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1.
The ability of aortae from young and mature swine to produce prostacyclin (PGI2) has been determined. PGI2 was measured as its hydration product, 6-keto-PGF and assayed by stable isotope dilution GC-MS. There was no significant difference in 6-keto-PGF production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PGF as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary differences an unlikely explanation for the age-related decrease in PGI2 synthesis. Both young and mature vascular tissues produced essentially only PGI2; insignificant amounts of PGE2 and PGF were found.  相似文献   

2.
Conversion of 1-14C-arachidonic acid (AA) to 6-keto-PGF, the stable metabolite of prostacyclin (PGI2) was assayed kinetically by employing an aqueous sampling technique. In this way, one can arrive at a kinetic view of PGI2 synthesis from AA in intact tissue. The assay appears to be particularly suitable to tissues such as the aorta where PGI2 constitutes the major metabolite of AA. The assay avoids the need for organic solvent extraction and relies on the essential absence of tissue binding of 6-keto-PGF. The disappearance of AA can also be followed in this system but quantitation is complicated by avid tissue binding of the fatty acid. The assay, as described should be applicable to other vascular tissues and should greatly simplify kinetic analyses of prostacyclin synthesis.  相似文献   

3.
The stable breakdown product of prostacyclin, 6-keto-PGF, was estimated in plasma samples after incubation with rat aortic rings. The 6-keto-PGF concentration obtained with the male aortae was two-fold higher than that of the female. Ovariectomy markedly increased 6-keto-PGF six-fold, but castration had no effect. Estradiol and progesterone treatment of the ovariectomized female suppressed (by 50%) and enhanced (two-fold) 6-keto-PGF. Testosterone was without effect in gonadectomized males and females. Castrate males did not respond to gonadal steroid treatment.  相似文献   

4.
The transformation of 6-keto-PGF to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF (II) by Mycobacterium rhodochrous UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ13 reductase enzyme systems.  相似文献   

5.
A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B2 chromatographs as two species and coelutes with PGF. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF, 6-keto-PGE1, 13,14-dihydro-6,15-diketo-PGF). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.  相似文献   

6.
Prostaglandin E2 (PGE2) and 6 keto-PGF, the stable metabolite of prostacyclin (PGI2), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE2 and 6-keto-PGF were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE2 and 6-keto-PGF was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI2 is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE2 and 6-keto-PGF release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.  相似文献   

7.
The TxA2 synthetase inhibitor, dazoxiben, and the TxA2 antagonist, ±SQ 29, 548, were examined for effects on release and vasoactivity of TxA2 and prostacyclin. Isolated perfused guinea pig lungs were used as the enzyme source from which TxA2 and prostacyclin were released in response to injections of arachidonic acid or bradykinin. Both dazoxiben and ±SQ 29, 548 inhibited contraction of the superfused rat aorta and bovine coronary artery after arachidonic acid injection through the lung. ±SQ 29, 548 abolished contractions of the rat aorta, but significant aorta contracting activity persisted during dazoxiben treatment. Dazoxiben significantly inhibited arachidonate-induced release of TxA2 (immunoreactive TxB2)iinto the superfusate, but TxA2 release was significantly potentiated by ±SQ 29, 548. Thus, in the presence of enhanced TxA2 concentrations, ±SQ 29, 548 effectively antagonized the vasospastic effect of TxA2. Dazoxiben diverted a significantly greater amount of arachidonic acid into prostacyclin synthesis (immunoreactive 6-keto-PGF), changing original coronary vasoconstriction into relaxation. ±SQ 29, 548 did not significantly modify lung prostacyclin synthesis. Moreover, with ±SQ 29, 548, the absence of TxA2-mediated coronary contraction unmasked active relaxation of the superfused bovine coronary artery, coincident with thromboxane and prostacyclin release. Dazoxiben consistently inhibited TxA2 synthesis and enhanced prostacyclin synthesis. ±SQ 29, 548 augmented TxB2 release in response to arachidonate, but not bradykinin, and did not significantly alter 6-keto-PGF release in response to either arachidonate or bradykinin. In terms of vasoactivity measured , ±SQ 29, 548 and dazoxiben produced similar anti-vasospastic effects, although this was accomplished by completely different mechanisms.  相似文献   

8.
Biosynthesis of prostaglandins in ob17 preadipose cells was studied in culture. Dihomo-γ-linolenic acid is exclusively converted to PGE1. Arachidonic acid behaves quantitatively as a more potent precursor, leading to the synthesis of PGE2 and 6-keto-PGF (stable product of prostacyclin). In all cases prostaglandin synthesis was confirmed directly by radioimmunoassay. This synthesis is maximal during the growth phase and decreases dramatically after confluence at a time where adipose conversion occurs, suggesting a possible relationship between both events.  相似文献   

9.
Metabolism of [9-3H]-PGI2 was studied in the isolated Tyrode's perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF, 6-keto-PGF, dinor-6-keto-PGF, pentanor PGF and a 6-keto-PGE1-like substance. The first two, 19-hydroxy-6-keto-PGF and 6-keto-PGF, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI2. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE1 was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE1, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE1, may arise from oxidation of 6-keto-PGF via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI2 or its hydrolysis product, 6-keto-PGF.  相似文献   

10.
This study examines the hypothesis that PAF stimulates release of PGI2 from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI2 release.  相似文献   

11.
Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF. According to the typical calibration curve of our ELISA, 6-keto-PGF can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF. The resulting ELISA was applied to the quantification of 6-keto-PGF generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.  相似文献   

12.
The plasma concentration of 6-keto-PGF, the stable degradation product of prostacyclin, was similar in the radial and pulmonary arteries and in the coronary sinus before and after the induction of the anesthesia in patients undergoing coronary artery bypass surgery. After the beginning of the mechanical ventilation and anesthesia the pulmonary vascular resistance decreased although no changes were detected in the plasma levels of 6-keto-PGF or TXB2. During the prebypass period after the sternotomy and cannulation of the large vessels the plasma level of 6-keto-PGF was increased similarly in the radial and pulmonary arteries and even more in the coronary sinus. During the cardiopulmonary bypass the concentration of 6-keto-PGF remained at the increased level as compared to the values before the anesthesia. This indicates that pulmonary circulation is perhaps not the main source of prostacyclin in man. The plasma level of TXB2 was increased during the prebypass period significantly only in the coronary sinus, but during the bypass also in the radial artery. The concentration ratio of 6-keto-PGF/TXB2 was increased significantly during the prebypass period in the radial and pulmonary arteries. At the same time the pulmonary vascular resistance was, however, returned to the preanesthesia level and was thus not decreased. The vascular resistance in the systemic circulation was increased during the prebypass period. The plasma level of 6-keto-PGF or TXB2 in the radial and pulmonary arteries did not correlate significantly with the total vascular resistance in the systemic and pulmonary circulation, respectively. The vascular resistance in the coronary circulation did not correlate significantly with TXB2 level in the radial artery or coronary sinus. There was, however, a slight positive correlation between the blood flow and the concentration of TXB2 in the coronary sinus (r=0.76, P < 0.01). Coronary sinus flow did, however, not correlate with the plasma level of 6-keto-PGF in the radial artery or coronary sinus. These results indicate that the detected plasma concentrations of prostacyclin and thromboxane A2 have no significant effects on the total vascular resistance in vivo.  相似文献   

13.
Eicosanoid production by intrauterine tissues from control and neonatal-steptozotocin induced diabetic rats during late pregnancy was evaluated. In diabetic placenta the release of 6-keto-PGF was found diminished when compared to controls. In addition, LTB4 generation was increased in diabetic placenta. No alterations in the production of TXA2, PGE2, PGE1 and PGF was found when diabetic and control placenta were compared. In amnion tissue a decreased generation of 6-keto-PGF was observed in the diabetic group, but no alteration in any other eicosanoid evaluated was found. Oxytocin (5 mU/ml, in vitro), which increases prostaglandin synthesis in rabbit and human amnion tissues, did not modify eicosanoid generation in control rat amnion. In contrast, in diabetic amnion the presence of oxytocin further decreased the release of 6-keto-PGF and diminished PGE1 generation. The present results suggest that this mildy diabetic state induces alterations in eicosanoid production in intrauterine tissues, abnormalities probably enhanced during parturition, when endogenous concentrations of oxytocin are elevated.  相似文献   

14.
There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.  相似文献   

15.
Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF1α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF1α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE2 were devoid of effect. The time-dependent in vitro production of 6-keto-PGF1α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.  相似文献   

16.
Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF1α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF1α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE2 were devoid of effect. The time-dependent in vitro production of 6-keto-PGF1α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.  相似文献   

17.
Acetylcholine causes pulmonary vasodilation, but its mechanism of action is unclear. We hypothesized that acetylcholine-induced pulmonary vasodilation might be associated with prostacyclin formation. Therefore, we used isolated rat lungs perfused with a recirculating cell- and plasma-free physiological salt solution to study the effect of acetylcholine infusion on pulmonary perfusion pressure, vascular responsiveness and lung prostacyclin production. Acetylcholine (20 ug infused over 1 minute) caused immediate vasodilation during ongoing hypoxic vasoconstriction and prolonged depression of subsequent hypoxic and angiotensin II-induced vasoconstrictions. Both effects of acetylcholine were abolished by atropine pretreatment. The prolonged acetylcholine effect, but not the immediate response, was blocked by meclofenamate, an inhibitor of cyclooxygenase. The prolonged effect, but not the immediate response, of acetylcholine was associated with an increase in perfusate 6-keto-PGF concentration. The acetylcholine stimulated increase in 6-keto-PGF production was inhibited by meclofenamate and by atropine. Thus, blockade of prostacyclin production corresponded with blockade of the prolonged acetylcholine effect. In conclusion, acetylcholine caused in isolated rat lungs an immediate vasodilation and a prolonged, time-dependent depression of vascular responsiveness. Whereas both acetylcholine effects were under muscarinic receptor control, only the prolonged effect depended on the cyclooxygenase pathway and, presumably, protacyclin synthesis.  相似文献   

18.
The effect of sex steroids, 17β-estradiol and testosterone, on the production of 6-keto-prostaglandin F, prostaglandin F and prostaglandin E2 was studied in cultures of piglet aorta endothelial cells. In cells isolated from female animals both steroids stimulated the secretion of prostaglandins. In contrast, sex steroids did not affect prostaglandin synthesis by endothelial cells taken from male animals. In addition, female endothelial cells convert testosterone into Estriol, estrone and estradiol. estradiol-induced stimulation of prostacyclin production may explain in part the beneficial role generally attributed to naturally occuring estrogens in cardiovascular diseases.  相似文献   

19.
PGI2 synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI2 produced were shown as 6-keto-PGF per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI2 synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI2 production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI2 production by atherosclerotic aorta was also significantly higher than that of controls. PGI2 producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI2 production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI2 than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI2 production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.  相似文献   

20.
Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI2. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4–10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of β-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF (6-keto, PGI2 metabolite), PGE2 and thromboxane B2 (TXB2) by EIA (hormone stimulated — basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF release, whereas estrogen increased RAEC 6-keto-PGF release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI2 release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI2.  相似文献   

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