Prostanoid TP receptor stimulation enhances contractile activities in guinea pig urinary bladder smooth muscle through activation of Ca2+ entry channels: Potential targets in the treatment of urinary bladder contractile dysfunction.

AIMS: We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity. MAIN METHODS: Changes in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence. KEY FINDINGS: U46619, prostaglandin (PG) E2, PGF2α, and PGA2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca2+ channels (ROCCs)/store-operated Ca2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs). SIGNIFICANCE: Prostanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Thromboxane A2 receptor signaling in endothelial cells attenuates monocrotaline-induced liver injury.

Sinusoidal obstruction syndrome (SOS) is a major complication of chemotherapy and hematopoietic stem cell transplantation. The early stage of SOS is characterized by liver sinusoidal endothelial cell (LSEC) injury accompanied by platelet aggregation. Thromboxane A2 (TxA2) induces platelet aggregation through the thromboxane prostanoid (TP) receptor. In this study, we explored the role of TP signaling in a monocrotaline (MCT)-induced mouse model of SOS. Relative to wild-type (WT) mice, TP-deficient (TP-/-) mice exhibited more severe MCT-liver injury, as indicated by elevated levels of alanine aminotransferase (ALT) and coagulative necrosis. Extensive accumulation of platelets in the liver was observed in both WT and TP-/- mice. TP expression co-localized with CD31-positive LSECs. MCT treatment caused LSEC destruction, concomitant with elevated expression of matrix metalloproteinases (MMPs) and adhesion molecules in WT mice, and LSEC damage was further exacerbated in TP-/- mice. Viability of isolated LSECs was lower in cells from TP-/- mice, whereas mRNA levels of MMPs and adhesion molecules were higher; U46619, a TxA2 agonist, reduced these levels in WT mice. These data suggest that TP signaling has no effect on platelet accumulation during MCT-induced liver injury, but instead prevents injury by suppressing LSEC damage.

Thromboxane-Induced α-CGRP Release from Peripheral Neurons Is an Essential Positive Feedback Loop in Capsaicin-Induced Neurogenic Inflammation.

α-CGRP is synthesized by sensory nerves in the dermis and its release can cause vasodilation and local inflammation. Its vasorelaxant effects are based on the direct activation of smooth muscle and endothelial cells, as well as the activation of mast cells causing the release of vasoactive and proinflammatory mediators. Here, we show that in the capsaicin model for neurogenic inflammation, capsaicin-induced edema formation is mediated by α-CGRP and mast cells, but is absent in thromboxane receptor-deficient mice. Capsaicin treatment of mice induced a thromboxane synthesis, which was mediated by α-CGRP and mast cells. Fittingly, α-CGRP induced thromboxane synthesis in mast cells and the thromboxane receptor agonist I-BOP caused edema formation independently of mast cells, suggesting that mast cells are the source of thromboxane. Most importantly, I-BOP-induced edema formation was mediated by α-CGRP and I-BOP was able to stimulate through calcineurin the α-CGRP release from peripheral neurons. Likewise, the signaling pathway, including α-CGRP, thromboxane receptor, and mast cells, also mediated capsaicin-induced mechanical hypersensitivity, a common symptom of capsaicin treatment. Taken together, the thromboxane-induced α-CGRP release from neurons forms a positive feedback loop causing prolonged α-CGRP release and edema formation during capsaicin-induced neurogenic inflammation.

Anticonvulsant-like effect of thromboxane receptor agonist U-46619 against pentylenetetrazol-induced seizures.

Increasing evidence suggests that prostanoid receptors and their ligands may constitute valuable tools for development of new antiepileptic drugs. Thromboxane A2 (TXA2) is a major eicosanoid in cardiovascular homeostasis. TXA2 exerts its action through the specific G protein-coupled TXA2 receptor (TP). In addition to its crucial role in the cardiovascular system, TXA2 and TPs play a role in the brain. Nevertheless, previously identified roles have been limited to cell protection of neurotoxicity, and the role of TPs on seizure activity was not investigated. Here we evaluated the effect of potent and selective TP agonist U-46619 on seizures induced by pentylenetetrazol (PTZ). Adult C57BL/6 mice received increasing doses of U-46619 (0, 30, 100 or 300 µg/kg). After 30 min we measured the latencies to myoclonic and generalized seizures induced by PTZ (60 mg/kg). We found that U-46619 increased the latency to PTZ-induced myoclonic jerks and tonic-clonic seizures. Moreover, U-46619 increased the immunocontent of phosphorylated Ser657 at protein kinase C (PKC) alpha subunit, indicating PKC activation in the hippocampus and cerebral cortex. Levels of TPs were not altered by the agonist. Administration of a TP antagonist, SQ 29,548, did not alter seizures and did not blunt the anticonvulsant-like effect of the agonist. In summary, we showed that a potent and selective TP agonist, U-46619, increased seizure latency in mice. Activation of PKC signaling pathways may underlie the anticonvulsant-like effect. Further investigation is needed to understand the potential of TPs in seizure treatment.

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPß homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations.

Protective effect of prostacyclin against pre-cardiac edema caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin and a thromboxane receptor agonist in developing zebrafish.

The role of prostaglandin pathways has been suggested in some toxicological responses to dioxins. Cyclooxygenase type 2b (COX2b), thromboxane synthase, and the thromboxane receptor (TP) pathway have been implicated in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced pre-cardiac edema in developing zebrafish at 55 h post fertilization (hpf). Pre-cardiac edema refers to edema located in a small cavity between the heart and body wall of zebrafish eleutheroembryos. In the present study, we assessed the role of prostacyclin, which counteracts some biological effects of thromboxane, in TCDD-induced pre-cardiac edema. Pre-cardiac edema induced by TCDD exposure (0.5 and 1 ppb) beginning at 24 hpf was markedly inhibited by exposure to beraprost (5 and 10 µM), a prostacyclin receptor (IP) agonist, beginning at 33 hpf. The preventive effect of beraprost was reduced by exposure to CAY10441 (10 µM), an IP antagonist starting at 33 hpf. Knockdowns of the IP receptor (IP-KD) with two different morpholinos caused edema by themselves and enhanced pre-cardiac edema caused by the low concentration of TCDD (0.5 ppb). On the other hand, short exposure beginning at 48 hpf to U46619 (7.5-30 µM), a thromboxane receptor agonist caused pre-cardiac edema, which was inhibited by exposure beginning at 48 hpf to both ICI-192,605 (24 µM), a TP antagonist, and beraprost. Expression of prostacyclin synthase was increased from fertilization, plateaued by 48 hpf, and was maintained until at least 96 hpf. Overall, the results demonstrate a preventive effect of prostacyclin on TCDD-induced pre-cardiac edema in developing zebrafish.

Decreased bioavailability of nitric oxide in aorta from ovariectomized senescent mice. Role of cyclooxygenase.

This study investigates the effects of aging and/or ovariectomy on vascular reactivity to thromboxane A2 (TXA2) receptor stimulation with U46619, and the modulation by nitric oxide (NO) and cyclooxygenase (COX) in aorta from female senescence-accelerated mice (SAMP8) and from senescence resistant mice (SAMR1). Five-month-old female SAMR1 and SAMP8 were divided into three groups: sham-operated, ovariectomized and ovariectomized plus estradiol. Twenty-eight days after surgery, thoracic aortic rings were mounted for isometric recording of tension and concentration-response curves for U46619 (10(-10)-3 × 10(-7) M) were performed in the absence and in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and/or COX inhibitor indomethacin (10(-5)M). Vascular superoxide production was detected by dihydroethidium staining on sections of thoracic aorta. NO bioavailability in response to U46619 was suppressed by estrogen withdrawn in young and senescent mice and was restored by the administration of estradiol. In the presence of indomethacin, contractions to U46619 decreased in all groups indicating an aging- and estrogen-dependent modulation of contractile prostanoids. The simultaneous incubation of L-NAME and indomethacin did not change the maximal responses and sensitivities to TXA2 in any group in comparison with untreated aortic segments. The superoxide generation induced by TXA2 was greater in aorta from SAMP8 than in SAMR1. Moreover, in ovariectomized groups superoxide production was further increased and treatment with 17ß-estradiol reverted the effects of the ovariectomy. Inhibition of COX with indomethacin prevented the U46619-induced increase in superoxide formation. Our results indicate that NO bioavailability in response to TP receptor activation is both estrogen- and aging-dependent. TXA2 induced contractions are partially mediated by COX activation. Both aging and ovariectomy enhanced COX-dependent component of the TXA2-induced contraction. It is noteworthy that in the absence of estrogen, COX inhibition induces an increase of NO bioavailability. Therefore, in senescent female mice with an experimental menopause, TP-receptor stimulation is responsible for COX activation and enhanced superoxide generation, which may result in reduced NO bioavailability. These effects were reversed by estrogen administration.

Prostaglandin E2 inhibits mast cell-dependent bronchoconstriction in human small airways through the E prostanoid subtype 2 receptor.

BACKGROUND: Inhaled prostaglandin (PG) E2 might inhibit asthmatic responses, but the mechanisms involved remain undefined. OBJECTIVE: We sought to characterize the direct and indirect effects of PGE2 on human small airways with particular reference to the receptors mediating the responses. METHODS: Contraction and relaxation were studied in isolated human bronchi with an inner diameter of 1 mm or less. RESULTS: Low concentrations of PGE2 (0.01-1 µmol/L) relaxed the bronchi precontracted by histamine. The bronchodilator response was inhibited by the E prostanoid (EP) subtype 4 receptor antagonist ONO-AE3-208 but unaffected by the EP2 receptor antagonist PF-04418948. Higher concentrations of PGE2 (10-100 µmol/L) contracted the small airways. However, the TP receptor agonists U-46,619, PGF2α, and PGD2 were more potent than PGE2. Moreover, the bronchoconstrictor responses to PGE2 and all other tested prostanoids, including the EP1/EP3 receptor agonist 17-phenyl trinor PGE2 and the partial FP receptor agonist AL-8810, were uniformly abolished by the TP receptor antagonist SQ-29,548. In the presence of TP and EP4 antagonists, PGE2 inhibited the mast cell-mediated bronchoconstriction resulting from anti-IgE challenge. Measurement of the release of histamine and cysteinyl leukotrienes documented that this bronchoprotective action of PGE2 was mediated by the EP2 receptor, unrelated to bronchodilation, and increased with time of exposure. CONCLUSION: The pharmacology of PGE2 in isolated human small airways was different from its profile in animal models. This first demonstration of powerful EP2 receptor-mediated inhibition of IgE-dependent contractions in human airways introduces a new selective target for the treatment of asthma. This EP2 control of mast cell-mediated bronchoconstriction is presumably exaggerated in patients with aspirin-exacerbated respiratory disease.

Involvement of the TP receptor in TNF-α-induced endothelial tissue factor expression.

BACKGROUND: Thromboxane (TX) A2, prostaglandin endoperoxides and F2-isoprostanes exert their effects through a TX-prostanoid (TP) receptor, also expressed in endothelial cells. We investigated a role of the TP receptor in the endothelial expression of tissue factor (TF), a key trigger to thrombosis. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) exposed to the TP receptor agonist U46619 featured a concentration-dependent increase in TF surface exposure and procoagulant activity. HUVEC pre-incubation with the TP receptor antagonist S18886, followed by stimulation with either U46619 or tumor necrosis factor-α (TNF-α), attenuated TF surface exposure and activity compared with stimulated control. Aspirin or indomethacin, while inhibiting cyclooxygenase (COX)-1 and -2 activities, did not mimic this effect. Probing of underlying mechanisms by selective pharmacological and gene silencing experiments showed that S18886 reduced U46619- or TNF-α-induced TF expression inhibiting ROS production, NAD(P)H oxidase and PKC activation. In addition, S18886 also inhibited ERK activation in the presence of both U46619 and TNF-α alone, while inhibition of JNK activation only occurred in the presence of U46619. CONCLUSION: The endothelial TP receptor contributes to TF surface exposure and activity induced not only by known TP receptor agonists, but also by TNF-α. Such findings expand the therapeutic potential of TP receptor inhibition.

Obesity and heterozygous endothelial overexpression of prepro-endothelin-1 modulate responsiveness of mouse main and segmental renal arteries to vasoconstrictor agents.

AIMS: Levels of the endothelium-derived peptide endothelin-1 (ET-1) are elevated in obese humans, and ET-1 mediated vascular tone is increased. Renal arterial smooth muscle is highly responsive to ET-1. Whether or not endothelium-derived ET-1 affects contractions of the renal artery under normal conditions or in obesity is unknown. The present study was designed to investigate whether or not overexpression of endogenous ET-1 in the endothelium affects the responsiveness of the main and segmental renal arteries differently in obesity. MAIN METHODS: Mice with tie-1 promoter-driven endothelium-restricted heterozygous overexpression of preproendothelin-1 were used (TET(het)). Obesity was induced in TET(het) mice and wild-type (WT) littermates by feeding a high fat diet for 30 weeks; lean controls were kept on standard chow. The renal arteries were studied in wire myographs testing contractions (in the presence of l-NAME) to ET-1, serotonin, and U46619. KEY FINDINGS: Contractions to ET-1 were comparable between groups in main renal arteries, but augmented in segmental preparations from obese mice. Serotonin-induced responses were enhanced in obese TET(het) mice renal arteries compared to lean controls. Concentration-contraction curves to U46619 were shifted significantly to the left in main renal arteries of obese animals, and the maximal response was significantly increased between lean and obese TET(het) mice. SIGNIFICANCE: These results indicate an augmented responsiveness of main renal arteries in obesity particularly to TP receptor activation. When combined with endothelial ET-1 overexpression this effect is even more pronounced, which may help to gain further insights into the mechanisms of hypertension in obesity.

Thromboxane A2 exerts promoting effects on cell proliferation through mediating cyclooxygenase-2 signal in lung adenocarcinoma cells.

BACKGROUND: Lung cancer concerns a worldwide health problem and the efficacy of available treatments is unsatisfactory. Recently, thromboxane A2 (TXA2) synthase (TXAS) and receptor (TXA2R) have been documented to play a role in lung cancer development. Therefore, dual TXA2R modulator (i.e., the dual blocker of TXAS and TXA2R) may be more efficacious to kill lung tumor cells than single TXAS inhibitor or TXA2R antagonism. The close relationship between cyclooxygenase (COX)-2 and TXAS also raises whether or how TXA2 contributes to the oncogenic activity of COX-2. This study is therefore conducted to answer these questions. METHODS: Various inhibitors and siRNA were used to evaluate the roles of TXA2 and COX-2 in the proliferation and apoptosis of lung adenocarcinoma cells. Cell proliferation was detected using both MTS ELISA and BrdU labeling ELISA. Cell cycle distribution and apoptosis were examined by flow cytometric analysis. TXB2 level, reflecting the biosynthesis of TXA2, was detected by peroxidase-labeled TXB2 conjugates using an enzyme immunoassay kit. Western blotting was performed to evaluate many biomarkers for cell cycles, apoptosis and proliferation. The levels of COXs were screened by reverse transcriptase and real-time quantitative PCR. RESULTS: We found either single TXAS inhibitor/TXA2R antagonist or the dual TXA2 modulators offered a similar inhibition on cell proliferation. Moreover, inhibition of TXA2 arrested cells at the G2/M phase and induced apoptosis. It is further demonstrated that TXA2 was able to function as a critical mediator for tumor-promoting effects of COX-2 in lung adenocarcinoma cells. CONCLUSION: The present study has for the first shown that dual TXA2 modulators and the single blocker of TXAS or TXA2R offer a similar inhibitory role in lung adenocarcinoma cell proliferation and that the tumor-promoting effects of COX-2 can largely be relayed by TXA2. Thus, TXA2 should be regarded as a critical molecule in COX-2-mediated tumor growth and a valuable target against lung cancer.

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).

Acute effects of prostaglandin E1 and E2 on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane.

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.

Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery.

During pregnancy, reduced vascular responses to constrictors contribute to decreased uterine and total vascular resistance. Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor that exerts its actions via diverse signaling pathways, and its biosynthesis increases in preeclampsia. In this study, we hypothesized that maternal vascular responses to TxA(2) will be attenuated via Rho kinase, PKC, p38 MAPK, and ERK1/2 signaling pathways. Isolated ring segments of uterine and small mesenteric arteries from late pregnant (19-21 days) and virgin rats were suspended in a myograph, and isometric force was measured. Pregnancy did not affect uterine and mesenteric artery responses to the TxA(2) analog U-46619 (10(-9)-10(-5) M), but transduction signals associated with these contractions were different between pregnant and nonpregnant rats. Inhibition of Rho kinase (10(-6) M Y-27632) reduced sensitivity to U-46619 in virgin uterine vessels but did not inhibit these contractions in pregnant uterine arteries and had no effect on mesenteric vessels. Treatment of arterial segments with a PKC inhibitor (10(-6) M bisindolylmaleimide I) reduced U-46619-induced contractions in virgin uterine and mesenteric arteries and in pregnant mesenteric arteries. Pregnant uterine arteries, however, were unresponsive to PKC inhibition. Inhibition of ERK1/2 (10(-5) M PD-98059) and p38 MAPK (10(-5) M SB-203580) reduced U46619-induced contractions in nonpregnant vessels and in pregnant uterine and mesenteric vessels. These data suggest that normal pregnancy does not affect uterine and mesenteric contractile responses to TxA(2) but reduces the contribution of Rho kinase and PKC signaling pathways to these contractions in the uterine vasculature. In contrast, the role of ERK1/2 and p38 MAPK in U-46619-induced uterine contractions remains unchanged with pregnancy. TxA(2)-associated transduction signals and its regulators might present potential targets for the development of new treatments for preeclampsia and other pregnancy-associated vascular diseases.

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.

Improved bioavailability of epoxyeicosatrienoic acids reduces TP-receptor agonist-induced tension in human bronchi.

Epoxyeicosatrienoic acid (EET) and thromboxane A(2) are arachidonic acid derivatives. The former has initially been defined as an epithelium-derived hyperpolarizing factor displaying broncho-relaxing and anti-inflammatory properties, as recently demonstrated, whereas thromboxane A(2) induces vaso- and bronchoconstriction upon binding to thromboxane-prostanoid (TP)-receptor. EETs, however, are quickly degraded by the soluble epoxide hydrolase (sEH) into inactive diol compounds. The aim of this study was to investigate the effects of 14,15-EET on TP-receptor activation in human bronchi. Tension measurements performed on native bronchi from various species, acutely treated with increasing 14,15-EET concentrations, revealed specific and concentration-dependent relationships as well as a decrease in the tension induced by 30 nM U-46619, used as a synthetic TP-receptor agonist. Interestingly, acute treatments with 3 µM N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, an epoxygenase inhibitor, which minimizes endogenous production of EET, resulted in an increased reactivity to U-46619. Furthermore, we demonstrated that chronic treatments with trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), a sEH inhibitor, reduced human bronchi reactivity to U-46619. During our tension measurements, we also observed that human bronchi generated small-amplitude contractions; these spontaneous activities were reduced upon acute 14,15-EET treatments in the presence of t-AUCB. Altogether, these data demonstrate that endogenous and exogenous 14,15-EET could interfere with the activation of TP-receptors as well as with spontaneous oscillations in human airway smooth muscle tissues.

Thromboxane prostanoid receptor activation amplifies airway stretch-activated contractions assessed in perfused intact bovine bronchial segments.

A deep inspiration (DI) produces bronchodilation in healthy individuals. Conversely, in asthmatics, DIs are less effective in producing bronchodilation and can cause more rapid airway renarrowing and even bronchoconstriction in moderate to severe asthmatics. It is noteworthy that the manner by which a DI is able to cause bronchoconstriction via a stretch-activated contraction (R(stretch)) is thought to correlate positively with airway inflammation. Asthmatic airway inflammation is associated with increased production of thromboxane A(2) (TxA(2)) and subsequent thromboxane prostanoid (TP) receptor activation, causing the heightened contractility of airway smooth muscle. In this study, we sought to investigate the effect of TxA(2) on airway R(stretch) by using bovine bronchial segments. In brief, these intact bronchial segments (2 mm in diameter) were dissected, side branches were ligated, and the tissues were mounted horizontally in an organ bath. R(stretch) was elicited by varying the transmural pressure under isovolumic conditions. Using a pharmacological approach, we showed a reduced R(stretch) response in tissues pretreated with indomethacin, a cyclooxygenase inhibitor, a result mimicked by pretreatment with the TP-selective receptor antagonist 4-(Z)-6-(2-o-chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl)hexenoic acid (ICI 192605) and the selective p42/p44 mitogen-activated protein kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 95089) and by airway epithelial denudation. 9,11-Dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), a TP receptor agonist, elicited enhanced R(stretch) responses in a dose-dependent manner. Pretreatment with 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a prostaglandin E (EP) receptor 1/prostaglandin D2 (DP)-selective receptor antagonist, and 9α,15R-dihydroxy-11.ß-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta-5Z,13E-dien-1-oic acid (AL 8810), a prostaglandin F (FP)-selective receptor antagonist, had no effect, suggesting EP, DP, and FP receptor activation is not involved in amplifying airway smooth muscle R(stretch). These data suggest a role for TP receptor activation and epithelial release of TxA(2) in amplifying airway R(stretch), thus providing novel insights into mechanisms regulating the DI-induced bronchoconstriction seen in asthmatics.

Contractile effects of 15-E2t-isoprostane and 15-F2t-isoprostane on chicken embryo ductus arteriosus.

Isoprostanes (IsoPs) are prostaglandin (PG)-like compounds produced nonenzymatically by free radical-catalyzed peroxidation of arachidonate. Cyclooxygenase-derived PGs play a major role in ductus arteriosus (DA) homeostasis but the putative role of IsoPs has not been studied so far. We investigated, using wire myography, the vasoactive effects of 15-E(2t)-IsoP and 15-F(2t)-IsoP in the chicken embryo DA, pulmonary artery (PA) and femoral artery (FA). 15-E(2t)-IsoP and 15-F(2t)-IsoP contracted DA, PA, and FA rings in a concentration-dependent manner. 15-E(2t)-IsoP was equally efficacious (mean±SE E(max)=1.25±0.06 mN/mm) as and more potent (-log of molar concentration producing 50% of E(max)=pEC(50)=7.00±0.04) than the thromboxane-prostanoid (TP) receptor agonist U46619 (E(max)=1.49±0.11 mN/mm; pEC(50)=6.48±0.05) in contracting chicken DA (pulmonary side). 15-F(2t)-IsoP was less potent (pEC(50)=5.74±0.11) and less efficacious (E(max)=0.96±0.11) than U46619. Concentration-dependent contractions to 15-E(2t)-IsoP and U46619 in DA rings were competitively inhibited by the TP receptor antagonist SQ29548 (0.1 µM to 10 µM) with no decrease in the E(max) values. SQ29548 also inhibited concentration-dependent contraction to 15-F(2t)-IsoP but this inhibition was associated with a decrease in E(max). Pre-incubation of DA rings with 15-F(2t)-IsoP inhibited responses to U46619 and, in vessels contracted with U46619 (1 µM), 15-F(2t)-IsoP (>1 µM) evoked a relaxant response. Enzyme immunoassay did not show a measurable release of 15-F(2t)-IsoP by DA rings. In conclusion, 15-E(2t)-IsoP is a potent and efficacious constrictor of chicken DA, acting through TP receptors. In contrast, 15-F(2t)-IsoP is probably acting as a partial agonist at TP receptors. We speculate that IsoPs play a role in the control of chicken DA tone and could participate in its closure.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

BACKGROUND AND PURPOSE: Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH: We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS: Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS: TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone.

Release of sphingosine-1-phosphate from human platelets is dependent on thromboxane formation.

BACKGROUND: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. OBJECTIVES: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. METHODS: S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. RESULTS: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 µM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. CONCLUSIONS: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.