sq-29548 and ramatroban

G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.

Topics: Amino Acid Substitution; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; Carbazoles; Drug Evaluation, Preclinical; Fatty Acids, Unsaturated; HEK293 Cells; Humans; Hydrazines; Inositol 1,4,5-Trisphosphate; Mutagenesis, Site-Directed; Receptors, Thromboxane A2, Prostaglandin H2; Sulfonamides

The present study examined the hypothesis that prostaglandin E2 (PGE2) through activation of prostaglandin E (EP) receptor contributes to endothelium-dependent contractions.. Western blotting revealed that the protein expression of EP1 receptor was significantly down-regulated in the aorta of the spontaneously hypertensive rat (SHR), but there was no significant difference in the expression of EP2, EP4, and total EP3 receptors between preparations of Wistar Kyoto rats (WKY) and SHR. Isometric tension studies showed that low concentrations of PGE2 caused endothelium-dependent relaxations in WKY but not in aortas of the SHR. High concentrations of PGE2 evoked contractions predominately through the activation of thromboxane-prostanoid (TP) receptors in the WKY, but involves the dual activation EP and TP receptors in the SHR. SQ29,548, BAYu3405 and Terutroban (TP receptor antagonists), and AH6809 (non-selective EP receptor antagonist) abolished, while SC19220 (preferential EP1 receptor antagonist) did not inhibit endothelium-dependent contractions. Both SC19220 and AH6809 significantly inhibited contractions to U46619 (TP receptor agonist).. The present study demonstrates that the contraction caused by PGE2 in the SHR aorta is dependent on the activation of EP1 and TP receptors, but that endothelium-dependent contractions do not require the former. Thus, PGE2 is unlikely to be an endothelium-derived contracting factor in this artery. The ability of AH6809 to inhibit endothelium-dependent contractions can be attributed to its partial antagonism at TP receptors. Nevertheless, the impairment of PGE2-mediated relaxation may contribute to endothelial dysfunction in the aorta of the SHR.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta, Thoracic; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Fatty Acids, Unsaturated; Hydrazines; Hypertension; Immunohistochemistry; Naphthalenes; Phenylephrine; Potassium Chloride; Propionates; Prostaglandin Antagonists; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Thromboxane; Sulfonamides; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Xanthones

We previously showed that ramatroban (Baynastrade mark), a thromboxane A(2) (TxA(2)) antagonist, had inhibited prostaglandin D(2) (PGD(2))-stimulated human eosinophil migration mediated through activation of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). However, detailed pharmacological characterization of its inhibitory activity has not been described. In the present study, we showed that [(3)H]ramatroban bound to a single receptor site on CRTH2 transfectants with a similar K(d) value (7.2 nM) to a TxA(2) receptor (8.7 nM). We also demonstrated that ramatroban inhibited PGD(2)-, 15-deoxy-Delta(12, 14)-PGJ(2) (15d-PGJ(2))- and indomethacin-induced calcium responses on CRTH2 transfectants in a competitive manner with similar pA(2) values (8.5, 8.5, and 8.6, respectively). This is the first report showing the evidence for direct binding of ramatroban to CRTH2, revealing its competitive inhibitory effects and another interesting finding that PGD(2), indomethacin and 15d-PGJ(2) share the same binding site with ramatroban on CRTH2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Binding, Competitive; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Carbazoles; Cell Line; Cell Movement; Cyclic AMP; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Humans; Hydrazines; Indomethacin; Models, Biological; Pentanoic Acids; Prostaglandin D2; Pyridines; Receptors, Immunologic; Receptors, Prostaglandin; Sulfonamides; Transfection; Tritium

PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells. In the present study, we investigated whether PGD2 has the ability to stimulate Th2 cytokine production in the absence of costimulation. At concentrations found at sites of allergic inflammation, PGD2 preferentially elicited the production of IL-4, IL-5, and IL-13 by human Th2 cells in a dose-dependent manner without affecting the level of the anti-inflammatory cytokine IL-10. Gene transcription peaked within 2 h, and protein release peaked approximately 8 h after stimulation. The effect of PGD2 was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD2 but not by the selective DP agonist BW245C, suggesting that the stimulation is mediated by CRTH2 and not DP. Ramatroban, a dual CRTH2/thromboxane-like prostanoid receptor antagonist, markedly inhibited Th2 cytokine production induced by PGD2, while the selective thromboxane-like prostanoid receptor antagonist SQ29548 was without effect. These data suggest that PGD2 preferentially up-regulates proinflammatory cytokine production in human Th2 cells through a CRTH2-dependent mechanism in the absence of any other costimulation and highlight the potential utility of CRTH2 antagonists in the treatment of allergic diseases.

Topics: Base Sequence; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cells, Cultured; Cytokines; DNA; Fatty Acids, Unsaturated; Humans; Hydantoins; Hydrazines; Inflammation; Inflammation Mediators; Interleukin-13; Interleukin-4; Interleukin-5; Prostaglandin D2; Receptors, Immunologic; Receptors, Prostaglandin; Sulfonamides; Th2 Cells; Up-Regulation

Thromboxane (TX) A(2), a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB(2), which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB(2), but not the TXA(2) analogue U46,619 or TXB(2), activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB(2) was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D(2) has been shown to be its principal ligand. 11-Dehydro-TXB(2) induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD(2) but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB(2) were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB(2)- and PGD(2)-induced shape change. 11-Dehydro-TXB(2) also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB(2) had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB(2) is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB(2)/CRTH2axis may be of therapeutic relevance.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Basophils; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Carbazoles; Cell Size; Chemotaxis, Leukocyte; Enzyme Inhibitors; Eosinophils; Fatty Acids, Unsaturated; Flow Cytometry; Humans; Hydrazines; Pertussis Toxin; Prostaglandin D2; Receptors, Immunologic; Receptors, Prostaglandin; Sulfonamides; Thromboxane B2; Transfection; Type C Phospholipases

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Fatty Acids, Unsaturated; Female; Humans; Hydrazines; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Muscle, Smooth, Vascular; Myometrium; Receptors, Thromboxane; Sulfonamides; Thromboxane A2; Umbilical Arteries

Human platelet membranes were used to characterize the receptor binding properties of the specific thromboxane receptor antagonist 3H-SQ 29548 and the displacement of 3H-SQ 29548 from its binding site by the new thromboxane receptor antagonist Bay u 3405. The specific binding of 3H-SQ 29548 was saturable with an association rate constant of 1 x 10(-11) mol-1 min-1 and a dissociation rate constant of 0.032 min-1. Nonspecific binding of 3H-SQ 29548 was below 10%. When Scatchard plot analysis was performed on equilibrium saturation binding the kD was 69 nmol/l and the Bmax was calculated as 3.9 pmol/mg membrane protein. 3H-SQ 29548 was dose dependently displaced from its binding site by addition of increasing concentrations of Bay u 3405 yielding an IC50 value of 68 +/- 12 nmol/l, being not significantly different from the IC50 of nonlabelled SQ 29548 (38 +/- 13 nmol/l). The results show that Bay u 3405 is a potent and specific thromboxane receptor antagonist, displacing 3H-SQ 29548 from its binding site on human platelet membranes with IC50 values being not significantly different. These receptor binding properties and the long biological half life reported in vivo make Bay u 3405 a promissing compound for the treatment of human cardiovascular diseases.

Topics: Binding, Competitive; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Fatty Acids, Unsaturated; Humans; Hydrazines; In Vitro Techniques; Kinetics; Membranes; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane A2; Thromboxanes