Thromboxane A2 Receptor Research Articles

Chronic swimming training resulted in more relaxed coronary arterioles in male and enhanced vasoconstrictor ability in female rats.

Exercise training is associated with hypertrophy of left ventricle (LV). The aim of the present study is to evaluate sex differences in the adaptation of the coronary contractile function in physiological left ventricular hypertrophy induced by long-term swim training. Thirty-two Wistar rats were randomly divided into 4 groups: exercised male (ExM), exercised female (ExF), untrained control male (CoM), and untrained control female (CoF). The trained animals underwent a 12-week-long swim training program. After finishing the training program, LV morphology and function were checked by echocardiography. The spontaneous tone, thromboxane (TxA<inf>2</inf>) agonist-induced vascular contractility and non-endothelial dilatation of the isolated intramural coronary resistance artery were examined by pressure microangiometry. The thromboxane receptor (TxA<inf>2</inf>R) protein expression in the wall of coronary arteries was examined using immunohistochemistry. The LV mass index was significantly higher in the ExM and ExF groups, furthermore the LV mass index was significantly higher in female than in male animals. ExM animals had lower spontaneous tone than ExF. TxA<inf>2</inf> agonist-induced tone was raised only in ExF animals. The resistance coronary artery of exercised male animals had a significantly lower level of TxA<inf>2</inf>R positivity compared to exercised females. Both sexes broaden their range of contractility following chronic swimming, but the vessel tone shifted toward contraction in exercised female rats, while these values shifted toward relaxation in males. These observations underline the significance of identifying potential gender differences in the chronic exercise-induced coronary vascular remodeling in human athletes.

Microarray analysis of long non-coding RNA expression profiles in low high-density lipoprotein cholesterol disease

BackgroundLow high-density lipoprotein cholesterol (HDL-C) disease with unknown etiology has a high prevalence in the Xinjiang Kazak population. In this study, long noncoding RNAs (lncRNAs) that might play a role in low HDL-C disease were identified.MethodsPlasma samples from 10 eligible individuals with low HDL disease and 10 individuals with normal HDL-C levels were collected. The lncRNA profiles for 20 Xinjiang Kazak individuals were measured using microarray analysis.ResultsDifferentially expressed lncRNAs and mRNAs with fold-change values not less than 1.5 and FDR-adjusted P-values less than 0.05 were screened. Bioinformatic analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses, were used to determine relevant signaling pathways and predict potential target genes. In total, 381 lncRNAs and 370 mRNAs were differentially expressed based on microarray analysis. Compared with those in healthy individuals, several lncRNAs were upregulated or downregulated in patients with low HDL-C disease, among which TCONS_00006679 was most significantly upregulated and TCONS_00011823 was most significantly downregulated. GO and KEGG pathway analyses as well as co-expression networks of lncRNAs and mRNAs revealed that the platelet activation pathway and cardiovascular disease were associated with low HDL-C disease.ConclusionsPotential target genes integrin beta-3 (ITGB3) and thromboxane A2 receptor (TBXA2R) were regulated by the lncRNAs AP001033.3–201 and AC068234.2–202, respectively. Both genes were associated with cardiovascular disease and were involved in the platelet activation pathway. AP001033.3–201 and AC068234.2–202 were associated with low HDL-C disease and could play a role in platelet activation in cardiovascular disease. These results reveal the potential etiology of dyslipidemia in the Xinjiang Kazakh population and lay the foundation for further validation using large sample sizes.

MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity.

Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expression of the receptors in human megakaryopoiesis. Our study aimed to establish a flow cytometry (FC) method for the measurement of P2Y12, TXA2R, and CLEC-2 on platelets of healthy donors and to monitor receptor expression in ex vivo megakaryopoiesis. We determined mean fluorescence intensity (MFI) values of FITC, PE, or APC labeled antibodies binding to the receptors on platelets of 90 healthy donors. For cord blood-derived megakaryopoiesis (CBMK) differentiation of CD34+ cells was induced by IL-3, SCF, and TPO. At 6 time points between day 0 and day 21 of cell culture the MFI values for CD34, CD41, CD61, P2Y12, TXA2R, and CLEC-2 were measured. Quantitative PCR was used for relative quantification of the corresponding mRNA. Transcription factor (TF) binding sites were predicted by in silico analysis of the genes. Platelets showed expectable high MFI values for the platelet marker CD41 (13,716 median MFI). Lower MFI was found for P2Y12 (2,847 median MFI) and CLEC-2 (1,211 median MFI), whereas, binding of the TXA2R antibody revealed even higher values (21,458 median MFI) than CD41. In CBMK the CD34+ cells were negative for P2Y12, TXA2R, and CLEC-2 at day 0. A maximum of 21-fold and 6-fold increase of P2Y12 and TXA2R MFI values, respectively, was found on day 14 to 17. MFI for CLEC-2 increased by 58-fold within the first week and reached a maximum of 1,572-fold increase within the first two weeks of CBMK. Very similar results were obtained on the RNA level. The differential regulation of receptor expression in CBMK was further supported by significant differences in the numbers and types of TF binding sites. P2Y12 and TXA2R, both upregulated only to a low extent in CBMK, probably, are dispensable for megakaryopoiesis. Furthermore, we speculate that CLEC-2 strongly upregulated in early CMBK is important for megakaryopoiesis.

Thromboxane A2 receptor contributes to the activation of rat pancreatic stellate cells induced by 8-epi-prostaglandin F2α.

BackgroundPancreatic stellate cells (PSCs) activation plays a critical role in the development of chronic pancreatitis. Previous studies confirmed that thromboxane A2 receptor (TxA2r) was overexpressed in activated PSCs in rats. The purpose of this study was to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-prostaglandin F2α (8-epi-PGF2α).MethodsTxA2r expression in both quiescent and activated PSCs was detected by immunocytochemistry and immunoblot assay. Isolated PSCs were treated with 8-epi-PGF2α (10−6, 10−7, 10−8 mol/L) for 48 h, and SQ29548 (10−4, 10−6, and 10−7 mol/L), a TxA2r-specific antagonist for 48 h, respectively, to identify the drug concentration with the best biological effect and the least cytotoxicity. Then isolated PSCs were treated with SQ29548 (10−4 mol/L) for 2 h, followed by 10−7 mol/L 8-epi-PGF2α for 48 h. Real-time polymerase chain reaction was performed to detect the messenger RNA (mRNA) levels of α-smooth muscle actin (α-SMA) and collagen I. Comparisons between the groups were performed using Student's t test.ResultsTxA2r was up-regulated in activated PSCs in vitro compared with quiescent PSCs (all P < 0.001). Compared with the control group, different concentrations of 8-epi-PGF2α significantly increased mRNA levels of α-SMA (10−6 mol/L: 2.23 ± 0.18 vs. 1.00 ± 0.07, t = 10.70, P < 0.001; 10−7 mol/L: 2.91 ± 0.29 vs. 1.01 ± 0.08, t = 10.83, P < 0.001; 10−8 mol/L, 1.67 ± 0.07 vs. 1.00 ± 0.08, t = 11.40, P < 0.001) and collagen I (10−6 mol/L: 2.68 ± 0.09 vs. 1.00 ± 0.07, t = 24.94, P < 0.001; 10−7 mol/L: 2.12 ± 0.29 vs. 1.01 ± 0.12, t = 6.08, P < 0.001; 10−8 mol/L: 1.46 ± 0.15 vs. 1.00 ± 0.05, t = 4.93, P = 0.008). However, different concentrations of SQ29548 all significantly reduced the expression of collagen I (10−4 mol/L: 0.55 ± 0.07 vs. 1.00 ± 0.07, t = 10.47, P < 0.001; 10−6 mol/L: 0.56 ± 0.10 vs. 1.00 ± 0.07, t = 6.185, P < 0.001; 10−7 mol/L: 0.27 ± 0.04 vs. 1.00 ± 0.07, t = 15.41, P < 0.001) and α-SMA (10−4 mol/L: 0.06 ± 0.01 vs. 1.00 ± 0.11, t = 15.17, P < 0.001; 10−6 mol/L: 0.28 ± 0.03 vs. 1.00 ± 0.11, t = 11.29, P < 0.001; 10−7 mol/L: 0.14 ± 0.04 vs. 1.00 ± 0.11, t = 12.86, P < 0.001). After being treated with SQ29548 (10−4 mol/L) and then 8-epi-PGF2α (10−7 mol/L), the mRNA levels of α-SMA (0.20 ± 0.08 vs. 1.00 ± 0.00, t = 17.46, P < 0.001) and collagen I (0.69 ± 0.13 vs. 1.00 ± 0.00, t = 4.20, P = 0.014) in PSCs were significantly lower than those of the control group.ConclusionsThe results show that 8-epi-PGF2α promoted PSCs activation, while SQ29548 inhibited PSCs activation induced by 8-epi-PGF2α. The result indicated that TxA2r plays an important role during PSC activation and collagen synthesis induced by 8-epi-PGF2αin vitro. This receptor may provide a potential target for more effective antioxidant therapy for pancreatic fibrosis.

Pharmacological Activity, Pharmacokinetics, and Toxicity of Timosaponin AIII, a Natural Product Isolated From Anemarrhena asphodeloides Bunge: A Review.

Anemarrhena asphodeloides Bunge is a famous Chinese Materia Medica and has been used in traditional Chinese medicine for more than two thousand years. Steroidal saponins are important active components isolated from A. asphodeloides Bunge. Among which, the accumulation of numerous experimental studies involved in Timosaponin AIII (Timo AIII) draws our attention in the recent decades. In this review, we searched all the scientific literatures using the key word “timosaponin AIII” in the PubMed database update to March 2020. We comprehensively summarized the pharmacological activity, pharmacokinetics, and toxicity of Timo AIII. We found that Timo AIII presents multiple-pharmacological activities, such as anti-cancer, anti-neuronal disorders, anti-inflammation, anti-coagulant, and so on. And the anti-cancer effect of Timo AIII in various cancers, especially hepatocellular cancer and breast cancer, is supposed as its most potential activity. The anti-inflammatory activity of Timo AIII is also beneficial to many diseases. Moreover, VEGFR, X-linked inhibitor of apoptosis protein (XIAP), B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1), thromboxane (Tx) A2 receptor, mTOR, NF-κB, COX-2, MMPs, acetylcholinesterase (AChE), and so on are identified as the crucial pharmacological targets of Timo AIII. Furthermore, the hepatotoxicity of Timo AIII was most concerned, and the pharmacokinetics and toxicity of Timo AIII need further studies in diverse animal models. In conclusion, Timo AIII is potent as a compound or leading compound for further drug development while still needs in-depth studies.

1730-P: Thromboxane Signaling and Obesity-Related Insulin Resistance

Background: Thromboxane A2 (TxA2), a lipid mediator plays a critical role in cardiovascular disease and chronic inflammatory disorders. TxA2 signaling is regulated by TxA2 synthase (Tbxas1), an enzyme that produces TxA2, and TxA2 receptor (Tbxa2r). Objective: To determine if Tbxa2r gene expression in adipose tissue (AT) is higher in obese insulin resistant (IR) subjects compared to overweight non-IR subjects. Methods: Insulin resistance was measured via a homeostatic model assessment of insulin resistance (HOMA-IR). Subcutaneous AT biopsies were obtained from overweight (BMI between ≥25 to &amp;lt;30) no IR and obese (BMI ≥30) IR (HOMA-IR&amp;gt;2.5) subjects. Gene expression of Tbxa2r and Tbxas1 were analyzed in tissue samples and compared between groups. Results: The mRNA levels of Tbxa2r and Tbxas1 were significantly higher in obese IR subjects compared to overweight subjects (P&amp;lt;0.05). A positive correlation was observed between AT expression of Tbxa2r and Tbxas1 (Spearman’s r=0.49, n=35, P&amp;lt;0.01). We noted a weak positive correlation between AT Tbxa2r and HOMA-IR (Spearman’s r=0.31, n=35, P&amp;lt;0.07). A positive correlation was also noted between AT Tbxas1 and HOMA-IR (Spearman’s r=0.42, n=37, P&amp;lt;0.05). Conclusion: Our data suggest that TxA2 signaling is activated in AT in obesity and is associated with the development of insulin resistance in obesity. Tbxas1 and/or Tbxa2r may be a potential therapeutic target for obesity-linked metabolic diseases. Disclosure D. Alcaraz Alvarez: None. S. Viswanathan: None. T. Thangavelu: None. N. Kumar: None. K. Kelley: None. C. Desouza: Consultant; Self; AstraZeneca, Bayer AG, Novo Nordisk A/S.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar–Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

Antiplatelet properties of Pim kinase inhibition are mediated through disruption of thromboxane A2 receptor signaling.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumor development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 kinase is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired hemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function were non-additive to inhibition caused by the cyclo-oxygenase inhibitor indomethacin or the thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase-dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of cyclo-oxygenase-1 inhibition or direct antagonism of the thromboxane A2 receptor that, while attenuating thrombosis, does not increase bleeding.

Early cerebrovascular and long-term neurological modifications ensue following juvenile mild traumatic brain injury in male mice

Clinical evidence suggests that a mild traumatic brain injury occurring at a juvenile age (jmTBI) may be sufficient to elicit pathophysiological modifications. However, clinical reports are not adequately integrated with experimental studies examining brain changes occurring post-jmTBI. We monitored the cerebrovascular modifications and assessed the long-term behavioral and electrographic changes resulting from experimental jmTBI.In vivo photoacoustic imaging demonstrated a decrease of cerebrovascular oxygen saturation levels in the impacted area hours post-jmTBI. Three days post-jmTBI oxygenation returned to pre-jmTBI levels, stabilizing at 7 and 30 days after the injury. At the functional level, cortical arterioles displayed no NMDA vasodilation response, while vasoconstriction induced by thromboxane receptor agonist was enhanced at 1 day post-jmTBI. Arterioles showed abnormal NMDA vasodilation at 3 days post-jmTBI, returning to normality at 7 days post injury. Histology showed changes in vessel diameters from 1 to 30 days post-jmTBI. Neurological evaluation indicated signs of anxiety-like behavior up to 30 days post-jmTBI. EEG recordings performed at the cortical site of impact 30 days post-jmTBI did not indicate seizures activity, although it revealed a reduction of gamma waves as compared to age matched sham. Histology showed decrease of neuronal filament staining. In conclusion, experimental jmTBI triggers an early cerebrovascular hypo‑oxygenation in vivo and faulty vascular reactivity. The exact topographical coherence and the direct casualty between early cerebrovascular changes and the observed long-term neurological modifications remain to be investigated. A potential translational value for cerebro-vascular oxygen monitoring in jmTBI is discussed.

A sex-specific, COX-derived/thromboxane receptor activator causes depolarization and vasoconstriction in male mice mesenteric resistance arteries.

We investigated whether sex differences exist in cyclooxygenase-dependent effects on membrane potential and relaxation in mice mesenteric resistance arteries. Mesenteric small arteries of 9- to 12-week-old, male and female, wild-type mice, db/+ mice and diabetic db/db mice were mounted in myographs for measurements of isobaric diameter and smooth muscle cell membrane potential. Acetylcholine caused smaller dilation of arteries from male db/+ mice compared to arteries from female db/+ mice. In the presence of the NO synthase inhibitor Nω -nitro-L-arginine methyl ester (L-NAME), acetylcholine-induced dilation of arteries from males increased in the presence of indomethacin and the COX-1-specific inhibitor FR122047. The presence of indomethacin was also associated with a more negative membrane potential in arteries from males. In arteries from db/db mice, no sex differences were seen. In arteries from male but not female wild-type mice, the thromboxane receptor blocker SQ29,548 increased relaxation to acetylcholine. In contrast to arteries from female mice, COX (most likely COX-1)-derived prostanoids and activation of thromboxane receptors counteract acetylcholine vasodilatation probably through increased smooth muscle depolarization in arteries from male mice. In mice with diabetes and pronounced endothelial dysfunction, inhibition of COX did not enhance acetylcholine vasodilatation.

NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension

BackgroundNTP42 is a novel antagonist of the thromboxane prostanoid receptor (TP), currently in development for the treatment of pulmonary arterial hypertension (PAH). PAH is a devastating disease with multiple pathophysiological hallmarks including excessive pulmonary vasoconstriction, vascular remodelling, inflammation, fibrosis, in situ thrombosis and right ventricular hypertrophy. Signalling through the TP, thromboxane (TX) A2 is a potent vasoconstrictor and mediator of platelet aggregation. It is also a pro-mitogenic, pro-inflammatory and pro-fibrotic agent. Moreover, the TP also mediates the adverse actions of the isoprostane 8-iso-prostaglandin F2α, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat most of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, inflammation and fibrosis. This study aimed to investigate the efficacy of NTP42 in the monocrotaline (MCT)-induced PAH rat model, alongside current standard-of-care drugs.MethodsPAH was induced by subcutaneous injection of 60 mg/kg MCT in male Wistar–Kyoto rats. Animals were assigned into groups: 1. ‘No MCT’; 2. ‘MCT Only’; 3. MCT + NTP42 (0.25 mg/kg BID); 4. MCT + Sildenafil (50 mg/kg BID), and 5. MCT + Selexipag (1 mg/kg BID), where 28-day drug treatment was initiated within 24 h post-MCT.ResultsFrom haemodynamic assessments, NTP42 reduced the MCT-induced PAH, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP), being at least comparable to the standard-of-care drugs Sildenafil or Selexipag in bringing about these effects. Moreover, NTP42 was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals.ConclusionsThese findings suggest that NTP42 and antagonism of the TP signalling pathway have a relevant role in alleviating the pathophysiology of PAH, representing a novel therapeutic target with marked benefits over existing standard-of-care therapies.

Signal transduction pathways of detrusor smooth muscle contraction evoked by prostanoids and isoprostanes in murine urinary bladder

The overactive bladder (OAB) is a clinical condition characterized by symptoms of frequency, urgency with/without incontinence, with a prevalence of 16% in the developed countries. The current pharmacological treatment mainly consists of anticholinergic drugs with several side effects. According to prior experimental results the arachidonic acid (AA) derivate prostanoids and isoprotanes, the latter produced non‐enzymatically during oxidative stress, are not only biomarkers of systemic oxidative stress, but are excreting in the urine and might act directly on the detrusor muscle leading to detrusor overactivity.Our aim was to examine the effects and the signal transduction pathways of prostanoids and isoprotanes in the urinary bladder smooth muscle, and potentially provide theoretical basis for the development of more specific medication of OAB with less adverse effects.Detrusor muscle strips were prepared from wild type (C57BL/6) and knockout mice, deficient for the thromboxane receptor (TP) or the α‐subunits of heterotrimeric G proteins (Gαq/11‐KO, Gα12/13‐KO) without urothelium under dissection microscope. Contraction force was measured by myograph under isometric conditions and normalized to the reference contractions evoked by 124 mM K+.The prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), as well as the isoprostane 8‐epi‐PGE2 and 8‐iso‐PGF2α evoked contraction in the urinary bladder strips. The effect of the prostanoids was decreased, and the effect of the isoprostanes was abolished in the strips of TP KO mice, suggesting that the effect of the prostanoids is mediated partially, whereas that of the isoprostanes mainly by the TP. The TP receptor selective agonist U‐46619 evoked dose‐dependent contraction in the bladder strips, and neither the cholinerg antagonist atropine, nor the purinerg P2X‐antagonist pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS) decreased the responses, indicating that the TP agonist has a direct effect on the detrusor muscle. The responses evoked by U‐46619 were comparable to the effect evoked by cholinergic agonist, carbachol. The contraction responses were decreased in the strips of the Gα12/13‐KO mice. Correspondingly, the responses evoked by the prostanoids and isoprostanes were reduced by the Rho‐kinase (ROCK) inhibitor Y‐27632. In the strips of the Gαq/11‐KO mice, the responses were also decreased and in the presence of Y‐27632 abolished completely.In conclusion, the examined prostanoids and isoprostanes evokes contraction acting directly on the detrusor muscle. These responses are mediated mainly by the TP receptor and are linked to the Gαq/11 and to the Gα12/13‐Rho‐ROCK intracellular signaling pathways in the murine urinary bladder. The Gα12/13‐Rho‐ROCK signaling pathway may provide a novel, more specific pharmacological target with less adverse effects in the treatment of OAB.Support or Funding InformationK‐112964, K‐125174 and NVKP‐16‐1‐2016‐0042 from the Hungarian National Research, Development and Innovation Office and EFOP‐3.6.3‐VEKOP‐16‐2017‐00009.

Vasoreactivity of the Murine External Jugular Vein and Carotid Artery

Few studies have characterized vasoreactivity of the murine external jugular vein. This segment often is used in preclinical models to evaluate vascular remodeling in response to arteriovenous fistula (AVF) maturation. When an AVF is established, the venous segment is exposed to arterial pressures and disturbed flow patterns that have strong potential to impact vasoreactivity. Before testing this hypothesis in the context of AVF, we sought to establish procedures to comprehensively characterize endothelium‐dependent and vascular smooth muscle function of the carotid artery and external jugular vein from healthy mice. External jugular veins and ipsilateral common carotid arteries were isolated from five‐month‐old male C57BL/6 mice and vasomotor responses were evaluated using isometric tension procedures. External jugular veins developed tension (p&lt;0.05) to the voltage‐gated Ca2+ channel opener potassium chloride (KCl) and the thromboxane A2 receptor agonist U‐46619, but not to the α‐1 receptor agonist phenylephrine (PE), whereas common carotid arteries responded to all three vasocontractile agents in a more robust (p&lt;0.05) manner. While maximal responses (~70%) to the endothelium‐dependent vasodilator acetylcholine (ACh) were similar between the venous and arterial segments, the dose required to achieve this value was lower (p&lt;0.05) in the artery versus the vein. Nitric oxide synthase (NOS) inhibition using L‐NMMA attenuated (p&lt;0.05) but did not abolish ACh‐evoked vasorelaxation in both vascular segments. Endothelium‐independent vasorelaxation to sodium nitroprusside (SNP) was similar in the artery and the vein. Endothelium‐dependent and vascular smooth muscle function can be reliably assessed in the murine external jugular vein of healthy male C57Bl/6 mice. Compared to common carotid arteries, external jugular veins are resistant to PE‐evoked vasocontraction, less responsive to KCl and U‐46619‐evoked vasocontraction, less sensitive to ACh‐evoked vasorelaxation, and equally responsive to NOS inhibition and guanylate cyclase activation via SNP. Ongoing studies will determine if and how external jugular vein function is altered by arterial pressures and disturbed flow patterns that are encountered subsequent to establishing an AVF.Support or Funding InformationJMC (University of Utah Graduate Research Fellowship); YTS (R01DK100505; I01BX004133); JDS (AHA16GRNT31050004, NIH RO3AGO52848, RO1HL141540); TL (R44DK109789, R01HL139692, I01BX003387).

Activation of metabolite receptor GPR91 promotes platelet aggregation and transcellular biosynthesis of leukotriene C4

BackgroundSuccinate is a Krebs cycle intermediate whose formation is enhanced under metabolic stress, and for which a selective sensor GPR91 has been identified on various cell types including platelets. Platelet‐derived eicosanoids play pivotal roles in platelet activation/aggregation, which is key to thrombus formation and progression of atherothrombosis. ObjectivesThis study aims to decipher the molecular mechanism(s) and potential involvement of eicosanoids in succinate enhanced platelet activation/aggregation. MethodsWe used liquid chromatography‐mass spectrometry (LC‐MS)/MS‐based lipid mediator profiling to identify eicosanoids regulated by succinate. We ran light transmittance aggregometry and flow cytometry to assess platelet aggregation, P‐selectin expression, and platelet‐polymorphonuclear leukocyte (PMN) adherence. Various pharmacological tools were used to assess the contributions of GPR91 signalling and eicosanoids in platelet aggregation. ResultsSuccinate and two types of synthetic non‐metabolite GPR91 agonists—cis‐epoxysuccinate (cES) and Cmpd131—potentiated platelet aggregation, which was partially blocked by a selective GPR91 antagonist XT1. GPR91 activation increased production of 12‐hydroxy‐eicosatetraenoic acid (12‐HETE), thromboxane (TX) A2, and 12‐hydroxy‐heptadecatrienoic acid (12‐HHT) in human platelets, associated with phosphorylation of cytosolic phospholipase A2 (cPLA2), suggesting increased availability of free arachidonic acid. Blocking 12‐HETE and TXA2 synthesis, or antagonism of the TXA2 receptor, significantly reduced platelet aggregation enhanced by GPR91 signalling. Moreover, platelet‐PMN suspensions challenged with succinate exhibited enhanced transcellular biosynthesis of leukotriene C4 (LTC4), a powerful proinflammatory vascular spasmogen. ConclusionSuccinate signals through GPR91 to promote biosynthesis of eicosanoids, which contribute to platelet aggregation/activation and potentially vascular inflammation. Hence, GPR91 may be a suitable target for pharmacological intervention in atherothrombotic conditions.

Loss‐ And Gain of Function Mutations in the Type 1 IP3 Receptor (IP3R1) Modify Agonist‐Evoked Vasoactive Responses in Cerebral and Skeletal Muscle Resistance Arteries From Adult Mice

Calcium mobilization within vascular smooth muscle and endothelial cells of small resistance arteries plays a critical role in agonist‐evoked vasoconstriction and dilation, respectively. Despite the recognized contribution of IP3‐mediated intracellular Ca2+ release for agonist‐evoked vasoactive responses, it has been difficult to study this process acutely in native arteries, due to the lack of selective pharmacological agents or experimental strategies to target the predominant IP3R1. In humans, heterozygous mutations in the IP3R1 are known to produce either loss‐of‐function (LOF) or gain‐of‐function phenotypes for Ca2+ release that impact cerebellar activity (e.g. ataxia), yet it remains unclear how these global mutations affect the cardiovascular system. To facilitate such investigation, we have engineered novel genetic knock‐in mice harbouring a single amino acid mutation at position Asp2594 in the transmembrane S6 helix of the endogenous IP3R1 Ca2+ release channel that confers either a LOF (Asp2594Ala) or GOF (Asp2594Lys) phenotype, respectively. In permeabilized HEK293 cells expressing these mutant receptors, the LOF mutation shifted IP3‐mediated ER Ca2+ release ~2‐fold to the right, whereas the GOF mutation produced a 2.3‐fold leftward shift compared with wild‐type (WT) IP3R1. In endothelium‐denuded cerebral arteries pressurized to 10 mmHg, the sensitivity of serotonin‐evoked vasoconstriction was reduced in vessels from heterozygous LOF mice compared with WT, whereas agonist responsiveness was enhanced in arteries from heterozygous GOF mice. Vasoconstriction in response to the thromboxane receptor agonist U46619 was modestly enhanced by the GOF mutation, but largely unaffected in arteries from LOF mice. In cannulated cremaster skeletal muscle arteries from LOF and GOF mice pressurized to 70 mmHg, the sensitivity of endothelium‐dependent, acetylcholine‐mediated inhibition of myogenic tone was right shifted (3.7‐fold) and left shifted (2‐fold), respectively, compared with vessels from WT. Comparable changes in functional sensitivity were observed in response to the endothelium‐dependent vasodilator bradykinin. In contrast, dilation evoked by the NO donor sodium nitroprusside was not different among the three genotypes. Collectively, these data demonstrate that genetic modification of IP3R1‐mediated Ca2+ mobilization in small resistance arteries functionally impacts agonist‐evoked vasoactive responses. These observations highlight the importance of IP3R1 activity in the vascular wall, and further implicate vascular impairment as a contributing factor to the neurological dysfunction observed in patients with LOF and GOF IP3R1 mutations.Support or Funding InformationThis work was funded by the following research grants from the Canadian Institutes of Health Research: MOP‐97988 (WCC), MOP‐142467 (APB) and PJT‐155963 (WC, WCC and APB).

Refined pharmacophore features for virtual screening of human thromboxane A2 receptor antagonists

For a long time, the structural basis of TXA2 receptor is limited due to the lack of crystal structure information, till the release of the crystal structure of TXA2 receptor, which deepens our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor. In this research, we report the successful implementation in the discovery of an optimal pharmacophore model of human TXA2 receptor antagonists through virtual screening. Structure-based pharmacophore models were generated based on two crystal structures of human TXA2 receptor (PDB entry 6IIU and 6IIV). Docking simulation revealed interaction modes of the virtual screening hits against TXA2 receptor, which was validated through molecular dynamics simulation and binding free energy calculation. ADMET properties were also analyzed to evaluate the toxicity and physio-chemical characteristics of the hits. The research would provide valuable insight into the binding mechanisms of TXA2 receptor antagonists and thus be helpful for designing novel antagonists.

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.

Neuroprotective effects of thromboxane receptor antagonist SQ 29,548 after pilocarpine-induced status epilepticus in mice

Thromboxane A2 (TXA2) is an important eicosanoid in the cardiovascular system, and increasing evidence suggests that TXA2 receptors (TPs) and their ligands may constitute valuable tools for the development of neuroprotective drugs. However, the role of TPs on seizure-induced damage has not been investigated. Therefore, we evaluated the effects of SQ 29,548, a potent and selective TP antagonist—on neuromotor performance, neurodegeneration, reactive astrocytosis, and c-Fos protein immunoreactivity after pilocarpine-induced status epilepticus (SE) in mice. Adult C57BL/6 mice received intracerebroventricular SQ 29,548 injections 90 min and 24 h after pilocarpine-induced SE. We found that SQ 29,548 prevented the impairment of neuromotor performance (Neuroscore test) 48 h after pilocarpine-induced SE. Data analysis suggested the existence of two subgroups of SQ 29,548-treated post-SE animals. Eight out of 12 SQ 29,548-treated animals displayed Neuroscore values identical to those of vehicle-treated controls, and were considered SQ 29,548 responders. However, 4 out of 12 SQ 29,548-treated animals did not show any improvement in Neuroscore values, and were considered SQ 29,548 non-responders. Treatment with SQ 29,548 attenuated SE-induced increase in the number of FJC- or GFAP-positive cells in the hippocampus of SQ 29,548 responders. In addition, SQ 29,548 prevented the SE-elicited increase of c-Fos immunoreactivity in the hippocampus. In summary, our results suggest that the TP antagonist (SQ 29,548) improves neurological outcome after pilocarpine-induced SE in mice. The existence of SQ 29,548 responders and non-responders was suggested by results from the Neuroscore test. Additional studies are needed to understand the mechanisms underlying these findings, as well as the potential uses of TP antagonists in the treatment of seizure-induced damage.

Transcriptome-based screening of intracellular pathways and angiogenesis related genes at different stages of thiram induced tibial lesions in broiler chickens

BackgroundThe Tibial dyschondroplasia (TD) in fast-growing chickens is mainly caused by improper blood circulation. The exact mechanism underlying angiogenesis and vascularization in tibial growth plate of broiler chickens remains unclear. Therefore, this research attempts to study genes involved in the regulation of angiogenesis in chicken red blood cells. Twenty-four broiler chickens were allotted into a control and thiram (Tetramethyl thiuram disulfide) group. Blood samples were collected on day 2, 6 (8- and 14-days old chickens) and 15 (23 days old chickens).ResultsHistopathology and hematoxylin and eosin (H&E) results showed that angiogenesis decreased on the 6th day of the experiment but started to recover on the 15th day of the experiment. Immunohistochemistry (IHC) results confirmed the expressions of integrin alpha-v precursor (ITGAV) and clusterin precursor (CLU). Transcriptome sequencing analysis evaluated 293 differentially expressed genes (DEGs), of which 103 up-regulated genes and 190 down-regulated genes were enriched in the pathways of neuroactive ligand receptor interaction, mitogen-activated protein kinase (MAPK), ribosome, regulation of actin cytoskeleton, focal adhesion, natural killer cell mediated cytotoxicity and the notch signalling pathways. DEGs (n = 20) related to angiogenesis of chicken erythrocytes in the enriched pathways were thromboxane A2 receptor (TBXA2R), interleukin-1 receptor type 1 precursor (IL1R1), ribosomal protein L17 (RPL17), integrin beta-3 precursor (ITGB3), ITGAV, integrin beta-2 precursor (ITGB2), ras-related C3 botulinum toxin substrate 2 (RAC2), integrin alpha-2 (ITGA2), IQ motif containing GTPase activating protein 2 (IQGAP2), ARF GTPase-activating protein (GIT1), proto-oncogene vav (VAV1), integrin alpha-IIb-like (ITGA5), ras-related protein Rap-1b precursor (RAP1B), tyrosine protein kinase Fyn-like (FYN), tyrosine-protein phosphatase non-receptor type 11 (PTPN11), protein patched homolog 1 (PTCH1), nuclear receptor corepressor 2 (NCOR2) and mastermind like protein 3 (MAML3) selected for further confirmation with qPCR. However, commonly DEGs were sarcoplasmic/endoplasmic reticulum calcium ATPase 3 (ATP2A3), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), centriole cilia and spindle-associated protein (CCSAP), coagulation factor XIII A chain protein (F13A1), shroom 2 isoform X6 (SHROOM2), ras GTPase-activating protein 3 (RASA3) and CLU.ConclusionWe have found potential therapeutic genes concerned to erythrocytes and blood regulation, which regulated the angiogenesis in thiram induced TD chickens. This study also revealed the potential functions of erythrocytes.Graphical abstract1. Tibial dyschondroplasia (TD) in chickens were more on day 6, which started recovering on day 15. 2. The enriched pathway observed in TD chickens on day 6 was ribosome pathway, on day 15 were regulation of actin cytoskeleton and focal adhesion pathway. 3. The genes involved in the ribosome pathways was ribosomal protein L17 (RPL17). regulation of actin cytoskeleton pathway were Ras-related C3 botulinum toxin substrate 2 (RAC2), Ras-related protein Rap-1b precursor (RAP1B), ARF GTPase-activating protein (GIT1), IQ motif containing GTPase activating protein 2 (IQGAP2), Integrin alpha-v precursor (ITGAV), Integrin alpha-2 (ITGA2), Integrin beta-2 precursor (ITGB2), Integrin beta-3 precursor (ITGB3), Integrin alpha-IIb-like (ITGA5). Focal adhesion Proto-oncogene vav (Vav-like), Tyrosine-protein kinase Fyn-like (FYN).