Isoprostanes evoke contraction of the murine and human detrusor muscle via activation of the thromboxane prostanoid TP receptor and Rho kinase.

Local or systemic inflammation can severely impair urinary bladder functions and contribute to the development of voiding disorders in millions of people worldwide. Isoprostanes are inflammatory lipid mediators that are upregulated in the blood and urine by oxidative stress and may potentially induce detrusor overactivity. The aim of the present study was to investigate the effects and signal transduction of isoprostanes in human and murine urinary bladders in order to provide potential pharmacological targets in detrusor overactivity. Contraction force was measured with a myograph in murine and human urinary bladder smooth muscle (UBSM) ex vivo. Isoprostane 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the murine UBSM, which was abolished in mice deficient in the thromboxane prostanoid (TP) receptor. The responses remained unaltered after removal of the mucosa or incubation with tetrodotoxin. Smooth muscle-specific deletion of Gα12/13 protein or inhibition of Rho kinase by Y-27632 decreased the contractions. In Gαq/11-knockout mice, responses were reduced and in the presence of Y-27632 abolished completely. In human UBSM, the TP agonist U-46619 evoked dose-dependent contractions. Neither atropine nor the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid decreased the effect, indicating that TP receptors directly mediate detrusor muscle contraction. 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the human UBSM, and these responses were abolished by the TP antagonist SQ-29548 and were decreased by Y-27632. Our results indicate that isoprostanes evoke contraction in murine and human urinary bladders, an effect mediated by the TP receptor. The G12/13-Rho-Rho kinase pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target in detrusor overactivity.NEW & NOTEWORTHY Voiding disorders affect millions of people worldwide. Inflammation can impair urinary bladder functions and contribute to the development of detrusor overactivity. The effects and signal transduction of inflammatory lipid mediator isoprostanes were studied in human and murine urinary bladders ex vivo. We found that isoprostanes evoke contraction, an effect mediated by thromboxane prostanoid receptors. The G12/13-Rho-Rho kinase signaling pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target.

Altered cyclooxygenase-1 and enhanced thromboxane receptor activities underlie attenuated endothelial dilatory capacity of omental arteries in obesity.

AIMS: Obesity is a risk factor for endothelial dysfunction, the severity of which is likely to vary depending on extent and impact of adiposity on the vasculature. This study investigates the roles of cyclooxygenase isoforms and thromboxane receptor activities in the differential endothelial dilatory capacities of arteries derived from omental and subcutaneous adipose tissues in obesity. MAIN METHODS: Small arteries were isolated from omental and subcutaneous adipose tissues obtained from consented morbidly obese patients (n = 65, BMI 45 ±â€¯6 kg m-2 [Mean ±â€¯SD]) undergoing bariatric surgery. Relaxation to acetylcholine was studied by wire myography in the absence or presence of indomethacin (10 µM, cyclooxygenase inhibitor), FR122047 (1 µM, cyclooxygenase-1 inhibitor), Celecoxib (4 µM, cyclooxygenase-2 inhibitor), Nω-Nitro-L-arginine methyl ester (L-NAME, 100 µM, nitric oxide synthase inhibitor) or combination of apamin (0.5 µM) and charybdotoxin (0.1 µM) that together inhibit endothelium-derived hyperpolarizing factor (EDHF). Contractions to U46619 (thromboxane A2 mimetic) were also studied. KEY FINDINGS: Acetylcholine relaxation was significantly attenuated in omental compared with subcutaneous arteries from same patients (p < 0.01). Indomethacin (p < 0.01) and FR122047 (p < 0.001) but not Celecoxib significantly improved the omental arteriolar relaxation. Cyclooxygenase-1 mRNA and U46619 contractions were both increased in omental compared with subcutaneous arteries (p < 0.05). L-NAME comparably inhibited acetylcholine relaxation in both arteries, while apamin+charybdotoxin were less effective in omental compared with subcutaneous arteries. SIGNIFICANCE: The results show that the depot-specific reduction in endothelial dilatory capacity of omental compared with subcutaneous arteries in obesity is in large part due to altered cyclooxygenase-1 and enhanced thromboxane receptor activities, which cause EDHF deficiency.

TP and/or EP3 receptors mediate the vasoconstrictor and pressor responses of prostaglandin F2α in mice and/or humans.

The vasoconstrictor and/or pressor effects of prostaglandin (PG)F2α participate in the development of vascular pathologies and limit the clinical use of the agent. This study aimed to determine the receptor types responsible for the vasoconstrictor activity of PGF2α and whether they mediate the pressor response evoked by the prostanoid under in vivo conditions. Experiments were performed on genetically altered mice and/or on vessels from these mice or humans. Here we show that deletion of the thromboxane-prostanoid receptor (TP-/-) abolished or drastically diminished the contraction to PGF2α in isolated mouse vessels (some of which were resistance arteries) and reduced the elevation in blood pressure evoked by the prostanoid under in vivo conditions. In accordance, TP antagonism abolished the contraction in small arteries of human omentum. Further deletion of E prostanoid receptor type 3 (EP3-/-) removed the PGF2α-evoked contraction that remained in some TP-/- arteries and added to the effect of TP-/- on the elevation in blood pressure evoked by the prostanoid under in vivo conditions. In contrast, the uterine contraction to PGF2α mediated via the F prostanoid receptor (FP) was unaltered in TP-/-/EP3-/- mice. These results demonstrate that the non-FP receptors TP and/or EP3 mediate the vasoconstrictor and pressor effects of PGF2α, which are still of concern under clinical conditions.-Liu, B., Li, J., Yan, H., Tian, D., Li, H., Zhang, Y., Guo, T., Wu, X., Luo, W., Zhou, Y. TP and/or EP3 receptors mediate the vasoconstrictor and pressor responses of prostaglandin F2α in mice and/or humans.

Molecular mechanisms of target recognition by lipid GPCRs: relevance for cancer.

Over the past decade the importance of lipids for cancer cell metabolism and cancer-related processes such as proliferation, metastasis and chemotherapy resistance has become more apparent. The mechanisms by which lipid signals are transduced are poorly understood, but frequently involve G-protein Coupled Receptors (GPCRs), which can be explored as druggable targets. Here, we discuss how GPCRs recognize four classes of cancer-relevant lipids (lysophospholipids, phospholipids, fatty acids and eicosanoids). We compare the ligand-binding properties of >50 lipid receptors, we examine how their dysregulation contributes to tumorigenesis and how they may be therapeutically exploited.

Receptors involved in the modulation of guinea pig urinary bladder motility by prostaglandin D2.

BACKGROUND AND PURPOSE: We have described a urothelium-dependent release of PGD2 -like activity which had inhibitory effects on the motility of guinea pig urinary bladder. Here, we have pharmacologically characterized the receptors involved and localized the sites of PGD2 formation and of its receptors. EXPERIMENTAL APPROACH: In the presence of selective DP and TP receptor antagonists alone or combined, PGD2 was applied to urothelium-denuded diclofenac-treated urinary bladder strips mounted in organ baths. Antibodies against PGD2 synthase and DP1 receptors were used with Western blots and for histochemistry. KEY RESULTS: PGD2 inhibited nerve stimulation -induced contractions in strips of guinea pig urinary bladder with estimated pIC50 of 7.55 ± 0.15 (n = 13), an effect blocked by the DP1 receptor antagonist BW-A868C. After blockade of DP1 receptors, PGD2 enhanced the contractions, an effect abolished by the TP receptor antagonist SQ-29548. Histochemistry revealed strong immunoreactivity for PGD synthase in the urothelium/suburothelium with strongest reaction in the suburothelium. Immunoreactive DP1 receptors were found in the smooth muscle of the bladder wall with a dominant localization to smooth muscle membranes. CONCLUSIONS AND IMPLICATIONS: In guinea pig urinary bladder, the main effect of PGD2 is an inhibitory action via DP1 receptors localized to the smooth muscle, but an excitatory effect via TP receptors can also be evoked. The urothelium with its suburothelium might signal to the smooth muscle which is rich in PGD2 receptors of the DP1 type. The results are important for our understanding of regulation of bladder motility.

Modulation of AD neuropathology and memory impairments by the isoprostane F2α is mediated by the thromboxane receptor.

Beside amyloid-ß plaques and neurofibrillary tangles, brain oxidative damage has been constantly implicated in Alzheimer's disease (AD) pathogenesis. Numerous studies demonstrated that F2-isoprostanes, markers of in vivo lipid peroxidation, are elevated in AD patients and mouse models of the disease. Previously, we showed that the 8-isoprostaneF2α, (8ISO) increases brain amyloid-ß levels and deposition in the Tg2576 mice. However, no data are available on its effects on behavior and tau metabolism. To this end, we characterize the behavioral, biochemical, and neuropathologic effects of 8ISO in the triple transgenic mouse model. Compared with controls, mice receiving 8ISO showed significant memory deficits, increase in tau phosphorylation, activation of the cyclin kinase 5 pathway, and neuroinflammation. All these effects were blocked by pharmacologic blockade of the thromboxane receptor. Our findings establish the novel functional role that oxidative stress via the formation of this isoprostane plays in the development of cognitive impairments and AD-related tau neuropathology. It provides important preclinical support to the neurobiological importance of the thromboxane receptor as an active player in the pathogenesis of AD.

Prenatal exposure to hyperoxia modifies the thromboxane prostanoid receptor-mediated response to H2O2 in the ductus arteriosus of the chicken embryo.

O2 tension plays a critical role in the control of prenatal patency and postnatal closure of the ductus arteriosus (DA). We hypothesized that exposure of chicken embryos to hyperoxia alters the morphology and function of DA. Hyperoxia was induced by incubating fertilized eggs at 60% O2 from day 15 to 19 of the 21-d incubation period. DA reactivity (assessed by wire myography), morphometry and mRNA expression of antioxidant enzymes were studied on day 19. Hyperoxic incubation neither affected embryonic growth nor induced signs of DA constriction or changed the mRNA expression of superoxide dismutase and catalase. The contractions induced by O2 (21%), KCl, 4-aminopyridine, phenylephrine, and endothelin-1 and the relaxations induced by acetylcholine (ACh), sodium nitroprusside, isoproterenol, and hydroxyfasudil were similar in DA from embryos incubated under normoxic or hyperoxic conditions. In contrast, hyperoxic incubation impaired the thromboxane prostanoid (TP) receptor-mediated contractions evoked by U46619, 15-E2t-Isoprostane and high concentrations (≥3 µM) of ACh. Exogenous hydrogen peroxide (H2O2) evoked endothelium-dependent contraction in the normoxic DA and endothelium-dependent relaxation in the hyperoxic group. The presence of the TP receptor antagonist SQ 29548 unmasked a relaxant response to H2O2 in the normoxic DA and the cyclooxygenase (COX) inhibitor indomethacin blocked H2O2-induced contraction (in the normoxic group) and relaxation (in the hyperoxic group). Altogether our functional data suggest that, in the chicken DA, exogenous H2O2 induces the release of endothelium-derived COX metabolite(s) with contractile and relaxant properties. Under normal conditions H2O2-induced contraction prevails and relaxation is unmasked after pharmacological or functional (i.e.hyperoxia) TP receptor impairment.

Enhanced uridine adenosine tetraphosphate-induced contraction in renal artery from type 2 diabetic Goto-Kakizaki rats due to activated cyclooxygenase/thromboxane receptor axis.

The dinucleotide uridine adenosine tetraphosphate (Up4A), which has both purine and pyrimidine moieties, was reported as a novel endothelium-derived contracting factor. Recently, growing evidence has suggested that Up4A plays an important role in regulation of the cardiovascular function. We previously demonstrated that Up4A-induced vasoconstrictions are altered in arteries from DOCA-salt hypertensive rats. We have assessed responses to Up4A shown by renal arteries from type 2 diabetic Goto-Kakizaki (GK) rats (42-46 weeks old) and identified the molecular mechanisms involved. Concentration-dependent contractions to Up4A were greater in renal arterial rings from the GK than age-matched control Wistar group. In both groups, the inhibition of nitric oxide synthase (with N (G)-nitro-L-arginine) increased the response to Up4A, whereas the inhibition of cyclooxygenase (COX) (with indomethacin) decreased the response. Specific inhibitors of COX-1 (valeroyl salicylate) and COX-2 (NS398), a thromboxane (TX) receptor (TP) antagonist (SQ29548), and P2 receptor antagonist (suramin) also decreased the response to Up4A. Protein expressions of COXs in renal arteries were greater in the GK than Wistar group. The production of TXB2 (a metabolite of TXA2) by Up4A did not differ between these groups. Concentration-dependent contractions to U46619, an agonist of the TP receptor, were greater in renal arteries from the GK than Wistar group. The expression of P2X1 and P2Y2 receptors did not differ between these groups. These results suggest that enhancement of the Up4A-induced contraction in renal arteries from GK rats may be attributable to the increased activation of COXs/TP receptor signaling.

Thromboxane-induced renal vasoconstriction is mediated by the ADP-ribosyl cyclase CD38 and superoxide anion.

The present renal hemodynamic study tested the hypothesis that CD38 and superoxide anion (O2(·-)) participate in the vasoconstriction produced by activation of thromboxane prostanoid (TP) receptors in the mouse kidney. CD38 is the major mammalian ADP-ribosyl cyclase contributing to vasomotor tone through the generation of cADP-ribose, a second messenger that activates ryanodine receptors to release Ca(2+) from the sarcoplasmic reticulum in vascular smooth muscle cells. We evaluated whether the stable thromboxane mimetic U-46619 causes less pronounced renal vasoconstriction in CD38-deficient mice and the involvement of O2(·-) in U-46619-induced renal vasoconstriction. Our results indicate that U-46619 activation of TP receptors causes renal vasoconstriction in part by activating cADP-ribose signaling in renal resistance arterioles. Based on maximal renal blood flow and renal vascular resistance responses to bolus injections of U-46619, CD38 contributes 30-40% of the TP receptor-induced vasoconstriction. We also found that the antioxidant SOD mimetic tempol attenuated the magnitude of vasoconstriction by U-46619 in both groups of mice, suggesting mediation by O2(·-). The degree of tempol blockage of U-46619-induced renal vasoconstriction was greater in wild-type mice, attenuating renal vasoconstriction by 40% compared with 30% in CD38-null mice. In other experiments, U-46619 rapidly stimulated O2(·-) production (dihydroethidium fluorescence) in isolated mouse afferent arterioles, an effect abolished by tempol. These observations provide the first in vivo demonstration of CD38 and O2(·-) involvement in the vasoconstrictor effects of TP receptor activation in the kidney and in vitro evidence for TP receptor stimulation of O2(·-) production by the afferent arteriole.

Antioxidants condition pleiotropic vascular responses to exogenous H(2)O(2): role of modulation of vascular TP receptors and the heme oxygenase system.

AIMS: Hydrogen peroxide (H(2)O(2)), a nonradical oxidant, is employed to ascertain the role of redox mechanisms in regulation of vascular tone. Where both dilation and constriction have been reported, we examined the hypothesis that the ability of H(2)O(2) to effect vasoconstriction or dilation is conditioned by redox mechanisms and may be modulated by antioxidants. RESULTS: Exogenous H(2)O(2) (0.1-10.0 µM), dose-dependently reduced the internal diameter of rat renal interlobular and 3rd-order mesenteric arteries (p<0.05). This response was obliterated in arteries pretreated with antioxidants, including tempol, pegylated superoxide dismutase (PEG-SOD), butylated hydroxytoluene (BHT), and biliverdin (BV). However, as opposed to tempol or PEG-SOD, BHT & BV, antioxidants targeting radicals downstream of H(2)O(2), also uncovered vasodilation. INNOVATIONS: Redox-dependent vasoconstriction to H(2)O(2) was blocked by inhibitors of cyclooxygenase (COX) (indomethacin-10 µM), thromboxane (TP) synthase (CGS13080-10 µM), and TP receptor antagonist (SQ29548-1 µM). However, H(2)O(2) did not increase vascular thromboxane B(2) release; instead, it sensitized the vasculature to a TP agonist, U46619, an effect reversed by PEG-SOD. Antioxidant-conditioned dilatory response to H(2)O(2) was accompanied by enhanced vascular heme oxygenase (HO)-dependent carbon monoxide generation and was abolished by HO inhibitors or by HO-1 & 2 antisense oligodeoxynucleotides treatment of SD rats. CONCLUSION: These results demonstrate that H(2)O(2) has antioxidant-modifiable pleiotropic vascular effects, where constriction and dilation are brought about in the same vascular segment. H(2)O(2)-induced oxidative stress increases vascular TP sensitivity and predisposes these arterial segments to constrictor prostanoids. Conversely, vasodilation is reliant upon HO-derived products whose synthesis is stimulated only in the presence of antioxidants targeting radicals downstream of H(2)O(2).

Prostanoid receptors involved in regulation of the beating rate of neonatal rat cardiomyocytes.

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F(2α) and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD(2) and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP(1) and EP(3) receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP(1) antagonist). Butaprost (a selective prostanoid EP(2) receptor agonist), misoprostol (a prostanoid EP(2) and EP(3) receptor agonist), 11-deoxy-PGE(1) (a prostanoid EP(2), EP(3) and EP(4) receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP(1) receptors are involved in positive regulation of the beating rate. Prostanoid EP(1) receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP(1) and EP(1) receptors (which positively regulate the spontaneous beating rate).

Thromboxane receptors antagonists and/or synthase inhibitors.

Atherothrombosis is the major cause of mortality and morbidity in Western countries. Several clinical conditions are characterized by increased incidence of cardiovascular events and enhanced thromboxane (TX)-dependent platelet activation. Enhanced TX generation may be explained by mechanisms relatively insensitive to aspirin. More potent drugs possibly overcoming aspirin efficacy may be desirable. Thromboxane synthase inhibitors (TXSI) and thromboxane receptor antagonists (TXRA) have the potential to prove more effective than aspirin due to their different mechanism of action along the pathway of TXA(2). TXSI prevent the conversion of PGH(2) to TXA(2), reducing TXA(2) synthesis mainly in platelets, whereas TXRA block the downstream consequences of TXA(2) receptors (TP) activation.TXA(2) is a potent inducer of platelet activation through its interaction with TP on platelets. TP are activated not only by TXA(2), but also by prostaglandin (PG) D(2), PGE(2), PGF(2α), PGH(2), PG endoperoxides (i.e., 20-HETE), and isoprostanes, all representing aspirin-insensitive mechanisms of TP activation. Moreover, TP are also expressed on several cell types such as macrophages or monocytes, and vascular endothelial cells, and exert antiatherosclerotic, antivasoconstrictive, and antithrombotic effects, depending on the cellular target.Thus, targeting TP receptor, a common downstream pathway for both platelet and extraplatelet TXA(2) as well as for endoperoxides and isoprostanes, may be a useful antiatherosclerotic and a more powerful antithrombotic intervention in clinical settings, such as diabetes mellitus, characterized by persistently enhanced thromboxane (TX)-dependent platelet activation through isoprostane formation and low-grade inflammation, leading to extraplatelet sources of TXA(2). Among TXRA, terutroban is an orally active drug in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. Despite great expectations on this drug supported by a large body of preclinical and clinical evidence and pathophysiological rationale, the PERFORM trial failed to demonstrate the superiority of terutroban over aspirin in secondary prevention of cerebrovascular and cardiovascular events among ~20,000 patients with stroke. However, the clinical setting and the design of the study in which the drug has been challenged may explain, at least in part, this unexpected finding.Drugs with dual action, such as dual TXS inhibitors/TP antagonist and dual COXIB/TP antagonists are currently in clinical development. The theoretical rationale for their benefit and the ongoing clinical studies are herein discussed.

Role of thromboxane receptor in C-reactive protein-induced thrombosis.

OBJECTIVE: Thromboxane A(2) and prostacyclin are thromboregulatory prostaglandins. The inflammatory C-reactive protein (CRP) promotes thrombosis after vascular injury, presumably via potentiation of thromboxane activity. Using a genetic approach, we investigated the role of thromboxane receptor (TP) pathway in CRP-induced thrombosis. METHODS AND RESULTS: Four genetically engineered mice strains were used: C57BL/6 wild-type, human CRP transgenic (CRPtg), thromboxane receptor-deficient (Tp(-/-)), and CRPtgTp(-/-) mice. CRP and TP expression were correlated, and suppression of CRP expression using small interfering RNA/CRP led to reduction in TP expression. Platelet-endothelial adherence was increased in CRPtg and suppressed in CRPtgTP(-/-)and CRPtg cells that were suppressed with TP small interfering RNA. TP deficiency in both platelets and endothelial cells was synergistic in affecting platelet-endothelial interactions. Time until arterial occlusion, measured after photochemical injury, was significantly shorter in CRPtg and prolonged in CRPtgTp(-/-) compared with controls (n=10-15, 35±3.4, 136±13.8, and 67±8.9 minutes, respectively; P<0.05). CONCLUSIONS: TP pathway is of major importance in CRP-induced thrombosis. The expression of TP is increased in CRPtg endothelial cells, and its blockade significantly suppresses the prothrombotic effect of CRP.

Prostaglandin D2 induces contractions through activation of TP receptors in peripheral lung tissue from the guinea pig.

Prostaglandin D(2) (PGD(2)), released through mast cell activation, is used as a non-invasive biomarker in patients with asthma. Since PGD(2) can elicit opposing effects on airway tone via activation of the PGD(2) receptors DP(1) and DP(2) as well as the thromboxane receptor TP, the aim of this study was to characterize the receptors that are activated by PGD(2) in the guinea pig lung parenchyma. PGD(2) and the thromboxane analog U46619 induced concentration-dependent contractions. U46619 was more potent and caused stronger effect than PGD(2). The specific TP receptor antagonist SQ-29548 and the combined TP and DP(2) receptor antagonist BAYu3405 concentration-dependently shifted the curves for both agonists to the right. The DP(1) receptor agonist BW245 induced a weak relaxation at high concentrations, whereas the DP(1) receptor antagonist BWA868C did not affect the PGD(2) induced contractions. The specific DP(2) receptor agonist 13,14-dihydro-15-keto-PGD(2) showed neither contractile nor relaxant effect in the parenchyma. Furthermore, studies in precision-cut lung slices specified that airways as well as pulmonary arteries and veins contracted to both PGD(2) and U46619. When the lung parenchyma from ovalbumin sensitized guinea pigs were exposed to ovalbumin, both thromboxane B(2) and PGD(2) were released. Ovalbumin also induced maximal contractions at similar level as PGD(2) in the parenchyma, which was partly reduced by SQ-29548. These data show that PGD(2) should be recognized as a TP receptor agonist in the peripheral lung inducing contraction on airways, arteries and veins. Therefore, a TP receptor antagonist can be useful in combination treatment of allergic responses in asthma.

TP receptor activation and inhibition in atherothrombosis: the paradigm of diabetes mellitus.

Patients with type 2 diabetes mellitus are characterized by increased incidence of cardiovascular events and enhanced thromboxane-dependent platelet activation. Urinary enzymatic TXA(2) metabolites (such as 11-dehydro-TXB(2)), reflecting the whole TXA(2) biosynthesis by platelet and extra-platelet sources, are significantly increased in diabetes with the absolute post-aspirin values of 11-dehydro-TXB(2) in diabetics being comparable to non-aspirated controls and such residual TXA(2) biosynthesis despite low-dose aspirin treatment is predictive of vascular events in high-risk patients. Thus, elevated urinary 11-dehydro-TXB(2) levels identify patients who are partially insensitive to aspirin and who may benefit from alternative antiplatelet therapies or treatments that more effectively block in vivo TXA(2) production or activity. Potential mechanisms relatively insensitive to aspirin include extraplatelet, nucleate sources of TXA(2) biosynthesis, possibly triggered by inflammatory stimuli, or lipid peroxidation with enhanced generation of F2-isoprostane (reflecting ongoing in vivo oxidative stress) than can activate platelets via the platelet TP receptor thus escaping inhibition by aspirin. In fact, aspirin does not inhibit isoprostane formation. Moreover, intraplatelet or extraplatelet thromboxane generation may be only partly inhibited by aspirin under certain pathological conditions, at least at the usual low doses given for cardiovascular protection. TXA(2) receptors (TP) are expressed on several cell types and exert antiatherosclerotic, antivasoconstrictive and antithrombotic effects, depending on the cellular target. Thus, targeting TP receptor, a common downstream pathway for both platelet and extraplatelet TXA(2) as well as for isoprostanes, may be an useful antithrombotic intervention in clinical settings, such as diabetes mellitus characterized by persistently enhanced thromboxane-dependent platelet activation.

Prostanoid receptors as possible targets for anti-allergic drugs: recent advances in prostanoids on allergy and immunology.

Prostanoids, consisting of prostaglandins and thromboxane, are cyclooxygenase metabolites of arachidonic acid released in various pathophysiological conditions which exert a range of actions mediated through their respective receptors expressed on target cells. Although it has been difficult to analyze the physiological role of prostanoids, recent developments in both the disruption of the respective gene and receptor selective compounds have enabled us to investigate the physiological roles for each receptor. It has been demonstrated that each prostanoid receptor has multiple functions, and that their expression is regulated in a context-dependent manner that sometimes results in opposite, excitatory and inhibitory, outcomes. The balance of prostanoid production and receptor expression has been revealed to be important for homeostasis of the human body. Here, we review new findings on the roles of prostanoids in allergic and immune diseases, focusing on contact dermatitis, atopic dermatitis, asthma, rheumatoid arthritis, and encephalomyelitis, and also discuss the clinical potentials of receptor-selective drugs.

Role of thromboxane TP and angiotensin AT1 receptors in lipopolysaccharide-induced arterial dysfunction in the rabbit: an in vivo study.

Inflammation plays a major role in pathological conditions leading to cardiovascular events. Administration of lipopolysaccharide to animals decreases arterial blood flow, in contrast to the dilatations that occur in microvessels. The purpose of the present study was to determine whether or not lipopolysaccharide, in vivo, evokes arterial constriction and if so the underlying mechanisms. Rabbits were anaesthetized, blood pressure monitored and femoral artery diameter continuously recorded with an echotracking device. Lipopolysaccharide induced leucopenia, thrombocytopenia, acidosis and a progressive hypotension with a decrease in femoral artery diameter (-30.7+/-2.4% after 3 h) and an increase in arterial rigidity. Three hours after lipopolysaccharide administration, the arterial dilatations to acetylcholine, arachidonic acid and iloprost were inhibited while that to sodium nitroprusside was not altered; the constrictions to norepinephrine, angiotensin II, U46619 (thromboxane analog) and serotonin were not modified. Under control conditions endothelin-1 produced an endothelin ET(B) dependent dilatation, reversed after lipopolysaccharide to an endothelin ETA dependent constriction. The thromboxane TP receptor antagonist S 18886 partially blocked the constriction; the angiotensin AT1 receptor antagonist candesartan prevented it. S 18886 normalized the impaired dilatations to acetylcholine, antagonists of 5-HT-receptors partially restored them while candesartan was ineffective. Antagonists of the endothelin or the histamine receptors had no effect. The present data show that lipopolysaccharide-induced inflammation causes 1) a strong constriction of the femoral artery in which activation of both thromboxane and angiotensin AT1 receptors is involved 2) a reduction of the endothelium-dependent dilatation to acetylcholine attributed to the activation of thromboxane TP receptors.

Endothelial dysfunction: a strategic target in the treatment of hypertension?

Endothelial dysfunction is a common feature of hypertension, and it results from the imbalanced release of endothelium-derived relaxing factors (EDRFs; in particular, nitric oxide) and endothelium-derived contracting factors (EDCFs; angiotensin II, endothelins, uridine adenosine tetraphosphate, and cyclooxygenase-derived EDCFs). Thus, drugs that increase EDRFs (using direct nitric oxide releasing compounds, tetrahydrobiopterin, or L-arginine supplementation) or decrease EDCF release or actions (using cyclooxygenase inhibitor or thromboxane A2/prostanoid receptor antagonists) would prevent the dysfunction. Many conventional antihypertensive drugs, including angiotensin-converting enzyme inhibitors, calcium channel blockers, and third-generation beta-blockers, possess the ability to reverse endothelial dysfunction. Their use is attractive, as they can address arterial blood pressure and vascular tone simultaneously. The severity of endothelial dysfunction correlates with the development of coronary artery disease and predicts future cardiovascular events. Thus, endothelial dysfunction needs to be considered as a strategic target in the treatment of hypertension.

RhoA-Rho kinase signaling mediates endothelium- and endoperoxide-dependent contractile activities characteristic of hypertensive vascular dysfunction.

Hypertensive vasomotor dysfunction is defined by endothelium-dependent contractions involving prostaglandins and ROS. Since both thromboxane-prostanoid receptor (TPr) signaling and ROS activate RhoA-Rho kinase (ROCK) in vascular smooth muscle (VSM) preparations, we hypothesized that enhanced endothelium-dependent contraction in the common carotid artery (CCA) of spontaneously hypertensive rats (SHRs) is ROCK mediated. ACh-stimulated contractions were approximately twofold greater in SHRs versus normotensive Wistar-Kyoto (WKY) rats, abolished by endothelial denudation or cyclooxygenase (COX)-1 inhibition, and nearly eliminated by TPr blockade. RhoA but not ROCK-II protein expression was increased ( approximately 50%) in the SHR CCA. Inhibition of ROCK, but not protein kinase C, caused a dose-dependent reduction in endothelium-dependent contractions to ACh across strains, with the highest dose mirroring the effect of high-dose TPr antagonism. Conversely, ROCK inhibition caused dose-dependent and endothelium- and nitric oxide-independent relaxation in CCAs precontracted with the TPr agonist U-46619. Prostacyclin was the predominant prostaglandin produced by ACh-stimulated CCAs, with greater than twofold more prostacyclin released from SHR versus WKY rats, and its production was unaffected by ROCK inhibition. RhoA activation was approximately twofold higher in quiescent SHR CCAs compared with those from WKY rats and was significantly increased by ACh stimulation. Augmentation of chemical superoxide quenching with tiron or inhibition of the NADPH oxidase-derived superoxide-producing pathway with apocynin reduced ACh-stimulated contractile activity in SHR more than in WKY rats, whereas the SOD mimetic tempol amplified the response. Exposure of CCAs to exogenous H(2)O(2) caused contractions, similar to ACh stimulation, that were greater in SHR than in WKY rats, abolished by COX-1 inhibition, and highly attenuated by TPr blockade or ROCK inhibition. These results indicate that RhoA-ROCK may act as a molecular switch, transducing signals from endothelium-derived prostaglandin(s) and ROS, which are overproduced in SHR CCAs, to "turn on" VSM contractile pathways, thus mediating the enhanced endothelium- and endoperoxide-dependent vascular contractions characteristic of hypertension, among other cardiovascular disease states, such as diabetes and aging.

Hypoxia-induced hyperreactivity of pulmonary arteries: role of cyclooxygenase-2, isoprostanes, and thromboxane receptors.

AIMS: This study investigates the role of the cyclooxygenase (COX)/prostanoid pathway in chronic hypoxia-induced hyperreactivity of pulmonary arteries. METHODS AND RESULTS: Pulmonary arteries were removed from normoxic or hypoxic (0.5 atm for 21 days) mice and studied for protein expression/localization of COX-1, COX-2, and thromboxane A2 (TXA2)-synthase, release of TXA2, prostacyclin (PGI2) and the isoprostane 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), and vasomotor responses. COX-2 expression was increased in all layers of pulmonary arteries from hypoxic mice. In contrast, COX-1 expression was not significantly modified following chronic hypoxia, whereas TXA2-synthase was decreased. Chronic hypoxia differentially affected prostanoid release from pulmonary arteries: TXA2 secretion was not significantly modified; PGI2 secretion was decreased, whereas 8-iso-PGF2alpha secretion was increased. A selective COX-2 inhibitor decreased 8-iso-PGF2alpha release. Arachidonic acid elicited an endothelium- and COX-1-dependent relaxation in pulmonary arteries from normoxic mice. In contrast, arachidonic acid induced an endothelium-independent contraction in pulmonary arteries from hypoxic mice that was partially reduced by catalase, COX-1, COX-2, or TXA2-synthase inhibitors and was totally abolished by blockade of the thromboxane (TP) receptor. Hyperresponsiveness to phenylephrine (PE) of pulmonary arteries from hypoxic mice was also decreased by COX-2 inhibitors, TP receptor antagonists or catalase, but not by TXA2-synthase inhibitors. Finally, 8-iso-PGF2alpha induced a TP receptor-dependent contraction in pulmonary arteries and markedly potentiated the contractile response to PE. CONCLUSION: Chronic hypoxia up-regulates COX-2 expression, increases 8-iso-PGF2alpha release, and shifts arachidonic acid-induced, endothelium-dependent relaxation to an endothelium-independent and TP receptor-dependent contraction in pulmonary arteries. COX-2-dependent production of 8-iso-PGF2alpha, by activating TP receptors, participates in hypoxia-induced hyperreactivity of pulmonary arteries.