8-epi PGF2alpha Research Articles

Determination of 8‐epi PGF2α concentrations as a biomarker of oxidative stress using triple‐stage liquid chromatography/tandem mass spectrometry

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples.

Effect of raisin consumption on oxidative stress and inflammation in obesity

Oxidative stress can initiate increased inflammation that elevates risk for cardiovascular disease. The objective of this study was to determine the effects of daily consumption of raisins on markers of oxidative stress, inflammation and endothelial activation in response to an acute high-fat meal in overweight individuals. Seventeen overweight men and women consumed 90 g raisins or isocaloric placebo (264 kcal/day) for 14 days in a randomized, crossover design while following a low-flavonoid diet. The oxidative [urinary 8-iso-prostaglandin-F(2alpha) (8-epi PGF(2alpha)) and serum oxygen radical absorbance capacity (ORAC)], inflammatory (serum C-reactive protein and interleukin-6), endothelial (serum soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1, sVCAM-1) and metabolic [free fatty acids (FFAs), triacylglycerol, glucose and insulin] response to four high-fat (53%) meals was tested pre- and postintervention. Urinary 8-epi PGF(2alpha) decreased (-22%) and fasting ORAC increased (+3%) after both interventions combined. Fasting protein-free ORAC was modestly (+3.5%) higher during the raisin than the placebo intervention. Neither the meals nor the raisins consistently induced fasted markers of inflammation or endothelial dysfunction. Gender influenced postprandial metabolic responses in that males responded with higher serum FFAs, sVCAM-1 and glucose compared with females. Serum antioxidant capacity was modestly increased by daily raisin consumption, but this did not alter fasted or postprandial inflammatory response in these relatively healthy but overweight individuals. Providing all food in regular pattern reduced measures of oxidative stress.

The effects of propofol on neutrophil function, lipid peroxidation and inflammatory response during elective coronary artery bypass grafting in patients with impaired ventricular function

Coronary artery bypass grafting (CABG) with cardiopulmonary bypass elicits a potent reperfusion injury and inflammatory response, more intense in patients with impaired myocardial function. Propofol has antioxidant properties which may attenuate such a response. In total, 27 patients with impaired left ventricular function undergoing CABG were randomly allocated to receive either target-controlled infusion propofol (P) or saline (S) immediately before aortic cross-clamp release until 4 h after reperfusion. Troponin-I, Urinary 8-epi PGF-2alpha isoprostane, coronary sinus and systemic malondialdehyde concentrations, Interleukin-6 (IL-6), -8 and -10 concentrations and leucocytes function studies (neutrophil respiratory burst, phagocytosis, CD-11b and CD-18 expression) were measured. Propofol decreased MDA coronary sinus concentration at 1, 3 and 5 min after reperfusion (P<0.01); 60 min after reperfusion a significant difference between the two groups in systemic MDA concentrations was also seen. IL-6 concentration increases were significantly greater in Group S than Group P, 4 h after reperfusion [1118 (1333) pg ml(-1) vs 228 (105) pg ml(-1), P<0.01]. Serum IL-8 concentrations did not increase significantly in either group. Compared with baseline values IL-10 concentrations decreased after reperfusion but the values were higher in the propofol group than in the control group [22 (16) vs 11 (4) pg ml(-1), P<0.05]. No difference in leucocyte function or urinary isoprostane concentrations was demonstrated. Propofol attenuates free-radical-mediated lipid peroxidation and systemic inflammation in patients with impaired myocardial function undergoing CABG.

Over‐expression of heme oxygenase‐1 promotes oxidative mitochondrial damage in rat astroglia

Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions.

Consumption of High-Pressurized Vegetable Soup Increases Plasma Vitamin C and Decreases Oxidative Stress and Inflammatory Biomarkers in Healthy Humans

Current evidence supports a significant association between fruit and vegetable intake and health. In this study, we assessed the effect of consuming a vegetable-soup "gazpacho" on vitamin C and biomarkers of oxidative stress and inflammation in a healthy human population. We also examined the association between vitamin C and F(2)-isoprostanes (8-epiPGF(2alpha)), uric acid (UA), prostaglandin E(2) (PGE(2)), monocyte chemotactic protein-1 (MCP-1), and the cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6. Gazpacho is a Mediterranean dish defined as a ready-to-use vegetable soup, containing approximately 80% crude vegetables rich in vitamin C. Subjects (6 men, 6 women) enrolled in this study consumed 500 mL/d of gazpacho corresponding to an intake of 72 mg of vitamin C. On d 1, subjects consumed the gazpacho in one dose; from d 2 until the end of the study, d 14, 250 mL was consumed in the morning and 250 mL in the afternoon. Blood was collected before drinking the soup (baseline) and on d 7 and 14. Baseline plasma vitamin C concentrations did not differ between men and women (P = 0.060). Compared with baseline, the vitamin C concentration was significantly higher on d 7 and 14 of the intervention in both men and women (P < 0.05). Baseline plasma levels of UA and F(2)-isoprostanes were higher (P < or = 0.002) in men than in women. The F(2)-isoprostanes decreased on d 14 in men and women (P < or = 0.041), and UA decreased in men (P = 0.028). The concentrations of vitamin C and 8-epiPGF(2alpha) were inversely correlated (r = -0.585, P = 0.0002). Plasma PGE(2) and MCP-1 concentrations decreased in men and women (P < or = 0.05) on d 14, but those of TNF-alpha, IL-1beta, and IL-6 did not change. Consumption of the vegetable soup decreases oxidative stress and biomarkers of inflammation, which indicates that the protective effect of vegetables may extend beyond their antioxidant capacity.

Oxidative stress and its association with coronary artery disease and different atherogenic risk factors

It is well known that free radicals contribute to endothelial dysfunction and are involved in the pathogenesis and development of cardiovascular diseases, such as atherosclerosis. The aim of this study was to provide evidence for enhanced oxidative stress in coronary artery disease (CAD). Plasma levels of 8-isoprostane (8-epiPGF(2alpha)), marker of lipid peroxidation, were measured in 68 subjects (age: 60 +/- 2 years, mean +/- SEM). Subjects included 30 healthy control subjects and 38 patients with angiographically proven CAD. In addition, the total antioxidant power (PAO) was evaluated in a subgroup (40 subjects, 12 healthy and 28 CAD). Levels of 8-epiPGF(2alpha) increased with the number of affected vessels (one- and multi-vessel disease versus control subjects, P < 0.001) and considering different risk determinants for atherosclerosis (i.e. hypertension, gender, hypercholesterolaemia, P < 0.01). In multivariate regression models the number of affected vessels was independently correlated with 8-epiPGF(2alpha) (P < 0.05). PAO values significantly decreased with increased number of affected vessels (P < 0.05) and in hypertensive patients when compared with those without hypertension (P < 0.05). In multivariate regression models the number of affected vessels resulted an independent determinant for PAO (P < 0.05). Concentration of 8-epiPGF(2alpha) and PAO also correlated with the number of cardiovascular risk factors (P < 0.01 and P = 0.07, respectively). These findings indicate that elevated levels of plasma 8-epiPGF(2alpha) and reduced antioxidant capacity are associated with the extent and the severity of CAD and with the occurrence and number of different atherogenic risk factors. This observation may assist in providing more information as to how oxidative stress may predispose to atherogenesis and suggest attractive therapeutic strategies in the prevention and treatment of cardiovascular disease.

High-Pressurized Orange Juice Consumption Affects Plasma Vitamin C, Antioxidative Status and Inflammatory Markers in Healthy Humans

We examined the bioavailability of vitamin C in orange juice processed using high pressure (HP) and its effects on plasma levels of vitamin C, uric acid (UA), F2-isoprostanes (8-epiPGF(2alpha)), C-reactive protein (CRP) and prostaglandin E(2) (PGE(2)) in a healthy human population. Subjects (6 men, 6 women) enrolled in the study consumed 500 mL/d of HP orange juice for 14 d, corresponding to an intake of 250 mg of vitamin C. On d 1 of the study, subjects drank the juice in one dose; on d 2 until the end of the study, d 14, they drank 250 mL in the morning and 250 mL in the afternoon. Blood was collected every h for 6 h, on d 1, and then on d 7 and 14 of the study. Baseline plasma vitamin C concentration was higher (P = 0.014) in women (55.8 +/- 3.8 micro mol/L) than in men (42.8 +/- 2.1 micro mol/L). The maximum plasma vitamin C increase occurred 3 h after drinking the juice, and it remained elevated on d 7 and 14. Plasma 8-epiPGF(2alpha) concentration did not differ between men and women at baseline. However, it was lower at the end of the study in both men (P = 0.044) and women (P = 0.034). Plasma levels of vitamin C and 8-epiPGF(2alpha) were inversely correlated (r = -0.615, P = 0.001). Plasma CRP concentrations tended to be lower on d 14 than at baseline in men (P = 0.317) and women (P = 0.235). Plasma PGE(2) was lower at the end of the study in both men and women (P <or= 0.037). Drinking orange juice increases plasma vitamin C, and decreases 8-epiPGF(2alpha) and PGE(2) levels in humans, which may help reduce the risk of chronic diseases.

8-Epi-PGF2alpha and 6-oxo-PGF1alpha in human (varicose) veins: influence of age, sex, and risk factors.

The isoprostane 8-epi PGF2alpha is a vasoconstrictive, mitogenic, proliferative, and mild proaggregatory agent. We examined 8-epi-PGF2alpha and 6-oxo-PGF1alpha from venous tissue derived from varicose (venous) surgery by means of a specific radioimmunoassay. A total of 336 samples from 82 patients (50 females, 32 males; aged 22-68 years) were examined. Tissue samples were classified according to normal, dilated, and varicose. Of these, 94 samples from 31 patients (20 females, 11 males; aged 29-64 years) with additional risk factors (cigarette smoking, hyperlipidemia, diabetes mellitus) were determined in the same way. Mean absolute values for 6-oxo-PGF1alpha are not significantly higher for dilated segments followed by varicose and intact samples. No significant age and sex differences can be monitored. Presence of risk factors, however, results in a significantly diminished 6-oxo-PGF1alpha, irrespective of morphology. 8-Epi-PGF2alpha again showed no age and sex dependence, its presence in varicose segments, however, was significantly (p<0.01) decreased. Risk factors resulted in a significantly increased 8-epi-PGF2alpha. These data indicate that the influence of risk factors on vasomodulatory (iso-)eicosanoids of human veins is more pronounced than the actual morphologic stage. Lower 8-epi-PGF2alpha in varicose veins may shift the venous tone toward vasodilatation and contribute to development and progression of varicosis.

Prostaglandins and lipid modification.

Postaglandins(PG) and low-density lipoproteins (LDL) both are playing a key role in atherogenesis. Their interaction at the local vascular level is of central relevance in plaque formation and progression. Details of these complex actions however, still need to be elucidated. Lipoproteins are influencing the PG-production of arterial wall cells and platelets, while PGs in turn have been shown to regulate lipoprotein receptor binding and entry into the arterial wall. Modification of LDL severely influences arterial wall trapping and foam cell formation. During LDL-modification, isoprostanes, a new family of compounds generated by free radical catalysed action, independent of cyclooxygenase, are formed. 8-epi PGF(2alpha) the most well known member exerts a great variety of proatherogenic actions, among them vasoconstriction and platelet activation; it also serves as a mitogen and stimulator of endothelin release. The influence of various eicosanoids on lipoprotein modification, however, has not been assessed yet.

Isoprostane 8-epi PGF2alpha, a product of oxidative stress, is synthesized in the bladder and causes detrusor smooth muscle contraction.

Isoprostane 8-epi PGF2alpha is a product of oxidative stress that causes potent smooth muscle contraction. Its production increases in conditions associated with oxidative stress such as in diabetes, smoking, and aging. The aim was to study whether the urinary bladder synthesizes isoprostane 8-epi PGF2alpha and releases to the urine and whether isoprostane 8-epi PGF2alpha causes bladder smooth muscle contraction. Urine samples were obtained transurethrally from 12 male New Zealand white rabbits for measurement of isoprostane 8-epi PGF2alpha levels. To examine whether bladder synthesizes isoprostane 8-epi PGF2alpha, both ureters were ligated, then the bladder was washed 5 times by filling and emptying with normal saline. Bladder was refilled with normal saline, and at 5 minutes a bladder washout sample was taken. After this, the bladder was contracted by nerve stimulation periodically for 30 minutes, and then another washout sample was taken. Strips of bladder tissues were processed for study of isoprostane 8-epi PGF2alpha production in tissue culture chambers and for isometric tension measurements in the organ bath. Enzyme immunoassay (EIA) revealed a remarkable amount of isoprostane 8-epi PGF2alpha in the rabbit urine. EIA of washout samples showed that the bladder synthesizes isoprostane 8-epi PGF2alpha and its production increases with nerve stimulation-induced contractions. EIA of samples from the tissue culture media showed that bladder strips synthesize isoprostane 8-epi PGF2alpha in vitro. Electrical field stimulation (EFS) significantly increased the synthesis and release of isoprostane 8-epi PGF2alpha by the bladder strips. In the organ bath, isoprostane 8-epi PGF2alpha caused concentration-dependent contraction of bladder tissue. While the threshold contraction required smaller concentration of isoprostane 8-epi PGF2alpha (3 nmol) than carbachol (10 nmol), the amplitude of contraction to carbachol was greater than isoprostane 8-epi PGF2alpha. Our studies show that the rabbit bladder synthesizes isoprostane 8-epi PGF2alpha and releases it to the urine. Production of isoprostane 8-epi PGF2alpha in the bladder increases with nerve stimulation-induced contraction. Exogenous isoprostane 8-epi PGF2alpha causes significant bladder smooth muscle contraction. Our findings necessitate further studies to evaluate the possible role of oxidative stress and increased isoprostane 8-epi PGF2alpha production in bladder dysfunction. Neurourol. Urodynam. 19:43-51, 2000.

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.

Actions of 8 epi prostaglandin F2alpha on isolated rat aorta.

8-epi prostaglandin F2alpha(8-epi PGF2alpha) contracted rat thoracic aorta rings in a concentration-dependent manner in the presence or absence of functional endothelium [median effective concentration (EC50) values, 455+/-52 and 268+/-34 nM, respectively; Student's t test; p=0.006]. U46619 was a more potent agonist with or without functional endothelium (EC50 values, 6.8+/-1.6 and 4.5+/-1.0 nM, respectively). SQ29548 [a thromboxane (TP)-receptor antagonist] inhibited contractions to both 8-epi PGF2alpha and U46619 in a competitive manner, with mean pA2 values of 8.3 and 7.9, respectively. 8-Epi PGF2alpha had a further contractile effect in vessels that had been contracted with noradrenaline and had been shown to possess a functional endothelium. Inhibition of thromboxane synthesis with OKY-046 or blockade of endothelin receptors with bosentan had no effect on responses to 8-epi PGF2alpha or U46619. Preincubation with 8-epi PGF2alpha or noradrenaline shifted the concentration-response curves to U46619 upward at low concentrations of U46619 with no significant change in EC50 values or maximal responses. Reduction of TP-receptor number in rat aorta with dithiothreitol caused a concentration-dependent inhibition of responses to both U46619 and 8-epi PGF2alpha, with no effect on maximal responses and or on the responses to U46619 after the preincubation with 8-epi PGF2alpha. These results indicate that 8-epi PGF2alpha is a potent vasoconstrictor in the rat aorta and are suggestive of an action of 8-epi PGF2alpha at the TP receptor.

Simultaneous Quantitative Analysis of Prostaglandins and Thromboxane after Low-Dose X Irradiation

The appearance of prostaglandins and thromboxane in mouse serum after X irradiation was observed by simultaneous quantitative analysis using gas chromatography/mass spectrometry/selected ion monitoring with stable isotope dilution methods. Mice of two strains (C57BL/CN Jcl and BALB/c) showed similar responses to X irradiation. In C57BL/6N Jcl mice, 0.2 Gy irradiation elicited a significant increase in generation of prostanoids: Immediately after irradiation, the 6-keto PGF1 alpha:TXB2 ratio and the level of PGE2 increased, after 20 min 6-keto PGF1 alpha and PGE2 increased, and after 4 h PGE1 and PGE2 increased. In BALB/c mice, generation of prostanoids was increased significantly immediately after irradiation (6-keto PGF1 alpha, 6-keto PGF1 alpha:TXB2 ratio, PGE2), and the increase was maintained from 20 min to 4 h (PGE1, PGE2) after 0.2 Gy irradiation. In C57BL/6N Jcl mice, a significant increase in production of 9alpha,11beta-PGF2 was observed at 20 min after irradiation. In BALB/c mice, a significant increase in 9alpha,11beta-PGF2 was seen immediately after irradiation and was maintained for 20 min. In C57BL/6N Jcl mice, the level of 8-epi PGF2 alpha was clearly increased 4 h after 4 Gy irradiation. A slight and slow increase was also seen after 0.2 Gy irradiation. In BALB/c mice, 8-epi PGF2 alpha was increased significantly at 20 min and 4 h after 4 Gy irradiation. These results show that 0.2 Gy irradiation stimulates production of prostanoids related to the inflammatory response in mice.

Localization of distinct F2-isoprostanes in human atherosclerotic lesions.

F2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

Coronary artery constriction by the isoprostane 8-epi prostaglandin F2 alpha.

1. This study was undertaken to compare the effects of 8-epi prostaglandin F2 alpha (8-epi PGF2 alpha) to those of prostaglandin F2 alpha (PGF2 alpha) and U46619, a thromboxane mimetic, on ovine, bovine and porcine coronary arteries. 2. 8-epi PGF2 alpha constricted porcine and bovine coronary arteries in a concentration-dependent manner with EC50 values of 689.0 +/- 229.3 and 1361.0 +/- 272.3 nM, respectively, but had no effect on ovine coronary arteries. 3. U46619 was a potent vasoconstrictor of porcine, ovine and bovine coronary arteries with EC50 values of 33.0 +/- 23.5, 373.3 +/- 69.7 and 254.1 +/- 134.3 nM, respectively. Emax values were significantly greater than those obtained with 8-epi PGF2 alpha. 4. PGF2 alpha constricted procine and bovine coronary arteries in a concentration-dependent manner with EC50 values of 1631.0 +/- 207.6 and 3644.0 +/- 344.8 nM, respectively, but had no effect on ovine coronary arteries. 5. Concentration-dependent constriction to U46619 in porcine coronary arteries was competitively inhibited by SQ29548 (10(-8) M to 10(-7) M) and BM13505 (10(-8) M to 10(-6) M) with no decrease in maximal responses. 6. Concentration-dependent constriction to 8-epi PGF2 alpha in porcine coronary arteries was inhibited in a concentration-dependent manner by SQ29548 (10(-8) M to 10(-7) M) and BM13505 (10(-8) M to 10(-6) M). However, the inhibition was associated with a decrease in maximal response. 7. Maximal responses of porcine coronary artery to U46619 (1 microM) and 8-epi PGF2 alpha (30 microM) were inhibited in a concentration-dependent manner by SQ29548 with IC50 values 99 +/- 12.36 nM and 46.5 +/- 18.67 nM, respectively. 8. Although ovine coronary arteries did not constrict to 8-epi PGF2 alpha pre-incubation of these vessels with 8-epi PGF2 alpha caused a rightward shift of the U46619 response curve in a concentration-dependent manner. 9. Pre-incubation of porcine coronary arteries with 8-epi PGF2 alpha competitively inhibited responses to U46619 with a Schild slope of 0.99 and a pA2 of 6.13. 10. We conclude that 8-epi PGF2 alpha is a vasoconstrictor within porcine and bovine coronary arteries, with a potency approximately twice that of PGF2 alpha but 5-20 times lower than U46619. The data suggest that 8-epi PGF2 alpha is acting as a partial agonist on the TP-receptor in the coronary vasculature.

8-Epi PGF2 alpha: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo.

1. Excessive free radical generation is thought to contribute to tissue injury in a broad spectrum of diseases. A particular constraint in addressing this hypothesis has been the inability to assess free radical generation in vivo and the lack of information on drugs or vitamins which act as effective antioxidants in vivo. 2. Traditional approaches have relied upon measures of substrate oxidizability or spin trapping of free radical adducts ex vivo. It is unknown how these measurements might relate, in a quantitative fashion, to the generation of reactive oxygen species in vivo. Isoeicosanoids are free radical catalyzed products of arachidonic acid. One of these compounds, 8-epi prostaglandin F2 alpha (8-epi PGF2 alpha) exhibits biological activity and may function as an autacoid. Specific analysis of this 8-epi PGF2 alpha isomer indicates that it is elevated in certain syndromes thought to be associated with oxidant stress. These include vascular reperfusion, paracetamol poisoning and liver cirrhosis. Apparently healthy individuals who smoke cigarettes or consume alcohol exhibit dose dependent increments in excretion of 8-epi PGF2 alpha. Excretion is depressed by antioxidant vitamins, although not by the nonspecific cyclooxygenase (COX) inhibitor, aspirin, even though 8-epi PGF2 alpha may be formed by either COX-1 or COX-2. 3. Specific analysis of this and other isoeicosanoids may afford an opportunity to evaluate the effects of antioxidant interventions in human diseases characterized by excessive lipid peroxidation in vivo.

Local Amplification of Platelet Function by 8-Epi Prostaglandin F2α Is Not Mediated by Thromboxane Receptor Isoforms

8-epi-Prostaglandin (PG) F2alpha may be formed by cyclooxygenases 1 and 2 or by a free radical catalyzed process as an isoprostane. Concentrations of 8-epi-PGF2alpha in the range 1 nM to 1 microM induce a dose-dependent increase in platelet shape change, in calcium release from intracellular stores [Ca2+]iand in inositol phosphates; it also causes irreversible platelet aggregation, dependent on thromboxane generation, when incubated with subthreshold concentrations of ADP, thrombin, collagen, and arachidonic acid. Much higher concentrations of 8-epi-PGF2alpha (10-20 microM) alone induce weak, reversible aggregation. Although these effects are prevented by pharmacological thromboxane receptor antagonists, they are unlikely to be mediated by thromboxane receptors. Thus, 8-epi-PGF2alpha does not compete for binding at the stably expressed placental or endothelial isoforms of the thromboxane receptor or for binding of thromboxane ligands to human platelets. Furthermore, the response to 8-epi PGF2alpha exhibits structural specificity versus 8-epi PGF3alpha and PGF2alpha. Concentrations in the range that evoke its effects on platelets do not desensitize the aggregation response stimulated by thromboxane or PGH2 analogs. Unlike primary prostaglandins, which are rapidly metabolized to inactive products, 8-epi PGF2alpha circulates in plasma. However, the systemic concentrations found in healthy volunteers (median 48 pmol/liter) and in patients with hepatic cirrhosis (median 147 pmol/liter), a syndrome of oxidant stress in vivo, fall well below those which modulate platelet function. 8-Epi PGF2alpha may amplify the response to platelet agonists in syndromes where oxidant stress and platelet activation coincide. Despite blockade by thromboxane antagonists, 8-epi PGF2alpha does not activate either of the thromboxane receptor isoforms described in platelets. Activation of a distinct receptor would be consistent with the enzymatic formation of 8-epi PGF2alpha by cyclooxygenases. However, incidental activation of such a receptor by systemic concentrations of 8-epi PGF2alpha is unlikely to occur, even in syndromes of excessive free radical generation in vivo.

Cloning of a receptor for prostaglandin F2 alpha from the ovine corpus luteum.

A complementary DNA clone encoding a functional receptor for prostaglandin F2 alpha (PGF2 alpha) has been isolated from an ovine large luteal cell complementary DNA library (prepared from day 10 mid-luteal phase RNA). This receptor, which has been designated FP, consists of 362 amino acids (M(r) = 40,982) and is a member of the family of G protein-coupled receptors. Radioligand binding studies with membranes prepared from transfected COS cells demonstrated specific 17-[3H]phenyl-trinor-PGF2 alpha binding that was displaced by prostanoids in the order of 17-phenyl-trinor-PGF2 alpha > PGF2 alpha > fluprostenol > PGD2 > PGE2 >> 8-epi PGF2 alpha. Xenopus laevis oocytes injected with RNA encoding the ovine FP receptor responded to 17-phenyl-trinor-PGF2 alpha with increased membrane chloride conductance in calcium-free medium. Northern blot analysis with RNA from day 10 corpus luteum showed a major band of approximately 6.1 kilobases. On day 14, when luteolysis usually starts, the abundance of this 6.1-kilobase band was variable between individual ewes, and on day 16, when luteolysis is underway, the message was uniformly less abundant. This variability appeared to correlate with circulating progesterone. Thus, when the progesterone level was high (days 10 and 14 depending on whether luteolysis had started), the amount of FP receptor message was high, whereas when the progesterone level was low or falling (day 16), the amount of FP receptor message decreased. We have cloned an ovine FP receptor whose expression confers appropriate functional activity in COS cells and Xenopus oocytes. Furthermore, the level of messenger RNA encoding the FP receptor is high in the midluteal phase ovine corpus luteum and decreases during luteolysis.

Cylooxygenase-dependent Formation of the Isoprostane, 8-Epi Prostaglandin F2α

Isoprostanes are a family of prostaglandin (PG) isomers formed in an enzyme-independent manner. They circulate in plasma and are excreted in urine. One of them, 8-epi PGF2 alpha is a vasoconstrictor and mitogen, effects which are prevented by thromboxane antagonists. Given that 8-epi PGF2 alpha may be formed by cyclooxygenase (COX) (Corey, E. J., Shih, C., Shig, N-Y., and Shimoji, K. (1984) Tetrahedron Letts. 44, 5013-5016; Hecker, M., Ullrich, V., Fischer, C., and Meese, C.O. (1987) Eur J. Biochem. 169, 113-123) and that this might confound its use as an index of free radical generation, we sought to characterize the mechanism of its formation by human platelets. Activation of platelets by threshold concentrations of collagen, thrombin, and arachidonic acid resulted in formation of 8-epi PGF2 alpha coincident with that of the COX product, thromboxane, and the 12 lipoxygenase product, 12-hydroxyeicosatetraenoic acid, as detected by selected ion monitoring assays using gas chromatography-mass spectrometry. The effect appeared selective for 8-epi PGF2 alpha among the F2 isoprostanes. Pretreatment of platelets with aspirin or indomethacin abolished 8-epi PGF2 alpha formation. COX-independent activation of platelets by high doses of collagen or thrombin, by the phorbol ester, phorbol 12-myristate 13-acetate, or the prostaglandin endoperoxide analog, U 46619 was not associated with 8-epi PGF2 alpha formation. Confirmation of the nature of the material formed by platelet COX as 8-epi PGF2 alpha included its cochromatography over three highly resolving high performance liquid chromatography systems, identification by electron impact mass spectrometry, and its formation by partially purified COX. Inhibition of platelet thromboxane formation was associated with augmented 8-epi PGF2 alpha formation. A major component of 8-epi PGF2 alpha formed in serum by healthy volunteers was shown to be sensitive to inhibition by aspirin ex vivo. In addition to its generation by free radical catalyzed mechanisms, 8-epi PGF2 alpha may be formed as a PG by human platelets. Given that activation of platelet COX characterizes many of the human syndromes which are putatively associated with free radical generation, assessment of the contribution of this pathway is relevant to the use of 8-epi PGF2 alpha as an index of lipid peroxidation in vivo.