TP Receptor Research Articles

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

AimsWe 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 methodsChanges 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 findingsU46619, 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). SignificanceProstanoids 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.

PDI Cleavage of Disulfide Bonds within the TP Receptor Inhibits Signaling through Gα 13

G-protein coupled receptors (GPCRs) are the most abundant superfamily of cell surface receptors. Approximately 35% of approved drugs target GPCRs and class A GPCRs account for ~85% of this superfamily. Class A GPCRs are characterized in part by two highly conserved disulfides in their extracellular domains that are thought to stabilize protein structure. Whether these disulfides can be enzymatically modified to influence G-protein signaling is not known. We and others have previously shown that disulfide bond modification by thiol isomerases such as protein disulfide isomerase (PDI) represent a previously unrecognized level of control of thrombus formation. We therefore evaluated the ability of recombinant PDI (rPDI) to modulate signaling through modification of the canonical disulfides within platelet GPCRs. Exposure of platelets to rPDI had no effect on stimulation through PAR1, PAR4, or the α 2A-adrenergic receptor. In contrast, rPDI exposure dramatically decreased platelet activation induced by the TP receptor agonists U46619 or arachidonic acid, implicating rPDI-mediated modulation of TP receptor signaling. Consistent with this finding, rPDI blocked U46619-mediated activation of α IIbβ 3, α-granule release, and dense granule release. Conversely, inhibition of endogenous PDI using either inhibitory antibodies or PDI-targeted small molecules enhanced TP receptor-mediated platelet aggregation and granule release, indicating that endogenous platelet PDI influences TP receptor signaling. Inhibition of TP receptor-mediated signaling required PDI active site cysteines since rPDI mutants lacking these cysteines lost inhibitory activity. Evaluation of the inhibitory activity of different PDI fragments showed that the PDI substrate binding domain is also critical for inhibitory activity. The ability to inhibit signaling through the TP receptor was specific for rPDI since incubation with other recombinant thiol isomerases including ERp57, ERp5, and ERp72 had no effect. To determine the specific modifications to TP receptor canonical disulfides induced by PDI, HEK cells were transfected with TP receptor and exposed to rPDI. TP receptor was subsequently immunopreciptated and disulfide-linked peptide analysis was performed using mass spectrometry. Compared to untreated controls, TP receptor exposed to rPDI demonstrated cleavage of Cys11-Cys102 and Cys105-Cys183 bonds and the generation of a new Cys102-Cys183 bond. To determine how modification of the disulfide bonding pattern affected signaling through the TP receptor, we evaluated signaling through specific Gα subunits in platelets. The platelet TP receptor signals through Gα q (which couples to phospholipase and increases calcium flux) and Gα 13 (which couples to RhoA and myosin light chain kinase). PDI-mediated cleavage of the platelet TP receptor resulted in biased signaling, with substantial inhibition of G α13-mediated RhoA-GTP activation and myosin light chain phosphorylation and little effect on Gα q-mediated calcium flux. These results show how PDI can modify platelet signaling and represent the first demonstration that a thiol isomerase can modulate the function of a GPCR via rearrangement of canonical disulfide bonds. DisclosuresNo relevant conflicts of interest to declare.

Comparative study on the effect of aspirin, TP receptor antagonist and TxA2 synthase inhibitor on the vascular tone of human saphenous vein and internal mammary artery

AimsThromboxane (TxA2) is synthesized from arachidonic acid (AA) via thromboxane synthase (TxS) enzyme and induces vasoconstriction via TP receptor. Our aim is to compare the effects of aspirin, TxS inhibitor and TP receptor antagonist on vascular reactivity of bypass grafts (saphenous vein and internal mammary artery). Main methodsUsing isolated organ bath, saphenous vein and internal mammary artery preparations were incubated with TP receptor antagonist, TxS inhibitor, aspirin, IP or EP4 receptor antagonist. Then prostaglandin (PG)E2, PGF2α, phenylephrine and AA were administered in concentration-dependent manner. The expression of prostanoid receptor and PGI2 synthase (PGIS) enzyme was determined by Western Blot. Key findingsTP receptor antagonist inhibited the contraction induced by PGE2, PGF2α, and AA but not that induced by phenylephrine in both types of vessels. Aspirin increased phenylephrine-induced contraction only in internal mammary artery and decreased AA-induced contraction in saphenous vein. TxS inhibitor decreased both PGE2 and AA-induced contraction in both types of vessels. This decrease was reversed by co-incubation of TxS inhibitor and IP/EP4 receptor antagonists. The expressions of EP3 receptor and PGIS enzyme were greater in internal mammary artery compared to saphenous vein while IP and TP receptors expressed at similar levels. SignificanceTP receptor antagonist and TxS inhibitor are more effective to reduce contraction induced by different spasmogens in comparison to aspirin. Our results suggest that TP receptor antagonist and TxS inhibitor might have an advantage over aspirin due to their preventive effect on increased vascular reactivity observed in post-operative period of coronary artery bypass grafting.

2-Methoxyestradiol Ameliorates Angiotensin II-Induced Hypertension by Inhibiting Cytosolic Phospholipase A2α Activity in Female Mice.

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6β-Hydroxytestosterone Promotes Angiotensin II-Induced Hypertension via Enhanced Cytosolic Phospholipase A2α Activity.

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Thromboxane-prostanoid receptor activation blocks ATP-sensitive potassium channels in rat aortas.

K+ channel activation is one of the major mechanisms involved in vasodilation. Vasoconstrictor agonists such as angiotensin II promote ATP-dependent potassium channels (KATP ) dysfunction. This study evaluates whether thromboxane-prostanoid (TP receptor) activation by the agonist U46619 increases reactive oxygen species (ROS) production in rat aortas, which could contribute to KATP channel dysfunction and impaired NO-dependent vasodilation. TP receptor activation with the selective agonist U46619 increased ROS in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but the TP receptor antagonist SQ29548 abolished this effect. ECs and VSMCs incubation with ROS scavengers like Tiron or PEG-Catalase impaired U46619-induced ROS production. U46619 at the concentrations of 0.1 and 1µmol/L induced contractions with similar amplitude. KATP channel activation with pinacidil-induced relaxation was lower for the contractions induced with 0.1 or 1µmol/L U46619 than with 10nmol/L U46619. Acetylcholine-induced relaxation provided similar results. In aortas pre-contracted with 10nmol/L U46619, neither Tiron (100µmol/L) nor catalase (300U/mL) affected pinacidil-induced relaxation. However, in aortas pre-contracted with 0.1µmol/L U46619, catalase potentiated pinacidil-induced relaxation. Pinacidil potentiated acetylcholine-induced relaxation in aortas pre-contracted with 0.1 and 1µmol/L U46619. Incubation with 10nmol/L U46619 increased NO concentration in ECs. Taken together, these results show that high concentrations of the TP receptor agonist U46619 impair KATP channels, which is probably due to ROS production. It is likely that hydrogen peroxide is the ROS.

Docosahexaenoic acid inhibits U46619- and prostaglandin F2α-induced pig coronary and basilar artery contractions by inhibiting prostanoid TP receptors

Docosahexaenoic acid (DHA, an n-3 polyunsaturated fatty acid) inhibits U46619 (a TP receptor agonist)- and prostaglandin F2α-induced contractions in rat aorta and mesenteric arteries. However, whether these effects could be replicated in vasospasm-prone vessels, such as coronary and cerebral arteries, remains unknown. Here, we evaluated the changes in pig coronary and basilar artery tensions and intracellular Ca2+ concentrations in human prostanoid TP or FP receptor-expressing cells. We aimed to clarify whether DHA inhibits U46619- and prostaglandin F2α-induced contractions in spasm-prone blood vessels and determine if the TP receptor is the primary target for DHA. In both pig coronary and basilar arteries, DHA suppressed U46619- and prostaglandin F2α-induced sustained contractions in a concentration-dependent manner, but did not affect contractions induced by 80 mM KCl. SQ 29,548 (a TP receptor antagonist) suppressed U46619- and prostaglandin F2α-induced contractions by approximately 100% and 60%, respectively. DHA suppressed both U46619- and prostaglandin F2α-induced increases in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, DHA did not affect prostaglandin F2α-induced increases in intracellular Ca2+ concentrations in human FP receptor-expressing cells. These findings suggest that DHA potently inhibits TP receptor-mediated contractions in pig coronary and basilar arteries, and the primary mechanism underlying its inhibitory effects on arterial contractions involves inhibiting TP receptors.

Molecular Pathomechanisms of Impaired Flow-Induced Constriction of Cerebral Arteries Following Traumatic Brain Injury: A Potential Impact on Cerebral Autoregulation.

(1) Background: Traumatic brain injury (TBI) frequently occurs worldwide, resulting in high morbidity and mortality. Here, we hypothesized that TBI impairs an autoregulatory mechanism, namely the flow-induced constriction of isolated rat middle cerebral arteries (MCAs). (2) Methods: TBI was induced in anaesthetized rats by weight drop model, and then MCAs were isolated and transferred into a pressure-flow chamber. The internal diameter was measured by a video-microscopy. (3) Results: In MCAs from intact rats, increases in flow and pressure + flow elicited constrictions (−26 ± 1.9 µm and −52 ± 2.8 µm, p < 0.05), which were significantly reduced after TBI or in the presence of thromboxane-prostanoid (TP receptor) antagonist SQ 29,548. Flow-induced constrictions were significantly reduced by HET0016, inhibitor of cytochrome P450 4A (CYP450 4A). Arachidonic acid, (AA, 10−7 M), and CYP-450 4A metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) elicited constrictions of intact MCA (−26 ± 2.3% and −31 ± 3.6%), which were significantly reduced after TBI (to 11 ± 1.3% and −16 ±2.5%). The TP receptor agonist U46619 (10−7 M) elicited substantial constrictions of MCA from intact rats (−21 ± 3.3%), which were also significantly reduced, after TBI (to −16 ± 2.4%). (4) Conclusions: Flow-induced constrictor response of MCA is impaired by traumatic brain injury, likely due to the reduced ability of cytochrome P450 4A to convert arachidonic acid to constrictor prostaglandins and the mitigated sensitivity of thromboxane-prostanoid receptors.

Chronic cyclooxygenase-2 inhibition prevents the worsening of hypertension and endothelial dysfunction induced by ouabain in resistance arteries of spontaneously hypertensive rats

AimPrevious studies raise cyclooxygenase (COX) activation as a possible mechanism involved in the pathophysiology of ouabain-induced hypertension. We hypothesized that inhibition of COX-2 activity might prevent ouabain-induced vascular dysfunction and worsening of hypertension in spontaneously hypertensive rats (SHR). MethodsSHR were exposed to ouabain or vehicle and treated or not with the selective COX-2 inhibitor nimesulide for 5 weeks. Systolic blood pressure was measured by plethysmography. Vascular reactivity by wire myograph and protein expression by Western-blot were assessed in mesenteric resistance arteries (MRA) of groups. Thromboxane A2 (TXA2) production by ELISA was evaluated in MRA supernatants of groups. ResultsNoradrenaline-induced maximal contraction (Emax) was greater in MRA from SHR receiving ouabain than those of vehicle group. In situ inhibition of COX-2, TXA2 synthase, or TP receptor reduced the Emax to noradrenaline in MRA of ouabain to vehicle levels. TXA2 production was higher in ouabain than in vehicle group. Ouabain enhanced expression of cytoplasmic tyrosine kinase Src (c-Src)/ERK1/2/COX-2/TXA2 synthase/TP receptor in SHR MRA, but did not change NFkB/iKB ratio. Anticontractile effect of nitric oxide (NO) was smaller in MRA from ouabain- than vehicle-treated SHR, as well as eNOS and nNOS expression. Nimesulide co-treatment prevented the ouabain-induced worsening of hypertension and noradrenaline MRA hypercontractility in SHR. ConclusionOuabain worsen hypertension and induce MRA hypercontractility in SHR associated with upregulated c-Src/ERK1/2/COX-2/TXA2 synthase/TXA2/TP receptor axis. These effects were prevented by COX-2 inhibition.

Signalling pathway of U46619-induced vascular smooth muscle contraction in mouse coronary artery.

Thromboxane A2 (TXA2 ) participates in many pathophysiological processes of coronary artery disease. However, its mechanism of TXA2 -induced contraction in the coronary artery remains to be clarified. A multi myograph system was used to measure the isometric tension of the mouse coronary arteries and identify the effect and pathway of TXA2 analogues U46619. Confocal laser scanning microscopy was used to measure the intracellular calcium concentration ([Ca2+ ]i ) in mouse coronary artery smooth muscle cells. Results from the experiment had shown that contraction in coronary artery was generated by U46619 in a concentration-dependent manner, which was completely abolished by a specific TXA2 receptor blocker, GR32191. PI-PLC inhibitors U73122 and D609 and Rho-Kinase inhibitor Y-27632 can block the U46619 elicited coronary artery contraction in a dose-dependent manner. Then, the vasoconstriction response to U46619 was obviously inhibited by two pan-PKC inhibitors chelerythrine or Gӧ6983, and a selective PKCδ inhibitor rottlerin, but was not blocked by a selective PKCζ inhibitor PKC-PS or a selective PKCβ inhibitor hispidin. Meanwhile, the PKC activator PDBu-induced vasoconstriction was significantly inhibited by 1μmol/L nifedipine, then mostly inhibited by 100μmol/L 2-APB and 10μmol/L Y27632. We further found that the response to U46619 was inhibited, respectively, by three calcium channel blockers nifedipine, SKF96356 or 2-APB in a concentration-dependent manner. Although Store-operated Ca2+ (SOC) channels generated the increase of [Ca2+ ]i in mouse coronary artery smooth muscle cells, SOC channels did not contribute to the vasoconstriction in mouse coronary arteries. Caffeine-induced sarcoplasmic reticulum (SR) Ca2+ release could obviously induce coronal vasoconstriction. In addition, NPPB, a cell membrane Ca2+ activated C1- channel blocker, could obviously inhibit the U46619-induced vasoconstriction. The U46619-induced mouse coronary artery contraction was involved in the increase in [Ca2+ ]i mediated by Cav1.2, TRPC channels and SR release through the activation of G-protein-coupled TP receptors and the kinases signalling pathway in TP downstream proteins, while SOC channels did not participate in the vasoconstriction.

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.

Larger endothelium-dependent contractions in iliac arteries of adult SHRs are attributed to differential downregulation of TP and EP3 receptors in the vessels of WKYs and SHRs during the transition from adolescence to adulthood

This study was to determine how endothelium-dependent contractions (EDCs) change in iliac arteries of Wistar-Kyoto (WKYs) and spontaneously hypertensive rats (SHRs) during the transition from adolescence to adulthood and the underlying mechanism(s). We also aimed to elucidate effects of L-798106, an EP3 receptor antagonist, on EDCs and the blood pressure increase in adolescent SHRs. Blood vessels were isolated for functional and biochemical analyses. EDCs were comparable in adolescent iliac arteries of both strains, and contractions to ACh, prostacyclin (PGI2), the EP3 receptor agonist sulprostone and the TP receptor agonist U46619 in adult vessels were less prominent compared with those in the adolescents, while the attenuation of vasoconstrictions to ACh, PGI2 or U46619 with age was to a lesser extent in SHRs. PGI2 production was decreased to a similar level in adult arteries. TP and EP3 expressions were downregulated in adult vessels, whereas the extent of TP downregulation was less in SHRs. L-798106 partially suppressed the vasoconstrictions to U46619 and attenuated EDCs to a greater extent than SQ29548, and administration of L-798106 blunted the blood pressure increase with age in prehypertensive SHRs. These results demonstrate the comparable EDCs in iliac arteries of the adolescents are decreased in the adults, but relatively larger EDCs in adult SHRs can be a reflection of differential downregulation of TP and EP3 receptors during the transition from adolescence to adulthood. Also, our data suggest that blockade of both TP and EP3 receptors starting from the prehypertensive stage suppresses EDCs and the development of hypertension in SHRs.

Expression of receptors in cow’s oviductal epithelial cells to prostaglandins E2, D2 and F2α

The significance of studying a condition of the reproductive system in productive animals, especially in dairy cattle, is directly proportional to the population’s needs in dairy, lactic acid, and other livestock products. The article presents the results of studies conducted at the Agricultural university of Hohhot city, China. The aim of the study was to identify the expression of receptors for commercial prostaglandins. The object of research was the biological material obtained from slaughter cattle, namely, the section of the cow’s oviducts. The subject of research was isolated epithelial cells. Expression was assessed by the level of intracellular Ca2+ and cAMP when exposed to PGE2, PGD2 and PGF2 of various concentrations from 10−5 to 10−9, also was using an agonist Butaprost. As a result, the concentration of PGE2 at 10−5 mol/L, PGF2α at 10−5 mol/L and 10−6 mol/L significantly contributed to an increase in the level of intracellular calcium (Ca2+) in the epithelial cells of the cow’s oviducts. The increase in the concentration of PGE2, PGD2, and Butaprost led to an increase in the level of cAMP concentration in epithelial cells of cow’s oviducts. EP1, EP2, EP4, FP, DP, TP receptors are available on cow’s oviductal epithelial cells. EP3 and IP receptors were not found on cow’s oviductal epithelial cells. Expression of the EP2, EP4, DP, FP receptor genes is well expressed, unlike EP1 and TP.

Mechanism of thromboxane receptor-induced vasoconstriction in human saphenous vein

Saphenous vein (SV) is one of the most widely used graft material in patients undergoing coronary artery bypass graft surgery (CABG). Thromboxane A2 (TXA2) is implicated in graft failure by inducing vasoconstriction and platelet aggregation. The aim of this study is to investigate the mechanism involved in TXA2-induced vasoconstriction in human SV. The role of different inhibitors and blockers on U46619 (TXA2-mimetic)-induced vasoconstriction is investigated by using an isolated organ bath system. Relaxation responses to several mediators are evaluated in SV pre-contracted with U46619 and compared with those pre-contracted with phenylephrine. Our results demonstrate that U46619-induced contraction is completely blocked by myosin light chain kinase inhibitor ML-9 or TP receptor antagonist BAY u3405. Furthermore, U46619-induced contraction is partially inhibited by phospholipase C inhibitor U73122, protein kinase C inhibitor calphostin C, Rho-kinase inhibitor Y-27632, L-type calcium channel blocker nifedipine, store-operated channel inhibitor SKF96365 or removal of extracellular calcium. Relaxation responses to NO donor (sodium nitroprusside), guanylate cyclase (GC) stimulator (riociguat), phosphodiesterase (PDE) inhibitors (sildenafil, IBMX), adenylate cyclase (AC) activator (forskolin) and acetylcholine (ACh) are markedly reduced when U46619 is used as a pre-contraction agent. Our results demonstrate that influx of extracellular Ca2+ (through L-type calcium channels and store-operated calcium channels) and intracellular Ca2+ release together with Ca2+ sensitization (through Rho-kinase activation) are necessary components for TXA2-induced vasoconstriction in SV. Moreover, more pronounced decrease in vasorelaxation induced by several mediators (SNP, riociguat, sildenafil, IBMX, forskolin, and ACh) in the presence of U46619 when compared with phenylephrine suggests that there is a crosstalk between the TP receptor signaling pathway and PDE, AC, GC enzymes. We believe that the investigation of mechanism of the TXA2-induced vasoconstriction in SV will provide additional information for the prevention of SV graft failure.

8-iso prostaglandin E2 causes nasal congestion in allergic rhinitis of mice

Abstract Allergic rhinitis (AR) is a complex nasal disease accompanied with mast cell and eosinophils infiltration into nasal cavity. Nasal congestion is a typical symptom of allergic rhinitis that is difficult to treat. Thromboxane A2 (TXA2)-TP receptor axis is reported to be involved in nasal congestion; however, its detailed mechanism is unknown. Here, we assessed the involvement of TP-mediated signaling in nasal congestion of mice. Intraperitoneal sensitization and intranasal administration of ovalbumin induced rhinitis with nasal congestion in mice. Pharmacological TP blockade but not TXA2 synthase inhibition abolished the nasal congestion accompanied with vascular hyper-permeability. These data suggest the presence of some other TP ligand than TXA2 in nasal cavity. LC-MS/MS based comprehensive analysis identified 15 types of lipid metabolites in nasal lavage fluid (NALF) of AR. The levels of almost all metabolites were higher in the NALF of AR than that in non-AR. Among these, we detected a possible TP ligand, 8-iso PGE2 in the AR NALF. Both in vitro and in vivo experiments showed that treatment of 8-iso PGE2 caused vascular hyper-permeability which was inhibited by pharmacological TP blockade. In addition, intranasal administration of 8-iso PGE2 induced nasal congestion which was significantly inhibited by TP blockade. We identified 8-iso PGE2 as a novel TP ligand which exacerbate nasal congestion in rhinitis mice.

Puerarin Inhibits Endothelium-Dependent Contractions in Mouse Carotid Arteries.

BackgroundMany bioactive ingredients of medicinal plants are known to produce vaso-protective benefits. Puerarin is one of the major isoflavone glucosides found in the root of kudzu vine and it exerts an anti-inflammatory effect and many other pharmacological actions. However, the mechanism underlying the vascular effect of puerarin is incompletely understood. Therefore, the present study aims to examine how puerarin reduces endothelium-dependent contractions (EDCs) in mouse arteries.Material/MethodsEDCs were evoked by acetylcholine (ACh) in isolated mouse carotid arteries with intact endothelium pretreated with Nω-NO2-L-Arg-OMe (L-NAME). The arteries were pretreated with puerarin and other pharmacological inhibitors before the addition of cumulative concentrations of ACh. The concentration of several prostaglandins (PGs) was measured by high performance liquid chromatography-coupled spectrometry (HPLC-MS).ResultsEDCs induced by ACh only presented in endothelium-intact arteries pretreated by L-NAME and EDCs were prevented by the treatment with cyclooxygenase (COX) inhibitor indomethacin (3 μmol/L) or thromboxane prostanoid receptor (TP receptor) antagonist S18886 (30 nmol/L). Acute 40-minute treatment with puerarin reduced EDCs in a concentration-dependent manner without affecting U46619-induced contraction. However, treatment with puerarin did not inhibit ACh-induced production of prostaglandins (PGs) in endothelium-intact arteries.ConclusionsThe present results show that puerarin is able to suppress EDCs in mouse carotid arteries, independent of inhibition of TP receptor or COX2-derived PGs.

Traumatic Brain Injury Impairs Flow‐Dependent Constrictions of Isolated Rat Middle Cerebral Arteries, Primarily by Interfering with the Flow Sensing Mechanisms

BackgroundTraumatic brain injury (TBI) is a major health problem worldwide due to its high mortality (35–40%), and morbidity (headache, brain edema, cognitive, behavioral, and communicative disabilities). Previously we have shown that mechanotransductions of two hemodynamic factors, pressure and flow are importantly involved in the autoregulation of cerebral blood flow, which may become inefficient after TBI. Indeed, previously we have shown that TBI diminishes pressure‐induced myogenic constriction of middle cerebral arteries (MCA). In the present study we hypothesized that TBI effects also flow‐induced constrictor response of MCA.MethodsTBI was induced in anaesthetized rat by Marmarou’s weight drop model, then MCA was isolated and transferred into pressure‐flow chamber. Inflow and outflow pressures were controlled and measured. The internal diameter was continuously measured by a videomicroscopy equipped with a microangiometer and recorded digitally by PowerLab system (AD Instruments). Data are mean±SEM.ResultsThe active (242,2±6,1 μm) and passive (277,9±8,8) diameters (p&lt;0.05) of isolated MCAs of control rats (n=39) indicate that a substantial myogenic tone developed in response to pressure (80 mmHg: Δ−35,7 ±2,7 μm, p&lt;0.05). Arachidonic acid (AA) ‐ after initial dilations (+6±3 and +9±2,5 μm) ‐ elicited dose dependent constrictions (−9±2,1 and −20±3,2 μm) of MCA of control rats. Step increases in flow elicited gradual constrictions of MCA of control rats (from max.: 229,3±6,6 to 191±6 μm, Δ−38,3 ±0,6 μm, p&lt;0.05), which was eliminated after TBI (from 227,3±14,1 to 227,4±13,3 μm). U46619, a thromboxane A2 (TXA2) / prostaglandin H2 (PGH2) receptor (TP receptor) agonist elicited similar constrictions of MCA of control and TBI rats (max.: Δ−66,1 ±0,7 μm vs. Δ−49 ±4 μm) rats, which were eliminated by SQ 29,548 a TP receptor antagonist. CYP‐450 metabolite 20‐HETE elicited similar constrictions of MCA in control (Δ−18 ±0,9 μm and TBI rats (Δ−15 ±0,1 μm) which were eliminated by HET0016, an inhibitor of CYP‐450. Paxilline (an inhibitor of potassium channel blocker) inhibited flow‐induced constrictions of MCA of control (max.: Δ−37,5 ±3 μm vs. max.: Δ−2,8 ±1 μm, p&lt;0.05), whereas did not affect the diameters of MCA after TBI (max.: Δ−0,1 ±1,6 μm vs. Δ−2,5 ±3 μm).ConclusionsThese findings suggest that 1) flow‐induced constrictor response of MCA depends on AA‐metabolites acting on TP receptors, 2) constrictor responses to TP agonists remain intact after TBI, 3) thus TBI interferes primarily with the flow sensing mechanisms of the vascular wall, which 4) likely leads to diminished autoregulation of cerebral blood flow.Support or Funding InformationScientific Excellence Program 2019., at the University of Physical Education, Innovation and Technology Ministry, Hungary TUDFO/51757/2019‐ITM and Higher Education Institutional Excellence Program at Semmelweis University and the National Bionika Program ED_17‐1‐2017‐0009, Hungary, and the University of Pecs within the framework of the 5th. thematic programme, National Research Development and Innovation Office NKFI‐FK123798.

Voltage Dependence of Prostanoid Receptors.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 to +60 mV in the presence of U46619, a stable analog of prostaglandin H2 Half-maximal effective potential (V0.5) determined for TP receptor was -46 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R2957.40, which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E2 receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R2957.40 in the agonist-binding pocket.

Low-dose Btk inhibitors selectively block platelet activation by CLEC-2.

Inhibitors of Bruton tyrosine kinase (Btk) have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC- 2 relative to GPVI signaling is explained by the positive feedback role involving Btk itself, as well as ADP- and thromboxane A2-mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in eight out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at low doses in patients to target CLEC-2 in thrombo-inflammatory disease.

Ramatroban as a Novel Immunotherapy for COVID-19.

SARS-CoV-2 virus suppresses host innate and adaptive immune responses, thereby allowing the virus to proliferate, and cause multiorgan failure, especially in the elderly. Respiratory viruses stimulate cyclooxygenase-2 (COX-2) to generate prostanoids including Prostaglandin D2 (PGD2) and thromboxane A2. Furthermore, PGD2 concentrations in the airways increase with aging. PGD2 action mediated via DP2 receptors suppresses both innate and adaptive immune responses, by inhibiting interferon-λ and stimulation of myeloid monocyte-derived suppressor cells respectively. PGD2 and thromboxane A2 actions via the TP receptors activate platelets leading to a prothrombotic state. Ramatroban, a small-molecule antagonist of DP2 and TP receptors, reverses viremia-associated proinflammatory, immunosuppressive5 and prothrombotic processes which are similar to those induced by SARS-Cov-2. Ramatroban, used for the treatment of allergic rhinitis in Japan for the past 20 years has an excellent safety profile. Therefore, Ramatroban merits investigation as a novel immunotherapy for the treatment of COVID-19 disease.