Thromboxane Metabolites Research Articles

Pharmacological characterization of SC-57461A (3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl), a potent and selective inhibitor of leukotriene A(4) hydrolase I: in vitro studies.

Leukotriene (LT) B(4) is an inflammatory mediator that has been implicated in the pathogenesis of various diseases, including inflammatory bowel disease and psoriasis. As the rate-limiting step for LTB(4) production, LTA(4) hydrolase represents an attractive target for therapeutic agents that interfere with LTB(4) production. In the present study we evaluated a chemically novel compound designated SC-57461A (3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl) as an inhibitor of LTA(4) hydrolase. Pharmacological comparisons are made to its free acid SC-57461. SC-57461A is a potent competitive inhibitor of recombinant human LTA(4) hydrolase when either LTA(4) (IC(50) = 2.5 nM, K(i) = 23 nM) or peptide substrates (IC(50) = 27 nM) are used. In human whole blood, the IC(50) for calcium ionophore-induced LTB(4) production was 49 nM, indicative of good cell penetration. Whole blood production of the cyclooxygenase metabolite thromboxane B(2) was not affected. SC-57461A was also active in several other species, including mouse, rat, dog, and rhesus monkey. The data indicate that SC-57461A is a potent and selective inhibitor of LTA(4) hydrolase.

Effect of two oral contraceptives with different ethinyl estradiol and levonorgestrel concentrations on the urinary excretion of biochemical vasoactive markers

In the present study the effect on the urinary excretion of vasoactive markers of two oral contraceptives (OCs), i.e., Leios, containing 0.02 mg ethinyl estradiol and 0.1 mg levonorgestrel, and Stediril 30, containing 0.03 mg ethinyl estradiol and 0.15 mg levonorgestrel, was investigated. cGMP, prostacyclin and its antagonist thromboxane, serotonin, and urodilatin, a natriuretic and diuretic peptide formed in the kidney, were measured as markers. In a comparative, double-blind, randomized, parallel group study, 34 women received Leios and 33 women Stediril 30. Nocturnal urine was collected before treatment and during cyclic treatment after 3 and 12 cycles. Both contraceptives significantly enhanced cGMP excretion after 12 cycles. The prostacyclin metabolite remained unchanged for both formulations, but the excretion of the thromboxane metabolite was significantly decreased after 12 cycles. Thus, the ratio of prostacyclin to thromboxane, crucial for the resulting effect on vascular tone, increased significantly. For the serotonin metabolite, no changes were observed for both contraceptives. The excretion of urodilatin significantly increased for both preparations after 12 cycles compared to the pretreatment values. These results indicate that the low-dose OCs Leios and Stediril 30 may stimulate the production of some vasoactive markers, at least after 12 cycles of treatment. The positive influence of these contraceptives on the various markers investigated may improve vascular tone, impede development of atherosclerosis and arterial thrombosis, and improve water and electrolyte homeostasis. These effects most likely can be attributed to the estrogenic component. Levonorgestrel may elicit no impact on these estrogen-induced changes that, however, seem only to be manifested after a longer treatment period.

Effect of endotoxin on oleic acid lung injury does not depend on priming.

Recent studies have demonstrated significant synergistic physiological and biochemical effects between low-dose endotoxin (Etx) administration and oleic acid (OA)-induced canine lung injury. To evaluate whether this interaction depends on Etx priming of some key cell population, we compared the effects of giving low-dose Etx both after as well as before inducing lung injury with OA. In addition to hemodynamic and blood-gas measurements, positron emission tomographic imaging was used to measure edema accumulation and intrapulmonary blood flow distribution. Biochemical measurements of the stable metabolites of prostacyclin and thromboxane were obtained as well as measurements of isoprostanes and reactive sulfhydryls as evidence for possible concomitant oxidant production. We found that the physiological and biochemical effects of low-dose Etx developed 30-45 min after its administration, regardless of whether Etx was administered before or after OA. No increase in either isoprostane or reactive sulfhydryl production after Etx and/or OA was detected. These data suggest that the synergistic effect of low-dose Etx and OA-induced lung injury is not due to a priming effect of Etx.

Smoking-Induced Vascular Disease

Polycyclic aromatic hydrocarbons (PAHs) are recognized widely as potential environmental procarcinogens and have attracted their share of attention from both funding agencies and scientists interested in biomedical research. Curiously, their potential relevance to cardiovascular disease, rather than cancer, has stirred less emotion. This imbalance of attention is reflected also in society’s response to that most intimate form of self-pollution with PAHs (and much else), cigarette smoking. While the States have chosen to allocate varying proportions of their Tobacco Settlements to research, cancer tends to dominate the agenda. Yet, tobacco-related cardiovascular disease yields an even more striking burden on the public health than cancer. It has been estimated that cardiovascular morbidity and mortality are increased 4-fold in smokers and by one third in passive smokers.1 One might think that such daunting figures would prompt advocacy groups, such as the American Heart Association, to lobby vigorously at the State level for an appropriate allocation of the Tobacco Settlements to cardiovascular research. Our understanding of the mechanisms by which smoking mediates cardiovascular injury and the factors that determine interindividual susceptibility to smoking-induced tissue injury is remarkably limited. In this issue of Circulation Research , Kerzee and Ramos2 draw our attention to a mechanism of potential relevance to acceleration of atherogenesis by PAHs, including those in cigarette smoke. The aryl hydrocarbon receptor (Ahr) is a member of the family of basic helix loop helix proteins (bHLHs) that contain PAS domains. This family includes proteins related to differentiation, such as MyoD, the response to hypoxia, such as Hif1α, and circadian rhythms, such as CLOCK, …

Effect of Hypoxia, Oxidative Stress and Lipopolysaccharides on the Release of Prostaglandins and Cytokines from Human Term Placental Explants

Placental hypoxia, ischaemia, reperfusion and resultant oxidative stress, with the release of various factors into the maternal vasculature acting as mediators of endothelial cell dysfunction, play an important role in the development of pre-eclampsia. Human term placental tissue explants were exposed to different stressors, e.g. hypoxia, oxidative stress and lipopolysaccarides, and the effect on the release of prostanoids and cytokines was determined. The hypoxic environment consisted of 2 per cent O 2, 5 per cent CO 2and 93 per cent N 2. Oxidative stress was induced by addition of xanthine together with xanthine oxidase to the incubation medium. As a third experimental variable, lipopolysaccharide was added to the medium. Prostaglandins (8-iso-PGF 2α, or 6-keto-PGF 1αand TXB 2as stable metabolites of prostacyclin and thromboxane, respectively) and cytokines (TNF-α, IL-1α, IL-1β, IL-6) were measured using commercial ELISA assays. Under control conditions, the production of prostaglandins in ng/24 h (mean±s.d.) was 6±3 for 8-iso-PGF 2α, 19±9 for 6-keto-PGF 1αand 5±2 for TXB 2. The production of cytokines was 13±6 pg for TNF-α, 7±2 pg for IL-1α, 5±3 pg for IL-1β and 18±9 ng for IL-6. Under hypoxia the production of prostaglandins remained unchanged and of the cytokines only IL-1β showed a 15-fold increase. Oxidative stress resulted in an increase in the release of prostaglandins and of cytokines of 4- to 15- and 3- to 130-fold, respectively. Lipopolysaccharides and oxidative stress had a similar effect on the production of prostaglandins, whereas the stimulatory effect of lipopolysaccharides on cytokines was significantly higher than that of oxidative stress.

Urinary thromboxane, prostacyclin, cortisol, and 8-hydroxy-2'-deoxyguanosine in nonsmokers exposed and not exposed to environmental tobacco smoke.

This study tested the hypotheses that (1) increased platelet aggregation, as measured by 2,3-dinor-thromboxane B(2) (Tx-M) and 2,3-dinor-6-keto-prostaglandin F(1alpha) (PGI-M), and (2) increased oxidative stress, as measured by 8-Hydroxy-2'-deoxyguanosine (8-OHdG), would occur in ETS-exposed nonsmokers as compared with non-ETS-exposed nonsmokers. The concentrations of the stable urinary metabolites of thromboxane (Tx-M) and prostacyclin (PGI-M), cortisol and 8-OHdG were measured in a 24-h urine sample from 3 groups of subjects: 21 nonsmokers with minimal (15 min or less per day) ETS exposure (termed non-ETS-exposed), 22 nonsmokers with at least 5 h per day of ETS exposure (termed ETS-exposed), and 20 cigarette smokers who served as a positive control group. The self-reported levels of ETS exposure were verified by personal air monitors. As compared with either group of nonsmokers, cigarette smokers excreted significantly more urinary Tx-M. Non-ETS-exposed nonsmokers showed a statistically significantly higher level of urinary Tx-M over that seen in nonsmokers with considerably more ETS exposure. Urinary concentrations of PGI-M were marginally higher in the smokers and did not differ between the nonsmoker groups. Nonsmokers exposed to at least five h of ETS per day did not have significantly higher excretion of 8-OHdG than non-ETS-exposed nonsmokers. The results from this study suggest that platelet aggregation, as measured by the thromboxane metabolite Tx-M and prostacyclin metabolite PGI-M, is not associated with ETS exposure. Therefore, platelet aggregation is not a plausible or quantitatively consistent mechanism to explain the nonlinear dose-response hypothesis of cardiovascular disease and ETS exposure.

Thromboxane A 2 causes retarded clearance of aggregated protein in glomeruli of nephritic mice

Recently, it has been demonstrated that the production of prostaglandins and thromboxane is increased in patients with chronic glomerulonephritis and lupus nephritis. We recently demonstrated that thromboxane A 2 delayed the clearance of heat-aggregated bovine serum albumin deposited in glomeruli. In the present study, we investigated the effect of thromboxane A 2 on the clearance of macromolecules in nephritic glomeruli. First, we attempted to clarify the conditions for the clearance of heat-aggregated bovine serum albumin in nephritic glomeruli, using glomeruli isolated from control and anti-glomerular basement membrane nephritic mice. Heat-aggregated bovine serum albumin was injected twice into each mouse. The glomeruli were then isolated and incubated in culture medium. The heat-aggregated bovine serum albumin content of control glomeruli gradually diminished with incubation time up to 24 h. The heat-aggregated bovine serum albumin content of nephritic glomeruli was 69% higher than that of control glomeruli at 24 h incubation. The production of thromboxane B 2 (the stable metabolite of thromboxane A 2) in nephritic glomeruli showed about a sevenfold increase compared with control. DP-1904 [6-(1-imidazolylmethyl)-5,6,7,8-tetrahydro-naphthalene-2-carboxylic acid hydrochloride], a thromboxane A 2 synthase inhibitor, and KT2-962 [sodium 3-(4-(4-chlorophenyl-butylsulfonamido) butyl)-6-isopropylazulene-1-sulfonate], a selective thromboxane A 2 receptor antagonist, significantly reduced the heat-aggregated bovine serum albumin content in nephritic glomeruli. Normal glomeruli treated with U-46619 [15 S-hydroxy-11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid], a stable analogue of thromboxane A 2, had significantly more heat-aggregated bovine serum albumin than control glomeruli. We next investigated whether thromboxane A 2 could affect the uptake/disposal of heat-aggregated bovine serum albumin by cultured rat mesangial cells. U-46619 significantly enhanced the uptake and inhibited the disposal of heat-aggregated bovine serum albumin by mesangial cells. Finally, we performed experiments to elucidate the role of the thromboxane A 2 receptor (TP receptor) in the clearance of heat-aggregated bovine serum albumin using TP-deficient mice. The glomerular heat-aggregated bovine serum albumin content of TP-receptor knockout [TP(−/−)] mice was lower than that of wild-type [WT(+/+)] mice. U-46619 dose dependently increased the uptake of heat-aggregated bovine serum albumin by mesangial cells in WT(+/+) mice, but not in the TP(−/−) mice. These findings suggest that thromboxane A 2 retards the clearance of aggregated protein in nephritic glomeruli and may contribute to the pathophysiology of glomerulonephritis.

Aspirin dose and thromboxane metabolism in African American stroke patients

P214 Purpose: To evaluate thromboxane metabolism in African American stroke patients on different doses of aspirin. Methods: Consecutive African American patients within 3 month of non-cardioembolic stroke and not being treated with anticoagulation were recruited. Antithrombotic therapy at the time of sample collection was not prespecified and varied according to the practice patterns of the different attending physicians who treated the patients during their acute stroke. The thromboxane metabolite 11-dehydrothroboxane B2 (11-DTB2) was measured by enzyme immunoassay in random urine samples collected at the time of enrollment. Results: 99 patients were enrolled but 15 could not give a urine sample at the time of enrollment. Data on the remaining 84 patients were evaluable. There were 49 men and 35 women aged 36–87 (mean 62) years. Based on antithrombotic treatment during sample collections, we divided patients into four groups: no aspirin N=16 (no antithrombotic drugs N=4 or ticlopidine N=12), aspirin 81–325 mg/d N=19 (81 mg/day N=1, 325 mg/day N=18), aspirin 650 mg/d N=19, aspirin 975–1300 mg/d N=30 (975 mg/d N=2 and 1300 mg/d N=28). After log transformation of the 11-DTB2 values the geometric means in the no aspirin, 81–325 mg/d, 650 mg/d, and 975–1300 mg/d groups were 1355, 912, 758, and 731 pg/mg creatinine, respectively. Based on one way ANOVA, the mean 11-DTB2 concentration was significantly different between the 4 groups (p=0.03). There was a significant negative relationship between aspirin dose and 11-DTB2 concentration (Pearson Correlation, r = -0.26, P=0.02). Conclusion: In African American stroke patients thromboxane production appears to be progressively inhibited by aspirin doses up to 650 mg/d. The clinical significance of this effect by aspirin doses ≤650 mg/d remains to be determined.

Endogenous biosynthesis of thromboxane and prostacyclin in 2 distinct murine models of atherosclerosis

Thromboxane A2 is a potent vasoconstrictor and platelet agonist; prostacyclin is a potent platelet inhibitor and vasodilator. Altered biosynthesis of these eicosanoids is a feature of human hypercholesterolemia and atherosclerosis. This study examined whether in 2 murine models of atherosclerosis their levels are increased and correlated with the evolution of the disease. Urinary 2,3-dinor thromboxane B2 and 2,3-dinor-6-keto prostaglandin F1α, metabolites of thromboxane and prostacyclin, respectively, were assayed in apoliprotein E (apoE)-deficient mice on chow and low-density lipoprotein receptor (LDLR)-deficient mice on chow and a Western-type diet. Atherosclerosis lesion area was measured by en face method. Both eicosanoids increased in apoE-deficient mice on chow and in LDLR-deficient mice on a high-fat diet, but not in LDLR-deficient mice on chow by the end of the study. Aspirin suppressed ex vivo platelet aggregation, serum thromboxane B2, and 2,3-dinor thromboxane B2, and significantly reduced the excretion of 2,3-dinor-6-keto prostaglandin F1α in these animals. This study demonstrates that thromboxane as well as prostacyclin biosynthesis is increased in 2 murine models of atherogenesis and is secondary to increased in vivo platelet activation. Assessment of their generation in these models may afford the basis for future studies on the functional role of these eicosanoids in the evolution and progression of atherosclerosis.

Endogenous biosynthesis of thromboxane and prostacyclin in 2 distinct murine models of atherosclerosis

AbstractThromboxane A2 is a potent vasoconstrictor and platelet agonist; prostacyclin is a potent platelet inhibitor and vasodilator. Altered biosynthesis of these eicosanoids is a feature of human hypercholesterolemia and atherosclerosis. This study examined whether in 2 murine models of atherosclerosis their levels are increased and correlated with the evolution of the disease. Urinary 2,3-dinor thromboxane B2 and 2,3-dinor-6-keto prostaglandin F1α, metabolites of thromboxane and prostacyclin, respectively, were assayed in apoliprotein E (apoE)-deficient mice on chow and low-density lipoprotein receptor (LDLR)-deficient mice on chow and a Western-type diet. Atherosclerosis lesion area was measured by en face method. Both eicosanoids increased in apoE-deficient mice on chow and in LDLR-deficient mice on a high-fat diet, but not in LDLR-deficient mice on chow by the end of the study. Aspirin suppressed ex vivo platelet aggregation, serum thromboxane B2, and 2,3-dinor thromboxane B2, and significantly reduced the excretion of 2,3-dinor-6-keto prostaglandin F1α in these animals. This study demonstrates that thromboxane as well as prostacyclin biosynthesis is increased in 2 murine models of atherogenesis and is secondary to increased in vivo platelet activation. Assessment of their generation in these models may afford the basis for future studies on the functional role of these eicosanoids in the evolution and progression of atherosclerosis.

Directed vascular expression of the thromboxane A2 receptor results in intrauterine growth retardation.

Thromboxane (Tx) A2 is a platelet agonist, smooth muscle cell constrictor, and mitogen. Urinary Tx metabolite (Tx-M) excretion is increased in syndromes of platelet activation and early in both normal pregnancies and in pregnancy-induced hypertension. A further increment occurs in patients presenting with severe preeclampsia, in whom Tx-M correlates with other indices of disease severity. TxA2 exerts its effects through a membrane receptor (TP), of which two isoforms (alpha and beta; refs. 5,6) have been cloned. Overexpression of TP in the vasculature under the control of the pre-proendothelin-1 promoter results in a murine model of intrauterine growth retardation (IUGR), which is rescued by timed suppression of Tx synthesis with indomethacin. IUGR is commonly associated with maternal diabetes or cigarette smoking, both conditions associated with increased TxA2 biosynthesis.

Levels of stable metabolites of prostacyclin and thromboxane A2and their ratio in normotensive and preeclamptic pregnant women during the antepartum and postpartum periods

Levels of stable metabolites of prostacyclin and thromboxane A₂and their ratio in normotensive and preeclamptic pregnant women during the antepartum and postpartum periods

Changes of the thromboxane A 2/prostacyclin balance in the urine of patients with renal diseases analyzed by gas chromatography/selected ion monitoring

The thromboxane A 2/prostacyclin (TX/PGI) ratios were measured in patients with renal diseases to elucidate the relationship between the ratios and the pathological changes of the diseases. Urinary stable metabolites of thromboxane A 2 and prostacyclin, 11-dehydro-thromboxane B 2 and 2,3-dinor-6-keto-prostaglandin F 1α, respectively, were converted to 1-methyl ester-propylamide9,12,15- tris-dimethylisopropylsilyl ether derivative and 1-methyl ester-6-methoxime-9,12,15- trisdimethylisopropylsilyl ether derivative, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of 10 outpatients and 6 inpatients with chronic glomerulonephritis were higher than those of 13 healthy volunteers. In an inpatient with systemic lupus erythematoides, the TX/PGI ratios were gradually lowered to the normal level with the therapies. Furthermore, the ratios seemed to change in advance of the changes of the levels of urinary protein and hematuria. These observations suggested that the TX/PGI ratio was a useful index to assess the pathological condition of renal diseases and the effects of treatment.

Aspirin dose and thromboxane metabolism in African American stroke patients

P214 Purpose: To evaluate thromboxane metabolism in African American stroke patients on different doses of aspirin. Methods: Consecutive African American patients within 3 month of non-cardioembolic stroke and not being treated with anticoagulation were recruited. Antithrombotic therapy at the time of sample collection was not prespecified and varied according to the practice patterns of the different attending physicians who treated the patients during their acute stroke. The thromboxane metabolite 11-dehydrothroboxane B2 (11-DTB2) was measured by enzyme immunoassay in random urine samples collected at the time of enrollment. Results: 99 patients were enrolled but 15 could not give a urine sample at the time of enrollment. Data on the remaining 84 patients were evaluable. There were 49 men and 35 women aged 36–87 (mean 62) years. Based on antithrombotic treatment during sample collections, we divided patients into four groups: no aspirin N=16 (no antithrombotic drugs N=4 or ticlopidine N=12), aspirin 81–325 mg/d N=19 (81 mg/day N=1, 325 mg/day N=18), aspirin 650 mg/d N=19, aspirin 975–1300 mg/d N=30 (975 mg/d N=2 and 1300 mg/d N=28). After log transformation of the 11-DTB2 values the geometric means in the no aspirin, 81–325 mg/d, 650 mg/d, and 975–1300 mg/d groups were 1355, 912, 758, and 731 pg/mg creatinine, respectively. Based on one way ANOVA, the mean 11-DTB2 concentration was significantly different between the 4 groups (p=0.03). There was a significant negative relationship between aspirin dose and 11-DTB2 concentration (Pearson Correlation, r = -0.26, P=0.02). Conclusion: In African American stroke patients thromboxane production appears to be progressively inhibited by aspirin doses up to 650 mg/d. The clinical significance of this effect by aspirin doses ≤650 mg/d remains to be determined.

Optimization of an Enzyme Immunoassay for 11-Dehydro-Thromboxane B 2 in Urine : Comparison with GC-MS

The urinary excretion of stable metabolites of thromboxane A 2, such as 11-dehydro-thromboxane B 2, reflects platelet activity in vivo. Efficient sample purification is required before analysis of thromboxane metabolites, due to the presence of large amounts of interfering material in urine. Analysis by gas chromatography–mass spectrometry after extensive sample work-up procedures provides the most reliable data, but detection by enzyme immunoassay may be reliable if sample cleanup is adequate. We describe an improved immunoassay procedure for 11-dehydro-thromboxane B 2, which is based on a simple one-step solid phase extraction, by using Bond-Elut Certify II columns, followed by enzyme immunoassay by using commercially available reagents. 11-Dehydro-thromboxane B 2 exists in two forms, with different chemical and immunological characteristics, which are in pH-dependent equilibrium. We kept 11-dehydro-thromboxane B 2 in its open ring form throughout the assay, by incubating and handling samples at pH 8.6. The extraction step achieved a recovery of 83% (95% confidence interval 74–92%), the sensitivity of the enzyme immunoassay was doubled, and the reproducibility of the assay improved under these conditions. Intra- and interassay coefficients of variation were 3 and 13.8%, respectively. A single 500-mg dose of aspirin reduced the excretion of 11-dehydro-thromboxane B 2 by 77±14%, suggesting good specificity. Comparison with gas chromatography–mass spectrometry in 28 urine samples showed excellent agreement between the two methods ( r 2=0.94; p<0.0001), and a regression line with a slope close to 1.0. The presently modified enzyme immunoassay for 11-dehydro-thromboxane B 2 is suitable for clinical studies evaluating platelet function in vivo and has the advantage of being simpler and less expensive to use than gas chromatography–mass spectrometry.

Urinary thromboxane A 2/prostacyclin balance reflects the pathological state of a diabetic

Levels of the stable urinary metabolites of thromboxane A 2 and prostacyclin, 11-dehydro-thromboxane B 2 (11-dehydro-TXB 2) and 2,3-dinor-6-keto-prostaglandin F 1α (2,3-dinor-6-keto-PGF 1α) were measured in diabetics to elucidate the relation between the thromboxane A 2/prostacyclin (TX/PGI) balance and pathological states of diabetes mellitus. 11-Dehydro-TXB 2 and 2,3-dinor-6-keto-PGF 1α were derivatized to methyl ester-propylamide-dimethylisopropylsilyl ether and methyl ester-methoxime-dimethylisopropylsilyl ether derivatives, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of diabetics were higher than those of healthy volunteers, suggesting the hypercoagulative states of this disease. The ratios showed positive correlations with the levels of blood glucose. The levels of hemoglobin A 1c and triglyceride were correlated weakly with the ratio. Some of the patients who had relatively low levels of blood glucose also showed high TX/PGI ratios. Furthermore, the ratio increased in the order of the groups 1, 2, and 3; group 1 contained patients who did not take medicine for diabetes, group 2 contained those who took oral hypoglycemic agents, and group 3 contained those who received insulin therapy. These observations indicate that the TX/PGI ratio reflects the pathological conditions of diabetes and is a useful marker, having few different features from other markers that are presently used.

Monitoring of the thromboxane A 2/prostacyclin ratio in the urine of patients with retinal vascular occlusion through the low-dose-aspirin therapy using the gas chromatography/selected ion monitoring method

We determined the levels of the stable urinary metabolites of thromboxane A 2 and prostacyclin, 11-dehydro-thromboxane B 2 (11-dehydro-TXB 2) and 2,3-dinor-6-keto-prostaglandin F 1α (2,3-dinor-6-keto-PGF 1α) in patients with retinal vascular occlusion (RVO) to elucidate the change of the thromboxane A 2/prostacyclin (TX/PGI) ratio with this disease and the effect of low-dose-aspirin therapy. 11-Dehydro-TXB 2 and 2,3-dinor-6-keto-PGF 1α were converted to 1-methyl ester-propylamide-9,12,15- tris-dimethylisopropylsilyl ether derivative and 1-methyl ester-6-methoxime-9,12,15- tris-dimethylisopropylsilyl ether derivative, respectively, and applied to a gas chromatography/selected ion monitoring. The average level of 11-dehydro-TXB 2 in 30 patients with RVO was 1038 ± 958 pg/mg creatinine. It was significantly higher than that of 27 healthy volunteers, which was 616 ± 294 pg/mg creatinine ( p < 0.05 with unpaired t-test). However, 2,3-dinor-6-keto-PGF 1α levels were not significantly different between these two groups. The average ratio of TX/PGI in the RVO patients was 32 ± 26 and it was significantly higher than that of healthy volunteers, 17 ± 10 ( p < 0.01). Patients with central retinal artery occlusion or branch retinal artery occlusion showed greatly high 11-dehydro-TXB 2 levels and TX/PGI ratios, although the number of patients was limited in the current study. After the administration of low-dose aspirin (40 mg/day) for about 1 month, the TX/PGI ratio decreased to around the normal level. Following the levels for up to 10 months, they also remained at the normal level. These observations suggested that the 11-dehydro-TXB 2 levels and the TX/PGI ratio reflect the pathological conditions of RVO and are useful markers of the treatment.

Cyclic nucleotides and production of prostanoids in human varicose veins

Objective: Experiments were designed to determine the production of prostacyclin and thromboxane and the activation of cyclic nucleotides in human varicose and nonvaricose veins and to determine whether these second messenger pathways were differentially activated by the venotropic extract of Ruscus aculeatus . Methods: The experiments were designed to characterize the activity of cyclic nucleotides and the production of prostaglandins in human varicose and nonvaricose veins. Segments of the greater saphenous veins and the adjacent tributaries were obtained from patients who underwent vein stripping and excision of primary varicose veins. The saphenous veins from the patients who underwent peripheral arterial bypass grafting were used as controls. The segments of veins were incubated in Krebs-Ringer bicarbonate solution in the presence of venotropic extract of Ruscus aculeatus (10 –3 g/mL) or in water-miscible organic solvent (dimethyl sulfoxide, 10 –3 g/mL), for 1, 5, and 10 minutes at 37°C. The nonspecific phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, 10 –4 g/mL) was used to block cyclic nucleotide degradation in some samples. Tissue and media samples were collected. Tissue concentrations of both cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP and cGMP, respectively) and media concentrations of 6-ketoprostaglandin-F 1 α (the stable metabolite of prostacyclin) and thromboxane B 2 (the stable metabolite of thromboxane A 2) were measured by means of radioimmunoassay. Cyclooxygenase 2 was measured with Western blot analysis. Results: The varicose veins showed greater levels of cAMP but not of cGMP at all time points as compared with the control veins. Prostanoid production was not significantly altered in the varicose veins. Stimulation with Ruscus aculeatus increased the cAMP concentration in the varicose veins but did not affect the cGMP levels. The ratio between 6-ketoprostaglandin-F 1 α and thromboxane B 2 was two-fold greater in the varicose veins as compared with the control veins. In the presence of the extract, the ratio of 6-ketoprostaglandin-F 1 α and thromboxane B 2 was identical in both types of veins. Cyclooxygenase 2 was not present in either the control or the varicose veins. Conclusion: These results suggest that cAMP levels are elevated in varicose veins and that they can be altered with drug treatment in varicose veins. This chemical pathway may be considered as a modulatory target to affect contraction with venotropic drugs. (J Vasc Surg 1999;30:876-84.)

Mild myocardial stunning affects platelet aggregation and certain hemostatic factors in swine.

Myocardial stunning is characterized by transient contractile dysfunction occurring subsequent to an episode of ischemia followed by reperfusion. Platelet activation and hemostatic abnormalities have been described in patients with unstable angina and acute myocardial infarction, however, their role in the pathogenesis of myocardial stunning is unknown. The purpose of this study was to determine if platelet aggregation and certain hemostatic factors change during myocardial stunning following brief coronary arterial occlusion. Nine Yorkshire swine underwent left anterior descending coronary artery occlusion for 8 minutes followed by 90 minutes of reperfusion. Blood samples were obtained at baseline, at 4 and 8 minutes of occlusion, and at 60 and 90 minutes of reperfusion. Platelet aggregability and concentrations of antithrombin III, protein C, protein S, fibronectin, endothelin 1, and the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a) were measured in systemic circulation. The occlusion phase was associated with a decline of endothelin 1 (-13.6%), and TxB2 (-19.6%), and elevation of antithrombin III (+40.2%) and protein C (+22.9%). Mild myocardial stunning was associated with a significant increase in platelet aggregation (+33.7%), endothelin-1 (+24.7%), 6-keto-PGF1a (+41.5%), TxB2 (+11.9%), and protein C (+42.3%) during the reperfusion phase. There were no changes in plasma fibronectin and total protein S. Thus, mild myocardial stunning following brief coronary artery occlusion is associated with substantial dynamic changes in platelet aggregability and certain hemostatic factors. These results may be relevant to understanding the mechanisms determining myocardial stunning and coronary arterial patency following reperfusion.

Stimulation of bradykinin B2-receptors on endothelial cells induces relaxation and contraction in porcine basilar artery in vitro.

1. The aim of the present study was to characterize the subtypes of bradykinin (BK) receptors that evoke the relaxation and contraction induced by BK and to identify the main contracting and relaxing factors in isolated porcine basilar artery by measuring changes in isometric tension and a thromboxane (TX) metabolite. 2. Endothelial denudation completely abolished both responses. [Thi5,8, D-Phe7]-BK (a B2-receptor antagonist) inhibited the BK-induced relaxation and contraction, whereas des-Arg9, [Leu8]-BK (a B1-receptor antagonist) had no effect. 3. L-nitro-arginine (L-NA, a nitric oxide synthase inhibitor) completely inhibited BK-induced relaxation. Indomethacin (a cyclo-oxygenase inhibitor) completely and ONO-3708 (a TXA2/prostaglandin H2 receptor antagonist) partially inhibited BK-induced contraction, whereas OKY-046 (a TXA2 synthase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) did not. 4. In the presence of L-NA, the contractile response to BK was inhibited by indomethacin or ONO-3708 and was competitively antagonized by [Thi5,8, D-Phe7]-BK (pA2=7.50). In the presence of indomethacin, the relaxant response to BK was inhibited by L-NA and was competitively antagonized by [Thi5,8, D-Phe7]-BK (pA2=7.59). 5. TXA2 release was not induced by BK-stimulation. 6. These results suggest that the endothelium-dependent relaxation and contraction to BK in the porcine basilar artery is mediated via activation of endothelial B2-receptors. The main relaxing factor may be NO and the main contracting factor may be prostaglandin H2.