintain base-line pulmonary artery pressures in lungs perfused with platelets and AGEPC prevented the development of lung edema suggesting that platelet and AGEPC-induced edema was hydrostatic in nature. Additional experiments suggested that the increased pressure was a result of TXA2 release from platelets stimulated by AGEPC. Specifically, preincubation of platelets with imidazole, a thromboxane synthetase blocker, prior to infusion with AGEPC significantly diminished pulmonary hypertension and prevented lung edema. Furthermore, pretreating lung preparations with 13-azaprostanoic acid, a TXA2 antagonist, before infusion of AGEPC and untreated platelets also reduced the pulmonary hypertension and blocked the lung edema. The role of TXA2 was further suggested when perfusates from lungs infused with platelets and AGEPC developed high levels of TXA2, whereas perfusates from controls did not. These results suggest that platelet aggregation induced by AGEPC may contribute to ARDS by releasing TXA2, which raises microvascular pressure and increases edema formation, especially when an underlying permeability defect is present.

Coronary vascular occlusion mediated via thromboxane A2-prostaglandin endoperoxide receptor activation in vivo.

The use of enzyme inhibitors to clarify the role of thromboxane A2 in vasoocclusive disease has been complicated by their non-specific action. To address this problem we have examined the effects of thromboxane A2/prostaglandin endoperoxide receptor antagonism in a canine model of platelet-dependent coronary occlusion. Two structurally distinct thromboxane A2/prostaglandin endoperoxide receptor antagonists, 3-carboxyl-dibenzo (b, f) thiepin-5,5-dioxide (L636,499) and (IS-(1 alpha,2 beta(5Z),3 beta,4 alpha))-7-(3-((2-((phenylamino)-carbonyl)hydrazino)methyl)-7- oxabicy-clo(2.2.1)-hept-2-yl)-5-heptenoic acid (SQ 29,548), were studied to ensure that the effects seen in vivo were mediated by receptor antagonism and did not reflect a nonspecific drug effect. Both compounds specifically inhibited platelet aggregation induced by arachidonic acid and by the prostaglandin endoperoxide analogue, U46619, in vitro and ex vivo, and increased the time to thrombotic vascular occlusion in vivo. When an antagonist (L636,499) was administered at the time of occlusion in vehicle-treated dogs, coronary blood flow was restored. In vitro L636,499 and a third antagonist, 13-azaprostanoic acid, specifically reversed endoperoxide-induced platelet aggregation and vascular smooth muscle contraction. Neither compound altered cyclic AMP in platelet-rich plasma before or during disaggregation. Therefore, reversal of coronary occlusion may reflect disaggregation of platelets and/or relaxation of vascular smooth muscle at the site of thrombus formation through specific antagonism of the thromboxane A2/prostaglandin endoperoxide receptor. Thromboxane A2/prostaglandin endoperoxide receptor antagonists are compounds with therapeutic potential which represent a novel approach to defining the importance of thromboxane A2 and/or endoperoxide formation in vivo.

The thromboxane antagonist, 13-azaprostanoic acid, inhibits arachidonic acid-induced Ca2+ release from isolated platelet membrane vesicles.

In the present study we investigated the ability of the arachidonic acid metabolites, prostaglandin H2 and thromboxane A2, to release Ca2+ from isolated platelet vesicles. The vesicles were prepared through modification of previously described procedures. 45Ca uptake and release were determined by Millipore filtration and isotope counting of the filter paper. Incubation of the vesicles (25 degrees C) with 50 microM CaCl2 (plus 45Ca) resulted in the accumulation of 13 nmol Ca2+ per mg of protein under steady-state conditions. Addition of arachidonic acid (25 microM) resulted in a 42% release of the accumulated Ca2+ and the production of 150 ng thromboxane B2/mg protein. Pretreatment of the vesicles with indomethacin (4 microM) completely inhibited arachidonic acid-induced Ca2+ release and reduced thromboxane B2 synthesis by 82%. Pretreatment of the vesicles with the specific thromboxane A2/prostaglandin H2 antagonist, 13-azaprostanoic acid (20 microM), also resulted in complete inhibition of Ca2+ release but no inhibition of thromboxane B2 production. Addition of prostaglandin H2 (0.3 microM) to the platelet vesicles produced a significant release of Ca2+ only in the presence of the adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (100 microM). This Ca2+ release was totally blocked by 13-azaprostanoic acid (20 microM). The thromboxane synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I, 3.6 microM), in the presence of 2',5'-dideoxyadenosine, only slightly inhibited Ca2+ release in response to added prostaglandin H2, even though thromboxane B2 production was blocked by 95%.

Specific binding of the thromboxane A2 antagonist 13-azaprostanoic acid to human platelet membranes.

In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.

Increased platelet arachidonic acid metabolism in diabetes mellitus.

Platelets obtained from some diabetic patients show enhanced in vitro platelet aggregation. This study sought to determine if platelet obtained from insulin-dependent diabetic subjects synthesize increased quantities of the labile aggregating substance, thromboxane A2 (TXA2), and if it may play a role in the enhanced platelet aggregation. Arachidonic acid (1 mM)-stimulated TXA2 synthesis, as determined via radioimmunoassay of its stable metabolite TXB2, was significantly greater (P less than 0.01, N = 12) in platelet-rich plasma obtained from diabetics compared with matched controls. Arachidonic acid-stimulated TXB2 synthesis in the diabetic platelet-rich plasma was positively correlated with the ambient fasting plasma glucose (r = 0.61, P less than 0.02, N = 15). Platelet aggregation induced by arachidonic acid (0.4-0.8 mM) was inhibited significantly less by 13-azaprostanoic acid (P less than 0.04, N = 14), a competitive antagonist of the actions of prostaglandin H2 or TXA2 on platelets, compared with matched controls. The results support the notion that platelets obtained from some insulin-dependent diabetic subjects manifest increased synthesis of TXA2, which may contribute to the enhanced platelet aggregation.

Enhancement of collagen-induced phosphoinositide turnover by thromboxane A2 analogue through Ca2+ mobilization in human platelets.

In human washed platelets, collagen-induced phosphoinositide turnover was inhibited by indomethacin, an inhibitor of thromboxane A2 (TXA2) formation, particularly at lower doses of collagen. This inhibition was counteracted by the addition of 9,11-epithio-11,12-methano-TXA2 (STA2), a stable analogue of TXA2 as well as by the Ca2+ ionophore A23187. STA2 and A23187 did not stimulate phosphoinositide turnover markedly, but significantly increased cytoplasmic free Ca2+ concentrations. The actions of STA2 were blocked by 13-azaprostanoic acid, a TXA2 receptor antagonist. The results suggest that TXA2 is generated during the action of collagen and increases cytoplasmic free Ca2+ which then stimulates phosphoinositide turnover in cooperation with collagen.

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone. A potent and time-dependent inhibitor of platelet aggregation.

Two new azaprostanoids, a hydrazone (3) and hydrazide (4), have been prepared by the condensation of 2-(6-carboxyhexyl)cyclopentanone with n-hexylhydrazine and caproic acid hydrazide. Preliminary results with the stable hydrazide 4 indicate that it inhibits arachidonic acid (AA) induced human platelet aggregation and that, unlike 13-azaprostanoic acid (1), its site of action is at the cyclooxygenase level. Results with the unstable hydrazone derivative 3 indicate it to be a potent and time-dependent inhibitor of AA-induced human platelet aggregation, with its site of action also at the cyclooxygenase level.

Azaprostanoic acid derivatives. Inhibitors of arachidonic acid induced platelet aggregation.

A series of 13-azaprostanoic acids (4a-h) and a 15-azaprostanoic acid (11a) have been prepared. Synthesis of the 15-aza derivative is based on a novel transformation of a ketone to an N-substituted ethylenamine using a formylmethylimino phosphate derivative. Several of the azaprostanoic acid derivatives were found to be potent inhibitors of platelet aggregation induced by arachidonic acid, whereas no effect was observed on ADP-induced primary aggregation, indicating blockade of the platelet arachidonic acid cascade. The compounds do not inhibit bovine cyclooxygenase activity and are postulated as acting beyond the synthesis of the prostaglandin endoperoxides. The inhibitory effect of the 13-aza series is highly sensitive to both stereochemistry and length of the amino side chain. Any deviation from the natural prostaglandin skeletal arrangement results in decreased biological activity.

Prostaglandin isosteres. 1. (8-Aza-, 8,10-diaza-, and 8-aza-11-thia)-9-oxoprostanoic acids and their derivatives.

A series of novel (8-aza-, 8,10-diaza-, and 8-aza-11-thia)-9-oxoprostanoic acids has been synthesized and evaluated for their ability to mimic the E series prostaglandins in stimulating cAMP formation in the mouse ovary and in binding to the rat kidney plasma prostaglandin receptor. 7-[2-(3-Hydroxyoctyl)-1,1,4-trioxo-3-thiazolidinyl]heptanoic acid markedly stimulates cAMP formation at reasonable pharmacological concentrations and avidly binds to the rat kidney prostaglandin receptor.

Preparation and biological evaluation of a potential photoaffinity label for the prostaglandin H2/thromboxane A2 receptor.

Two aromatic azides (24 and 26) were prepared as potential photoaffinity probes for the PGH2/TXA2 receptor. The compounds are based on the well-characterized PGH2/TXA2 receptor antagonist 13-azaprostanoic acid, with the terminus of its lower side chain replaced with phenoxy (24) or benzyl (26) azide functionality. The two compounds were shown to irreversibly inhibit platelet function after photolysis and resuspension. However, of the two aromatic azides, only the benzyl derivative 26 appeared to be selective for the prostaglandin pathway. The latter compound was also prepared as the aromatic 125I (29) derivative, which may ultimately prove useful as a labeled probe for the identification and isolation of the putative TXA2/PGH2 receptor.

Prostaglandin D2 interacts at thromboxane receptor-sites on guinea-pig platelets.

The anti-aggregatory prostanoid, prostaglandin D2 (PGD2) does not completely inhibit ADP-induced aggregation of guinea-pig platelets and thus produces a bell-shaped dose-inhibition curve. The nature of this bell-shaped curve has now been investigated in guinea-pig platelet-rich plasma. Two selective thromboxane receptor antagonists, 13-aza-prostanoic acid (13-AZA; 16-64.4 microM) and BM 13.177 (5.9-29.8 microM), converted PGD2 to a full inhibitor of aggregation in a dose-related manner. The putative platelet PGD2 receptor antagonist, N-0164 (75 microM) also converted PGD2 to a full inhibitor of platelet aggregation. In contrast to 13-AZA and BM 13.177, higher concentrations of N-0164 (380 and 760 microM) caused a dose-related rightward shift of the PGD2 dose-inhibition curve. The thromboxane receptor antagonism of N-0164 was confirmed in studies in which the dose-aggregation curve to U-46619, a thromboxane mimetic, was competitively antagonized with a pA2 value of 4.67 and a slope of 1.13, comparable to that of 13-AZA. The results show that N-0164 acts as both a platelet PGD2 and thromboxane-receptor antagonist in both human and guinea-pig platelet-rich plasma. The results further indicate that PGD2 can interact at thromboxane receptors in guinea-pig platelets.

Characterization of the inhibitory prostanoid receptors on human neutrophils.

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.

Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-alpha generation.

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists. 2. In human monocytes that were adherent to plastic, neither prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F(2 alpha) (PGF(2 alpha)) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNF alpha-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells. 3. Exposure of human monocytes to LPS (3 ng ml-1, approximately EC84) resulted in a time-dependent elaboration to TNF alpha which was suppressed in cells pretreated with prostaglandin E1 (PGe1), PGE2 and cicaprost. This effect was concentration-dependent with mean pIC50 values of 7.14, 7.34 and 8.00 for PGE1, PGE2 and cicaprost, respectively. PGD2, PGF(2 alpha) and U-46619 failed to inhibit the generation of TNF alpha at concentrations up to 10 microM. 4. With respect to PGE2, the EP-receptor agonists, 16,16-dimethyl PGE2 (non-selective), misoprostol (EP2/EP3-selective), 11-deoxy PGE1 (EP2-selective) and butaprost (EP2-selective) were essentially full agonists as inhibitors of LPS-induced TNF alpha generation with mean pIC50 values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE1, another EP2-selective agonist, AH 13205, inhibited TNF alpha generation by only 21% at the highest concentration (10 microM) examined. EP-receptor agonists which have selectively for the EP1- (17-phenyl-omega-trinor PGE2) and EP3-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNF alpha generation. 5. Pretreatment of human monocytes with the TP/EP4-receptor antagonist, AH 23848B, at 10, 30 and 100 microM suppressed LPS-induced TNF alpha generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves. 6. Given that AH 13205 was a poor inhibitor of TNF alpha generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 and 30 microM AH 13205 inhibited the generation of TNF alpha by 31% and 53%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves at either concentration examined. 7. Since PGD2 and 17-phenyl-omega-trinor PGE2 (EP1-agonist) did not suppress TNF alpha generation, the EP1/EP2/DP-receptor antagonist, AH 6809, was employed to assess if EP2-receptors mediated the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 microM AH 6809 did not affect LPS-induced TNF alpha generation but produced a parallel 3.5 fold rightwards shift of the PGE2 concentration-response curve. 8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNF alpha generation. One of these probably represents i.p. receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP2- and/or EP-4- and/or i.p.) or novel EP-receptors mediate the inhibitory effect of PGE2 on TNF alpha generation.

Characterization of the prostaglandin E2 sensitive (EP)-receptor in the rat isolated trachea.

1. Using a range of natural and synthetic prostanoid receptor agonists and antagonists, we have shown that the rat isolated trachea contains a heterogeneous population of prostaglandin receptor sub-types mediating both relaxation and contraction of the smooth muscle. Prostaglandin E2 (PGE2) elicits smooth muscle relaxation of pre-contracted preparations, the responses being well defined, with a mean potency (p[A50]) of 7.81 +/- 0.05. 2. 11-deoxy PGE1 16,16-dimethyl PGE2 and misoprostol were all full agonists at this receptor, whilst AH13205 was a low potency agonist, and sulprostone was inactive. 3. The EP1 receptor antagonist, AH6809 (5 microM), and the selective DP receptor antagonist, BW A868C (0.1 microM), had no significant effect on the concentration-effect (E/[A]) curves to PGE2. 4. The putative EP4-receptor antagonist, AH23848B, produced non-competitive antagonism of the PGE2 response curves; pA2 values of 5.07 +/- 0.15 and 5.24 +/- 0.19 were obtained at concentrations of 30 microM and 100 microM respectively. 5. The synthetic thromboxane A2 mimetic, U46619, caused smooth muscle contractions, with a mean p[A50] of 6.90 +/- 0.11. This response was antagonized by the TP receptor antagonist, GR32191B, yielding a mean pA2 of 8.31. 6. At concentrations of 1 microM and above, prostaglandin D2 (PGD2) and the IP-receptor agonist, cicaprost, generally elicited concentration-dependent relaxations of the rat trachea. Prostaglandin F2 alpha (PGF2 alpha) was without affinity or efficacy. 7. These data suggest that the rat isolated trachea contains EP-receptors, TP-receptors, and few, if any DP, IP or FP-receptors. The inactivity of sulprostone (EP3/EPj receptor selective) and the low potency of AH1 3205 (EP2-receptor selective) suggest that the rat trachea contains an atypical EP-receptor that does not conform to the current classification system.

Characterization of the prostanoid receptors mediating inhibition of PAF-induced aggregation of guinea-pig eosinophils.

1. Prostanoids induce a wide range of biological actions which are mediated by specific membrane-bound receptors. We have recently shown that the E-type prostaglandins, PGE1 and PGE2, effectively inhibit eosinophil aggregation induced by platelet-activating factor (PAF). In an attempt to determine which prostanoid receptor(s) were involved, we investigated the effects of a range of selective prostanoid agonists and antagonists on eosinophil homotypic aggregation induced by PAF. 2. Both PGE1 and PGE2 (10(-10) to 10(-6) M) induced a concentration-related inhibition of the aggregation response induced by PAF. PGE1 was more effective than PGE2 but PGE2 was slightly more potent than PGE1 (approximate IC50 values for PGE1 and PGE2 of 1.5 x 10(-8) M and 5 x 10(-9) M, respectively). 3. The EP2-selective agonists, 11-deoxy-PGE1, butaprost and AH13205, and the EP2/EP3-selective agonist, misoprostol, also inhibited PAF-induced aggregation. The rank order of potency for EP2-selective agonists was 11-deoxy-PGE1 > misoprostol > butaprost = AH13205. The protein kinase A inhibitor, KT5720 (10(-6) M), reversed the inhibitory effects of 11-deoxy-PGE1 (10(-6) M) and AH13205 (10(-5) M). 4. The EP1/EP3-selective agonist, sulprostone, and the EP1-selective agonist, 17-phenyl-omega-trinor PGE2, had no significant inhibitory activity when tested at concentrations up to 10(-6) M. The EP4-receptor antagonist, AH23848B, had no effect on PAF-induced aggregation and did affect the inhibitory activity of PGE1. 5. The IP-selective agonist, cicaprost (up to 10(-6) M), and the IP/EP1-receptor agonist, iloprost (up to 10(-5) M), had no significant effect on PAF-induced eosinophil aggregation. However, iloprost significantly augmented the inhibitory effects of a maximally inhibitory concentration of PGE2. 6. PGD2 (10(-5) M) had no effect on eosinophil aggregation and the inhibitory activity of PGE1 on PAF-induced eosinophil aggregation was not altered by the DP-selective receptor antagonist, BWA868C. 7. The results presented here suggest that the inhibition of PAF-induced eosinophil aggregation by prostanoids is mediated by the occupation of EP2-receptors. It is important to note that the effects of naturally occurring prostanoids, such as PGE2, on eosinophil aggregation occur at low concentrations highlighting a potential role for EP2 receptors in regulating eosinophil function in vivo.

Antagonism of prostaglandin-mediated responses in platelets and vascular smooth muscle by 13-azaprostanoic acid analogs. Evidence for selective blockade of thromboxane A2 responses.

Studies were undertaken to examine the pharmacological properties and stereochemical requirements of a limited series of prostanoic acid analogs for inhibition of arachidonic acid (AA) and/or endoperoxide (U46619)-mediated responses in human platelets and rat aorta. To assess the role of stereochemistry, a set of trans- and cis-isomers of 13-azaprostanoic acid (APA) and 11a-homo-13-azaprostanoic acid (HAPA) were prepared. Each prostanoic acid analog blocked AA- or U46619-induced aggregatory and secretory responses in platelets, and U46619-mediated contractions of rat aorta in a concentration-dependent manner (0.1 to 100 microM). The azaprostanoic acid analogs blocked responses to both inducers of platelet activation with IC50 values ranging from 3.4 to 27.5 microM. Trans-APA was about 2- to 3-fold more active as an antagonist of serotonin release induced by AA or U46619 than the remaining analogs. The rank order of inhibitory potency (IC50; microM) for these analogs against U46619-induced serotonin release in human platelets was trans-APA (3.4) greater than cis-APA (8.9) = cis-HAPA (8.7) = trans-HAPA (9.1). Concentrations of the prostanoic acid analogs required to block these responses to AA and U46619 were similar, and the highest concentration used (100 microM) did not modify AA-induced malondialdehyde production in human platelet preparations. In contrast, the isomers of APA and HAPA were equally active as antagonists of U46619-induced contractions of rat vascular tissue, possessing KB values varying from 7.1 to 13.2 microM. Each azaprostanoic acid analog shifted the concentration-response curve of U46619 in rat aorta to the right, indicating a competitive-type inhibition. In addition, the azoprostanoic acid analog (U51605) was a more potent competitive antagonist of U46619 in this preparation and possessed an average pKB value of 6.18. In summary, the results show that (1) expansion of the five-membered ring of APA to the six-membered ring analogs (HAPA) led to a retention of potent inhibitory activity against U46619 in human platelets and rat vascular smooth muscle, (2) the antiaggregatory and antisecretory actions of the azaprostanoic acid analogs were mediated by a blockade of the responses to AA and U46619, and not by an inhibition of AA metabolism, (3) the blocking activity for the APA isomers was stereoselective (trans greater than cis) whereas the isomers of HAPA were equally effective as inhibitors of platelet function; and (4) these azaprostanoic acid analogs act as selective endoperoxide (U46619)/thromboxane A2 antagonists in these two tissues.

Prostaglandins and human platelet aggregation. Implications for the anti-aggregating activity of thromboxane-synthase inhibitors.

Selective pharmacological blockade of thromboxane-synthase in human platelets by dazoxiben resulted in the reorientation of cyclic-endoperoxides towards PGE2, PGD2 and PGF2 alpha. At concentrations which can be reached when thromboxane-synthase is inhibited, PGE2 (100-500 nM) exerted a marked, concentration-dependent pro-aggregatory effect. This required the formation of endogenous or the addition of exogenous endoperoxides and was prevented by PGD2 or 13-aza-prostanoic acid, a selective antagonist of PGH2/TxA2 receptors. The anti-aggregating effect of PGD2 was evident at concentrations lower than those obtained in dazoxiben-treated platelets. It is proposed that in the absence of TxA2 generation, a combination of endoperoxides and PGE2 may result in normal aggregation. The latter may be inhibited by PGD2. No interference of PGF2 alpha on platelet function could be shown.