首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
Formation of 12L-hydroxy-5,8,10,14-eicosatetraenoic acid from [10L-3H; 3-14C]arachidonic acid in suspensions of human platelets occurred with extensive loss of tritium and was accompanied by an isotope effect. These experiments showed that there is an antarafacial relation between the elimination of hydrogen from C-10 and insertion of oxygen at C-12 by human platelet lipoxygenase, and that the hydrogen elimination probably occurs as the initial step of the conversion. (Endo) peroxide intermediates formed by the fatty acid cyclo-oxygenase pathway activated platelet lipoxygenase.  相似文献   

2.
Lipoxygenase Metabolism of Arachidonic Acid in Brain   总被引:14,自引:13,他引:1  
When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.  相似文献   

3.
Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B2 both from [1-14C]arachidonic acid added to washed platelets and from [1-14C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B2 from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B2 in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B2 (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B2 and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B2 formation by the elastin.  相似文献   

4.
The oxygenation of arachidonic acid into thromboxane B2 (TXB2), 12-hydroxy-heptadecatrienoic (HHT) and 12-hydroxy-eicosatetraenoic (12-HETE) acids has been examined in human platelets in the absence or presence of 1mM calcium. From endogenous arachidonic acid, external calcium did not affect the formation of cyclo-oxygenase products (TXB2 and HHT) but enhanced that of 12-HETE when thrombin at high concentrations was the agonist. Dose-response curves performed with thrombin and collagen revealed that increased stimulation resulted in higher ratios of 12-HETE/HHT. On the other hand external calcium did not alter significantly the synthesis of either products from exogenous arachidonic acid and the total conversion of the substrate was unchanged. We conclude that extracellular calcium may facilitate the liberation of arachidonic acid from platelet phospholipids when induced by high thrombin concentrations. The excess of arachidonic acid liberated would then be diverted towards the lipoxygenase pathway.  相似文献   

5.
Interactions of human platelets with neutrophils were studied in suspensions of [3H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [3H]-labeled leukotriene B4 (LTB4), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [3H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [3H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB4 and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.  相似文献   

6.
Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.  相似文献   

7.
6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I1 (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both invitro and in vivo. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB4 synthesis in human peripheral neutrophils with an ID50 of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.  相似文献   

8.
In the present study, the effects of hypochlorous acid (HOCl), monochloramine (NH(2)Cl), glutamine-chloramine (Glu-Cl) and taurine-chloramine (Tau-Cl) on the formation of 12-lipoxygenase (LOX) metabolite, 12-HETE, and cyclooxygenase (COX) metabolites, TXB(2), and 12-HHT, from exogenous arachidonic acid (AA) in rat platelets were examined. Rat platelets (4x10(8)/ml) were preincubated with drugs for 5min at 37 degrees C prior to the incubation with AA (40microM) for 2min at 37 degrees C. HOCl (50-250microM) showed an inhibition on the formation of LOX metabolite (12-HETE, 5-67% inhibition) and COX metabolites (TXB(2), 33-73% inhibition; 12-HHT, 27-74% inhibition). Although Tau-Cl and Glu-Cl up to 100microM were without effect on the formation of 12-HETE, TXB(2) and 12-HTT, NH(2)Cl showed a strong inhibition on the formation of all three metabolites (10-100microM NH(2)Cl, 12-HETE, 21-92% inhibition; TXB(2), 58-94% inhibition; 12-HHT, 36-92% inhibition). Methionine reversed a reduction of formation of LOX and COX metabolites induced by NH(2)Cl, and taurine restoring that induced by both NH(2)Cl and HOCl. These results suggest that NH(2)Cl is a more potent inhibitor of COX and LOX pathways in platelets than HOCl, and taurine and methionine can be modulators of NH(2)Cl-induced alterations in the COX and LOX pathways in vivo.  相似文献   

9.
12-Lipoxygenase activity in platelets of spontaneously hypertensive rats was investigated. Enzyme activity was measured in the absence and the presence of reduced glutathione. In both assay conditions, 12-lipoxygenase activity in platelets of spontaneously hypertensive rats was significantly higher than that in platelets of normotensive rats. Since 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, has been reported to be a potent chemoattractant for aortic smooth muscle cells, increase in biosynthesis of 12-HETE in platelets of spontaneously hypertensive rats might contribute to the explanation of pathogenesis of vascular disorder commonly found in hypertension patients.  相似文献   

10.
The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.  相似文献   

11.
We used gas chromatography/mass spectrometry to measure brain 12-HETE (12-Hydroxy-5,8,10,14-eicosatetraenoic acid) formation from endogenous arachidonic acid in different species and different brain regions and in isolated brain microvessels. When blood-free brain slices were incubated for 20 minutes we found that the rabbit and cat brain incubates contained little 12-HETE when compared to rat and mouse brain incubates. Further in vitro studies of various rat brain regions showed a generally even distribution of 12-HETE. When isolated rat or rabbit microvessels were incubated and analyzed, we found 1 and 0.25 g, respectively, of 12-HETE/mg of microvessel protein. Also, rabbit brain had limited or no capacity to actively metabolize tritiated 12-HETE. In summary, these studies show substantial species variation with respect to brain formation of 12-HETE and indicate that the vasculature is a potentially significant contributor to the 12-HETE found in whole brain tissue.  相似文献   

12.
In clinical and pre-clinical research the pharmacodynamics of selective 5-lipoxygenase and dual 5-lipoxygenase/cyclo-oxygenase inhibitors may be studied by direct RIA of plasma LTB4. Although immunoreactive LTB4 in plasma from A23187 stimulated human blood has the characteristics of authentic LTB4 our results show, particularly in mice and rats, that exposure to A23187 produces large quantities of 12-HETE. Since in different species the levels of 12-HETE increase with platelet concentration we suggest that the 12(S)-HETE is produced by platelet lipoxygenase. However, we do not rule out the possibility that a proportion of 12-HETE may exist as the (R)-stereoisomer. The latter has greater potential for interference in the direct RIA of LTB4. Biosynthesis of 12-HETE may be measured either by RPHPLC/U.V. abs. (8) or by RIA (9) and LTB4 by a more specific antibody described in this report. We conclude that the combined ex vivo RIA of plasma TXB2, LTB4 and 12-HETE has utility in determining the selectivity of inhibitors of arachidonate metabolism and in distinguishing between selective 5-lipoxygenase inhibitors which interact directly with the enzyme and anti-oxidant or free radical scavenging types which may be less specific.  相似文献   

13.
The effects of procyanidins on platelet aggregation and arachidonate metabolism in platelets were studied. Nine procyanidins were used in this investigation. Procyanidins B-2-S, EEC and C-1 significantly induced the inhibition of platelet aggregation, and the potency of inhibition was comparable with aspirin. Procyanidin B-2-S was used as a representative of procyanidins for further studies on the effect on arachidonate metabolism. In arachidonate metabolism by fatty acid cyclooxygenase pathway, B-2-S inhibited TXB2 and HHT formation by intact platelets treated with exogenous arachidonic acid. It also inhibited TXB2 formation measured by a specific radioimmunoassay when the cells were challenged with calcium ionophore A23187. In cell-free system, B-2-S inhibited both TXB2 and 12-HETE bioxynthesis in platelet microsome and cytosol, respectively. The inhibitory effect on thromboxane biosynthesis might explain the inhibitory effect of procyanidins on platelet aggregation.  相似文献   

14.
AA-861, a selective 5-lipoxygenase inhibitor, suppressed A23187-induced formations of 5-HETE and LTB4 in rat peritoneal macrophages. Immunologically-stimulated generation of SRS-A was also inhibited in guinea pig lung and rat peritoneal cavity. AA-861 had no effects on histamine release from rat mast cels or passive cutaneous anaphylaxis in rats. Essentially no antagonistic activity to LDT4 or histamine was observed. This compound exerted an obvious inhibition of allergic bronchoconstriction in guinea pigs and a moderate reduction of carrageenin-induced paw edema and pleurisy in rats. These findins suggest that SRS-A plays an important role in asthmatic and inflammatory reactions.  相似文献   

15.
The important role of eicosanoids in pregnancy-induced hypertension is generally accepted. Because of the lack of innervation of the uteroplacental vessels, humoral vasoactive factors are important for the regulation of vascular tone. Until now, mainly the balance of vasodilatative and vasoconstrictive prostaglandins has been studied. We were able to confirm their intrauterine imbalance in hypertensive pregnancies. In addition, the placental production of less known lipoxygenase metabolites has been analyzed in this study. Intrauterine tissues (30–100mg wet weight) were examined for their release of eicosanoids. Short term tissue cultures were performed in Hanks balanced salts solution (HBSS) at 37°C in an atmosphere of 95% air/5% CO2 with and without incorporation of tritiated arachidonic acid. The arachidonate metabolites in culture media were analyzed by High Performance Liquid Chromatography (HPLC) with radioactivity detection or by enzymeimmunoassays or radioimmunoassays, respectively. All intrauterine tissues released more lipoxygenase metabolites than cyclooxygenase metabolites with 12-hydroxyeicosatetraenic acid (12-HETE) as their main metabolite. The placental release of 12-HETE was significantly decreased in hypertensive pregnancies. In hypertensive pregnancies the ratio TXB2/6-keto-PGF synthesis was increased.Lipoxygenase metabolites, especially 12-HETE, seem to have impotant physiological and pathophysiological functions in the intrauterine compartment. Their biological role in this context needs further investigation.  相似文献   

16.
This study was designed to evaluate the effects of different amounts of dietary vitamin E on platelet tocopherol levels and 12-lipoxygenase activity when exogenous arachidonic acid was used as substrate. Weanling male Sprague-Dawley rats were fed diets containing 0, 50, and 5000 ppm of D-alpha-tocopherol acetate for 4 months. Platelet tocopherol was increased with increasing concentrations of dietary vitamin E; however, the conversion of exogenously added arachidonate by platelet to 12-HETE (12-hydroxyeicosatetraenoic acid) and thromboxane B2 from these three dietary groups was essentially the same. This study provides direct evidence that platelet 12-lipoxygenase activity is independent of its vitamin E content when exogenously added arachidonate was used as substrate.  相似文献   

17.
The relationship between arachidonate metabolism and stimulated calcium fluxes in rabbit peritoneal neutrophils has been investigated. The addition of arachidonate to the neutrophils was found to cause a rapid and significant increase in the permeability of the plasma membrane to calcium. This effect is specific to calcium, concentration dependent and sensitive to inhibitors of the lipoxygenase mediated metabolic pathway(s). These results strongly suggest that arachidonate metabolites are directly involved in the mechanisms underlying calcium gating in the neutrophils.  相似文献   

18.
Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B (LTB), 6-trans-LTB4, 12-epi-6-trans-LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohydroxyeicosatetraenoic acids (i.e., 5-HETE) and omega-oxidation products (i.e., 2O -COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 microM), LTB4 but not 5-HETE formation was impaired. (1-14C)Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate, (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.  相似文献   

19.
Albumin is a major determinant of eicosanoid formation, affecting autacoids important in cell-cell interactions. We delineated three mechanisms by which albumin controlled platelet eicosanoid formation: 1) Albumin diverted free arachidonate toward 12-lipoxygenation. 2) Albumin enhanced release of arachidonate from phospholipids. 3) Albumin inhibited incorporation of arachidonate from the medium into platelet phospholipids. 12(S)-Hydroxyheptadecatrienoic acid (12-HHTrE) formation was reduced 70% by albumin as compared to that formed in albumin-free medium. In sharp contrast, formation of 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the platelet lipoxygenase product, was much less influenced by albumin. Moreover, 12-HETE production in the presence of albumin was markedly increased and prolonged after aspirin treatment. These data suggested that albumin redirected released endogenous arachidonate from cyclooxygenase to lipoxygenase. Therefore, the metabolic fate of arachidonate present in the medium of stimulated platelets was studied by adding tracer [3H]arachidonate 30 sec before thrombin. Albumin increased arachidonate metabolism by lipoxygenase 7-fold as compared to albumin-free controls, while cyclooxygenation increased 2.7-fold. Redirection of eicosanoid metabolism by albumin toward lipoxygenase products constitutes a heretofore undescribed and potentially important physiological role for albumin. In vitro utilization of albumin may reflect in vivo events in thrombosis and hemostasis more accurately than previous studies without albumin could appreciate.  相似文献   

20.
Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B4(LTB4). 6- -LTB4, 12- -6- -LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB4 but 5-HETE formation was impaired. (1-14C) Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate. (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号