Eicosapentaenoic acid (EPA)-induced inhibitory effects on porcine coronary and cerebral arteries involve inhibition of prostanoid TP receptors.

This study was performed to elucidate whether eicosapentaenoic acid (EPA) suppresses spasm-prone blood vessel contractions induced by a thromboxane mimetic (U46619) and prostaglandin F2α (PGF2α) and determine whether the primary target of EPA is the prostanoid TP receptor. Accordingly, we assessed: (1) the tension changes in porcine basilar and coronary arteries, and (2) changes in the Fura-2 (an intracellular Ca2+ indicator) fluorescence intensity ratio at 510 nm elicited by 340/380 nm excitation (F340/380) in 293T cells expressing the human TP receptor (TP-293T cells) and those expressing the human prostanoid FP receptor (FP-293T cells). EPA inhibited both porcine basilar and coronary artery contractions induced by U46619 and PGF2α in a concentration-dependent manner, but it did not affect the contractions induced by 80 mM KCl. EPA also inhibited the increase in F340/380 induced by U46619 and PGF2α in TP-293T cells. In contrast, EPA showed only a marginal effect on the increase in F340/380 induced by PGF2α in FP-293T cells. These findings indicate that EPA strongly suppresses the porcine basilar and coronary artery contractions mediated by TP receptor and that inhibition of TP receptors partly underlies the EPA-induced inhibitory effects on these arterial contractions.

Pharmacological strategies for targeting platelet activation in asthma.

The activation of platelets during host defence and inflammatory disorders has become increasingly documented. Clinical studies of patients with asthma reveal heightened platelet activation and accumulation into lung tissue. Accompanying studies in animal models of allergic lung inflammation, using protocols of experimentally induced thrombocytopenia proclaim an important role for platelets during the leukocyte recruitment cascade, tissue integrity, and lung function. The functions of platelets during these inflammatory events are clearly distinct to platelet functions during haemostasis and clot formation, and have led to the concept that a dichotomy (or polytomy, depending on what else platelets do) in platelet activation exists. The platelet, therefore, presents us with novel opportunities for modulating these inflammatory responses. This review discusses the rationale and effectiveness of current anti-platelet drugs in their use to supress inflammation with regard to asthma, and the need to consider novel possibilities for pharmacological modulation of platelet function associated with inflammation that are pharmacologically distinct to current anti-platelet therapies.

Genistein Ameliorates Non-alcoholic Fatty Liver Disease by Targeting the Thromboxane A2 Pathway.

Non-alcoholic fatty liver disease (NAFLD) is now a public health issue worldwide, but no drug has yet received approval. Genistein, an isoflavonoid derived from soybean, ameliorates high-fat-diet-induced NAFLD in mice, but the molecular underpinnings remain largely elusive. Arachidonic acid (AA) is a major ingredient of animal fats, and the AA cascade has been implicated in chronic inflammation. In this study, we investigated whether genistein was against NAFLD by targeting the AA cascade. Using a mouse model, we showed that genistein supplementation improved high-fat-diet-induced NAFLD by normalizing hepatomegaly, liver steatosis, aminotransferase abnormalities, and glucose tolerance. The thromboxane A2 (TXA2) pathway was aberrantly active in NAFLD, evidenced by an elevation of circulating TXA2 and hepatic thromboxane A2 receptor expression. Mechanistically, we found that genistein directly targeted cyclooxygenase-1 activity as well as its downstream TXA2 biosynthesis, while the TXA2 pathway might mediate NAFLD progression by impairing insulin sensitivity. Taken together, our study revealed a crucial pathophysiological role of the TXA2 pathway in NAFLD and provided an explanation as to how genistein was against NAFLD progression.

Timosaponin AIII induces antiplatelet and antithrombotic activity via Gq-mediated signaling by the thromboxane A2 receptor.

The thromboxane (Tx) A2 pathway is a major contributor to the amplification of initial platelet activation and is therefore a key drug target. To identify potent small-molecule inhibitors of the thromboxane prostaglandin (TP) receptor, we screened a small steroidal saponin library using U46619-induced rat platelet aggregation assays. Timosaponin AIII (TAIII) was identified as a potent inhibitor of U46619-induced rat platelet aggregation and exhibited superior selectivity for the TP receptor versus other G protein-coupled receptors and a PKC activator. TAIII inhibited U46619-induced rat platelet aggregation independent of increases in cAMP and cGMP and the inhibition of TxA2 production. Both PKC and PLC activators restored TAIII-inhibited platelet aggregation, whereas TAIII did not inhibit platelet aggregation induced by co-activation of the G12/13 and Gz pathways. Furthermore, TAIII did not affect the platelet shape change or ROCK2 phosphorylation evoked by low-dose U46619. In vivo, TAIII prolonged tail bleeding time, reduced the mortality of animals with acute pulmonary thromboembolism and significantly reduced venous thrombus weight. Our study suggests that TAIII, by preferentially targeting Gq-mediated PLC/PKC signaling from the TP receptor, induces stronger in vitro antiplatelet activity and in vivo antithrombotic effects and may be an excellent candidate for the treatment of thrombotic disorders.

Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.

Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the ß-isoform of thromboxane A2 receptor (TPß) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPß and ß2-adrenergic receptor (ß2AR) in HEK 293 cells, but also with endogenous ß2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPß than ß2AR. Interestingly, Hsp90 coimmunoprecipitated with ß2AR but virtually not with TPß, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPß C-terminus is critical for the CCT7 interaction and plays an important role in TPß maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPß. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs).

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets.We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -'good' outcome no relapse > 3 years; 'poor' outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in 'good' outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression.We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2.

BACKGROUND: The aim of the present study was to observe the concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery and explore the mechanism by which NO synthase inhibition attenuates EDHF-mediated relaxation in endothelium-intact rat pulmonary artery. METHODS: Tension experiments were conducted on the pulmonary artery from male Wistar rats. RESULTS: TRPV4 channel agonist GSK1016790A (GSK) caused concentration-dependent relaxation (Emax 86.9±4.6%; pD2 8.7±0.24) of the endothelium-intact rat pulmonary artery. Combined presence of apamin and TRAM-34 significantly attenuated the relaxation (Emax 61.1±6.0%) to GSK. l-NAME (100µM) significantly attenuated (8.2±2.9%) the relaxation response to GSK that was resistant to apamin plus TRAM-34. However, presence of ICI192605 or furegrelate alongwith l-NAME revealed the GSK-mediated EDHF-response (Emax of 28.5±5.2%; Emax 24.5±4.3%) in this vessel, respectively. Further, these two TxA2 modulators (ICI/furegrelate) alongwith l-NAME had no effect on SNP-induced endothelium-independent relaxation in comparison to l-NAME alone. This EDHF-mediated relaxation was sensitive to inhibition by K(+) channel blockers apamin and TRAM-34 or 60mMK(+) depolarizing solution. Further, combined presence of apamin and TRAM-34 in U46619 pre-contracted pulmonary arterial rings significantly reduced the maximal relaxation (Emax 71.6±6.9%) elicited by GSK, but had no effect on the pD2 (8.1±0.03) of the TRPV4 channel agonist in comparison to controls (Emax, 92.4±4.3% and pD2, 8.3±0.06). CONCLUSION: The present study suggests that NO and EDHF are released concomitantly and NO synthase inhibition attenuates GSK-induced EDHF response through thromboxane pathway in the rat pulmonary artery.

Activation of the Thromboxane A2 Receptor by 8-Isoprostane Inhibits the Pro-Angiogenic Effect of Vascular Endothelial Growth Factor in Scleroderma.

The pathogenesis of scleroderma (SSc) includes components of autoimmunity, vascular dysfunction, and accumulation of extracellular matrix. 8-isoprostane, an oxidized lipid created by oxidative stress, activates the thromboxane A2 receptor (TXAR) and the Rho-associated kinase (ROCK) pathway. In this study, we determined whether the TXAR was activated by 8-isoprostane in SSc endothelial cells (ECs) and whether this pathway inhibited VEGF-induced angiogenesis. Elevated 8-isoprostane was observed in plasma and conditioned media from SSc patients. SSc-conditioned media inhibited EC tube formation, whereas addition of vitamin E, by reducing 8-isoprostane, increased tube formation. VEGF did not induce angiogenesis in SSc ECs, but vitamin E or TXAR inhibition restored its effect. The expression of TXAR, RhoA, and ROCK1/2 was elevated in SSc ECs. ROCK activity and 8-isoprostane-induced ROCK activation were significantly higher in SSc ECs, whereas VEGF had no effect. The hyper-activation of the TXAR leads to inhibition of VEGF-induced angiogenesis, as inhibition of the TXAR pathway results in a blockade of 8-isoprostane-induced ROCK activation and restoration of VEGF activity. These results suggest that the TXAR pathway has a crucial role in angiogenesis and that 8-isoprostane is not just a by-product of oxidative stress but also has a significant role in the impaired angiogenesis that characterizes SSc.

Prostaglandin I2 and prostaglandin E2 modulate human intrarenal artery contractility through prostaglandin E2-EP4, prostacyclin-IP, and thromboxane A2-TP receptors.

Cyclooxygenase inhibitors decrease renal blood flow in settings with decreased effective circulating volume. The present study examined the hypothesis that prostaglandins, prostaglandin E2 (PGE2) and prostacyclin (PGI2), induce relaxation of human intrarenal arteries through PGE2-EP and PGI2-IP receptors. Intrarenal arteries were microdissected from human nephrectomy samples (n=53, median diameter ≈362 µm, 88% viable, 76% relaxed in response to acetylcholine). Rings were suspended in myographs to record force development. In vessels with K(+)-induced tension (EC70: -log [mol/L]=1.36±0.03), PGE2 and PGI2 induced concentration-dependent relaxation (-log EC50: PGE2=7.1±0.3 and PGI2=7.7). The response to PGE2 displayed endothelium dependence and desensitization. Relaxation by PGE2 was mimicked by an EP4 receptor agonist (CAY10598, EC50=6.7±0.2). The relaxation after PGI2 was abolished by an IP receptor antagonist (BR5064, 10(-8) mol/L). Pretreatment of quiescent arteries with PGE2 for 5 minutes (10(-6) mol/L) led to a significant right shift of the concentration-response to norepinephrine (EC50 from 6.6±0.1-5.9±0.1). In intrarenal arteries with K(+)-induced tone, PGE2 and PGI2 at 10(-5) mol/L elicited increased tension. This was abolished by thromboxane receptor (TP) antagonist (S18886, 10(-6) mol/L). A TP agonist (U46619, n=6) evoked tension (EC50=8.1±0.2) that was inhibited by S18886. Polymerase chain reaction and immunoblotting showed EP4, IP, and TP receptors in intrarenal arteries. In conclusion, PGE2 and PGI2 may protect renal perfusion by activating cognate IP and EP4 receptors associated with smooth muscle cells and endothelium in human intrarenal arteries and contribute to increased renal vascular resistance at high pathological concentrations mediated by noncognate TP receptor.

Selective and potent inhibitory effect of docosahexaenoic acid (DHA) on U46619-induced contraction in rat aorta.

Inhibitory effects of docosahexaenoic acid (DHA) on blood vessel contractions induced by various constrictor stimulants were investigated in the rat thoracic aorta. The inhibitory effects of DHA were also compared with those of eicosapentaenoic acid (EPA) and linoleic acid (LA). DHA exhibited a strong inhibitory effect on the sustained contractions induced by U46619, a TXA(2) mimetic. This inhibitory effect of DHA was not affected by removal of the endothelium or by treatment with either indomethacin or N(ω)-nitro-l-arginine. DHA also significantly diminished PGF(2α)-induced contraction but did not show any appreciable inhibitory effects on the contractions to both phenylephrine (PE) and high-KCl. Similarly, EPA exhibited significant inhibitory effects against the contractions induced by both U46619 and PGF(2α) without substantially affecting either PE- or high-KCl-induced contractions. However, both DHA and EPA generated more potent inhibitions against contractions induced by U46619 than those by PGF(2α). In contrast, LA did not show significant inhibitory effects against any contractions, including those induced by U46619. The present findings suggest that DHA and EPA elicit more selective inhibition against blood vessel contractions that are mediated through stimulation of prostanoid receptors than those through α-adrenoceptor stimulation or membrane depolarization. Although DHA and EPA have similar inhibitory potencies against prostanoid receptor-mediated contractions, they had a more potent inhibition against TXA(2) receptor (TP receptor)-mediated contractions than against PGF(2α) receptor (FP receptor)-mediated responses. Selective inhibition by either DHA or EPA of prostanoid receptor-mediated blood vessel contractions may partly underlie the mechanisms by which these ω-3 polyunsaturated fatty acids exert their circulatory-protective effects.

Role of thromboxane A2-activated nonselective cation channels in hypoxic pulmonary vasoconstriction of rat.

Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K(+) channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K(+) channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A(2) (TXA(2)) is required for induction of HPV, we hypothesize that TXA(2) receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K(+) channel (K(v)). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (I(NSC,TXA2)) with weakly outward rectifying current-voltage relation. I(NSC,TXA2) was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of I(NSC,TXA2) activation and K(v) inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA(2)-activated NSC in rat PASMC.

Thromboxane and isoprostane share the same prostanoid receptors to increase human placental tone.

As the placenta is devoid of autonomic innervation, umbilical-placental vascular tone should be under the control of tissue and humoral factors. Among the numerous stimuli capable of challenging the placental circulation, we propose that prostanoids could be responsible for the regulation of placental vascular tone. Consequently, we measured vasomotor responses to the thromboxane A(2) (TXA(2)) mimetic U-46619 and the isoprostane 8-iso-prostaglandin E(2) (8-isoPGE(2)) in the human placental vasculature. Placental tissues were collected from normotensive women after elective caesarean delivery. Cotyledons were set up in a perfusion system, whereas chorionic arteries were prepared as rings and installed in glass-jacketed tissue baths. The effects of U-46619 and 8-isoPGE(2) were measured in the absence and presence of blockers of TXA(2) receptors (TP), SQ29,548 and ICI192,605, and of PGE(2) receptors (EP), AH6809. The influence of nitric oxide (NO) was assessed with NG-nitro-L-arginine methyl ester (L-NAME). U-46619 and 8-isoPGE(2) markedly increased perfusion pressure in cotyledons and tension in chorionic arteries. Dose-response curves to both prostanoids were competitively shifted to the right by all antagonists, but to different extents. L-NAME had no significant impact on the dose-response curves to U-46619. The effects of U-46619 and 8-isoPGE(2) were found to be mediated by both TP and EP. The presence of these receptors and the actions exerted by their agonists support our postulate that prostanoids play an important regulatory role in placental vascular tone and resistance. NO, however, does not seem to be involved.

Thromboxane A2 induces contraction of human prostate smooth muscle by Rho kinase- and calmodulin-dependent mechanisms.

Thromboxane A(2) (TXA(2)) induces contraction in different smooth muscle types via its receptor (TXA(2) receptor). However, any motoric role of TXA(2) in prostate smooth muscle tone has not been studied to date. Here, we investigated whether TXA(2) induces contraction of human prostate tissue. After ethical approval, prostate tissue was obtained from 47 patients undergoing radical prostatectomy. Effects of the TXA(2) analogue U46619 ((5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptonic acid) in isolated human prostate strips were studied in organ bath experiments with or without the Rho kinase inhibitor, Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), or the calmodulin antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide hydrochloride). Expression of TXA(2) synthase and TXA(2) receptors were examined by Western blot analysis and immunohistochemistry. Endogenous TXA(2) was quantified by enzyme immunoassay. U46619 induced concentration-dependent contractions of human prostate strips, with a maximum contraction at 3 µM. U46619-induced prostate contraction was significantly inhibited by Y27632 (30 µM) and by W7 (100 µM). TXA(2) synthase and TXA(2) receptors were detected by Western blot analysis. Immunohistochemical stainings showed that expression of TXA(2) synthase in prostate tissue was located to glandular cells, while prostate TXA(2) receptors were located to smooth muscle and glandular cells. The stable TXA(2) metabolite TXB(2) was detected by enzyme immunoassay in the prostate. TXA(2) induces contraction of isolated human prostate tissue by TXA(2) receptor activation. Prostate smooth muscle TXA(2) receptors are coupled to Rho kinase and Ca(2+)-dependent mechanisms. The distribution of TXA(2) synthase and TXA(2) receptors in the human prostate suggests TXA(2)-mediated paracrine epithelial-stromal interactions.

Involvement of prostaglandin F2α in preeclamptic human umbilical vein vasospasm: a role of prostaglandin F and thromboxane A2 receptors.

OBJECTIVE: Preeclampsia is characterized by hypertension and proteinuria developing after 20 weeks of gestation. Increased vasoconstriction can be one of the major underlying pathophysiological event in this syndrome. We examined the role of vasoconstrictor prostanoid, prostaglandin F2α (PGF2α) in preeclamptic and normotensive human umbilical veins. METHODS: Umbilical veins were set up in organ bath. The concentration-response curves of PGF2α (endogenous agonist of prostaglandin F receptor) and fluprostenol (prostaglandin F receptor selective agonist) were determined in normal and preeclamptic veins either in the absence or presence of BAY u3405 (thromboxane A2 receptor selective antagonist). PGF2α and its major metabolite concentrations were measured by enzyme immunoassay kit. The expression of vasoconstrictor prostanoid receptors was determined by western blot. RESULTS: The concentration-dependent contractions to PGF2α and fluprostenol were significantly increased in umbilical vein preparations derived from preeclamptic women compared with those of normotensives. Increased reactivity was related with enhanced sensitivity to these spasmogens in preeclamptic veins. BAY u3405 (10 µmol/l) did not modify the responsiveness to PGF2α in normal umbilical veins whereas moderately reduced the contractions in preeclamptic preparations. Serum concentrations of PGF2α and its major metabolite, 13,14-dihydro-15-keto-PGF2α, were comparable between preeclamptics and normotensives whereas the metabolite concentration was elevated in umbilical cord serum of preeclamptics. 13,14-dihydro-15-keto-PGF2α, release was also increased in umbilical vein preparations of preeclamptic women. An increased prostaglandin F receptor protein expression was determined whereas EP3 and thromboxane A2 protein expressions were unchanged in preeclamptic umbilical veins. CONCLUSION: Prostaglandin F and thromboxane A2 receptors activation by PGF2α could be involved in umbilical vasospasm observed in preeclampsia.

[Regulation of the expression of inducible nitric oxide synthase by prostanoids].

Circulatory failure in septic shock is due to vascular hyporesponsiveness, in which a massive amounts of nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. In response to various inflammatory stimuli, prostanoids are also derived from inducible isoform of cyclooxygenase-2 (COX-2). Several reports on the cross talk between NO and prostanoids have been published; vasodilator prostanoids such as prostacyclin (PGI(2)) and prostaglandin E(2) enhance iNOS expression in cultured cells. However, the details of the cross talk between prostanoids and the iNOS-NO system remains unknown. We examined inflammatory cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the thromboxane A(2) (TXA(2)) receptor (TP(-/-) mice). The cytokine-induced iNOS expression and NO production were significantly augmented in TP(-/-) VSMCs. Furthermore, U-46619, a TP agonist, inhibited the cytokine-induced iNOS expression and NO production. The cytokine-induced hyporesponsiveness of aortas to vasoconstrictor was significantly augmented in TP(-/-) aorta. Finally, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo in wild-type mice, however, this effect was not observed in TP(-/-) mice. These results suggest that TXA(2) has a protective role against the development of the vascular hyporesponsiveness via its inhibitory action on iNOS-NO system under pathological conditions such as sepsis. Thus, it seems that the cross-talk between PG and NO works to maintain the vascular homeostasis in the systemic inflammatory reactions such as sepsis.

Thromboxane A2 receptor-mediated epidermal growth factor receptor transactivation: involvement of PKC-delta and PKC-epsilon in the shedding of epidermal growth factor receptor ligands.

We examined thromboxane A(2) receptor (TP)-mediated transactivation of epidermal growth factor receptor (EGFR) through the shedding of EGFR ligands. A TP agonist U46619 caused the phosphorylation of EGFR in 1321N1 human astrocytoma cells, which was inhibited by an EGFR selective inhibitor AG1478 and by a disintegrin and metalloproteinase (ADAM) inhibitor TAPI-2, indicating TP stimulation caused the EGFR transactivation through the EGFR ligand shedding. Since 1321N1 cells expressed heparin-binding EGF (HB-EGF) mRNA, the mechanism of TP-mediated EGFR transactivation was examined in HEK293 cells expressing alkaline phosphatase-conjugated HB-EGF and TP. U46619 caused the shedding of HB-EGF in a time- and concentration-dependent manner. The TP-mediated shedding was inhibited by a furin inhibitor CMK, TAP-2, dominant-negative G alpha(q), a G(q/11) inhibitor YM254890, and also by a non-selective PKC inhibitor GF109203X and PKC down-regulation, but not by a conventional PKC inhibitor Gö6976. Furthermore, siRNAs of PKC-delta and PKC-epsilon inhibited U46619-induced HB-EGF shedding. Although BAPTA/AM had no effect on U46619-induced shedding of HB-EGF, EGTA inhibited it. These results suggest that TP-mediated EGFR transactivation is partially caused by shedding of HB-EGF, which involves furin and ADAM via novel types of PKCs (PKC-delta and PKC-epsilon) through G alpha(q/11) proteins in an extracellular Ca(2+)-dependent manner.

Intermolecular cross-talk between the prostaglandin E2 receptor (EP)3 of subtype and thromboxane A(2) receptor signalling in human erythroleukaemic cells.

BACKGROUND AND PURPOSE: In previous studies investigating cross-talk of signalling between prostaglandin (PG)E(2) receptor (EP) and the TPalpha and TPbeta isoforms of the human thromboxane (TX)A(2) receptor (TP), 17-phenyl trinor PGE(2)-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP(1) subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells. EXPERIMENTAL APPROACH: Specificity of 17-phenyl trinor PGE(2) signalling and its possible cross-talk with signalling by TPalpha/TPbeta receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)(3) generation and intracellular calcium ([Ca(2+)](i)) mobilization. KEY RESULTS: While 17-Phenyl trinor PGE(2) led to activation of phospholipase (PL)Cbeta to yield increases in IP(3) generation and [Ca(2+)](i), it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA(2) mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE(2) was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G(i)/G(betagamma)-dependent activation of PLCbeta. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE(2)-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP(3), rather than the EP(1), EP(2) or EP(4) receptor subtype in HEL cells. CONCLUSIONS AND IMPLICATIONS: The EP(1) and EP(3) subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP(1) receptors impaired or desensitized, while that of EP(3) receptors augmented signalling through TPalpha/TPbeta receptors, in a cell type-specific manner.

Activation of thromboxane A(2) receptors induces orphan nuclear receptor Nurr1 expression and stimulates cell proliferation in human lung cancer cells.

Previous studies implicate that activation of thromboxane A(2) receptor (TP) induced cell proliferation and transformation in several cell lines. We report here that the activation of TP by its agonist, [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 (I-BOP), induced Nurr1 expression and stimulated proliferation of human lung cancer cells. Nurr1, an orphan nuclear receptor in the nuclear receptor subfamily 4A subfamily, has been implicated in cell proliferation, differentiation and apoptosis. I-BOP markedly induced Nurr1 messenger RNA and protein levels as compared with other subfamily members, Nur77 and Nor-1. The signaling pathways of I-BOP-induced Nurr1 expression were examined by using various inhibitors of signaling molecules. The induction of Nurr1 expression by I-BOP appeared to be mediated through protein kinase A (PKA)/cAMP response element binding (CREB), protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways and not related to epidermal growth factor receptor and prostaglandin E(2) pathways. Transcriptional activation of Nurr1 gene by I-BOP was further investigated at the promoter level in H157 cells. 5'-Deletion analysis, site-directed mutagenesis and luciferase reporter assay demonstrated that Nurr1 expression was induced by I-BOP in a PKA/CREB-dependent manner. Further studies have revealed that Nurr1 may mediate cyclin D1 expression and I-BOP-induced cell proliferation in H157 cells since small interfering RNA of Nurr1 blocked I-BOP-induced cyclin D1 expression and cell proliferation and also decreased cell growth rate. These results provide strong evidence that Nurr1 plays a significant role in cell proliferation and may mediate TP agonist-induced proliferation in lung cancer cells.

The effect of long-term hypoxia on tension and intracellular calcium responses following stimulation of the thromboxane A(2) receptor in the left anterior descending coronary artery of fetal sheep.

OBJECTIVE: The purpose of this study was to investigate the mechanisms of tension and intracellular calcium regulation following stimulation with the thromboxane A(2) receptor agonist U46619 in the left anterior descending coronary artery of fetal sheep exposed to long-term hypoxia. We hypothesized that there would be a reduction in intracellular calcium responses in long-term hypoxic left anterior descending coronary artery accompanied by an increase in calcium sensitivity of the contractile mechanism. METHODS: Pregnant sheep were kept at altitude (3820 m) from day 30 of gestation until day 140. Fetal hearts from long-term hypoxic and from a control, normoxic group were obtained and the left anterior descending coronary artery of the fetus was dissected, cleaned, and mounted in a bath (Jasco) in which tension and intracellular calcium [Ca(2+)](i), using Fura-2, could be measured simultaneously following stimulation of the thromboxane A(2) receptor with U46619. The role of intracellular calcium and the Rho kinase and protein kinase C pathways in the tension responses were investigated by maintaining intracellular calcium constant or by using the Rho kinase blocker, Y27632, or the protein kinase C blocker, GF109203-X. RESULTS: There was no difference in the tension dose-response to U46619 between the normoxic fetal and hypoxic fetal left anterior descending, although [Ca(2+)](i) was lower in the hypoxic fetal than normoxic fetal at the highest doses. When [Ca(2+)]( i) was maintained constant at baseline levels, U46619 produced the same tension dose-response in both normoxic fetal and hypoxic fetal left anterior descending as when [Ca(2+)](i) was allowed to rise. The tension response was abolished in both groups when the Rho kinase inhibitor, Y27632, was given either during or before stimulation with U46619. The protein kinase C blocker, GF109203-X, had no effect on the tension response in either group. CONCLUSIONS: Long-term hypoxia did not alter the tension response to thromboxane A(2) receptor stimulation in fetal left anterior descending. The contractions in response to U46619 were produced apparently completely by changes in calcium sensitivity through the Rho kinase pathway.