Thromboxane A2 Antagonist Research Articles

Investigating the possible mechanisms involved in adenosine preconditioning-induced cardioprotection in rats.

Adenosine is a breakdown product of adenosine triphosphate and plays an important role in pharmacological preconditioning. The cardioprotective effects of adenosine preconditioning are well established. However, the possible mechanisms need to be explored. This study was aimed to investigate the possible mechanisms involved in adenosine preconditioning-induced cardioprotection in rats. Rat heart was isolated and perfused on Langendorff apparatus. Global ischemia for 30minutes followed by reperfusion for 120minutes was employed to produce myocardial injury. Myocardial injury was assessed by measuring myocardial infarct size, release of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary effluent and hemodynamic parameters including left ventricular developed pressure (LVDP), dp/dtmax, and dp/dtmin . Serum nitrite levels were measured as an index of nitric oxide release in blood. Adenosine (4mg/kg) preconditioning significantly decreased ischemia-reperfusion-induced increase in LDH, CK release, infarct size, improved LVDP, dp/dtmax and dp/dtmin, and increased serum nitrite levels. Pretreatment with L-NAME, a specific NOS inhibitor, (5mg/kg) and montelukast, leukotriene receptor antagonist, (10mg/kg) significantly abrogated the cardioprotective effect of adenosine preconditioning. However, seratrodast, thromboxane A2 antagonist, (15mg/kg) had no effect on adenosine-induced cardioprotection. Sodium nitroprusside (SNP) preconditioning also produced cardioprotective effects. However, caffeine (20mg/kg) (adenosine receptor blocker) and seratrodast (15mg/kg) had no effect on SNP-induced cardioprotection. Administration of montelukast abrogated the cardioprotective effects of SNP preconditioning-induced cardioprotection. Adenosine preconditioning may increase the release of nitric oxide, which in turn may increase the release of cysteinyl leukotrienes to confer cardioprotection.

Pharmacological adjuncts for chronic venous ulcer healing: a systematic review.

The aim of this study was to systematically review the current evidence and determine whether there is a clinical benefit for using pharmacological agents as adjunctive treatment for chronic venous ulcers. A systematic review of the MEDLINE and EMBASE (from 1 January 1947 through 15 August 2013) and Cochrane databases (from inception through 15 August 2013) was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria were all randomised controlled trials investigating pharmacological adjuncts for the treatment of venous ulcers with a minimum sample size of 20 patients for each treatment arm. Ten relevant articles were identified; one pilot randomised controlled trial and four Cochrane reviews were included. Pentoxifylline, aspirin, sulodexide, mesoglycan, flavonoids, thromboxane A2 antagonist (ifetroban), zinc, prostaglandin and prostacyclin analogues were the drugs reviewed. Pentoxifylline was found to be more effective than placebo in terms of complete ulcer healing or in causing a significant improvement (greater than 60% reduction in ulcer size) (RR 1.70, 95% CI 1.30 to 2.24). Aspirin and flavonoids show potential to be effective adjuncts but methodological shortcomings and issues with bias limit the validity of results from trials involving each of these drugs, respectively. There was no significant difference between placebo and Ifetroban and likewise pooled results from trials investigating sulodexide and zinc showed no benefit in comparison to placebo. Many systemic pharmacological agents have been investigated as adjuncts to venous ulcer healing; however, pentoxifylline (400 mg, three times a day) is currently the only drug that has promising evidence to support its use. Other compounds are in early stage research.

Myeloperoxidase evokes substantial vasomotor responses in isolated skeletal muscle arterioles of the rat.

AimsMyeloperoxidase (MPO) catalyses the formation of a wide variety of oxidants, including hypochlorous acid (HOCl), and contributes to cardiovascular disease progression. We hypothesized that during its action MPO evokes substantial vasomotor responses.MethodsFollowing exposure to MPO (1.92 mU mL−1) in the presence of increasing concentrations of hydrogen peroxide (H2O2), changes in arteriolar diameter of isolated gracilis skeletal muscle arterioles (SMAs) and coronary arterioles (CAs) and in the isometric force in basilar arteries (BAs) of the rat were monitored.ResultsMyeloperoxidase increased vascular tone to different degrees in CAs, SMAs and BAs. The mechanism of increased vasoconstriction was studied in detail in SMAs. MPO-evoked vasoconstrictions were prevented by the MPO inhibitor 4-aminobenzhydrazide (50 μm), by endothelium removal in the SMAs. Surprisingly, the HOCl scavenger L-methionine (100 μm), the thromboxane A2 (TXA2) antagonist SQ-29548 (1 μm) or the non-specific cyclooxygenase (COX) antagonist indomethacin (1 μm) converted the MPO-evoked vasoconstrictions to pronounced vasodilations in SMAs, not seen in the presence of H2O2. In contrast to noradrenaline-induced vasoconstrictions, the MPO-evoked vasoconstrictions were not accompanied by significant increases in arteriolar [Ca2+] levels in SMAs.ConclusionThese data showed that H2O2-derived HOCl to be a potent vasoconstrictor upon MPO application. HOCl activated the COX pathway, causing the synthesis and release of a TXA2-like substance to increase the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells and thereby to augment H2O2-evoked vasoconstrictions. Nevertheless, inhibition of the HOCl–COX–TXA2 pathway unmasked the effects of additional MPO-derived radicals with a marked vasodilatory potential in SMAs.

Prostaglandin F2α modulates atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats

Endotoxemia induces various physiological adaptive responses such as tachycardia. There is evidence to show that inflammatory tachycardia might be linked to a direct action of prostanoids on the cardiac pacemaker cells. Recent reports have indicated that systemic inflammation may uncouple of cardiac pacemaker from cholinergic neural control in experimental animals; however, the exact mechanism of this phenomenon is uncertain. This study was aimed to explore the hypothesis that prostanoids modulate atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats. Male albino rats were given intraperitoneal injection of either saline or lipopolysaccharide (LPS, 1mg/kg). 3h after saline or LPS injection, the atria were isolated and chronotropic responsiveness to cholinergic stimulation was evaluated in an organ bath. The expression of atrial cyclooxygenases (COX)-1, COX-2 and COX-3 mRNA was assessed by quantitative real-time RT-PCR and cytosocalcium-dependent phospholipase A2 (cPLA2) activity was measured in the atria. The expression of atrial COX-2 mRNA and cPLA2 activity increased significantly in endotoxemic atria (P<0.05). Incubation with prostaglandin F2α (PGF2α, 100pM) could significantly decrease chronotropic response to cholinergic stimulation in vitro. Likewise, LPS injection could induce a significant hyporesponsiveness to cholinergic stimulation, and incubation of isolated atria with either indomethacin (5µM) or AL-8810 (a PGF2α antagonist, 10µM) could reverse it (P<0.01, P<0.05, respectively), while SQ29548 (a thromboxane A2 antagonist, 10nM) was failed (P>0.05). Our data showed that PGF2α may contribute to the atrial chronotropic hyporesponsiveness to cholinergic stimulation in endotoxemic rats.

Synthesis of Optically Active Thromboxane A2 and Leukotriene D4 Receptor Antagonists

Abstract Both (+)- and (−)-4-{[(2-{4-chlorophenylsulfonylamino}-1-{3-[(E)-2-(7-chloro-2-quinolyl)-vinyl]phenyl}ethyl)thio]methyl}benzoic acid (1), Thromboxane A2 and Leukotriene D4 receptor dual antagonist were synthesized. Racemic methyl 4-({[2-amino-1-(3-hydroxymethylphenyl)ethyl]thio}methyl)benzoate (2), and methyl 4-[({2-amino-1-[3-(1,3-dioxolan-2-yl)phenyl]ethyl}thio)methyl]benzoate (3) were derived from 2-(3-bromophenyl)-1,3-dioxolane (4). Compound 2 was resolved via di-p-toluoyl-d/l-tartaric acid salts to transform to (+)- and (−)-1, respectively. We also demonstrated that one optically active salt of compound 2 was converted to optically active (+)-1 without chromatography purification.

Mechanism of platelet activation induced by endocannabinoids in blood and plasma

Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that pharmacological CB1- and CB2-receptor ligands will not affect platelets and platelet-dependent progression and complications of cardiovascular diseases.

Impaired Contractile Response Of Human Peripheral Arterioles To TXA-2 After Cardiopulmonary Bypass

Introduction: Cardiopulmonary bypass (CPB) is often associated with peripheral vasomotor dysfunction. This is thought to lead to decreased vascular tone, hypotension, and an increased need for vasopressor support. We investigated the contractile response to the vasoconstrictor thromboxane A2 (TXA-2), the blockade of TXA-2 receptors and the inhibition of phospholipase-C (PLC) and phospholipase-A2 (PLA-2) in the human peripheral microvasculature pre- and post- CPB. We also examined the effect of CPB on the gene expression of skeletal muscle TXA-2 and TXA-2 synthase. Methods: Chest wall skeletal muscle tissue was harvested pre- and post-CPB from patients undergoing coronary artery bypass grafting. Skeletal muscle arterioles (90-180 micrometer in diameter) were dissected from the harvested tissue and placed in an organ bath containing Krebs solution. Constractile response of the peripheral arterioles to the TXA-2 analog U46619, with and without the highly selective TXA-2 receptor antagonist, SQ 29548, the specific PLC inhibitor U73122 or the selective PLA-2 inhibitor, quinacrine was assessed by videomicroscopy. TXA-2 receptor and TXA-2 synthase gene expression were examined using microarray analysis. Results: The post-CPB contractile response of peripheral arterioles to TXA-2 analog U46619 (10−6M) was significantly impaired compared with pre-CPB (28.5±2% vs. 42.7±3.6 %, P=0.002). The presence of TXA-2 antagonist SQ 29548 (10−6 M) prevented the contractile response to U46619. Pretreatment with the PLC inhibitor U73122 (10−5M) significantly inhibited the U46619-induced contractile response as well. Addition of the PLA-2 inhibitor,quinacrine (10−6M) failed to affect U46619-induced contraction. Microarray analysis showed no significant changes in the gene expression of TXA-2 receptor and TXA-2 synthase. Conclusions: CPB decreases the contractile response of human peripheral arterioles to TXA-2 analog U46619. The contractile response to the TXA-2 analog U46619 is via activation of TXA-2 receptors and PLC, but not of PLA-2. These results provide novel mechanisms for TXA-2-induced contraction and signaling in peripheral vasomotor dysfunction after CPB and cardiac surgery.

Role of arachidonic acid-derived metabolites in the control of pulmonary arterial pressure and hypoxic pulmonary vasoconstriction in rats

BackgroundThe roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. MethodsPulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. ResultsHypoxia (Po2, 3%)-induced pulmonary arterial pressure increases (ΔPAPhypox) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAPhypox was inhibited by 29%, 16%, and 28% by the thromboxane A2 (TXA2) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAPhypox by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAPhypox. Consistently, the TXA2 analogue U46619 increased ΔPAPhypox whereas prostacyclin abolished ΔPAPhypox. However, leukotriene D4 had no direct effect on ΔPAPhypox. In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. ConclusionsThe above results suggest that TXA2 and cysteinyl leukotrienes, other than leukotriene D4, are endogenous factors that facilitate HPV in rats. The indispensable role of TXA2-induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

Vascular Oxidative Stress: The Common Link in Hypertensive and Diabetic Vascular Disease

Vascular disease in hypertension and diabetes is associated with increased oxidants. The oxidants arise from NADPH oxidase, xanthine oxidase, and mitochondria. Superoxide anion and hydrogen peroxide are produced by both leukocytes and vascular cells. Nitric oxide is produced in excess by inducible nitric oxide synthase, and the potent oxidant, peroxynitrite, is formed from superoxide and nitric oxide. The damage to proteins caused by oxidants is selective, affecting specific oxidant-sensitive amino acid residues. With some important vascular proteins, for example, endothelial nitric oxide synthase, prostacycline synthase, and superoxide dismutase, oxidation of a single susceptible amino acid inactivates the enzyme. The beneficial effects of antioxidants, at least in animal models of hypertension and diabetes, can in part be ascribed to protection of these and other proteins. Mutant proteins lacking their reactive constituent can recapitulate some disease phenotypes suggesting a pathogenic role of the oxidation. Thus, many of the shared functional abnormalities of hypertensive and diabetic blood vessels may be caused by oxidants. Although studies using antioxidants have failed in patients, the successful treatment of vascular disease with HMG-CoA reductase inhibitors, thromboxane A2 antagonists, and polyphenols may depend on their anti-inflammatory effects and ability to decrease production of damaging oxidants.

Bronchial hyperresponsiveness to histamine induced by intravenous administration of thromboxane A2 (TXA2) in guinea-pigs

Thromboxane A2 (TXA2) has been suggested to play an important role in the pathogenesis of bronchial asthma. In this study the effects of intravenous administration of TXA2 on bronchial smooth muscle in guinea-pigs were investigated by measuring dynamic compliance and dynamic respiratory resistance, using a formula to exclude the effects of differences in airway wall thickness. Using this formula, the ratio of bronchial smooth muscle constriction by histamine can be estimated as an index of bronchial hyperresponsiveness. Administration of TXA2 did not induce airway wall edema. The ratio of bronchial smooth muscle constriction by histamine was significantly (p less than 0.01) enhanced by the administration of TXA2. Moreover, TXA2 antagonists, ONO-NT-126 and ONO-8809, inhibited the effect of TXA2 administration. These results suggest that TXA2 is an important mediator affecting bronchial hyperresponsiveness.

Thromboxane A2 receptor antagonism in man and rat by a sulphonylcyanoguanidine (BM-144) and a sulphonylurea (BM-500).

Torasemide, a loop diuretic, has been reported to relax dog coronary artery precontracted by thromboxane A2 (TXA2), an endogenous prostanoid involved in cardiovascular and pulmonary diseases. N-cyano-N'-[[4-(3'-methylphenylamino)pyrid-3-yl]sulphonyl] homopiperidinoamidine (BM-144) and N-isopropyl-N'-[5-nitro-2-(3'-methylphenylamino)-benzenesulphon yl]urea (BM-500), chemically related to torasemide, have been examined for their TXA2 antagonism. The affinity (IC50, the concentration resulting in 50% inhibition) of BM-144 and BM-500 for the TXA2 receptor of washed platelets from man was 0.28 and 0.079 microM, respectively. This is better than for sulotroban (IC50 = 0.93 microM) but less than for SQ-29548 (IC50 = 0.021 microM), two TXA2 antagonists used as reference. The aggregation of platelets from man induced by arachidonic acid was prevented by BM-144 (IC50 = 9.0 microM) and by BM-500 (IC50 = 14.2 microM). Similar results were obtained by use of U-46619, a TXA agonist, as aggregating agent (BM-144, IC50 = 12.9 microM and BM-500, IC50 = 9.9 microM). The contracting effect of U-46619 on rat stomach strip was abolished by BM-144 (IC50 = 1.01 microM) and BM-500 (IC50 = 2.54 microM). Both drugs (BM-144: IC50 = 0.12 microM and BM-500: IC50 = 0.19 microM) also relaxed rat aorta precontracted by U-46619; both were more potent than sulotroban (IC50 = 1.62 microM). The two torasemide derivatives (100 microM) did not significantly affect the myo-stimulating effect of some prostaglandins (PGE2, PGI2, PGF2alpha) or aorta contraction elicited by KCl (30 mM). They did not modify rat diuresis after administration of a 30-mg kg(-1) dose. In conclusion, BM-144 and BM-500 can be regarded as novel non-carboxylic TXA2 receptor antagonists and offer a novel template for the design of more potent molecules.

Design, synthesis and biological evaluation of a sulfonylcyanoguanidine as thromboxane A2 receptor antagonist and thromboxane synthase inhibitor

The synthesis and the structure of N-isopropyl-N'-[2-(3'-methylphenylamino)-5-nitrobenzenesulfonyl] urea (14) was drawn from two thromboxane A2 receptor antagonists structurally related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A2 (TXA2) receptor of human washed platelets. Compound 14 prevented platelet aggregation induced by arachidonic acid (0.6 mM) and U-46619 (1 microM) with an IC50 value of 0.45 and 0.15 microM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea precontracted by U-46619, a TXA2 agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively. Finally, 14 (1 microM) completely inhibited TXA2 synthase of human platelets. The pKa value and the crystallographic data of 14 were determined and used to propose an interaction model between the TXA2 antagonists related to torasemide and their receptor.

Circulating Microparticles from Patients with Septic Shock Exert Protective Role in Vascular Function

Sepsis is an archetypal condition with molecular links between inflammation and coagulation. Both events can be orchestrated by the interaction between circulating and vascular cells that under activation release microparticles. We characterized circulating microparticles from both nonseptic subjects and patients with septic shock and evaluated their contribution to vascular function. Circulating microparticles and their cell origin were measured in blood from 36 patients with septic shock and 18 nonseptic subjects by flow cytometry. Microparticles were then injected intravenously into mice and vascular reactivity was assessed in aorta. Expression and activity of enzymes involved in nitric oxide (NO) and cyclooxygenase metabolite production were analyzed. Circulating levels of microparticles and platelet- and endothelial-derived microparticles were increased in septic patients. Surprisingly, septic microparticles enhanced the sensitivity of contraction of mouse aorta in response to serotonin. Interestingly, septic microparticles enhanced the contraction of aorta from lipopolysaccharide-treated mice. This effect was linked neither to increased calcium entry nor to Rho kinase inhibitor-sensitive mechanisms. In addition, the effect of septic microparticles was not modified either by NO-synthase or cyclooxygenase-2 inhibitors, and was not associated with NO or O2- overproduction. The nonselective cyclooxygenase-2 inhibitor indomethacin reduced, and the specific thromboxane A2 antagonist SQ-29548 abolished, aortic contraction in mice treated with nonseptic and septic microparticles. The effect of septic microparticles was associated with increased thromboxane A2 production, and was sensitive to a selective thromboxane A2 antagonist. We provide evidence that increased circulating microparticles are protective against vascular hyporeactivity accounting for hypotension in patients with septic shock.

Analysis of Activity Space by Fragment Fingerprints, 2D Descriptors, and Multitarget Dependent Transformation of 2D Descriptors

The effect of multitarget dependent descriptor transformation on classification performance is explored in this work. To this end decision trees as well as neural net QSAR in combination with PLS were applied to predict the activity class of 5HT3 ligands, angiotensin converting enzyme inhibitors, 3-hydroxyl-3-methyl glutaryl coenzyme A reductase inhibitors, platelet activating factor antagonists, and thromboxane A2 antagonists. Physicochemical descriptors calculated by MOE and fragment-based descriptors (MOLPRINT 2D) were employed to generate descriptor vectors. In a subsequent step the physicochemical descriptor vectors were transformed to a lower dimensional space using multitarget dependent descriptor transformation. Cross-validation of the original physicochemical descriptors in combination with decision trees and neural net QSAR as well as cross-validation of PLS multitarget transformed descriptors with neural net QSAR were performed. For comparison this was repeated using fragment-based descriptors in combination with decision trees.

Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery.

In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). ACh (10(-6), 10(-5), and 10(-4) M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10(-8)-10(-6) M), and a relaxation followed by a contraction at higher concentrations (10(-5) M). Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (10(-4) M) enhanced the EDC to ACh. Catalase (1000 U ml(-1)) reduced the EDC to ACh. H2O2 (10(-6), 10(-5), and 10(-4) M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2. In isolated endothelial cells, ACh (10(-5) M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2',7'-dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 x 10(-4) M at 3 min and was prevented by catalase. Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.

F2-isoprostanes stimulate collagen synthesis in activated hepatic stellate cells: a link with liver fibrosis?

Carbon tetrachloride (CCl4)-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F2-isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological effects. By contrast with aldehydes, F2-isoprostanes act through receptors able to elicit definite signal transduction pathways. In a rat model of CCl4-induced hepatic fibrosis, plasma F2-isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content also increased. When hepatic stellate cells (HSCs) from normal liver were cultured with F2-isoprostanes in the concentration range found in the in vivo studies (10−9–10−8 M), a striking increase in DNA synthesis (reversed by the thromboxane A2 antagonist SQ 29 548), in cell proliferation and in collagen synthesis was observed. Total collagen content was similarly increased. Moreover, F2-isoprostanes markedly increased the production of transforming growth factor-β1 by U937 cells, considered a model of liver macrophages. The data provide evidence for the possibility that F2-isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen production seen in hepatic fibrosis.

INVOLVEMENT OF ENDOTHELIAL CYCLO‐OXYGENASE METABOLITES IN NORADRENALINE‐INDUCED CONTRACTION OF RAT CORONARY ARTERY

1. Noradrenaline (NA; 0.3 micromol/L) caused a contraction of the rat coronary artery that markedly increased in the presence of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 micromol/L) and arachidonic acid (1 micromol/L; P < 0.05). 2. The present experiments attempted to elucidate the endothelium dependency of the contraction and to pharmacologically characterize the factors involved in the contraction induced by NA (0.3 micromol/L) in the presence of L-NAME and arachidonic acid in ring preparations of the rat coronary artery. 3. The NA (0.3 micromol/L)-induced contraction was attenuated by a chemical remover of the endothelium (saponin at concentrations of 0.1 and 0.4 mg/mL) in a concentration-dependent manner (P < 0.05). 4. The cyclo-oxygenase (COX)-1 inhibitor flurbiprofen (0.01-1 micromol/L) and the COX-2 inhibitor nimesulide (0.01-1 micromol/L) attenuated the NA-induced contraction in a concentration-dependent manner and the inhibitory effect of flurbiprofen was significantly more potent than that of nimesulide (P < 0.05). The 5-lipoxigenase inhibitor ZM-230487 (1 micromol/L) did not affect the NA-induced contraction. 5. The thromboxane A2 (TXA2) synthetase inhibitor OKY-046 (30 micromol/L) and the TXA2 antagonist S-1452 (0.1-10 micromol/L) did not attenuate the NA-induced contraction. 6. These results indicate that the contraction induced by NA in the rat coronary artery in the presence of L-NAME and arachidonic acid is endothelium dependent and is due to endothelial COX metabolites of arachidonic acid.

Molecular Surface Point Environments for Virtual Screening and the Elucidation of Binding Patterns (MOLPRINT 3D)

A novel method (MOLPRINT 3D) for virtual screening and the elucidation of ligand-receptor binding patterns is introduced that is based on environments of molecular surface points. The descriptor uses points relative to the molecular coordinates, thus it is translationally and rotationally invariant. Due to its local nature, conformational variations cause only minor changes in the descriptor. If surface point environments are combined with the Tanimoto coefficient and applied to virtual screening, they achieve retrieval rates comparable to that of two-dimensional (2D) fingerprints. The identification of active structures with minimal 2D similarity ("scaffold hopping") is facilitated. In combination with information-gain-based feature selection and a naive Bayesian classifier, information from multiple molecules can be combined and classification performance can be improved. Selected features are consistent with experimentally determined binding patterns. Examples are given for angiotensin-converting enzyme inhibitors, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, and thromboxane A2 antagonists.

Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo.

Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI-evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in G(alpha)q or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen-induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin alpha2beta1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.

Effect of ramatroban, a thromboxane A2 antagonist, in the treatment of perennial allergic rhinitis

ABSTRACT Background It is known that the symptoms of allergic rhinitis can significantly reduce the quality of life of the patient. One of such typical symptoms of allergic rhinitis is nasal obstruction. Nasal obstruction is currently thought to be closely related to the presence and abundance of lipid mediators, such as leukotriene and thromboxane (TX) A 2 . The novel drug ramatroban, a TXA 2 receptor antagonist, which has been developed by Bayer Yakuhin Ltd. (Osaka, Japan), has been demonstrated, in clinical trials, to improve nasal obstruction in the treatment of patients with allergic rhinitis and it has recently become commercially available. Methods In the present study, ramatroban was administered for 28 days to 10 patients who were diagnosed with perennial allergic rhinitis but were untreated. Changes in self-reported symptom scores and in vivo allergic reaction parameters were assessed during three observational periods. Results From baseline scores, all three symptom scores after 28 days treatment with ramatroban declined clearly in all patients, except for one patient who suffered a cold during the study period and had aggravated rhinorrhea and nasal obstruction as a result. The concentrations of histamine and TXB 2 (a metabolite of TXA 2 ) in the nasal fluid induced by antigen challenge after the 28 day treatment period also decreased in most subjects compared with concentrations during the pretreatment period. The symptom scores for nasal obstruction during the pretreatment period were correlated with the concentration of TXB 2 in antigen-induced nasal fluid. Conclusions The present study reconfirmed the clinical efficacy of a post-marketed drug, namely ramatroban, in the treatment of allergic rhinitis. In addition, the results suggest that ramatroban suppressed the secretion of chemical mediators in nasal that are thought to be involved in the allergic reaction in patients with perennial allergic rhinitis.