Endoperoxide Receptor Antagonist Research Articles

Terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, prevents hypertensive vascular hypertrophy and fibrosis

Thromboxane A(2) and other eicosanoids such as isoprostanes contribute to vascular proliferation and atherosclerosis by binding to the thromboxane/prostaglandin endoperoxide receptors. The effects of terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, on aorta remodeling were evaluated in spontaneously hypertensive stroke-prone rats (SHRSPs), a model of severe hypertension, endothelial dysfunction, vascular inflammation, and cerebrovascular diseases. Male SHRSPs were allocated to three groups receiving a standard diet (n = 5) or a high-sodium permissive diet plus vehicle (n = 6) or plus terutroban (30 mg · kg(-1) · day(-1); n = 6). After 6 wk of dietary treatment, all of the animals were injected with bromodeoxyuridine and simultaneously euthanized for aorta collection. The aortic media thickness-to-lumen ratio significantly (P < 0.0001) increased in the salt-loaded rats compared with the rats fed a standard diet, whereas terutroban treatment completely prevented media thickening (P < 0.001). When compared with vehicle, terutroban was also effective in preventing cell proliferation in the media, as indicated by the reduced number of bromodeoxyuridine-positive (P < 0.0001) and proliferating cell nuclear antigen-positive cells (P < 0.0001). Severe fibrosis characterized by a significant accumulation of collagen and fibronectin in the vascular wall was observed in the vehicle-treated rats (P < 0.01) but was completely prevented by terutroban (P < 0.001). The latter also inhibited heat shock protein-47 (P < 0.01) and TGF-1β expression (P < 0.001), which were significantly increased by the high-salt diet. In conclusion, terutroban prevents the development of aorta hyperplasia and has beneficial effects on fibrotic processes by affecting TGF-β and heat shock protein-47 expression in SHRSPs. These findings provide mechanistic data supporting the beneficial effects of terutroban in preventing or retarding atherogenesis.

Thromboxane Antagonism with terutroban in Peripheral Arterial Disease: the TAIPAD study.

Terutroban is a selective prostaglandin endoperoxide (TP) receptor antagonist with antithrombotic, antivasoconstrictive and antiatherosclerotic properties and is currently in development for long-term cardiovascular secondary prevention. TAIPAD is an international, double-blind, randomized controlled study comparing the effects of five dosages of oral terutroban vs. aspirin and placebo on platelet aggregation in peripheral arterial disease (PAD) patients. After 10 day's placebo run-in, included patients (n = 435; ankle-brachial pressure index, 0.7 ± 0.1) were randomly allocated to aspirin 75 mg day(-1), terutroban 1, 2.5, 5, 10 or 30 mg day(-1) or placebo. On day 5, the placebo group was reallocated to one of the terutroban groups for the rest of the study (day 83). Ex vivo platelet aggregation induced by the thromboxane analog U46619 (7 μm) was measured 24 h after dosing, as well as platelet aggregation induced by arachidonic acid (AA), collagen and ADP. Terutroban dose-dependently inhibited U46619-induced platelet aggregation at days 5 and 83. At day 5, the inhibition was significant vs. placebo for all terutroban dosages (P < 0.001). Terutroban (5, 10 and 30 mg day(-1)) was at least as effective as aspirin in inhibiting platelet aggregation induced by arachidonic acid (AA), collagen and adenosine diphosphate (ADP). Terutroban was well tolerated, with a safety profile similar to aspirin. In PAD patients, terutroban dose-dependently inhibited platelet aggregation 24 h after dosing, and was at least as effective as aspirin at 5, 10 and 30 mg day(-1). Terutroban was well tolerated.

Terutroban, a Thromboxane/Prostaglandin Endoperoxide Receptor Antagonist, Increases Survival in Stroke-Prone Rats by Preventing Systemic Inflammation and Endothelial Dysfunction: Comparison with Aspirin and Rosuvastatin

This study investigated the efficacy of terutroban, a specific thromboxane/prostaglandin endoperoxide receptor antagonist, on stroke incidence in spontaneously hypertensive stroke-prone rats (SHRSP). The effects of terutroban were compared with those of aspirin, another antiplatelet agent, and rosuvastatin, known to exert end-organ protection in SHRSP. Salt-loaded male SHRSP were treated orally once a day with vehicle, terutroban (30 mg/kg/day), aspirin (60 mg/kg/day), or rosuvastatin (10 mg/kg/day). Compared with vehicle, and regardless of any effect on blood pressure or serum thromboxane B(2) levels, terutroban significantly increased survival (p < 0.001) as a consequence of a delayed brain lesion occurrence monitored by magnetic resonance imaging (p < 0.001), and a delayed increase of proteinuria (p < 0.001). Terutroban decreased cerebral mRNA transcription of interleukin-1beta, transforming growth factor-beta, and monocyte chemoattractant protein-1 after 6 weeks of dietary treatment. Terutroban also prevented the accumulation of urinary acute-phase proteins at high molecular weight, identified as markers of systemic inflammation, and assessed longitudinally by one-dimensional electrophoresis. Terutroban also has protective effects on the vasculature as suggested by the preservation of endothelial function and endothelial nitric-oxide synthase expression in isolated carotid arteries. These effects are similar to those obtained with rosuvastatin, and superior to those of aspirin. Terutroban increases survival in SHRSP by reducing systemic inflammation as well as preserving endothelial function. These data support clinical development of terutroban in the prevention of cerebrovascular and cardiovascular complications of atherothrombosis.

Cyclooxygenase-2-Linked Attenuation of Hypoxia-Induced Pulmonary Hypertension and Intravascular Thrombosis

Exogenous prostacyclin is effective in reducing pulmonary vascular resistance in some forms of human pulmonary hypertension (PH). To explore whether endogenous prostaglandins played a similar role in pulmonary hypertension, we examined the effect of deleting cyclooxygenase (COX)-gene isoforms in a chronic hypoxia model of PH. Pulmonary hypertension, examined by direct measurement of right ventricular end systolic pressure (RVESP), right ventricular hypertrophy (n = 8), and hematocrit (n = 3), was induced by 3 weeks of hypobaric-hypoxia in wild-type and COX-knockout (KO) mice. RVESP was increased in wild-type hypoxic mice compared with normoxic controls (24.4 +/- 1.4 versus 13.8 +/- 1.9 mm Hg; n = 8; p < 0.05). COX-2 KO mice showed a greater increase in RVESP following hypoxia (36.8 +/- 2.7 mm Hg; p < 0.05). Urinary thromboxane (TX)B(2) excretion increased following hypoxia (44.6 +/- 11.1 versus 14.7 +/- 1.8 ng/ml; n = 6; p < 0.05), an effect that was exacerbated by COX-2 gene disruption (54.5 +/- 10.8 ng/ml; n = 6). In contrast, the increase in 6-keto-prostacyclin(1alpha) excretion following hypoxia was reduced by COX-2 gene disruption (29 +/- 3 versus 52 +/- 4.6 ng/ml; p < 0.01). Tail cut bleed times were lower following hypoxia, and there was evidence of intravascular thrombosis in lung vessels that was exacerbated by disruption of COX-2 and reduced by deletion of COX-1. The TXA(2)/endoperoxide receptor antagonist ifetroban (50 mg/kg/day) offset the effect of deleting the COX-2 gene, attenuating the hypoxia-induced rise in RVESP and intravascular thrombosis. COX-2 gene deletion exacerbates pulmonary hypertension, enhances sensitivity to TXA(2), and induces intravascular thrombosis in response to hypoxia. The data provide evidence that endogenous prostaglandins modulate the pulmonary response to hypoxia.

Renal Effects of S18886 (Terutroban), a TP Receptor Antagonist, in an Experimental Model of Type 2 Diabetes

Thromboxane A(2) (TxA(2)) is assumed to contribute to the development of diabetes complications, including nephropathy. We investigated whether the selective thromboxane-prostanoid endoperoxide receptor antagonist, S18886, ameliorates renal damage in uninephrectomized (UNX) obese Zucker rats (OZR). S18886, at doses of 10 (S18886-10) and 30 (S18886-30) mg x kg(-1) x day(-1), was administered to UNX-OZR by gavage over 8 weeks (n = 8 each group). UNX lean rats (n = 12) and OZR rats that received placebo (OZR-PLAC, n = 8) served as controls. As compared with the OZR-PLAC, S18886 had no significant effect on the elevated blood pressure and the enhanced creatinine clearance, while augmented proteinuria was partially prevented (-12 and -37%, low and high dose, respectively; NS). The increased excretion of transforming growth factor beta(1) (TGF-beta(1)) and of the thromboxane metabolite 2,3-dinor thromboxane B(2) (TxB(2)) was lowered (P < 0.05). S18886 prevented both the enhanced mesangiolysis (P < 0.01) in the OZR-PLAC as well as enlargement and degeneration of podocytes. In the blood, S18886-30 augmented the antioxidant enzymes (P < 0.01) and lessened the increase of plasma advanced oxidation protein products (-25%, NS). Body weight, hyperglycemia, and dyslipidemia remained uninfluenced under both doses of treatment. S18886 has renoprotective properties in the model of UNX-OZR. It prevents mesangiolysis, reduces urinary TGF-beta(1) and 2,3-dinor-TxB(2) excretion, and enhances the antioxidative defense.

Intravascular Thrombosis After Hypoxia-Induced Pulmonary Hypertension

Background— Pulmonary hypertension induced by chronic hypoxia is characterized by thickening of pulmonary artery walls, elevated pulmonary vascular resistance, and right-heart failure. Prostacyclin analogues reduce pulmonary pressures in this condition; raising the possibility that cycloxygenase-2 (COX-2) modulates the response of the pulmonary vasculature to hypoxia. Methods and Results— Sprague-Dawley rats in which pulmonary hypertension was induced by hypobaric hypoxia for 14 days were treated concurrently with the selective COX-2 inhibitor SC236 or vehicle. Mean pulmonary arterial pressure (mPAP) was elevated after hypoxia (28.1±3.2 versus 17.2±3.1 mm Hg; n=8, P &lt;0.01), with thickening of small pulmonary arteries and increased COX-2 expression and prostacyclin formation. Selective inhibition of COX-2 aggravated the increase in mPAP (42.8±5.9 mm Hg; n=8, P &lt;0.05), an effect that was attenuated by the thromboxane (TX) A 2 /prostaglandin endoperoxide receptor antagonist ifetroban. Urinary TXB 2 increased during hypoxia (5.9±0.9 versus 1.2±0.2 ng/mg creatinine; n=6, P &lt;0.01) and was further increased by COX-2 inhibition (8.5±0.7 ng/mg creatinine; n=6, P &lt; 0.05). In contrast, urinary excretion of the prostacyclin metabolite 6-ketoprostaglandin F 1α decreased with COX-2 inhibition (8.6±3.0 versus 27.0±4.8 ng/mg creatinine; n=6, P &lt; 0.05). Platelet activation was enhanced after chronic hypoxia. COX-2 inhibition further reduced the PFA-100 closure time and enhanced platelet deposition in the smaller pulmonary arteries, effects that were attenuated by ifetroban. Mice with targeted disruption of the COX-2 gene exposed to chronic hypoxia had exacerbated right ventricular end-systolic pressure, whereas targeted disruption of COX-1 had no effect. Conclusions— COX-2 expression is increased and regulates platelet activity and intravascular thrombosis in hypoxia-induced pulmonary hypertension.

20-HETE-induced contraction of small coronary arteries depends on the activation of Rho-kinase.

20-HETE is a potent constrictor of small blood vessels and has been suggested to play a crucial role in the generation of myogenic tone and the development of hypertension. In the present study, we investigated the mechanisms by which exogenously applied 20-HETE modulates vascular tone in small porcine coronary arteries. In organ chamber experiments, 20-HETE elicited a concentration-dependent contraction of small porcine coronary artery rings that was partially inhibited by the cyclooxygenase inhibitor diclofenac, the thromboxane and endoperoxide receptor antagonist SQ29548, and the thromboxane A2 synthase inhibitor furegrelate. Removal of endothelium attenuated the response to 20-HETE, whereas preconstriction of endothelium-denuded vessels to 25% of the maximum response with KCl markedly enhanced the response to 20-HETE. This 20-HETE-induced contraction was not associated with a significant increase in the intracellular concentration of Ca2+. 20-HETE-induced contraction was also observed in beta-escin-permeabilized arteries precontracted with a submaximal concentration of Ca2+ and was abolished by the Rho-kinase inhibitor Y27632, but was insensitive to the PKC inhibitor RO 31-8220. 20-HETE elicited the phosphorylation of the myosin light chain (MLC20) in coronary artery rings, an effect that was sensitive to Y27632 and mimicked by the thromboxane analog U46619. These data suggest that in small porcine coronary arteries, 20-HETE can induce contraction by 2 mechanisms, one endothelium-dependent involving the cyclooxygenase-dependent generation of vasoconstrictor prostanoids, and the other endothelium-independent. The latter response is associated with the activation of Rho-kinase, phosphorylation of MLC20, and sensitization of the contractile apparatus to Ca2+.

Why Are Some Individuals Resistant to the Cardioprotective Effects of Aspirin?

It has been slightly more than 100 years since the synthesis, development, and commercialization of acetylsalicylic acid, the most widely consumed drug in the world. It is estimated that 35 000 kg are consumed daily in the United States and 6000 kg in the UK.1 Who would have ever thought that this widely consumed drug would achieve significant uses other than as an analgesic, antipyretic, and anti-inflammatory. The first report of a possible antithrombotic effect of aspirin appeared in 1953 in the Mississippi Valley Medical Journal .2 Dr Craven noticed that the patients who took Aspergum had a tendency to bleed more easily. He concluded that aspirin must be thinning the blood and because thrombosis of the coronary arteries led to myocardial infarction, if his patients took aspirin, they might be less prone to experiencing a myocardial infarction. See p 1650 These prescient observations were succeeded by the discovery by numerous investigators that aspirin could significantly decrease platelet function. In 1971, the mechanism by which aspirin inhibited platelet function began to unfold when Smith and Willis3 demonstrated that aspirin inhibited platelet prostaglandin synthesis. It was subsequently shown that aspirin irreversibly acetylated serine-529 close to the active site of the fatty acid cyclooxygenase (COX).4 In the case of the anucleate platelet, the enzyme is rendered inactive for its lifetime. Samuelsson and co-workers5 demonstrated that thromboxane A2 along with prostaglandin H2 were the arachidonic acid metabolites responsible for activation of the platelet. Thromboxane A2 is also a very effective vasoconstrictor and mitogenic substance. After the discovery of thromboxane A2, numerous studies followed that investigated its potential role in thrombotic cardiovascular diseases. Platelet thromboxane A2 synthesis is increased in acute myocardial infarction, unstable angina, thrombolysis therapy, percutaneous transluminal coronary artery angioplasty, and …

Synthesis and human beta-adrenoceptor activity of 1-(3,5-diiodo-4- methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ol derivatives in vitro.

Trimetoquinol (1, TMQ) is a potent nonselective beta-adrenergic receptor (AR) agonist and a thromboxane A(2)/prostaglandin endoperoxide (TP) receptor antagonist, while 3',5'-diiodo-TMQ (2) exhibits beta(3)-AR selectivity. In search of selective beta(3)-AR agonists as potential drugs for the treatment of human obesity and type II diabetes mellitus, a series of 1-(3, 5-diiodo-4-methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ols has been prepared and evaluated for their biological activities at human beta(1)-, beta(2)-, and beta(3)-ARs expressed in Chinese hamster ovary (CHO) cells. The compounds have been synthesized by the Bischler-Napieralski cyclization of corresponding amides followed by NaBH(4) reduction, and the halogens in the aromatic ring A were introduced by direct halogenation of protected compound 11. Whereas halogen substitution in ring A reduced either potency or intrinsic activity on beta(3)-AR, the non-halogen-substituted compounds 8 and 10 were potent, selective, nearly full agonists for beta(3)-AR.

Involvement of thromboxane A2 in the endothelium-dependent contractions induced by myricetin in rat isolated aorta.

1. The present study was undertaken to analyse the mechanism of the contractile response induced by the bioflavonoid myricetin in isolated rat aortic rings. 2. Myricetin induced endothelium-dependent contractile responses (maximal value=21+/-2% of the response induced by 80 mM KCl and pD2=5.12+/-0.03). This effect developed slowly, reached a peak within 6 min and then declined progressively. 3. Myricetin-induced contractions were almost abolished by the phospholipase A2 (PLA2) inhibitor, quinacrine (10 microM), the cyclo-oxygenase inhibitor, indomethacin (10 microM), the thromboxane synthase inhibitor, dazoxiben (100 microM), the putative thromboxane A2 (TXA2)/prostaglandin endoperoxide receptor antagonist, ifetroban (3 microM). These contractions were abolished in Ca2+-free medium but were not affected by the Ca2+ channel blocker verapamil (10 microM). 4. In cultured bovine endothelial cells (BAEC), myricetin (50 microM) produced an increase in cytosolic free calcium ([Ca2+]i) which peaked within 1 min and remained sustained for 6 min, as determined by the fluorescent probe fura 2. This rise in [Ca2+]i was abolished after removal of extracellular Ca2+ in the medium. 5. Myricetin (50 microM) significantly increased TXB2 production both in aortic rings with and without endothelium and in BAEC. These increases were abolished both by Ca2+-free media and by indomethacin. 6. Taken together, these results suggests that myricetin stimulates Ca2+ influx and subsequently triggers the activation of the PLA2 and cyclo-oxygenase pathways releasing TXA2 from the endothelium to contract rat aortic rings. The latter response occurs via the activation of Tp receptors on vascular smooth muscle cells.

Prostaglandin endoperoxide-dependent vasospasm in bovine coronary arteries after nitration of prostacyclin synthase.

In the present study we used a bioassay to study the effects of peroxynitrite (ONOO-) on angiotensin II (A-II)-triggered tension in isolated bovine coronary arteries in order to show the consequences of the previously reported PGI2-synthase inhibition by ONOO- in this model. The following results were obtained: 1. 1 micromol L(-1) ONOO- impaired A-II-induced vasorelaxation and caused a second long lasting constriction phase. Indomethacin (10(-5)M) prevented both effects. U51605, a dual blocker of PGI2-synthase and thromboxane (TX)A2-synthase mimicked the effects of ONOO-. 2. The selective TXA2/prostaglandin endoperoxide (PGH2) receptor antagonist SQ29548 antagonized the second vasoconstriction phase after ONOO- -treatment. Since a generation of TXA2 and 8-iso-prostaglandin F2alpha could be excluded a direct action of unmetabolized PGH2 on the TXA2/PGH2 receptor was postulated. 3. ONOO- dose-dependently inhibited the conversion of 14C-PGH2 into 6-keto-PGF1alpha in isolated bovine coronary arteries with an IC50-value of 100 nM. 4. Immunoprecipitation of 3-nitrotyrosine-containing proteins with a monoclonal antibody revealed PGI2-synthase as the only nitrated protein in bovine coronary arteries treated with 1 micromol 1(-1) ONOO-. 5. Using immunohistochemistry a co-localization of PGI2-synthase and nitrotyrosine-containing proteins was clearly visible in both endothelial and vascular smooth muscle cells. We concluded that ONOO- not only eliminated the vasodilatory, growth-inhibiting, antithrombotic and antiadhesive effects of PGI2 but also allowed and promoted an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2.

Functional response of the rat kidney to inhibition of nitric oxide synthesis: role of cytochrome p450-derived arachidonate metabolites.

1. We tested the hypothesis that nitric oxide (NO) exerts a tonic inhibitory influence on cytochrome P450 (CYP450)-dependent metabolism of arachidonic acid (AA). 2. N(omega)-nitro-L-Arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), increased mean blood pressure (MBP), from 91+/-6 to 137+/-5 mmHg, renal vascular resistance (RVR), from 9.9+/-0.6 to 27.4+/-2.5 mmHg ml(-1) min(-1), and reduced renal blood flow (RBF), from 9.8+/-0.7 to 6.5+/-0.6 ml min(-1)) and GFR from 1.2+/-0.2 to 0.6+/-0.2 ml 100 g(-1) min(-1)) accompanied by diuresis (UV, 1.7+/-0.3 to 4.3+/-0.8 microl 100 g(-1) min (-1)), and natriuresis (U(Na)V, 0.36+/-0.04 to 1.25+/-0.032 micromol 100 g(-1) min(-1)). 3. 12, 12 dibromododec-enoic acid (DBDD), an inhibitor of omega hydroxylase, blunted L-NAME-induced changes in MBP, RVR, UV and U(Na)V by 63+/-8, 70+/-5, 45+/-8 and 42+/-9%, respectively, and fully reversed the reduction in GFR by L-NAME. Clotrimazole, an inhibitor of the epoxygenase pathway of CYP450-dependent AA metabolism, was without effect. 4. BMS182874 (5-dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfo namide), an endothelin (ET)A receptor antagonist, also blunted the increases in MBP and RVR and the diuresis/natriuresis elicited by L-NAME without affecting GFR. 5. Indomethacin blunted L-NAME-induced increases in RVR, UV and U(Na)V. BMS180291 (1S-(1alpha,2alpha,3alpha,4alpha)]-2-[[3-[4-[(++ +pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl ]methyl]benzenepropanoic acid), an endoperoxide receptor antagonist, attenuated the pressor and renal haemodynamic but not the renal tubular effects of L-NAME. 6. In conclusion, the renal functional effects of the CYP450-derived mediator(s) expressed after inhibition of NOS with L-NAME were prevented by inhibiting either CYP450 omega hydroxylase or cyclooxygenase or by antagonizing either ET(A) or endoperoxide receptors. 20-hydroxyeicosatetraenoic acid (20-HETE) fulfils the salient properties of this mediator.

Thromboxane/Prostaglandin Endoperoxide–Induced Hypertrophy of Rat Vascular Smooth Muscle Cells Is Signaled by Protein Kinase C–Dependent Increases in Transforming Growth Factor-β

In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-beta (TGF-beta) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-beta in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-beta was supported by the following observations: (1) exogenous human TGF-beta 1 increased protein synthesis; (2) antibody to TGF-beta blocked both TGF-beta- and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-beta bioactivities; and (4) the actions of U46619 on protein content and TGF-beta bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-beta and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-beta (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-beta that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-beta bioactivity and stimulated protein synthesis. The hyper-trophic response to bFGF was blocked by anti-TGF-beta. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-beta-induced suppression of proliferation, as evidenced by the ability of antibody to TGF-beta to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis as well as active and total TGF-beta bioactivities. By contrast, the protein kinase C inhibitors did not prevent the increases in protein synthesis induced by exogenous TGF-beta. The results demonstrate that thromboxane/prostaglandin endoperoxide signals increased TGF-beta bioactivity via protein kinase C. Increases in both bFGF and TGF-beta are required for an optimal hypertrophic response to U46619. The hypertrophic response to TGF-beta occurs through a protein kinase C-independent pathway.

Effect of ifetroban, a thromboxane A2 receptor antagonist, in stroke-prone spontaneously hypertensive rats.

Experiments were performed to determine the effect of chronic therapy with the potent and long-acting thromboxane (TX) A2/prostaglandin endoperoxide (TP) receptor antagonist, ifetroban, on hypertension development and the incidence of stroke in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP instrumented with radiotelemetry probes, for continuous monitoring of arterial blood pressure, were given 1% NaCl to drink and Stroke-Prone Rodent Diet and were chronically treated with ifetroban (20 mg/kg/day, n = 10) or vehicle (n = 12) starting at 16.5 weeks of age. Ifetroban did not affect blood pressure or the development of proteinuria and cerebrovascular lesions. Chronic administration of a higher dose ifetroban (40 mg/kg/day) starting at 7 weeks of age was also without effect on blood pressure and stroke in noninstrumented saline-drinking SHRSP. These results do not support a major role for TXA2 and its endoperoxide precursors in the elevation of blood pressure and the development of cerebrovascular lesions in saline-drinking SHRSP.

Mechanisms of 3-Carene-lnduced Bronchoconstriction in the Isolated Guinea Pig Lung

Inhaled 3-carene at a concentration of 5,000 mg/m3 caused bronchoconstriction in isolated, ventilated and perfused guinea pig lungs. This effect was inhibited by the cyclooxygenase inhibitor diclofenac (100 microM) and the thromboxane/prostaglandin endoperoxide-receptor antagonist L-670,596 (1 microM). 3-Carene exposure also increased the amount of thromboxane in the perfusate from the lungs. In cultured calf pulmonary arterial endothelial cells 3-carene caused a dose-related release of arachidonic acid. Thus, the results obtained in this experimental model may have implications in the understanding of the pathophysiology of 3-carene-induced obstructive pulmonary disease in humans.

Dose-related cardioprotection by ifetroban in relation to inhibition of thrombosis and ex vivo platelet function.

The dose-related cardioprotective efficacy of the thromboxane A2/prostaglandin endoperoxide (TP) receptor antagonist, ifetroban (BMS-180291), was investigated in an anesthetized ferret model of myocardial ischemia (90 min) followed by reperfusion (5 h). Treatment was begun at either the 75th minute of ischemia or 5 min after initiating reperfusion. The magnitude of TP receptor blockade was evaluated by ex vivo platelet function. Additional experiments in ferrets tested the antithrombotic potency of ifetroban as an inhibitor of thrombotic cyclic flow reduction (CFR) in the stenosed abdominal aorta (Folts model). Continuous ifetroban infusions of 0.03, 0.1 and 0.3 mg/kg/h reduced myocardial infarct size from 22% of the left ventricle in vehicle-control ferrets to 20, 12 and 9%, respectively. These represented reductions in infarct size of 8, 43 and 56%. Delaying initiation of treatment with high-dose ifetroban until 5 min into reperfusion also significantly reduced infarct size by 45%. High-dose ifetroban treatment did not prevent neutrophil (PMNL) accumulation measured as tissue myeloperoxidase activity in infarcted tissue. At the end of the 5-hour reperfusion period, the low, medium and high doses produced 90, 98 and 98% blockade of platelet TP receptors, respectively, measured as inhibition of ex vivo platelet shape change responses to U-46,619. Ifetroban inhibited thrombotic CFR at a threshold dose of 0.03 +/- 0.004 mg/kg, which antagonized 92% of ferret platelet TP receptors. Thus, ifetroban exhibited cardioprotective and antithrombotic activities and was effective at doses producing > 90% TP receptor blockade. Cardioprotective activity was not associated with any reductions of PMNL accumulation in infarcted tissue and was demonstrable even when treatment was delayed until 5 min after initiation of reperfusion.

Effect of picotamide on the calcium mobilization and phospholipase C activation in human platelets

The effect of picotamide (G137 or N,N′-bis-3-picolyl-4-methoxy-isophthalamide), a dual thromboxane A 2 (TxA 2) synthetase inhibitor/TxA 2 endoperoxide receptor antagonist, on the phospholipase C (PLC) activation and calcium mobilization in human platelets stimulated by arachidonic acid (AA) and TxA 2 receptor synthetic agonist U46619, has been studied. Preincubation with picotamide (10 −4 M) for 1 and 3 min significantly reduced (p<0.03 and p<0.005 respectively) the calcium concentration changes induced in gel-filtered platelets (GFP s) by U46619 125 nM and 250 nM. Picotamide also reduced the calcium concentration changes induced in GFP s by AA 75 and 150 μM and by ADP 5 and 10 μM. In thrombin degranulated platelets picotamide inhibited the effect of U46619 up to 500 nM. The PLC activation, as indicated by inositol-1,3,4 P 3 (Ins 1,3,4 P 3) formation in response to U46619 250 nM and AA 150 μM was also inhibited by picotamide. These results may suggest a dual effect of picotamide on the receptor/effector systems through which TxA 2 mediates platelet activation.

Sensory neuropeptide-mediated bronchoconstriction of the guinea pig lung by diamide; a comparison to hydrogen peroxide

The effect of the thiol oxidizing agent diamide on airway conductance, dynamic compliance and perfusion flow of isolated perfused and ventilated guinea pig lungs was investigated. When infused in the pulmonary circulation, diamide (100 μM) induced bronchoconstriction, but no effect on perfusion flow was observed. Although diamide exposure induced the formation of thromboxane A 2, the thromboxane/prostaglandin endoperoxide receptor antagonist L-670,596 did not affect the decrease in conductance and compliance induced by diamide. Diamide induced the release of the sensory neuropeptide calcitonin gene-related peptide. The bronchoconstriction and the release of calcitonin gene-related peptide induced by diamide were abolished by capsaicin pretreatment of the guinea pigs. Combined pretreatment with the NK 1 and NK 2 receptor antagonists, CP-96,345 and SR-48968, attenuated the effect of diamide. Hydrogen peroxide-induced vaso- and bronchoconstriction was not affected by capsaicin-pretreatment, nor did hydrogen peroxide induce detectable release of calcitonin gene-related peptide. The results indicate that diamide activates sensory nerves and induces neuropeptide release and neurokinin receptor-mediated bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

Ridogrel improves maternal/fetal homeostasis in an ovine model of pregnancy-induced hypertension

The effects of ridogrel (a thromboxane synthetase inhibitor / endoperoxide receptor antagonist) were assessed in an ovine model of pregnancy-induced hypertension. Maternal serum prostacyclin and thromboxane levels were quanitiated using RIA, and maternal and neonatal coagulation status was assessed. Pregnancy and neonatal outcome were recorded. Ridogrel,(E)-5-[[[3-pyridinyl)[3-(tri-fluoromethyl)phenyl]methylen]amin]oxy]pentanoic acid, was administered in one bolus dose at 0.1 or 1.0mg/kg IV, three hours following the onset of a 27 hour magnesium sulfate infusion given hypertensive ewes to prevent maternal seizures. At both doses, ridogrel improved neonatal outcome (0% neonatal mortality in each ridogrel group versus 67% neonatal mortality in the magnesium sulfate group), and ridogrel at 0.1mg/kg IV normalized birth weights. Abnormalities of maternal platelet function (abnormal or no response to collagen), occurring during the ovine syndrome, resolved following ridogrel treatment. Ridogrel's effects on maternal and neonatal coagulation were more dramatic at the 0.1mg/kg IV dose. Ridogrel appeared to be beneficial in this model of pregnancy-induced hypertension.

Platelets Increase the Tone of Quiescent Rat Aortic Rings by Release of Serotonin and Potentiate the Subsequent Contractile Response to Norepinephrine

To examine the effect of platelets on the tone of quiescent vessels, rat aortic rings with intact endothelium and others with denuded endothelium were exposed to platelets in an organ bath. Suspension of washed platelets induced a mild but consistent contraction of rings with intact endothelium. The subsequent contractile response of these rings to norepinephrine (NE) was also potentiated. Platelets caused a marked contraction of quiescent rings without intact endothelium and potentiated the subsequent contractile effects of NE. The procontractile effect of platelets in rat aortic rings with and without intact endothelium was not modulated by the selective thromboxane A2 (TXA2)/endoperoxide receptor antagonist SQ29,548 or the cyclooxygenase inhibitor indomethacin, but was completely blocked by serotonin (S2) receptor antagonist LY53,857, suggesting that release of serotonin from aggregating platelets is perhaps more important than release of TXA2 in the procontractile effects of platelets on quiescent rat aortic rings. To examine the role of vascular endothelium in the procontractile effect of platelets further, we treated aortic rings with intact endothelium with the inhibitor of endothelium-derived relaxing factor (EDRF) NG-monomethyl-L-arginine (L-NMMA) or the adenine nucleotide scavenger apyrase. In rings treated with L-NMMA or apyrase, the contractile effect of platelets was enhanced (p < 0.01) and mimicked that in rings without intact endothelium. These observations indicate that although basal release of EDRF modulates the contractile effect of platelets, the dominant effect of platelets on quiescent rat aortic rings with intact endothelium is procontractile and this effect is mediated exclusively by release of serotonin.