Selective Thromboxane Receptor Antagonist Research Articles

TXA2 mediates LPA1-stimulated uterine contraction in late pregnant mouse

Lysophosphatidic acid (LPA) is known to increase uterine contraction in the estrus cycle and early pregnancy, however, the effect of LPA in late pregnant uterus and its mechanisms are not clear. In the present study, we show the LPA receptor subtypes expressed and the mechanism of LPA-induced contractions in late pregnant mouse uterus. We determined the relative mRNA expression of LPA receptor genes by quantitative PCR and elicited log concentration-response curves to oleoyl-L-α-LPA by performing tension experiments in the presence and absence of nonselective and selective receptor antagonists and inhibitors of the TXA2 pathway. LPA1 was the most highly expressed receptor subtype in the late pregnant mouse uterus and LPA1/2/3 agonist (Oleoyl-L-α LPA) elicited increased contractions in this tissue that had lesser efficacy compared to oxytocin. LPA1/3 antagonist, Ki-16425, and a potent LPA1 antagonist (AM-095) significantly inhibited the LPA-induced contractions. Further, the nonselective COX inhibitor, indomethacin, and potent thromboxane A2 synthase inhibitor, furegrelate significantly impaired LPA-induced contractions. Moreover, selective thromboxane receptor (TP) antagonist, SQ-29548, and Rho kinase inhibitor, Y-27632 almost eliminated LPA-induced uterine contractions. LPA1 stimulation elicits contractions in the late pregnant mouse uterus using the contractile prostanoid, TXA2 and may be targeted to induce labor in uterine dysfunctions/ dystocia.

Role of thromboxane-dependent platelet activation in venous thrombosis: Aspirin effects in mouse model

Recent trials suggest that Aspirin (ASA) reduces the incidence of venous thromboembolism in human. However, the molecular mechanisms underlying this effect are still unclear. In this study we assessed the effects of ASA in venous thrombosis mouse model induced by inferior vena cava (IVC) ligation and we investigated the mechanisms responsible for this effect.ASA (3mg/kg daily for 2 days) treatment decreased the thrombus size, the amounts of tissue factor activity in plasma microvesicles (TF-MP) and the levels of 2,3-dinor Thromboxane B2 (TXB-M) in urine compared to control mice. Interestingly, the thrombus size positively correlated with both TF-MP activity and TXB-M. In addition, positive correlation was observed between TF-MP activity and TXB-M. A reduced number of neutrophils and monocytes, and of TF-positive cells accompanied to a lower amount of fibrin and neutrophil extracellular traps (NETs) were also found in thrombi of ASA-treated mice. Similar results were obtained when mice were treated 24h before IVC ligation with SQ29548 (1mg/kg), a selective thromboxane receptor antagonist. In addition, transfusion of platelets in SQ29548 treated-mice excluded the likelihood of a redundant role of platelet-TP receptor in this context.Finally, incubation of macrophages and neutrophils with SQ29548 prevented TF activity and/or NETs formation induced by supernatant of activated platelets or by IBOP, a selective thromboxane analogue.In conclusion, ASA, suppressing TXA2, prevents macrophages and neutrophils activation and markedly reduces thrombus size with a mechanism most likely dependent of the inhibition of TF activity and NETs formation. These results provide a new link between platelet-produced thromboxane and the occurrence of venous thrombosis.

Menthol Modulates Pacemaker Potentials through TRPA1 Channels in Cultured Interstitial Cells of Cajal from Murine Small Intestine

Background/Aims: ICCs are the pacemaker cells responsible for slow waves in gastrointestinal (GI) smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. Methods: The effects of menthol on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) from mouse small intestine were studied using the whole cell patch clamp technique. Results: Menthol (1 - 10 μM) was found to induce membrane potential depolarization in a concentration-dependent manner. The effects of various TRP channel antagonists were examined to investigate the receptors involved. The addition of the TRPM8 antagonist, AMTB, did not block menthol-induced membrane potential depolarizations, but TRPA1 antagonists (A967079 or HC-030031) blocked the effects of menthol, as did intracellular GDPβS. Furthermore, external and internal Ca2+ levels were found to depolarize menthol-induced membrane potentials, whereas external Na+ was not. Y-27632 (a Rho kinase inhibitor), SC-560 (a selective COX 1 inhibitor), NS-398 (a selective COX 2 inhibitor), ozagrel (a thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective thromboxane receptor antagonist) were used to investigate the involvements of Rho-kinase, cyclooxygenase (COX), and the thromboxane pathway in menthol-induced membrane potential depolarizations, and all inhibitors were found to block the effect of menthol. Conclusions: These results suggest that menthol-induced membrane potential depolarizations occur in a G-protein-, Ca2+-, Rho-kinase-, COX-, and thromboxane A2-dependent manner via TRPA1 receptor in cultured ICCs in murine small intestine. The study shows ICCs are targeted by menthol and that this interaction can affect intestinal motility.

Brain changes associated with thromboxane receptor antagonist SQ 29,548 treatment in a mouse model.

The purpose of this study was to characterize behavioral and physiological effects of a selective thromboxane (TP) receptor antagonist, SQ 29,548, in the C57Bl/6 mouse model. At 6 months of age, male mice were given either sham or drug i.p. injections for 3 days at a dose of 2 mg/kg each day. On the day after the final injection, mice were subjected to behavioral testing before brain collection. Left hemisphere hippocampi were collected from all mice for protein analysis via Western blot. Right brain hemispheres were fixed and embedded in gelatin and then serially sectioned. The sections were immunostained with anti-c-Fos antibodies. Prostaglandin analysis was performed from remaining homogenized brain samples, minus the hippocampi. Injection of SQ 29,548 decreased selective brain prostaglandin levels compared with sham controls. This correlated with robust increases in limbic-region c-Fos immunoreactivity in the SQ 29,548-injected mice. However, drug-treated mice demonstrated no significant changes in relevant hippocampal protein levels compared with sham treatments, as determined from Western blots. Surprisingly, injection of SQ 29,548 caused mixed changes in parameters of depression and anxiety-like behavior in the mice. In conclusion, the results indicate that administration of peripheral TP receptor antagonists alters brain levels of prostanoids and influences neuronal activity, with only minimal alterations of behavior. Whether the drug affects neurons directly or through a secondary pathway involving endothelium or other tissues remains unclear.

Qualitative Determination of Known Thromboxane Receptors in Human Plasma by LC-MS (Quadrupole Time of Flight)

Thromboxane is a potent platelet aggregating agent which also contracts both vascular and non-vascular smooth muscle. Selective agents have been identified which inhibit these actions of thromboxane in a manner consistent with competitive receptor antagonism. Some of these antagonists have potential utility in the treatment of thromboxane-dependent pathologies, particularly within the cardiovascular/renal systems. Classically, receptor types are defined by use of selective antagonists and possible receptor differences derived from differential potency of these antagonists when measured in different tissues. Dazmegrel [3-(1H-imidazole-1-yl-methyl)2-methyl-1H-indole-1-propanoic acid] is persistence as enzyme inhibitors; platelet aggregation inhibitors and Vasodilator Agents. GR32191 (vapiprost) 7-(5-(((1,1'-biphenyl)-4-yl)methoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl)-4-heptanoic acid is a potent and selective thromboxane receptor antagonist. The thromboxane A2/PG endoperoxide receptor inhibitor. Its mechanism is Fibrinolytic Agents; platelet aggregation inhibitors and prostaglandin antagonists. SQ 29548 7-(3-((2((phenylamino)carbonyl)hydrazine)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid is a Thromboxane/antagonists & inhibitors. U-46619 (Z)-7-((1R,4S,5S,6R)-6-((E)-3hydroxyoct-1-enyl)-2-oxabicyclo[2.2.1]heptan-5-yl)hept-5Qualitative Determination of Known Thromboxane Receptors in Human Plasma by LC-MS (Quadrupole Time of Flight)

Determination of efficacy of linotroban by inducing a reduction of renal inulin/para-aminohippuric acid clearances with the thromboxane A2 mimetic U-46619 in the conscious female rat.

Linotroban (CAS 141443-73-4), a potent and selective thromboxane (TXA2) receptor antagonist, is known as a novel antithrombotic agent. It is suggested that pharmacological inhibition of TXA2 synthesis or action merits continued evaluation in the treatment of a variety of renal diseases. The aim of this study was the determination of efficacy of this new TXA2 receptor antagonist by assessing its effect on the reduction in inulin and para-aminohippuric acid (PAH) clearances induced by the TX/prostaglandin endoperoxide mimetic U-46619 in the conscious female rats. The following doses (3, 10, 30 mg/kg/24 h) of linotroban mixed with 720 micrograms TX-mimetic U-46619/kg/24 h were administered via osmotic pumps at a delivery rate of 10 microliters/h, implanted s.c. during 72 h. Rats of the U-46619 group were administered 720 micrograms U-46619/kg/24 h alone as described above, controls received 3.5% NaHCO3, respectively. Inulin/PAH clearances were determined at the end of the 4-h clearance period (68 h-72 h). Summarizing the data, glomerular filtration rate (GFR) and PAH clearances were reduced significantly by U-46619. When linotroban (3, 10 or 30 mg/kg/24 h) was added to U-46619 the GFR and PAH clearance were reversed to values that showed no significant differences to the controls.

Anti-thromboxane B2 antibodies protect against acetaminophen-induced liver injury in mice

Prostanoids are lipid compounds that mediate a variety of physiological and pathological functions in almost all body tissues and organs. Thromboxane (TX) A2 is a powerful inducer of platelet aggregation and vasoconstriction and it has ulcerogenic activity in the gastrointestinal tract. Overdose or chronic use of a high dose of acetaminophen (N-acetyl-paminophenol, APAP) is a major cause of acute liver failure in the Western world. We investigated whether TXA2 plays a role in host response to toxic effect of APAP. CBA/H Zg mice of both sexes were intoxicated with a single lethal or high sublethal dose of APAP, which was administered to animals by oral gavage. The toxicity of APAP was determined by observing the survival of mice during 48 h, by measuring concentration of alanine-aminotransferase (ALT) in plasma 20-22 h after APAP administration and by liver histology. The results have shown that anti-thromboxane (TX) B2 antibodies (anti-TXB2) and a selective inhibitor of thromboxane (TX) synthase, benzylimidazole (BZI), were significantly hepatoprotective, while a selective thromboxane receptor (TPR) antagonist, daltroban, was slightly protective in this model of acute liver injury. A stabile metabolite of TXA2, TXB2, and a stabile agonist of TPR, U-46619, had no influence on APAP-induced liver damage. Our findings suggest that TXA2 has a pathogenic role in acute liver toxicity induced with APAP, which was highly abrogated by administration of anti-TXB2. According to our results, this protection is mediated, at least in part, through decreased production of TXB2 by liver fragments<em> ex vivo</em>.

Attenuation of diabetes-induced retinal vasoconstriction by a thromboxane receptor antagonist

Retinal blood flow has been reported to decrease early in human diabetes as well as in diabetic animal models. The purpose of the present study is to investigate the role of thromboxane receptor binding in the decrease of flow. C57BL/6 mice were injected with streptozotocin (STZ) at 11–12 weeks of age and remained hyperglycemic for 4 weeks. The mice were treated with a selective thromboxane receptor antagonist, GR32191B (vapiprost), in drinking water for the final three weeks at a dose of 1 mg/kg/day. In separate experiments, vapiprost was administered only once, as an acute injection 25 min prior to the experimental measurements. The measurements included retinal arteriolar and venular diameters and red blood cell (RBC) velocities, from which retinal blood flow was calculated. STZ induced decreases in vascular diameters and RBC velocities, resulting in an approximate 30% decrease in overall retinal blood flow. However, these decreases were not seen in mice given the three-week administration of vapiprost. Acute administration to diabetic mice of 1 mg/kg vapiprost, but not 0.1 mg/kg, induced arteriolar vasodilation, with the dilation more substantial in smaller feed arterioles. In summary, STZ-induced decreases in retinal blood flow can be attenuated by the thromboxane receptor antagonist vapiprost.

Abstract 5447: Thromboxane A 2 Receptor Activation Attenuates Insulin Signaling and Angiogenesis by Upregulating Phosphatase PTEN in Cultured Endothelial Cells

Increasing evidence indicates that elevated levels of endogenous thromboxane receptor (TPr) agonists including thromboxane (Tx) A 2 , prostaglandin H 2 , and 8-iso-prostaglandin F 2a contribute to insulin resistance, endothelial dysfunction, and accelerated atherosclerosis in diabetes. However, the mechanisms by which TPr triggers insulin resistance in endothelial cells remaine poorly defined. Here we show how TPr activation impairs insulin signaling and angiogenesis in endothelial cells. Exposure of cultured human umbilical vein endothelial cells (HUVEC) to a structurally related TxA 2 mimetic (IBOP, U46619, or carboxy-TxA 2 ) for 3 or 16h significantly inhibited both basal- and insulin-stimulated Akt-Ser473 phosphorylation, Akt activity, the serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS-Ser1177), and angiogenesis. Concomitantly, IBOP significantly increased the levels of Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and PTEN phosphorylation at Ser380/Thr382/383. Further, Y27632, a Rho kinase (ROCK) inhibitor, abolished the effects of IBOP on the phosphorylation of PTEN, Akt and eNOS, and the PTEN level. Similarly, inhibition of PTEN by small interference RNA (siRNA), like the selective TPr antagonist, SQ29548, or knockdown of TPr by siRNA, abolished IBOP-induced inhibition on both the phosphorylation of the Akt-eNOS axis and angiogenesis. Furthermore, IBOP significantly increased the Ser428 phosphorylation of LKB1, a known upstream kinase for PTEN. Finally, suppression of LKB1 abolished IBOP-induced PTEN phosphorylation and PTEN accumulation in HUVEC. We conclude that TPr suppresses insulin signaling and angiogenesis by upregulating RhoA mediated but LKB1-dependent PTEN phosphorylation in endothelial cells. This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).

The Selective TP Receptor Antagonist, S18886 (Terutroban), Attenuates Renal Damage in the Double Transgenic Rat Model of Hypertension

Background/Aims: Thromboxane receptors play a decisive role in the renovascular actions of angiotensin II. We studied the efficacy of the selective thromboxane receptor antagonist, S18886, in the retardation of renal damage in the double transgenic rats (dTGR), harboring human renin and angiotensinogen genes. Methods: dTGR were gavaged daily with either S18886 (30 mg/kg/day, n = 12), or placebo (dTGR-Plac, tap water, n = 14) for 3 weeks. Matched Sprague-Dawley rats (n = 10) served as controls. Results: The dTGR-Plac had higher systolic blood pressure (1.7-fold) than controls, and developed profound renal damage with significantly higher proteinuria (6.9-fold), polyuria (2.3-fold), index of glomerulosclerosis (+58%), and tubulointerstitial (+47%) and vascular damage scores (+19%). Creatinine concentration and the mesangiolysis index remained unchanged. In dTGR, S18886 slightly lowered the blood pressure (162 ± 15 vs. 149 ± 13 mm Hg, not significant) and improved proteinuria (558 ± 218 vs. 136 ± 71 mg/µmol creatinine, p < 0.01), polyuria and renal morphology (glomerulosclerosis index: 0.79 ± 0.05 vs. 0.66 ± 0.13, p < 0.01; tubulointerstitial damage index: 1.82 ± 0.22 vs. 1.49 ± 0.27, p < 0.05; mesangiolysis index: 1.31 ± 0.18 vs. 0.36 ± 0.09, p < 0.01). Vascular damage score and plasma creatinine were not influenced. S18886 did not alter measured markers of oxidative stress. Conclusion: The data present the first evidence that thromboxane receptor inhibition ameliorates angiotensin II-induced nephropathy.

Endothelial Nitric Oxide Synthase–Dependent Tyrosine Nitration of Prostacyclin Synthase in Diabetes In Vivo

There is evidence that reactive nitrogen species are implicated in diabetic vascular complications, but their sources and targets remain largely unidentified. In the present study, we aimed to study the roles of endothelial nitric oxide synthase (eNOS) in diabetes. Exposure of isolated bovine coronary arteries to high glucose (30 mmol/l d-glucose) but not to osmotic control mannitol (30 mmol/l) switched angiotensin II-stimulated prostacyclin (PGI(2))-dependent relaxation into a persistent vasoconstriction that was sensitive to either indomethacin, a cyclooxygenase inhibitor, or SQ29548, a selective thromboxane receptor antagonist. In parallel, high glucose, but not mannitol, significantly increased superoxide and 3-nitrotyrosine in PGI(2) synthase (PGIS). Concurrent administration of polyethylene-glycolated superoxide dismutase (SOD), l-nitroarginine methyl ester, or sepiapterin not only reversed the effects of high glucose on both angiotensin II-induced relaxation and PGI(2) release but also abolished high-glucose-enhanced PGIS nitration, as well as its association with eNOS. Furthermore, diabetes significantly suppressed PGIS activity in parallel with increased superoxide and PGIS nitration in the aortas of diabetic C57BL6 mice but had less effect in diabetic mice either lacking eNOS or overexpressing human SOD (hSOD(+/+)), suggesting an eNOS-dependent PGIS nitration in vivo. We conclude that diabetes increases PGIS nitration in vivo, likely via dysfunctional eNOS.

Lysophosphatidylcholine Potentiates Phenylephrine Responses in Rat Mesenteric Arterial Bed through Modulation of Thromboxane A2

Lysophosphatidylcholine (LPC) plays important physiological and pathophysiological roles in the cardiovascular system. Despite this, there is little information about its effects on vasore-activity of resistance vessels. The present study was designed to characterize the effects of LPC in the isolated perfused rat mesenteric arterial bed (MAB) and to investigate the underlying mechanisms of the changes it produced. Perfusion with 10 microM LPC for 40 min did not significantly affect basal perfusion pressure or reactivity of MAB to the alpha(1)-adrenoceptor agonist phenylephrine (PE) but almost completely abolished the maximal endothelium-dependent relaxation to acetylcholine (Ach), reducing it from 93 +/- 5 to 7 +/- 4% (p < 0.001). After washout of LPC for 60 min, the vasodilator response to Ach partially recovered, whereas the vasoconstrictor response to PE was markedly enhanced, the pD(2) value increasing from 7.50 +/- 0.04 to 8.13 +/- 0.15 and maximum response to 199 +/- 24% of control (p < 0.001). Pretreatment with either indomethacin, a nonselective inhibitor of cyclooxygenase, or SQ-29548 [[1S-[1a,2a(Z),3a,4a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino] methyl]-7-oxabicyclo [2.2.1]hept-2-yl]-5-heptanoic acid], a selective thromboxane receptor antagonist, completely prevented the potentiation of the PE response after washout of LPC. In untreated MABs, only the highest concentration of PE produced a significant increase in thromboxane A(2) (TxA(2)) production (assessed by enzyme-immunoassay of thromboxane B(2) levels). This was prevented by perfusion with LPC but was significantly increased after LPC washout. The basal release of TxA(2) was not modified by LPC. These results demonstrate that LPC exerts both immediate and residual effects on the reactivity of the rat MAB and that these effects are at least partially due to modification of PE-induced TxA(2) production.

Enhanced vascular reactivity of small mesenteric arteries from diabetic mice is associated with enhanced oxidative stress and cyclooxygenase products

Vascular reactivity to the alpha-adrenoceptor agonist phenylephrine (PE) was enhanced in small mesenteric arteries (SMA) from diabetic (db/db) mice under both high and low in vitro oxygen conditions. Mechanical removal of the endothelium significantly attenuated the enhanced vascular reactivity of SMA from db/db mice. Acute incubation of the SMA with sepiapterin, a precursor of tetrahydrobiopterin, and N(omega)-nitro L-arginine (L-NA), an inhibitor of nitric oxide (NO) synthase (NOS), resulted in no significant change in the enhanced vascular reactivity to PE in db/db mice. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels in SMA were not different between db/+ and db/db mice. Acute incubation of SMA with a combination of polyethylene glycol superoxide dismutase and catalase significantly reduced the enhanced contraction to PE in db/db mice. There were higher levels of malondialdehyde, a marker of lipid peroxidation and basal superoxide as measured by dihydroethidium staining, in SMA from db/db mice compared to db/+ mice. Acute incubation with indomethacin, a nonselective inhibitor of cyclooxygenase, SQ 29548, a selective thromboxane receptor antagonist and furegrelate, a thromboxane synthesis inhibitor, significantly attenuated the enhanced contraction to PE in SMA from db/db mice. This study demonstrates that the enhanced contractility of SMA from db/db mice to PE was endothelium dependent and involves elevated reactive oxygen species, cyclooxygenase activity and thromboxane synthesis, but not changes in the eNOS/NO pathway.

The vasoconstrictor effect of 8-epi prostaglandin F2alpha in the hypoxic rat heart.

1. 8-epi prostaglandin (PG) F2alpha, a vasoconstrictor isoprostane, is synthesized under conditions of oxidative stress. This study was undertaken to investigate the vasoconstrictor effect of 8-epi PGF2alpha in the coronary circulation before and after a period of oxidative stress. 2. The effects of the isoprostane 8-epi PGF2alpha and the thromboxane mimetic U46619 were compared in the isolated rat heart perfused in the Langendorff mode at a constant pressure of 80 mmHg. 3. In normal hearts U46619 caused a dose-related reduction in coronary flow (ED50 4.7+/-2.2 nmol). In contrast, 8-epi PGF2alpha had no effect. 4. After reducing perfusion pressure to 20 mmHg for 30 min and reperfusing at 80 mmHg, the dose-response curve to U46619 was unaffected. In contrast, 8-epi PGF2alpha caused a dose-dependent drop in coronary flow (ED50 52.6+/-12.7 nmol), producing a similar maximal reduction to U46619. 5. Similarly, after perfusion with xanthine and xanthine oxidase for either 15 or 30 min there was little change in the response to U46619 in comparison to control hearts. In contrast, 8-epi PGF2alpha caused a reduction in coronary flow similar to that produced by U46619, the magnitude of the response being related to the length of xanthine/xanthine oxidase perfusion. 6. Responses to both U46619 and 8-epi PGF2alpha after xanthine/xanthine oxidase perfusion were blocked by the selective thromboxane receptor antagonist SQ29548 10(-7) M. 7. These results show that oxidative stress in the isolated perfused rat heart reveals a potent vasoconstrictor effect of the isoprostane 8-epi PGF2alpha by an action on the thromboxane receptor. 8. The data also suggest that, since 8-epi PGF2alpha is a partial agonist at the thromboxane receptor, thromboxane receptor reserve is increased by oxidative stress.

ADP-, PAF- and adrenaline-induced platelet aggregation and thromboxane formation are not affected by a thromboxane receptor antagonist at physiological external Ca ++ concentrations

Thromboxane (TX) receptor antagonists are of considerable clinical interest in prevention of acute thrombembolic vessel occlusion. This study demonstrates that the selective TX receptor antagonist, daltroban, at a concentration (10 μM) that does not inhibit TX synthesis, markedly inhibits ADP-, PAF- and adrenaline-induced platelet secretion and TX formation. With the exception of ADP-induced platelet secretion, these actions are only detectable in citrated platelet-rich plasma but not in plasma anticoagulated by hirudin. Since TX antagonists are supposed to act at physiological external Ca ++ concentrations in the clinics, it is questionable whether in vitro studies in Ca ++-deprived media are the optimum model to evaluate the clinical potential of these compounds.

Prevention of complement-induced pulmonary hypertension and improvement of right ventricular function by selective thromboxane receptor antagonism

The effect of complement activation on the pulmonary vascular system and on right ventricular function was studied in sheep ( n = 12) by injection of cobra venom factor. Animals were instrumented for measurement of pulmonary flow, mean pulmonary artery pressure, right ventricular stroke work, arterial blood gases, and systemic vascular resistance. Blood was sampled from the left atrium and pulmonary artery to measure thromboxane B2, the metabolite of thromboxane A2, by radioimmunoassay. After baseline measurements, animals were randomly assigned to receive a selective thromboxane receptor antagonist SQ30741 as a 10 mg/kg bolus with an infusion of 10 mg/kg per hour or else to receive vehicle. Cobra venom factor was then injected (30 U/kg) in all animals, and data were recorded at 15, 30, 60, 90, and 120 minutes. In control animals there was a 2.4-fold increase in mean pulmonary artery pressure and a 76% increase in right ventricular stroke work at 15 minutes from baseline ( p < 0.05); these values remained elevated for 30 minutes and returned to baseline by 1 hour with no change in systemic vascular resistance. Arterial oxygenation decreased by 124% at 15 minutes and remained depressed through the experiment, but in treated animals oxygen tension remained unchanged from baseline. Thromboxane B2increased 95% from baseline in the control group and 1.5 fold in treated animals and followed a similar time course as the functional measurements ( p < 0.05). A pulmonary vascular thromboxane B2gradient of approximately 1000 pg/ml was measured at 15 and 30 minutes in both control and treated groups ( p < 0.05) We conclude that after complement activation in this model pulmonary hypertension and decreased oxygen tension are mediated by thromboxane release from the pulmonary vascular bed. This increased afterload causes a stress on the right ventricle as demonstrated by the increased right ventricular stroke work. Selective thromboxane receptor antagonism may be a beneficial therapy for pulmonary hypertension in patients after cardiopulmonary bypass. (J THORAC CARDIOVASC SURG 1994;107:800-6)

Endothelin‐induced contraction and mediator release in human bronchus

1. To elucidate the role of acetylcholine and various autacoids in endothelin-1 (ET-1)-induced contraction in human bronchus, the effects of various receptor antagonists were examined. In addition, the ability of ET-1 to stimulate the release of histamine, peptidoleukotrienes and prostanoids was determined. 2. ET-1 was a potent and effective contractile agonist in human bronchus, possessing similar potency and efficacy to leukotriene D4 (LTD4); EC50 (-log M): ET-1 = 7.76 +/- 0.09, n = 7; LTD4 = 8.46 +/- 0.53, n = 7; P > 0.2; maximum response (% 10 microM pre-carbachol): ET-1 = 103.8 +/- 17.4, n = 7; LTD4 = 95.5 +/- 9.3, n = 7; P > 0.6. 3. The cyclo-oxygenase inhibitor, sodium meclofenamate (1 microM) or the potent and selective thromboxane receptor antagonist, SQ 29,548 (1 microM) were without significant effect on ET-1 concentration-response curves. 4. In the presence of sodium meclofenamate (1 microM), the muscarinic receptor antagonist, atropine (1 microM), the platelet activating factor (PAF) receptor antagonist, WEB 2086 (1 microM) or the combination of the H1-histamine receptor antagonist, mepyramine (10 microM) and the leukotriene receptor antagonist, SK&F 104353 (10 microM), were without marked effect on ET-1 concentration-response curves. In addition, the combination of all four receptor antagonists did not antagonize ET-1-induced contraction. 5. ET-1 (0.3 microM) did not stimulate the release of histamine or immunoreactive leukotrienes from human bronchus. 6. ET-1 (0.3 microM) significantly stimulated the release of prostaglandin D2 (PGD2), 9alpha, 11beta PGF2 (PGD2 metabolite), PGE2, 6-keto PGF1alpha (PGI2 metabolite), PGF2alpha, and thromboxane B2 (TxB2) a lower concentration, 10 nM, was without effect on prostanoid release. The production of PGD2 was increased 7.5 fold, whereas the release of the other prostanoids was stimulated only about 1.6 to 2.7 fold.7. These data provide evidence that ET-1 elicits contraction of human isolated bronchus predominantly via a direct mechanism with no significant involvement of the release of acetylcholine, leukotrienes,histamine or PAF. Although ET-1 increased the release of several prostanoids they did not have a significant modulatory effect on the smooth muscle contraction.

Effects of a selective thromboxane receptor antagonist (GR32191B) and of glyceryl trinitrate on bleeding time in man.

1. GR32191B is a potent and selective thromboxane receptor antagonist. Glyceryl trinitrate (GTN) also inhibits platelet function in vitro. Both have been reported to prolong bleeding time. Since they may be used together in patients with coronary artery disease, we have investigated the possibility of an interaction between them. 2. Twenty-four healthy male volunteers were treated with GR32191B using a double-blind placebo-controlled crossover design. Bleeding times were determined before and after GR32191B or placebo and during co-administration of GTN. Plasma GR32191B concentration was measured. Platelet aggregation in response to adenosine diphosphate and to a thromboxane mimetic, U46619, was measured ex vivo. Urinary excretion of thromboxane (TX)B2 and 2,3-dinor-TXB2 was determined in 24 h urine samples. 3. Twelve hours after GR32191B (80 mg; p.o.), the plasma concentration was 36.6 +/- 2.7 nM (mean +/- s.e. mean) and bleeding time was increased by 66%. Ninety minutes after a second dose (40 mg; p.o.) the plasma concentration was 431.9 +/- 23.6 nM but bleeding time was not further prolonged. 4. Responses of platelets to U46619 were selectively antagonised following GR32191B. Urinary excretion rates of TXB2 and 2,3-dinor TXB2 were similar after treatment with GR32191B or placebo. 5. GTN (1 mg sub-lingually) had no significant effect on bleeding time 90-100 min following either placebo or GR32191B. 6. We conclude that GR32191B, in a dose that causes profound yet specific blockade of thromboxane receptors, causes only a modest prolongation of bleeding time that is unaffected by a therapeutic dose of GTN. This may prove advantageous when GR32191B is used in patients with ischaemic heart disease.

The thromboxane receptor antagonist SQ 30,741 reduces myocardial infarct size in monkeys when given during reperfusion at a threshold dose for improving reflow during thrombolysis

The threshold dose of the selective thromboxane receptor antagonist SQ 30,741 for increasing reflow during thrombolysis was identified and then evaluated in a model of myocardial ischemia with reperfusion. In anesthetized cynomolgus monkeys, stenotic carotid arteries were occluded with a platelet-rich thrombus by electrical stimulation and recanalized with streptokinase (680 U/min intraarterially for 1 h) and heparin (200 U/kg + 120 U/h intravenously for 3 h). Concurrent administration of SQ 30,741 (2.1 mg/kg + 0.5 mg/kg per h intravenously for 3 h; n = 4) enhanced the extent of reflow 174% compared with saline solution (n = 4; p < 0.05) during the third hour, when lower doses were ineffective.This threshold dose was tested in anesthetized African green monkeys subjected to 90 min of left circumflex coronary artery occlusion and 5 h of reperfusion. SQ 30,741 (n = 8) or saline solution (n = 11) was administered 2 min before reperfusion and continued throughout reperfusion. The heart was removed on termination of reperfusion and perfused in vitro with Evans blue and triphenyltetrazolium chloride dyes to stain tissue at risk and infarcted tissue, respectively. The percent of left ventricle at risk did not differ between saline- (37 ± 4%) and SQ 30,741-treated (35 ± 3%) monkeys. In contrast, infarcted tissue expressed as percent of the left ventricle at risk was less (p < 0.01) in monkeys receiving SQ 30,741 (31 ± 2%) than in those receiving saline solution (49 ± 5%).This 36% reduction in infarct size occurred without significant differences between treatments in hemodynamic variables and myocardial blood flows (as assessed by radioactive microspheres) measured during control, occlusion and reperfusion intervals. Thus, a dose of SQ 30,741 that stabilizes reflow after thrombolysis also possesses antiischemic activity when administered only during coronary reperfusion in monkeys.

BM 13.505, a selective thromboxane receptor antagonist, reduces myocardial infarct size following coronary artery reperfusion

This study was designed to assess the effectiveness of the thromboxane receptor antagonist, BM 13.505, in limiting myocardial infarct size in rats subjected to 30 min of coronary artery occlusion followed by reperfusion for 5.5 hr ( MI R . Myocardial infarct size was determined histochemically with triphenyltetrazolium chloride staining of the left ventricle. BM 13.505 (30 mg/kg, i.p.) was administered 1 min prior to coronary artery occlusion. In MI R -vehicle treated animals, myocardial infarct size was 39 ± 6% of the left ventricle. In MI R-BM 13.505 treated animals, reperfusion injury was reduced by 50% to 19 ± 7% of the left ventricle (p < 0.05, compared to the MI R -vehicle group). There were no significant differences in mean arterial blood pressure, heart rate, platelet count or white blood cell count between the treatment groups. Incubation of cultured L929 cells with the thromboxane/endoperoxide mimetic U 46619 produced a cytolytic effect, with an EC 50 value of 125 μM. Addition of BM 13.505 at concentrations up to 30 μM did not protect against the the cytolytic effect of U 46619, suggesting a non-receptor-mediated mechanism. These data indicate that hemodynamic, hematologic or cytoprotective factors do not explain the cardioprotective effects of BM 13.505. These results provide further evidence that antagonism of thromboxane receptors is beneficial in myocardial ischemia/reperfusion injury.