TP Receptor Antagonist Research Articles

Atherosclerosis regression and TP receptor inhibition: effect of S18886 on plaque size and composition—a magnetic resonance imaging study

Endothelial dysfunction, platelet hyperactivity, and inflammation play a crucial role in atherogenesis. A growing body of evidence suggests that inhibition of the thromboxane A2 (TxA2 or TP) receptor may improve endothelial function and reduce the inflammatory component of atherosclerosis in addition to its demonstrated antiplatelet activity. Consequently, we sought to assess the effect of a novel TP receptor antagonist S18886, on atherosclerotic lesion progression and composition by serial non-invasive magnetic resonance imaging (MRI). S18886 was compared with control in an experimental model of established aortic atherosclerosis in New Zealand White rabbits (n=10). The animals underwent MRI of the abdominal aorta at the time of randomization and at the end of treatment. Subsequently, animals were euthanized and specimens were stained for histopathology and immunohistochemistry with anti-alpha-actin antibodies for vascular smooth muscle cells (VSMC), anti-RAM-11 for macrophages, anti-caspase-3 for apoptotic cells, anti-MMP-1 for metalloproteinases, and anti-endothelin-1 (ET-1) as a marker of endothelial dysfunction. MRI analysis revealed a significant reduction in total vessel area (TVA) and vessel wall area (VWA) in the S18886 group (P<0.05). Immunostaining analysis showed a significant decrease in RAM-11, caspase-3, MMP-1, ET-1 and an increase in alpha-actin in the treated group (P<0.05 vs. control). Inhibition of the TP receptor by S18886 causes a regression of advanced atherosclerotic plaques. In addition, the reduction in the markers for macrophages, apoptotic cells, metalloproteinases, and endothelin-1 and the increase in VSMC, suggests that S18886 may not only halt the progression of atherosclerosis, but also transform lesions towards a more stable phenotype. The possibility of combining antithrombotic and antiatherosclerotic activity by means of the administration of TP inhibitors deserves further investigation in a clinical setting.

Thromboxane A2 Up-Regulates Neutrophil Elastase Release in Syrian Hamsters With Trinitrobenzene Sulfonic Acid-Induced Colitis

Neutrophil elastase (NE) is a factor that aggravates colitis. We investigated the influence of thromboxane A2 (TXA2) and leukotriene B4 (LTB4) on NE release in Syrian hamsters with trinitrobenzene sulfonic acid-induced colitis. Colonic specimens with colitis were incubated with U-46619 (a TXA2 analogue) or LTB4 in vitro and NE release was examined. As a result, U-46619 increased NE release, while LTB4 had no effect. The NE release induced by U-46619 was inhibited by a TP-receptor antagonist. To demonstrate that TXA2 caused NE release in vivo as well, while LTB4 did not, colitis animals were treated with nordihydroguaiaretic acid (NDGA), a dual inhibitor of cyclooxygenase/lipoxygenase; and colonic luminal TXB(A)2 and LTB4 levels and NE activity were determined. The TXB(A)2 level was significantly correlated with NE activity, while no correlation was found between LTB4 and NE activity. An inhibitory effect of NDGA on the ulcer area was also observed, and NE activity was significantly correlated with the ulcer area. The suppression of TXA2 production by NDGA may result in the inhibition of NE release so that colonic tissue damage becomes less severe. Regulation of NE release is a new biological action of TXA2 that has not been reported before.

Evidence for the involvement of the cyclooxygenase-metabolic pathway in diclofenac-induced inhibition of spontaneous contraction of rat portal vein smooth muscle cells

The effects of diclofenac, a cyclooxygenase (COX) inhibitor, were investigated on spontaneous phasic contractions of longitudinal preparations of the rat portal vein. Diclofenac produced a concentration-dependent decrease in the amplitude of these spontaneous phasic contractions. Diclofenac (30 microM) decreased the amplitude of the spontaneous phasic increase in the F340/F380 ratio of Fura PE3, an indicator of intracellular Ca2+ concentration. It also reduced the number of action potentials in each burst discharge without changing the resting membrane potential of longitudinal smooth muscle cells. The extent of the distribution of Lucifer Yellow injected into a smooth muscle cell was decreased in the presence of diclofenac (30 microM). Both AH6809, a prostanoid EP receptor antagonist, and SQ22536, an adenylate cyclase inhibitor, decreased the amplitude of the spontaneous contractions. On the other hand, neither ozagrel, a thromboxane synthase inhibitor, nor SQ29548, a prostanoid TP receptor antagonist, significantly affected spontaneous contractions. These results indicate that diclofenac inhibits the amplitude of spontaneous contractions of the rat portal vein through inhibition of electrical activity, which may be related to an inhibition of the cyclooxygenase pathway.

In Vitro and in Vivo Pharmacological Characterization of BM-613 [N-n-Pentyl-N′-[2-(4′-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a Novel Dual Thromboxane Synthase Inhibitor and Thromboxane Receptor Antagonist

Thromboxane A2 (TXA2) is a key mediator of platelet aggregation and smooth muscle contraction. Its action is mediated by its G protein-coupled receptor of which two isoforms, termed TPalpha and TPbeta, occur in humans. TXA2 has been implicated in pathologies such as cardiovascular diseases, pulmonary embolism, atherosclerosis, and asthma. This study describes the pharmacological characterization of BM-613 [N-n-pentyl-N'-[2-(4'-methylphenylamino)-5-nitrobenzenesulfonyl]urea], a new combined TXA2 receptor antagonist and TXA2 synthase inhibitor. It exhibits a strong affinity for human platelet TP receptors (IC50 = 1.4 nM), TPalpha and TPbeta expressed in COS-7 cells (IC(50) = 2.1 and 3.1 nM, respectively), and TPs expressed in human coronary artery smooth muscle cells (IC50 = 29 microM). BM-613 shows a weak ability to prevent contraction of isolated rat aorta (ED50 = 1.52 microM) and guinea pig trachea (ED50 = 2.5 microM) induced by TXA2 agonist U-46619 (9.11-dideoxy-9.11-methanoepoxy-prostaglandin F2). Besides, BM-613 antagonizes TPalpha (IC50 = 0.11 microM) and TPbeta (IC50 = 0.17 microM) calcium mobilization induced by U-46619 and inhibits human platelet aggregation induced by U-46619 (ED50 = 0.278 microM), arachidonic acid (ED50 = 0.375 microM), and the second wave of ADP. BM-613 also dose dependently prevents TXA2 production by human platelets (IC50 = 0.15 microM). In a rat model of ferric chloride-induced thrombosis, BM-613 significantly reduces weight of formed thrombus by 79, 49, and 28% at 5, 2, and 1 mg/kg i.v., respectively. In conclusion, BM-613 is a dual and potent TP receptor antagonist and TXA2 synthase inhibitor characterized by a strong antiplatelet and antithrombotic potency. These results suggest that BM-613 could be a potential therapeutic drug for thrombotic disorders.

Thromboxane receptor mediates renal vasoconstriction and contributes to acute renal failure in endotoxemic mice.

Sepsis is a major cause of acute renal failure (ARF) and death. Thromboxane A2 (TxA(2)) may mediate decreases of renal blood flow (RBF) and/or GFR associated with LPS-induced sepsis. This study tested whether TxA(2) receptor blockade, with the use of TxA(2) receptor knockout (TP-KO) mice or a selective TP receptor antagonist (SQ29,548), would alleviate LPS-induced renal vasoconstriction and ARF. Under basal conditions, anesthetized TP-KO mice displayed a lower mean arterial pressure than wild-type (WT) mice (102 versus 94 mmHg; P < 0.05). RBF, renal vascular resistance (RVR), GFR, and urine flow did not differ among groups under basal conditions, suggesting little tonic influence of TxA(2) on renal TP receptors in health. In endotoxemic WT mice, 14 h after LPS (Escherichia coli LPS 8.5 mg/kg intraperitoneally), mean arterial pressure was reduced to 85 mmHg (P < 0.001), as were RBF (5.0 versus 9.3 ml/min per g kidney wt; P < 0.001) and GFR (0.38 versus 1.03 ml/min per g kidney wt; P < 0.001). Heart rate and RVR (71 versus 47 mmHg/ml per min; P < 0.05) increased. The decreases in RBF and GFR after LPS were attenuated in TP-KO mice versus WT mice (both P < 0.05). In both TP-KO and TP antagonist-treated mice, RVR remained stable in response to LPS versus WT mice that did not receive LPS. Delayed TP-antagonist treatment (12 h after LPS injection) ameliorated RBF and RVR but did not restore GFR. In other WT animals, TP-antagonist treatment for 2 h before intravenous LPS abolished the early renal vasoconstriction and alleviated the decrease in GFR. These results demonstrate that renal vasoconstriction during endotoxemic shock induced by LPS is mediated by TP receptors as indicated by pharmacologic blockade and genetic disruption of TP receptors.

Vasoconstrictor effects of 8-iso-prostaglandin E 2 and 8-iso-prostaglandin F 2α on human umbilical vein

The present study was undertaken to determine whether 8-iso-prostaglandin E 2 and 8-iso-prostaglandin F 2α posses contractile action on human umbilical vein and to evaluate the possible involvement of prostanoid TP receptors in this effect. Human umbilical vein rings were mounted in organ baths and concentration–response curves to 8-iso-prostaglandin E 2 or 8-iso-prostaglandin F 2α were constructed. Both isoprostanes evoked concentration-dependent contraction. 8-iso-prostaglandin E 2 (pEC 50=6.90±0.03) was significantly more potent than 8-iso-prostaglandin F 2α (pEC 50=6.10±0.04). However, both isoprostanes were equieffective. The prostanoid TP receptor antagonists, ICI-192,605 (4-( Z)-6-(2- o-Chlorophenyl-4- o-hydroxyphenyl-1,3-dioxan- cis-5-yl)hexenoic acid) and SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-[1 S(1α,2α( Z),3α,4α)]-5-Heptenoic acid) produced a competitive rightward shift of 8-iso-prostaglandin E 2 concentration–response curves with p K B values of 8.91±0.04 and 8.07±0.07, respectively. When ICI-192,605 (1 nM) and SQ-29548 (10 nM) were evaluated against 8-iso-prostaglandin F 2α they produced a parallel rightward displacement of 8-iso-prostaglandin F 2α concentration–response curves without affecting the maximum responses giving p A 2 values of 9.02±0.12 and 8.26±0.13, respectively. In conclusion, the present study describes for the first time the vasoconstrictor action of 8-iso-prostaglandin E 2 and 8-iso-prostaglandin F 2α in human umbilical vein. Furthermore, the affinity values obtained with ICI-192,605 and SQ-29548 provide strong pharmacological evidence of prostanoid TP receptors involvement in this effect.

Platelet Receptors for Adenine Nucleotides and Thromboxane A2

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.

Ramatroban (BAY u 3405): a novel dual antagonist of TXA2 receptor and CRTh2, a newly identified prostaglandin D2 receptor.

It is known that thromboxane A2 (TXA2) contributes to various diseases such as bronchial asthma, ischemic heart disease, cerebrovascular disorders and allergic rhinitis. A number of TXA2 synthase inhibitors and TXA2 receptor (TP receptor) antagonists have been developed to treat these diseases. Ramatroban (BAY u 3405) was developed as a potent TP receptor antagonist with excellent efficacy against allergic rhinitis in many animal models and patients. Recent studies also revealed that ramatroban can block the newly identified PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2). PGD2 induces migration and degranulation of eosinophils through CRTh2 and contributes to late-phase inflammation and cell damage. Accordingly, it was considered that ramatroban suppresses the late-phase inflammation via TP receptor and CRTh2 blockade. In terms of the efficacy on vascular systems, it was revealed that ramatroban can suppress the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in endothelial cells and prevent exacerbation of inflammation by blocking these responses. According to our recent studies in hypercholesterolemic rabbits ramatroban prevents macrophage infiltration through MCP-1 downregulation and neointimal formation after balloon injury and attenuates vascular response to acetylcholine. Therefore, ramatroban may be beneficial in the treatment of atherosclerosis.

Endothelium‐dependent contractions to acetylcholine, ATP and the calcium ionophore A 23187 in aortas from spontaneously hypertensive and normotensive rats

The present study was designed to determine whether or not an increase in endothelial intracellular concentration of calcium ([Ca2+]i) evokes endothelium-dependent contractions in the aorta from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Acetylcholine, adenosine triphosphate (ATP) and the calcium ionophore, A 23187, produced endothelium-dependent relaxations in isolated aortic rings of both WKY and SHR. These relaxations in response to the three agonists were significantly smaller in the SHR when compared with the WKY. Endothelium-dependent contractions to acetylcholine, ATP and A 23187 were observed only in the aorta isolated from the SHR. In the presence of NG-nitro-L-arginine, an NO synthase inhibitor, the endothelium-dependent contractions in response to acetylcholine, ATP and A 23187 were potentiated significantly in the aorta SHR and were unmasked in that of WKY. However, the contractions were still significantly greater in SHR than in WKY. These contractions were abolished by indomethacin and valeryl salicylate (two cyclo-oxygenase inhibitors) as well as by S 18886 (a TP-receptor antagonist), indicating that the endothelium-dependent contraction produced by the three agonists share the same characteristics. The results of the present study indicate that the release/generation of endothelium-derived contracting factor, requires an increase in endothelial [Ca2+]i.

Antithrombotic effects of S 18886, a novel orally active thromboxane A2 receptor antagonist

Platelet activation and thrombus formation play a critical role in the onset of acute coronary syndromes. Thromboxane A2 (TxA2) is among the different chemical modulators released by activated platelets. TxA2 is considered one of the most powerful agonists for platelet activation. In addition, TxA2 exerts a vasoconstrictor effect by serving as an agonist of the thromboxane receptor (TP) on the vascular smooth muscle cell membranes. The putative effect of TxA2 on thrombosis is demonstrated by the clinical effectiveness of acetylsalicylic acid (ASA) in the prevention of acute coronary syndromes. Among the clinically used antiplatelet agents, clopidogrel has shown to be slightly more effective than ASA in the prevention of atherothrombotic events in patients with peripheral arterial disease, and is one of the most widely used after aspirin. The aims of the study were to study the antithrombotic effects of escalating doses of the TP-receptor antagonist, S 18886 and to compare its effects with those achieved by the administration of ASA (5 mg kg-1 day-1), and clopidogrel (3 mg kg-1 day-1). The study was undertaken at high and low shear rate conditions using the Badimon perfusion chamber in a porcine model. Antithrombotic effects were assessed as changes on platelet and fibrin(ogen) deposition. The doses of 30 and 100 micro g kg-1 day-1 were selected based on a previous platelet aggregation study. S 18886 shows a dose-dependent antithrombotic response. The dose of S-100 develops similar antithrombotic effects to those of clopidogrel and superior to those of aspirin. The antithrombotic effects were statistically significant at both studied shear rate conditions. Therefore, the orally active TP-receptor antagonist, S 18886, appears to be a new and effective agent to prevent atherothrombotic complications.

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries.

1. Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E(2) (PGE(2)) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2. In the presence of indomethacin (3 microm) and the TP receptor antagonist GR32191 (1 microM), PGE(2) was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC(50) 8.0+/-0.1, n=5). 3. Establishment of a rank order of potency using the EP(4)>EP(2) agonist 11-deoxy PGE(1), and the EP(2)>EP(4) agonist PGE(1)-OH (mean pEC(50) of 7.6+/-0.1 (n=6) and 6.4+/-0.1 (n=4), respectively), suggested the presence of functional EP(4) receptors. Furthermore, the selective EP(2) receptor agonist butaprost at concentrations <1 microM failed to relax the tissues. 4. Blockade of EP(4) receptors with the EP(4) receptor antagonists AH23848 and EP(4)A caused significant rightward displacements in PGE(2) concentration-response curves, exhibiting pA(2) and pK(B) values of 5.7+/-0.1, n=3, and 8.4, n=3, respectively. 5. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC(50) values 8.3+/-0.1 (n=4) and 8.1+/-0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD(2) and PGF(2 alpha) failed to cause appreciable relaxation or contraction at concentrations of up to 30 microm. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC(50) 7.4+/-0.3, n=3). 6. These data demonstrate the presence of prostanoid EP(4) receptors mediating PGE(2) vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

Thromboxane A 2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity

Although there are species-related differences in uterine prostanoid receptor subtypes, functional prostanoid receptors in the porcine uterus are similar with those in the human uterus (FP, TP, EP 1, EP 2, EP 3, DP and IP) except for the TP receptor. These similarities promoted us to determine whether TP receptors are present in the non-pregnant porcine uterus. For this purpose, the effects of TP receptor agonists and antagonists were investigated by a contraction study and by a binding study. 9,11-Dideoxy-9α, 11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619, 1 nM–10 μM), a stable thromboxane A 2 mimetic, caused tetrodotoxin-resistant contraction in both longitudinal and circular muscles of the uterine cornu. The pEC 50 value in the longitudinal muscle (6.69) was lower than that in the circular muscle (7.62), but the maximum response in the longitudinal muscle was two times larger than that in the circular muscle. The longitudinal and circular muscles of other regions (corpus and cervix) also responded to U46619, and region-related difference in contractile responses was observed only in the longitudinal muscles. 4( Z)-6-(2- o-Chlorophenyl-4- o-hydroxyphenyl-1,3-dioxan- cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1 S-[1α,2α( Z),3α,4α]]-]5-heptenoic acid (SQ29548) inhibited the contractile responses to U46619 competitively. The longitudinal and circular muscles in the cornu contained a single class of [ 3H]SQ29548 binding site with similar K d values (30 nM), but B max in the circular muscle (90.9±8.6 fmol/mg protein) was two times higher than that in the longitudinal muscle (58.2±8.6 fmol/mg protein). The ranking order of competition by TP receptor agonists and antagonists (with p K i values in parentheses) was [1 S-[1,2( Z),3(1 E,3 S*),4]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 7.70)>SQ29548 (7.39)>7-[3-(3-Hydroxy-1-octenyl)bicycle[3.1.1]hept-2-yl]-,[2 S-[2α( Z),3β(1 E,3 R*)]]-5-heptenoic acid (CTA 2, 6.55)>7-[3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl-,[1 S-[1α,2β( Z),3α(1 E,3 R*),5α]]-5-heptenoic acid (PTA 2, 6.50)>U46619 (6.41)>7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1] hept-6yl]-,[1 S-[1α,4α,5α(1 E,3 R*),6β( Z)]]-5-heptenoic acid (U44069, 6.34), and this order is consistent with current TP receptors. Treatment with indomethacin (100 nM) and N- tert-butyl- N¢-[(2-cyclohexylamino-5-nitrobenzene) sulfonyl] urea (BM-531, 10 μM) inhibited the spontaneous contractile activities of both longitudinal and circular muscles. The present results indicate that contractile TP receptors are present in the non-pregnant porcine uterus. Therefore, the prostanoid receptor subtypes that exist in the porcine uterus (TP, IP, DP, FP, EP 1, EP 2 and EP 3) are the same as those present in the human uterus. The distribution of TP receptors in the porcine uterus differed depending on the type of myometrium (longitudinal and circular muscles) and region of the uterus. The endogenous thromboxane A 2-TP receptor pathway is thought to play a physiological role in regulation of spontaneous contractile activity in the porcine uterus.

Effects of TRA-418, a novel TP-receptor antagonist, and IP-receptor agonist, on human platelet activation and aggregation.

[4-[2-(1,1-Diphenylethylsulfanyl)-ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxy]-acetic acid N-Methyl-d-glucamine salt (TRA-418) has both thromboxane A2 (TP)-receptor antagonist and prostacyclin (IP)-receptor agonist properties. The present study examined the advantageous effects of TRA-418 based on the dual activities, over an agent having either activity alone and also the difference in the effects of TRA-418 and a glycoprotein alphaIIb/beta3 integrin (GPIIb/IIIa) inhibitor. TRA-418 inhibited platelet GPIIb/IIIa activation as well as P-selectin expression induced by adenosine 5'-diphosphate, thrombin receptor agonist peptide 1-6 (Ser-Phe-Leu-Leu-Arg-Asn-NH2), and U-46619 in the presence of epinephrine (U-46619+ epinephrine). TRA-418 also inhibited platelet aggregation induced by those platelet-stimulants in Ca2+ chelating anticoagulant, citrate and in nonchelating anticoagulant, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK). The TP-receptor antagonist SQ-29548 inhibited only U-46619+epinephrine-induced GPIIb/IIIa activation, P-selectin expression, and platelet aggregation. The IP-receptor agonist beraprost sodium inhibited platelet activation. Beraprost also inhibited platelet aggregation induced by platelet stimulants we tested in citrate and in PPACK. The GPIIb/IIIa inhibitor abciximab blocked GPIIb/IIIa activation and platelet aggregation. However, abciximab showed slight inhibitory effects on P-selectin expression. TRA-418 is more advantageous as an antiplatelet agent than TP-receptor antagonists or IP-receptor agonists separately used. TRA-418 showed a different inhibitory profile from abciximab in the effects on P-selectin expression.

The thromboxane receptor antagonist PBT-3, a hepoxilin stable analog, selectively antagonizes the TPalpha isoform in transfected COS-7 cells.

The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOP [[1S-[1alpha,2alpha (Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid] in human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist [3H]SQ 29,548 [[1S-[1alpha,2alpha (Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid], to the TP receptor isoforms in TPalpha- and TPbeta-transfected COS-7 cells. These isoforms possess a different tail length, the alpha being shorter than the beta isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC50 value for PBT-3 = 2.0 +/- 0.3 x 10-7 M was observed for TPalpha, whereas this was one-sixth less active on the TPbeta isoform (IC50 = 1.2 +/- 0.2 x 10-6 M), suggesting selectivity for the TPalpha isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC50 was similar to that of the TPalpha isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TPalpha isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.

Action of prostanoids on the emetic reflex of Suncus murinus (the house musk shrew)

Several prostanoids were investigated for a potential to induce emesis in Suncus murinus. The TP receptor agonist 11α,9α-epoxymethano-15 S-hydroxyprosta-5 Z,13 E-dienoic acid (U46619) induced emesis at doses as low as 3 μg/kg, i.p. but the DP receptor agonist 5-(6-Carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C) was approximately 1000 times less potent. The emetic action of U46619 (300 μg/kg, i.p.) was antagonized significantly by the TP receptor antagonist, vapiprost ( P<0.05). EP (prostaglandin E 2, 17-phenyl-ω-trinor prostaglandin E 2, misoprostol and sulprostone), FP (prostaglandin F 2α and fluprostenol) and IP (iloprost and cicaprost) receptor agonists failed to induce consistent emesis at doses up to 300–1000 μg/kg, i.p. Fluprostenol reduced nicotine (5 mg/kg, s.c.)-but not copper sulphate (120 mg/kg, intragastric)-induced emesis; the other inconsistently emetic prostanoids were inactive to modify drug-induced emesis. The results indicate an involvement of TP and possibly DP and FP receptors in the emetic reflex of S. murinus.

Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein.

1. This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV). 2. HUV rings were mounted in organ baths and concentration-response curves to U-46619 (TXA(2) mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC(50): 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pK(B) values of 7.96 and 9.07, respectively. 3. Concentration-response curves to EP receptor agonists: PGE(2), misoprostol and 17-phenyl-trinor-PGE(2) gave pEC(50) values of 5.06, 5.25 and 5.32, respectively. Neither pEC(50) nor maximum of PGE(2) and 17-phenyl-trinor-PGE(2) concentration-response curves were modified by the DP/EP(1)/EP(2) receptor antagonist AH 6809 (1 micro M). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA(2) estimated for ICI-192,605 against PGE(2) or misoprostol were 8.91 and 9.22, respectively. 4. Concentration-response curves to FP receptor agonists: PGF(2)(alpha) and fluprostenol gave pEC(50) values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF(2)(alpha) or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 micro M) against PGF(2)(alpha) was observed. 5. In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel.

TRA-418, a novel compound having both thromboxane A2 receptor antagonistic and prostaglandin I2 receptor agonistic activities: its antiplatelet effects in human and animal platelets

TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 micro g kg min-1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity.

Solution structure of the third extracellular loop of human thromboxane A 2 receptor

The extracellular domains of the thromboxane A 2 receptor (TP receptor) were found to be involved in the specific ligand recognition. Determination of the three-dimensional (3D) structure of the extracellular loops would help to explain the mechanism of the ligand binding to its receptor with regard to the tertiary structure. Based on our previous studies on the extracellular loop of the human TP receptor, the synthetic loop peptides, whose termini are constrained to 10 to 14-Å separations, are more likely to mimic the native structure of the extracellular loops. In this study, a peptide with the sequence of the third extracellular loop (eLP 3, residues 271–289) of the TP receptor was synthesized, and its termini were constrained by the formation of a disulfide bond between the additional homocysteines located at both ends. Fluorescence spectroscopic studies showed that the fluorescence intensity of this constrained loop peptide could be increased by the addition of SQ29,548, a TP receptor antagonist, which indicated the interaction between the peptide and the ligand. The structure of this peptide was then studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. 1H NMR assignments of the peptide were obtained and structure constraints were derived from nuclear Overhauser effects and J-coupling constants. The solution structure of the peptide was then calculated based on these constraints. The overall structure shows a β turn from residues 278 to 281. It also shows a distance of 9.45 Å between the ends of the N and C termini of the peptide, which agrees with the distance between the two residues at the ends of the transmembrane helices connecting the eLP 3 on the TP receptor working model generated using molecular modeling, based on the crystal structure of bovine rhodopsin. These results provide valuable information for the characterization of the complete 3D structure of the extracellular domains of the human TP receptor.

Effects of 8- iso-prostaglandin E 2 and 8- iso-prostaglandin F 2α on the release of noradrenaline from the isolated rat stomach

In the present experiment, we examined the effect of 8- iso-prostaglandin E 2 and 8- iso-prostaglandin F 2α on the release of noradrenaline from the isolated rat stomach. The postganglionic sympathetic nerves were electrically stimulated twice at 1 Hz for 1 min and test reagents were added during the second stimulation. 8-Iso-prostaglandin E 2 (10 −8–10 −6 M) and 8- iso-prostaglandin F 2α (10 −7–10 −5 M) dose-dependently reduced the evoked noradrenaline release, and these inhibitory potencies were as follows: 8- iso-prostaglandin E 2>8- iso-prostaglandin F 2α. The inhibitory effect of 8- iso-prostaglandin F 2α, but not 8- iso-prostaglandin E 2, was abolished by 10 −6 M SQ-29548 ([1 S-[1α,2α( Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino] methyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid) (a prostanoid TP receptor antagonist). On the other hand, the inhibitory effect of 8- iso-prostaglandin E 2 was abolished by 10 −5 M AH-6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) (a prostanoid EP receptor antagonist), which also attenuated the inhibitory effects of ONO-AE-248 (16 S-9-deoxy-9β-chloro-15-deoxy-16-hyfroxy-17,17-trimethylene 19, 20-didehydro prostaglandin F 2) (a selective EP 3 receptor agonist) on the evoked release of noradrenaline. The inhibitory effect of 8- iso-prostaglandin F 2α, but not 8- iso-prostaglandin E 2, was abolished by pertussis toxin. These results suggest that 8- iso-prostaglandin F 2α inhibits noradrenaline release through TP receptors, whereas 8- iso-prostaglandin E 2 seems to inhibit noradrenaline release through EP 3 receptors, located on the gastric sympathetic nerve terminals in rats.

Ramatroban, a TP receptor antagonist, improves vascular responses to acetylcholine in hypercholesterolemic rabbits in vivo

Recent studies show that 8- iso-prostaglandin F 2α, a member of F 2-isoprostane family, acts as a vasoconstrictor via TP receptor activation; and its local release may contribute to an abnormal vasomotor tone associated with hypercholesterolemia. The purpose of this study was to examine whether ramatroban, a TP receptor antagonist, improves abnormal vascular reactivity in vivo in hypercholesterolemic rabbits. The plasma 8- iso-prostaglandin F 2α levels in hypercholesterolemic groups were significantly higher than those in normal groups. The treatment by ramatroban reversed the attenuation of the vascular response to acetylcholine in hypercholesterolemic groups. However, l- N G-nitroarginine methyl ester, a nitric oxide synthase inhibitor, did not inhibit the protective effects of ramatroban. Attenuation of the vascular response to acetylcholine in hypercholesterolemic rabbits was significantly enhanced by 8- iso-prostaglandin F 2α. Attenuation of the vascular response to acetylcholine by a cholesterol-rich diet and 8- iso-prostaglandin F 2α was canceled by ramatroban. These findings suggest that ramatroban improves the vascular response in vivo to acetylcholine in hypercholesterolemic rabbits by blocking the action of 8- iso-prostaglandin F 2α.