Thromboxane A2 Receptor Research Articles

Expression of Concern: Thromboxane A2 receptor activates a Rho-associated kinase/LKB1/PTEN pathway to attenuate endothelium insulin signaling.

Expression of Concern: Thromboxane A2 receptor activates a Rho-associated kinase/LKB1/PTEN pathway to attenuate endothelium insulin signaling.

Prostaglandin F2α Facilitates Platelet Activation by Acting on Prostaglandin E2 Receptor Subtype EP3 and Thromboxane A2 Receptor TP in Mice.

Platelets play an important role in both physiological hemostasis and pathological thrombosis. Thromboxane (TX) A2 and prostaglandin (PG) I2 are well known as a potent stimulator and an inhibitor of platelet function, respectively. Recently, PGE2 has also been reported to regulate platelet function via PGE2 receptor subtypes. However, the effect of PGF2α on platelet function remains to be determined. The aim of the present study was to clarify the effect of PGF2α on murine platelet function both in vitro and in vivo. Platelets prepared from wild-type mice (WT platelets) expressed several types of prostanoid receptors, including the PGE2 receptor subtype EP3 and the TXA2 receptor TP, while expression of the PGF2α receptor FP was not detected. In WT platelets, PGF2α potentiated adenosine diphosphate-induced aggregation in a concentration-dependent manner, while PGF2α alone did not induce aggregation. In platelets prepared from mice lacking FP, however, PGF2α-induced potentiation was not significantly different from that in WT platelets. Interestingly, the potentiation was significantly blunted in platelets lacking EP3 or TP and disappeared completely in platelets lacking both EP3 and TP. Accordingly, PGF2α decreased the cyclic adenosine monophosphate level via EP3 and increased the inositol triphosphate level via TP in WT platelets. Intravenously administered PGF2α significantly shortened the bleeding time and aggravated arachidonic acid-induced acute thromboembolism in WT mice, suggesting that PGF2α works as a platelet stimulator also in vivo. In conclusion, PGF2α potentiates platelet aggregation in vitro via EP3 and TP but not FP. Accordingly, PGF2α facilitates hemostasis and thromboembolism in vivo.

Hyperglycemia Enhances Constriction of Retinal Venules via Activation of the Reverse-Mode Sodium-Calcium Exchanger.

Diabetes is associated with hyperglycemia and impairment of retinal microvascular function. However, the impact of hyperglycemia on retinal venular constriction remains unknown. We examined retinal venular responsiveness to endogenous vasoconstrictors and the contribution of the reverse-mode sodium-calcium exchanger (NCX) to these responses during hyperglycemia. Retinal venules were isolated from pigs with streptozocin-induced diabetes (2 weeks, in vivo hyperglycemia) and age-matched control pigs for vasoreactivity and molecular studies. For in vitro hyperglycemia, vessels from euglycemic pigs were exposed to high glucose (25 mmol/L) for 2 h, and 5 mmol/L glucose served as the control. Constrictions of venules from euglycemic pigs to endothelin-1 (ET-1), thromboxane analog U46619, and norepinephrine were mediated by ETA, thromboxane, and α2-adrenergic receptors, respectively, and were insensitive to reverse-mode NCX blockade (KB-R7943). In vivo hyperglycemia enhanced these vasoconstrictions without altering respective receptor mRNA expression. Similarly, in vitro hyperglycemia augmented venular constrictions. Enhanced vasoconstrictions during hyperglycemia were prevented by KB-R7943, while mRNA expression of venular NCX isoforms was unaltered. In vivo hyperglycemia increased vitreous levels of ET-1 but not thromboxane B2 In conclusion, both in vitro and in vivo hyperglycemia enhance retinal venular responses to endogenous vasoconstrictors by activating reverse-mode NCX. Therapies targeting this vascular molecule may alleviate retinal complications during diabetes.

Oligonucleotide Microarrays Identified Potential Regulatory Genes Related to Early Outward Arterial Remodeling Induced by Tissue Plasminogen Activator.

Constrictive vascular remodeling limiting blood flow, as well as compensatory outward remodeling, has been observed in many cardiovascular diseases; however, the underlying mechanisms regulating the remodeling response of the vessels remain unclear. Plasminogen activators (PA) are involved in many of the processes of vascular remodeling. We have shown previously that increased levels of tissue-type PA (tPA) contributes to outward vascular remodeling. To elucidate the mechanisms involved in the induction of outward remodeling we characterized changes in the expression profiles of 8799 genes in injured rat carotid arteries 1 and 4 days after recombinant tPA treatment compared to vehicle. Periadventitial tPA significantly increased lumen size and vessel area, encompassed by the external elastic lamina, at both one and 4 days after treatment. Among 41 differentially expressed known genes 1 day after tPA application, five genes were involved in gene transcription, five genes were related to the regulation of vascular tone [for example, thromboxane A2 receptor (D32080) or non-selective-type endothelin receptor (S65355)], and eight genes were identified as participating in vascular innervation [for example, calpain (D14478) or neural cell adhesion molecule L1 (X59149)]. Four days after injury in tPA-treated arteries, four genes, regulating vascular tone, were differentially expressed. Thus, tPA promotes outward arterial remodeling after injury, at least in part, by regulating expression of genes in the vessel wall related to function of the nervous system and vascular tone.

Mechanisms of U46619-induced contraction in mouse intrarenal artery.

Thromboxane A2 (TXA2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca2+ ]i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10μmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50μmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10μmol/L, and a selective PKCδ inhibitor rottlerin at 10μmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10μmol/L and was further completely inhibited together with a putative IP3 receptor antagonist and store-operated Ca2+ (SOC) entry inhibitor 2-APB at 100μmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1μmol/L and 2-APB at 50 and 100μmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca2+ activated C1- channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100μmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway.

SUN-LB039 Novel Use of Thromboelastography (TEG) to Investigate the Prothrombotic Effects of Testosterone Replacement Therapy (TRT) for Androgen Deficiency (AD) Established with Diagnostic Rigor (DR)

Background Endocrine Society recommends ≥2 low bioavailable (BAT) or free Testosterone (T) levels in the early AM by tandem mass spectroscopy to establish AD with DR. They also advise against TRT with a recent history of vascular events (VEs). Data from our center affirm these recommendations. TRT increased VE risk four-fold without DR versus with DR, and eight-fold with a history of recent VEs versus without, suggesting that inappropriate TRT might provoke VEs in the absence of AD, or with high vascular risk. The etiopathogenesis of VEs on TRT is not understood, nor is it known if TRT induces a prothrombotic state, but two putative mechanisms might be involved. First, T increases platelet receptors for thromboxane A2, a potent vasoconstrictor and platelet aggregant. Second, T is aromatized to estradiol (E2), a potent activator of the clotting cascade, inhibitor of antithrombin III, and platelet aggregant. Objective To determine (a) whether TEG, a clinically validated tool for assessing platelet function and clotting can be used to identify a prothrombotic state during TRT for AD proven with DR, and (b) if changes are related to estradiol levels. Methods Male veterans >18 years of age referred for suspected AD, and with no contraindications to TRT, were recruited for the study. After AD was established according to guidelines, blood samples were drawn for TEG and E2 before TRT initiation and after 3 months of TRT, and the following TEG parameters were assessed: Reaction Time for initial fibrin formation (R), reflecting the clotting cascade, Kinetic Time for fibrin cross linkage (K), reflecting fibrinogen and platelet number, Angle (α), reflecting the speed of clot strengthening, and Maximum Amplitude (MA), reflecting platelet function. Results In 7 subjects (median age 64 [IQR 38,68]), on TRT for 88±5d (Mean±SE), calculated BAT increased from 82±17ng/dl to 143±32ng/dl (p=0.046), but E2 did not change significantly (14.3±2.5 vs 19.6±4.2pg/ml, p=0.4). R increased 17% on TRT compared to before (∆=0.9±0.2min, p=0.012), showing that time to initial fibrin formation was prolonged, while remaining within the normal range. However, there was no significant change in K, α, or MA. ConclusionsOur data, based on the novel use of TEG to study a potentially prothrombotic effect of TRT, show for the first time that TRT inhibits, not activates the kinetics of the enzymatic process of coagulation. In other words, appropriate TRT had no prothrombotic effect in an admittedly small number of patients with AD established with DR. This accords with both the four-fold decrease in VEs we observed earlier on TRT prescribed appropriately with DR, as well as some prior reports of a lower risk of VEs on TRT. It is conceivable that TRT might induce a prothrombotic state when prescribed inappropriately in the absence of AD, given that many studies showing that TRT increases VE risk have not employed DR to establish AD conclusively. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

SUN-206 Acute Cardiovascular Complications FromTestosterone Therapy

Background: Some studies have suggested Testosterone (T), especially parenteral administration may increase the short-term risk of thromboembolic events. We report 2 patients who developed acute cardiovascular complications following treatment with intramuscular injection of T. Case 1: A 43-year-old man was evaluated for erectile dysfunction and fatigue. History was significant for hypercholesterolemia treated with atorvastatin. On physical examination, vital signs, heart and lung examinations were normal. Testicular volume was 15 mL bilaterally. Lab revealed normal CBC, liver function, creatinine, and iron studies. A serum T level at 7am was 140 ng/dL; FSH 4.2mIU/mL, LH 3.8 mIU/mL, prolactin and TFT were normal. Pituitary MRI was normal. Patient was started on T enanthate 200 mg IM every 2 weeks. 6 weeks later, serum T level (obtained 7 days after injection) revealed a value of 480 ng/dL. 2 months following treatment the patient noted significant improvement. Four months after T treatment patient developed acute chest pain followed by cardiac arrest. Despite resuscitation, the patient expired after 45 minutes. Autopsy confirmed obstructions of left anterior descending coronary artery and right coronary artery. Case 2: A 48-year-old man was evaluated for erectile dysfunction and generalized muscle weakness. Past history and family history were noncontributory. Physical examination revealed normal vital signs and examination of the heart, lungs and abdomen was normal. Right testis 10 mL and left testis 15 mL in size. Lipid panel showed LDL 110 mg/dL, HDL 48 mg/dL. Serum T level at 8am was 135 ng/dL, FSH 3.1 mIU/mL, LH 2.8 mIU/mL, prolactin and TFT were normal. A pituitary MRI showed an empty sella. Patient was started on T injection 100 mg every 10 days. Six weeks later follow-up serum T (5 days after injection) was 466 ng/dL and serum estradiol was 39 pg/mL. Six months later he developed acute dyspnea and tachycardia. D-dimer value was 3260 ng/mL. CT angiogram revealed a thrombus in one of the left lower lobe pulmonary artery branches. Patient was treated in the intensive care unit with supportive care and anticoagulation. Follow-up evaluation confirmed factor V Leiden mutation. Discussion: T replacement is commonly given for hypogonadism but clinicians need to be aware of the potential rare adverse side effects such as acute cardiac events, as described in these 2 otherwise healthy patients with no known risk factors. In the second patient serum estradiol which may contribute to thrombosis was normal. The mechanism of these CV effects are unknown, but may be due to increased expression of platelet thromboxane A2 receptor and/or via other mechanisms especially in patients with procoagulant predisposition.

Pharmacological inhibition of the F1 -ATPase/P2Y1 pathway suppresses the effect of apolipoprotein A1 on endothelial nitric oxide synthesis and vasorelaxation.

The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high-density lipoprotein (HDL), to endothelium-dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto-F1 -ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)-sensitive P2Y receptors (P2Y ADP receptors). Ecto-F1 -ATPase is involved in APOA1-mediated cell proliferation and HDL transcytosis. Here, we investigated the effect of lipid-free APOA1 and the involvement of ecto-F1 -ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. Nitric oxide synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. Physiological concentrations of lipid-free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto-F1 -ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto-F1 -ATPase and P2Y1 receptor. Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto-F1 -ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored.

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3' region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3' region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

Alterations in Nitric Oxide‐Mediated Signals in Coronary Arteries Following Spaceflight

Proportional cardiovascular disease mortality rates are not different between US astronauts that have flown in low Earth orbit (LEO) and those that have never flown orbital missions in space. While these data suggest that spaceflight (SF) in LEO holds nominal long‐term cardiovascular health consequences, to date the acute effect of SF on coronary artery function has not been explored. The purpose of this study was to examine murine coronary artery contractile, dilatory, and mechanical properties following SF in LEO aboard the International Space Station (ISS). Twenty C57BL/6J mice were housed in a specially designed rodent habitat and were launched to the ISS aboard a Falcon 9 rocket (Space‐X CRS‐12). Following ~33 days of SF in LEO, the Dragon capsule containing the mice splashed down in the Pacific Ocean. Within 33–41 hours of returning to Earth, the left anterior descending (LAD) coronary arteries were cleared of surrounding cardiac tissue and mounted in a Danish Myotech wire myograph. LADs were then exposed to agonists that allowed for the examination of endothelial (EC) and smooth muscle cell (SMC) function. Vivarium controls (VC), consisting of mice housed in conventional vivarium cages, and habitat controls (HC), which were housed in the same type cages as those flown on orbit and maintained under similar pCO2, temperature, humidity, and light conditions, were used for comparison. The passive pressure‐diameter relationship was not different between SF and either control. SMC‐dependent vasoconstriction to U46619, a thromboxane receptor agonist, was significantly greater in HC versus SF at only a single dose, while vasoconstriction to isosmotic KCl was unaltered. LADs from SF mice preconstricted with U46619 exhibited similar EC‐dependent vasodilation to the muscarinic receptor agonist acetylcholine when compared to VC and HC. Acetylcholine and ADPβS (a P2Y1 receptor agonist)‐mediated vasodilation were completely absent in all LADs preconstricted with KCl in the presence of the nitric oxide synthase inhibitor, L‐NAME. In vessels preconstricted with U46619 vasodilation to the EC‐dependent vasodilator ADPβS was similar between SF and both controls, with SF LADs exhibiting less dependence on nitric oxide (NO)‐mediated mechanisms as observed in the presence of L‐NAME. LADs from SF mice exhibited a right‐ward shift to the NO donor and SMC‐dependent vasodilator, DEA NONOate. Collectively, these data suggest that subtle changes in LAD function occur following spaceflight in LEO. In particular, NO‐mediated signals were the most affected, as indicated by decreased SMC sensitivity to exogenous NO. In addition, while the magnitude of vasodilation to ADPβS was unchanged, SF animals exhibited less dependence on NO‐mediated signals. These ADPβS responses suggest a shift away from the canonical NO‐mediated pathway of vasodilation. These data represent the first examination of coronary artery function following SF and suggest that function is well maintained due to subtle mechanistic adaptations.Support or Funding InformationThis work was supported by NASA Space Biology grant NNX16AC28G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aspirin-Dependent Effects on Purinergic P2Y1 Receptor Expression.

Chronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y1 receptor exerts its effects via a Gq-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y1 expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y1 mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y1 mRNA expression was higher than in mock culture. P2Y1 expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y1 expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y1 up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y1 is coupled to the same Gq-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway.

Alterations in cardiovascular function in an experimental model of lung fibrosis and pulmonary hypertension.

What is the central question of this study? We have evaluated changes in cardiovascular physiology using echocardiography in an experimental model of lung fibrosis. What is the main finding and its importance? Remarkably, we report changes in cardiovascular function as early as day7, concomitant with evidence of vascular remodelling. We also report that isolated pulmonary arteries were hypercontractile in response to a thromboxane A2 agonist. These findings are significant because the development of pulmonary hypertension is one of the most significant predictors of mortality in patients with lung fibrosis, where there are no available therapies and a lack of animal models. Group III pulmonary hypertension is observed in patients with chronic lung diseases such as chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. Pulmonary hypertension (PH) develops as a result of extensive pulmonary vascular remodelling and resultant changes in vascular tone that can lead to right ventricle hypertrophy. This eventually leads to right heart failure, which is the leading indicator of mortality in patients with idiopathic pulmonary fibrosis. Treatments for groupIII PH are not available, in part owing to a lack of viable animal models. Here, we have evaluated the cardiovascular changes in a model of lung fibrosis and PH. Data obtained from this study indicated that structural alterations in the right heart, such as right ventricular wall hypertrophy, occurred as early as day14, and similar increases in right ventricle chamber size were seen between days21 and 28. These structural changes were correlated with decreases in the systolic function of the right ventricle and right ventricular cardiac output, which also occurred between the same time points. Characterization of pulmonary artery dynamics also highlighted that PH might be occurring as early as day21, indicated by reductions in the velocity-time integral; however, evidence for PH is apparent as early as day 7, indicated by the significant reduction in pulmonary acceleration time values. These changes are consistent with evidence of vascular remodelling observed histologically starting on day 7. In addition, we report hyperactivity of bleomycin-exposed pulmonary arteries to a thromboxaneA2 receptor (Tbxa2r) agonist.

Evaluation of some prostaglandins modulators on rat corpus cavernosum in-vitro: Is relaxation negatively affected by COX-inhibitors?

IntroductionProstaglandins (PGs) play an important role in corpus cavernosum relaxation, as evidenced by alprostadil being used as a drug for erectile dysfunction. Reports about the effect of cyclooxygenase (COX) inhibitors on erectile function are highly contradictory. AimTo compare the potential effects of some COX inhibitors with varying COX-1/COX-2 selectivities (indomethacin, ketoprofen and diclofenac) with that of the selective COX-2 inhibitor (DFU) on corpus cavernosal tone in-vitro. The role played by PGE1, PGI2-analogue and PGE4 receptor (EP4)-agonist in controlling corpus cavernosum function and the modulation of their action by sildenafil is also studied. MethodsOrgan bath experiments were performed using isolated rat corpus cavernosum. Direct relaxations and changes to electric field stimulation (EFS, 2–16 Hz, 60 V, 0.8 ms, 10 s train)-induced relaxation by the effect of the selected drugs were studied. Strips were precontracted using phenylephrine (PE, 10−5 M). Results are expressed as mean ± SEM of 5–9 rats. ResultsAlprostadil, iloprost and L902688 (selective EP4 agonist) induced direct relaxation where L902688 showed greater relaxant effect. Sildenafil potentiated the Emax of alprostadil and iloprost but not L902688. EFS and acetylcholine (ACh)-induced relaxations were significantly potentiated in presence of indomethacin, ketoprofen and diclofenac (20, 100 μM) but not in presence of selective COX-2 inhibitor (DFU, 1 μM). GR32191B (Thromboxane A2 receptor antagonist, 10−6 M) significantly reduced the potentiatory effect of indomethacin. Only diclofenac succeeded to potentiate sodium nitroprusside (SNP)-induced relaxation. ConclusionsEP4 receptors may play an important nitric oxide (NO)/cGMP-independent role in corpus cavernosal relaxation. Nonselective COX inhibitors seem of no harm concerning cavernosal tissue relaxation, possibly because they inhibit the synthesis of the highly contracting mediator thromboxane A2.

Thromboxane A Synthase: A New Target for the Treatment of Cardiovascular Diseases.

Atherothrombosis-related diseases are one of the world's leading causes of mortality, and thus the search for new therapeutic approaches in this area remains a very urgent task. Modern pharmacogenomic technologies make it possible to obtain valuable data on disease pathogenesis and optimal therapeutic approaches. One promising research direction is the study of the thromboxane A2 - thromboxane A synthase - thromboxane A2 receptor axis. This review summarizes the recent evidence and suggests that systematic works in this area are creating new and promising opportunities in the treatment of patients with cardiovascular diseases.

β2-Microglobulin, interleukin-31, and arachidonic acid metabolites (leukotriene B4 and thromboxane A2) are involved in chronic renal failure-associated itch-associated responses in mice

Chronic renal failure (CRF) is a progressive disease with severe pruritus and dry skin as the major symptoms. However, the mechanisms of CRF-induced pruritus remain unclear. In this study, 5/6 nephrectomized (NPCT) mice were used as a mouse model of CRF. Serum concentrations of blood urea nitrogen and creatinine in 5/6 NPCT mice were increased. The stratum corneum water content in the skin of 5/6 NPCT mice was decreased. These findings suggest that 5/6 nephrectomy in mice results in a phenotype resembling human CRF. 5/6 NPCT mice showed spontaneous scratching, which was inhibited by μ-opioid receptor antagonist, suggesting that the scratching is an itch-related response. The number of cutaneous mast cells was not altered in 5/6 NPCT mice compared with sham-operated mice. The H1 histamine-receptor antagonist, proteinase-activated receptor 2-neutralizing antibody, and 5-HT3-receptor antagonist did not inhibit spontaneous scratching in 5/6 NPCT mice; therefore, the role of mast cells and serotonin in spontaneous scratching appears to be minimal. The anti-allergy agent azelastine, BLT leukotriene (LT) B4 receptor antagonist, and TP thromboxane (TX) A2 receptor antagonist inhibited spontaneous scratching in 5/6 NPCT mice, suggesting that LTB4 and TXA2 are involved in CRF-induced pruritus. Interestingly, in the skin of 5/6 NPCT mice, levels of two newly identified pruritogens (β2-microglobulin and interleukin-31) were increased. Taken together, these findings suggest that 5/6 NPCT mice are useful for the study of itching in CRF. In addition to LTB4 and TXA2, it is also suggested that β2-microglobulin and interleukin-31 are involved in itching associated with CRF-induced pruritus.

Gradient-dependent inhibition of stimulatory signaling from platelet G protein-coupled receptors.

As platelet activation is an irreversible and potentially harmful event, platelet stimulatory signaling must be tightly regulated to ensure the filtering-out of inconsequential fluctuations of agonist concentrations in the vascular milieu. Herein, we show that platelet activation via G protein-coupled receptors is gradient-dependent, i.e., determined not only by agonist concentrations per se but also by how rapidly concentrations change over time. We demonstrate that gradient-dependent inhibition is a common feature of all major platelet stimulatory G protein-coupled receptors, while platelet activation via the non-G protein-coupled receptor glycoprotein VI is strictly concentration-dependent. By systematically characterizing the effects of variations in temporal agonist concentration gradients on different aspects of platelet activation, we demonstrate that gradient-dependent inhibition of protease-activated receptors exhibits different kinetics, with platelet activation occurring at lower agonist gradients for protease-activated receptor 4 than for protease-activated receptor 1, but shares a characteristic bimodal effect distribution, as gradient-dependent inhibition increases over a narrow range of gradients, below which aggregation and granule secretion is effectively shut off. In contrast, the effects of gradient-dependent inhibition on platelet activation via adenosine diphosphate and thromboxane receptors increase incrementally over a large range of gradients. Furthermore, depending on the affected activation pathway, gradient-dependent inhibition results in different degrees of refractoriness to subsequent autologous agonist stimulation. Mechanistically, our study identifies an important role for the cyclic adenosine monophosphate-dependent pathway in gradient-dependent inhibition. Together, our findings suggest that gradient-dependent inhibition may represent a new general mechanism for hemostatic regulation in platelets.

Increase in the constrictor effects of Rho-kinase in skeletal muscle and coronary arteries of rats with chronic hypothyroidism

Aim. The deficit of thyroid function is known to be accompanied by an increase in the overall peripheral vascular resistance. This work tested the hypothesis that long-term hypothyroidism leads to an increase in the vasoconstrictor effect of Rho-kinase in skeletal muscle and heart resistance arteries of adult rats.Materials and methods.Male Wistar rats consumed the antithyroid drug propylthiouracil (PTU) in drinking water (0.025%), starting at 10 weeks of age. The rats of the control group received PTU-free water. After 14 weeks, the contractile responses of the gastrocnemius muscle arteries (to the α1-adrenoceptor agonist methoxamine) and the septal coronary artery (to the thromboxane A2 receptor agonist U46619) were isometrically recorded. The contribution of the Rho-kinase to the arterial contractile responses was assessed using inhibitor Y27632 (3 μM).Results.The consumption of propylthiouracil was accompanied by a marked decrease of thyroid hormone concentrations and an increase in total cholesterol serum level as well as a decrease in body weight. Maximal contractile responses of studied arteries were also reduced in hypothyroid rats. However, basal tone and reactivity to the moderate concentrations of agonists in arteries of hypothyroid rats were increased compared to control animals. Y27632 significantly weakened the contractile responses of the arteries and negated the differences between the two groups of rats.Conclusion.Chronic hypothyroidism leads to an increase in the activity of the Rho-kinase signaling pathway in the arteries of the gastrocnemius muscle and heart, which results in the increase of the spontaneous tone of the arteries and their reactivity to agonists.

Structural basis for ligand recognition of the human thromboxane A2 receptor.

Stimulated by thromboxane A2, an endogenous arachidonic acid metabolite, the thromboxane A2 receptor (TP) plays a pivotal role in cardiovascular homeostasis, and thus is considered as an important drug target for cardiovascular disease. Here, we report crystal structures of the human TP bound to two nonprostanoid antagonists, ramatroban and daltroban, at 2.5 Å and 3.0 Å resolution, respectively. The TP structures reveal a ligand-binding pocket capped by two layers of extracellular loops that are stabilized by two disulfide bonds, limiting ligand access from the extracellular milieu. These structures provide details of interactions between the receptor and antagonists, which help to integrate previous mutagenesis and SAR data. Molecular docking of prostanoid-like ligands, combined with mutagenesis, ligand-binding and functional assays, suggests a prostanoid binding mode that may also be adopted by other prostanoid receptors. These insights into TP deepen our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor.

RBMSCs/ITGA5B1 Promotes Human Vascular Smooth Muscle Cell Differentiation via Enhancing Nitric Oxide Production.

Background and ObjectivesPrevious studies have shown that integrins alpha5beta1 (ITGA5B1) gene-modified rat bone marrow mesenchymal stem cells (rBMSCs) could prevent cell anoikis and increase the nitric oxide (NO) production. Here we examined the capability of rBMSCs/ITGA5B1 on the phenotype modulation of Human Pulmonary Artery Smooth Muscle Cell (HPASMC) in vitro.Methods and ResultsThe synthetic (dedifferentiated) phenotype of HPASMC was induced by monocrotaline (MCT, 1μM) for 24 h and then co-cultured with rBMSCs/ITGA5B1 in a transwell culture system. The activation of NO/cGMP (nitric oxide/Guanosine-3′, 5′-cyclic monophosphate) signaling was investigated in HPASMC. The changes of pro-inflammatory factors, oxidative stress, vasodilator, vasoconstrictor, contractile and synthetic genes, and the morphological changes of HPASMC were investigated. The results of this study showed that the NO/cGMP signal, endothelial nitric oxide synthase (eNOS) expression, the expression of the vasoprotective genes heme oxygenase-1 (HMOX1) and prostaglandin-endoperoxide synthase 2 (PTGS2) were increased, but the expression of transforming growth factor-β1 (TGF-β1), CCAAT/enhancer-binding proteins delta (Cebpd), Krüppel-like factor 4 (KLF4), and activating transcription factor 4 (ATF4) were reduced in MCT treated HPASMC co-cultured with rBMSCs/ITGA5B1. The synthetic smooth muscle cells (SMCs) phenotype markers thrombospondin-1, epiregulin and the vasoconstrictor endothelin (ET)-1, thromboxane A2 receptor (TbxA2R) were down-regulated, whereas the contractile SMCs phenotype marker transgelin expression was up-regulated by rBMSCs/ITGA5B1. Furthermore, rBMSCs/ITGA5B1 promoted the morphological restoration from synthetic (dedifferentiation) to contractile (differentiation) phenotype in MCT treated HPASMC.ConclusionsrBMSCs/ITGA5B1 could inhibit inflammation and oxidative stress related genes to promote the HPASMC cell differentiation by activation NO/cGMP signal.