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1.
Altered lipoxygenase metabolism and decreased glutathione peroxidase activity in platelets from selenium-deficient rats 总被引:5,自引:0,他引:5
Washed platelets from selenium-deficient and control rats were incubated with [1-14C]-arachidonic acid and the lipoxygenase and cyclooxygenase products were identified by gas chromatography/mass spectrometry. Platelets from selenium-deficient rats showed a three to four-fold increased synthesis of the lipoxygenase-derived isomeric trihydroxy fatty acids, 8,9,12-trihydroxy-5,10,14-eicosatrienoic acid and 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid. A major reduction in glutathione peroxidase activity was also observed in platelets from deficient rats. These results support the interpretation that these trihydroxy fatty acids arise from breakdown of the primary platelet lipoxygenase product -12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) under conditions in which its reduction to the -12-hydroxy product (12-HETE) by a selenium-dependent glutathione peroxidase is limited. Further-more, these results indicate a specific function for selenium in platelet metabolism of essential fatty acids. 相似文献
2.
Keizo Sekiya Hiromichi Okuda 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,797(3):348-353
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
3.
Wen-Chang Chang Junko Nakao Hajime Orimo Sei-itsu Murota 《The Biochemical journal》1982,202(3):771-776
Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by the cytosol fractions of rat platelets. The more polar products are formed via the lipoxygenase pathways in the same way as are 12-HPETE and 12-HETE, since their formation is not inhibited by indomethacin but by eicosa-5,8,11,14-tetraynoic acid (ETYA). The presence of 0.5-1.5mm-reduced glutathione (GSH) in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. l-Cysteine has the same effect as GSH. However, oxidized glutathione (GSSG) and l-cystine are not able to prevent the formation of the more polar products. The results indicate that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE. Thus the presence of GSH in the reaction mixture offers a convenient and precise cell-free assay system for 12-lipoxygenase in rat platelets. Routine assays of 12-lipoxygenase are carried out in the presence of 1mm-GSH in the reaction mixture. The synthesis of 12-HETE by 12-lipoxygenase is linear during the first 4 min of incubation at 37 degrees C, and has a pH optimum of 7.7. The 12-lipoxygenase reaches half-maximal activity at an arachidonate concentration of 20mum. Fractionation of cell homogenates indicates that the cytosol fraction possesses almost all the 12-lipoxygenase activity, whereas the microsomal fraction exhibits little enzyme activity. 相似文献
4.
Lipoxygenase Metabolism of Arachidonic Acid in Brain 总被引:14,自引:13,他引:1
Sunday A. Adesuyi Carolyn S. Cockrell Daniel A. Gamache Earl F. Ellis 《Journal of neurochemistry》1985,45(3):770-776
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
5.
Y. Ashida T. Saijo H. Kuriki H. Makino S. Terao Y. Maki 《Prostaglandins & other lipid mediators》1983,26(6):955-972
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
6.
Fujimoto Y Ikeda M Sakuma S 《Biochemical and biophysical research communications》2006,344(1):140-145
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
7.
This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production. 相似文献
8.
Male rats were exposed to freshly generated cigarette smoke once daily for 4 to 8 weeks. Inhalation of smoke was verified by elevated level of carboxyhemoglobin. Arachidonate metabolism through lipoxygenase and cyclooxygenase pathways in platelets was determined. Cigarette smoking increased 12-lipoxygenase activity significantly without affecting the cyclooxygenase pathway. In view of platelet-leukocyte interactions and potent chemotactic activity of 12-HETE for aortic smooth muscle cell migration, increased 12-lipoxygenase activity may predispose individuals to atherosclerosis, thromboembolism and emphysema commonly found in smokers. 相似文献
9.
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 and . 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
10.
12-Hydroxyeicosatetraenoic acid (12-HETE) is formed from arachidonic acid either by 12-lipoxygenase or by a cytochrome P450 monooxygenase. 12-Lipoxygenase is generally localized in the soluble cytosolic fraction, and the cytochrome P450 monooxygenase is a microsomal enzyme. In this study, 12-HETE biosynthesis and the regulation of 12-HETE biosynthesis by epidermal growth factor (EGF) in A431 cells were investigated. 12-HETE was biosynthesized from arachidonic acid by the microsomal fraction of A431 cells, but not by the cytosolic fraction. The formation of 12-HETE was inhibited by 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and caffeic acid. Nordihydroguaiaretic acid at 10(-4) M and 5,8,11,14-eicosatetraynoic acid at 10(-5) M almost completely inhibited its formation. However, the formation of 12-HETE was not affected by the presence of an NADPH-generating system, carbon monoxide, or SKF 525A. The biosynthetic 12-HETE was analyzed by chiral stationary phase high performance liquid chromatography and was highly enriched in (12S)-HETE. We therefore concluded that the enzyme responsible for the formation of (12S)-HETE in the microsomes of A431 cells is a 12-lipoxygenase. The microsomal 12-lipoxygenase of A431 cells belongs to the "leukocyte-type" enzyme as determined by substrate specificity and enzyme kinetics studies. The microsomal 12-lipoxygenase oxygenated linoleic acid much faster than the cytosolic platelet 12-lipoxygenase and is a "self-catalyzed inactivation" enzyme. Treatment of cells with 50 ng/ml EGF significantly induced microsomal 12-lipoxygenase activity. The lag period for the expression of the stimulatory effect of EGF on 12-lipoxygenase activity was approximately 10 h. The stimulatory effect of EGF on 12-lipoxygenase activity was completely blocked by treatment with 35 microM cycloheximide, indicating a requirement for de novo protein biosynthesis. Furthermore, the presence of the endogenous inhibitor of 12-lipoxygenase (which masked (12S)-HETE biosynthesis in intact cells) was identified in the cytosolic fraction of A431 cells. The putative inhibitor was enzyme-selective. It inhibited the leukocyte-type 12-lipoxygenase, but not the "platelet-type" enzyme. 相似文献
11.
F Sekiya J Takagi K Sasaki K Kawajiri Y Kobayashi F Sato Y Saito 《Biochimica et biophysica acta》1990,1044(1):165-168
We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407-415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids. 相似文献
12.
Makoto Nishiyama Hisayo Okamoto Takashi Watanabe Tomokatsu Hori Takahiko Hada Natsuo Ueda Shozo Yamamoto Hideo Tsukamoto† Keiichi Watanabe† Takaaki Kirino‡ 《Journal of neurochemistry》1992,58(4):1395-1400
The cytosol fraction from a thoroughly irrigated canine cerebrum was subjected to immunoaffinity chromatography using a monoclonal antibody against porcine leukocyte 12-lipoxygenase. Arachidonate 12-lipoxygenase eluted from the column with some retardation. The enzyme, with a specific activity of 9 nmol/min/mg of protein, converted arachidonic acid to 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid. The enzyme was active not only with arachidonic acid, but also with linoleic and alpha-linolenic acids. In contrast, 12-lipoxygenase of canine platelets was almost inactive with linoleic and alpha-linolenic acids, and the platelet enzyme was also distinguished from the cerebral enzyme in terms of reactivity with the anti-12-lipoxygenase antibody. 12-Lipoxygenase activity was also detected in the cytosol fractions of other parts of canine brain: basal ganglia, hippocampus, cerebellum, olfactory bulb, and medulla oblongata. 相似文献
13.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1994,1210(3):288-296
Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-lipoxygenase and a 15-lipoxygenase. The 12-lipoxygenase was found in the microsomal fraction, while the 15-lipoxygenase was mainly present in the cytosol (100 000 × g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosa-tetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 μ M. IC50 for the 12-lipoxygenase was 0.3 μM. The bovine corneal 12-lipoxygenase was compared with the 12-lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized 14C-labelled linoleic acid and α-linolenic acid poorly (5–16%) in comparison with [l4C]arachidonic acid. [14C]Docosahexaenoic acid and [14C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyconal antibody against porcine leukocyte 12-lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [14C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-lipoxygenase and that bovine corneal epithelium contains both a 15-lipoxygenase and a 12-lipoxygenase. The corneal 12-lipoxygenase appears to differ catalytically from earlier described bovine 12-lipoxygenases. 相似文献
14.
Zhou W Wang XL Kaduce TL Spector AA Lee HC 《American journal of physiology. Heart and circulatory physiology》2005,288(5):H2210-H2218
Arachidonic acid (AA) is a precursor of important vasoactive metabolites, but the role of AA-mediated vasodilation in Type 2 diabetes is not known. Using Zucker diabetic fatty (ZDF) rats, we examined the effects of AA in small mesenteric arteries preconstricted with endothelin. In ZDF rat mesenteric arteries, 1 microM AA produced only one-third the amount of dilation as in vessels from lean control animals. In lean control rats, the effect of AA was significantly and predominantly inhibited by the lipoxygenase inhibitors baicalein and cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC). However, baicalein and CDC had no effect on AA-mediated dilation in ZDF rat mesenteric arteries. The major [3H]AA metabolite produced by isolated mesenteric arteries in both lean and ZDF rats was 12-hydroxyeicosatetraenoic acid (12-HETE), but the amount of [3H]12-HETE produced by ZDF rat vessels was only 36% of that of control vessels. In addition, 12-HETE produced similar amounts of dilation in lean and ZDF rat mesenteric arteries. Immunoblot analysis showed an 81% reduction in 12-lipoxygenase protein in ZDF rat mesenteric arteries. Immunofluorescence labeling showed strong nitrotyrosine signals in ZDF rat mesenteric arteries that colocalized with 12-lipoxygenase in endothelium, and 12-lipoxygenase coprecipitation with anti-nitrotyrosine antibodies was enhanced in ZDF rat vessels. We conclude that AA-mediated relaxation in ZDF rat small mesenteric arteries is impaired due to reduced 12-lipoxygenase protein and activity. Increased oxidative stress and nitration of 12-lipoxygenase may underlie the impairment of AA-mediated relaxation in small mesenteric arteries of diabetic rats. 相似文献
15.
Since mouse mast tumor P-815 cells produce the slow reacting substance of anaphylaxis, their 5-lipoxygenase activity was examined by determining the conversion of arachidonic acid to 5-hydroxyeicosatetraenoic acid (HETE). Mast tumor cells from mouse ascites fluid synthesized 12-HETE as a major and 5-HETE as a minor metabolite. Once the cells were transferred to an in vitro culture system, the predominant synthesis of 12-HETE was abolished and synthesis of 5-HETE was greater than that of 12-HETE. 2-E-6 cells, obtained by cloning the tumor cells, synthesized a negligible amount of 12-HETE, but produced a large amount of 5-HETE. When the 2-E-6 cells were inoculated into mice and harvested again from the ascites fluid, their ratio of 5-HETE to 12-HETE synthesis was similar to that of normal mouse peritoneal cells; that is, 12-HETE synthesis was much greater than 5-HETE synthesis. It is concluded that the predominant synthesis of 12-HETE in mast tumor cells was derived from natural peritoneal cells, which have very high 12-lipoxygenase activity. The cloned mastocytoma, 2-E-6 cells, should be useful in investigating regulation of 5-lipoxygenase activity. 相似文献
16.
H Sinzinger W Linkesch H Ludwig H Gisslinger J O'Grady B A Peskar 《Prostaglandins》1990,40(4):351-360
In the course of an investigation of cyclooxygenase and 12-lipoxygenase activity in platelets of patients with myeloproliferative syndrome receiving treatment with interferon-alpha 2 patients showed unusual results which have not been reported so far. Both patients had thrombocytosis, in one case associated with polycythaemia. In platelets of both patients, a reduced conversion of exogenous 14C arachidonic acid to TXB2 was observed accompanied by a shift in conversion to PGE2 and 12-HETE in one patient and to 12-HETE alone in the other before therapy. These findings were paradoxically associated with evidence of enhanced platelet activation in vivo. Treatment of both patients with interferon-alpha resulted in reversal of the biochemical abnormalities and in clinical remission. 相似文献
17.
In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands. 相似文献
18.
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
19.
Activation of a 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen 总被引:3,自引:0,他引:3
Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively. 相似文献
20.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1994,1210(3):361-367
Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A4 (LTA4) either by a specific LTC4 synthase to leukotriene C4 or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC4, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA4 to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC4, LTD4 and LTE4). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA4 and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100 000 × g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE. 相似文献