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1.
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
2.
Anna-Karin Johnsson Elin Rönnberg David Fuchs Johan Kolmert Jesper Säfholm Hans-Erik Claesson Mats Hamberg Craig E. Wheelock Gunnar Nilsson Sven-Erik Dahlén 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2021,1866(5):158886
15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined. 相似文献
3.
Substitution of 15-hydroxyeicosatetraenoic acid in the phosphoinositide signaling pathway 总被引:6,自引:0,他引:6
A B Legrand J A Lawson B O Meyrick I A Blair J A Oates 《The Journal of biological chemistry》1991,266(12):7570-7577
Consideration of how 15-hydroxyeicosatetraenoic acid (15-HETE) might exert its biological actions led us to investigate the consequences of its incorporation into bovine pulmonary arterial endothelial cell (BPAEC) phospholipids [3H]15(S)-HETE was incorporated mainly (89%) into phosphatidylinositols, predominantly as 1-stearoyl-2-(15-HETE) phosphatidylinositol. By contrast 5(S)- and 12(S)-HETE are incorporated largely into phosphatidylcholine. 15-HETE had a long persistence in the phosphatidylinositols of BPAEC with a half-life of 12 h; its uptake was concentration-dependent, and it accumulated so that 2-(15-HETE) phosphatidylinositol accounted for 10.9% of total phosphatidylinositol after four sequential 1-h incubations of cells with 1 microM 15(S)-HETE. After incubating BPAEC with 15(S)-HETE, stimulation of the cells with bradykinin led to an increase in the levels of 15-HETE. Following addition of bradykinin to cells exposed to [3H]15(S)-HETE, a radiolabeled diacylglycerol was isolated. A mass spectrum of its pentafluorobenzoyl (PFBO) trimethylsilyl (Me3Si) derivative obtained with direct electron capture negative ion chemical ionization mass spectrometry (DNICI/MS) revealed a molecular anion and fragment ions that were identical with those observed with the PFBO/Me3Si derivative of authentic 1-stearoyl-2-(15-HETE) diacylglycerol. There was a lesser quantity of 1-oleoyl-2-(15-HETE) diacylglycerol. An increase in the quantity of 1-stearoyl-2-(15-HETE) diacylglycerol from 6 +/- 1.4 pmol/10(7) cells in the basal state to 12.7 +/- 3.5 after bradykinin was measured by DNICI/MS utilizing a deuterium-labeled analog as an internal standard. Thus, incorporation of 15(S)-HETE into the phosphatidylinositol of these cells led to the release of altered second messengers. 相似文献
4.
Oxidation of 15-hydroxyeicosatetraenoic acid and other hydroxy fatty acids by lung prostaglandin dehydrogenase 总被引:4,自引:0,他引:4
J M Bergholte R J Soberman R Hayes R C Murphy R T Okita 《Archives of biochemistry and biophysics》1987,257(2):444-450
The oxidation of the 15-hydroxy group of prostaglandins of the A, E, and F series by the NAD+-dependent prostaglandin dehydrogenase (PGDH) has been well documented. In addition to prostaglandins, we have observed that the purified lung PGDH also will oxidize 15-HETE to a novel metabolite that was isolated by reverse-phase HPLC and identified by gas chromatography-mass spectrometry as the 15-keto-5,8,11-cis-13-trans-eicosatetraenoic acid (15-KETE). The Km for 15-HETE was 16 microM, which was 2.5 times lower than the value obtained for PGE1. In addition to 15-HETE, 5,15-diHETE and 8,15-diHETE also were substrates for the lung PGDH with Km values of 138 and 178 microM, respectively. Other hydroxy derivatives of eicosatetraenoic acid that did not have a hydroxy group at carbon atom 15 did not support the PGDH-mediated reduction of NAD+. In addition to the 15-hydroxy derivatives of eicosatetraenoic acid, 12-HHT also was a substrate for the lung enzyme with a Km of 12 microM. These data indicate that omega 6-hydroxy fatty acids, in addition to prostaglandins, are also substrates of the lung NAD+-dependent PGDH and that the enzyme does not require the cyclopentane ring of prostaglandins. 相似文献
5.
Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage. 相似文献
6.
J P Falgueyret Y Leblanc J Rokach D Riendeau 《Biochemical and biophysical research communications》1988,156(3):1083-1089
The possibility that 12-keto-5,8,10,14 eicosatetraenoic acid (12-KETE) could be used as substrate by reductase(s) to generate 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated using rat liver microsomes as a source of enzyme activity. Microsomes catalyzed the time-dependent reduction of 12-KETE to 12-HETE in a reaction that required NAD(P)H. The maximal specific activity of 12-HETE formation was 1.7 nmol/min/mg of protein in the presence of NADH. The reaction could not be detected in the absence of cofactor or by using heat inactivated microsomes. The identity of the 12-HETE product was established by U.V. spectroscopy and co-elution with 12-HETE in two different systems of RP-HPLC. Resolution of the methyl esters of reaction products by chromatography on chiral columns also indicated that the reduction of 12-KETE with either NADPH or NADH generated a mixture of 12(S)- and 12(R)-HETE in a ratio of about 2:1. The results demonstrate the presence of a 12-KETE reductase activity in rat liver microsomes which can form both the R and S isomers of 12-HETE. 相似文献
7.
Murine cerebral microvascular endothelium incorporate and metabolize 12-hydroxyeicosatetraenoic acid 总被引:3,自引:0,他引:3
S A Moore L J Prokuski P H Figard A A Spector M N Hart 《Journal of cellular physiology》1988,137(1):75-85
Cultured murine cerebromicrovascular endothelial cells were employed to study the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) in an in vitro model of the blood-brain barrier. These endothelial cells convert 12-HETE to at least four, more polar compounds. Analysis of the least polar and predominant metabolite by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives indicate that the compound is 8-hydroxyhexadecatrienoic acid (8-HHDTrE). The uptake of 12-HETE into cell phospholipids peaks at 2 hr, and is not saturable up to the highest concentration tested, 5 microM. Seventy-five to 92% of this 12-HETE is incorporated into phosphatidylcholine, while the remainder is divided between the inositol and ethanolamine phospholipids. Incorporation into neutral lipids is slower, with radioactivity gradually accumulating in triglycerides over 24 hr. Saponification of cell lipids demonstrated that not only 12-HETE, but also its major metabolite, 8-HHDTrE, is incorporated into the cell lipids. Prostacyclin and prostaglandin E2 production by the cerebral endothelial cells is inhibited by up to 56% with 1 microM and 90% with 5 microM 12-HETE. These data demonstrate that 12-HETE is actively metabolized by cerebral endothelium and suggest at least two mechanisms through which 12-HETE may alter cerebromicrovascular function: 1) incorporation into cerebral endothelial membranes and 2) inhibition of cerebral endothelial prostaglandin production. Conversion of 12-HETE to more polar compounds, particularly 8-HHDTrE, may be interpreted as either the inactivation of 12-HETE or the production of additional, biological mediators. 相似文献
8.
Ability of 15-hydroxyeicosatrienoic acid (15-OH-20:3) to modulate macrophage arachidonic acid metabolism 总被引:1,自引:0,他引:1
R S Chapkin C C Miller S D Somers K L Erickson 《Biochemical and biophysical research communications》1988,153(2):799-804
Mouse peritoneal macrophages metabolize dihomogammalinolenic acid (20:3n-6) primarily to 15-hydroxy-8,11,13-eicosatrienoic acid (15-OH-20:3). Since the biological properties of this novel trienoic eicosanoid remain poorly defined, the effects of increasing concentrations of 15-OH-20:3 and its arachidonic acid (20:4n-6) derived analogue. 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), on mouse macrophage 20:4n-6 metabolism were investigated. Resident peritoneal macrophages were prelabeled with [3H]-20:4n-6 and subsequently stimulated with zymosan in the presence of either 15-OH-20:3 or 15-HETE (1-30 microM). After 1 hr, the radiolabeled soluble metabolites were analyzed by reverse phase high performance liquid chromatography. 15-OH-20:3 inhibited zymosan-induced leukotriene C4 (IC50 = 2.4 microM) and 5-HETE (IC50 = 3.1 microM) synthesis. In contrast to the inhibition of macrophage 5-lipoxygenase, 15-OH-20:3 enhanced 12-HETE synthesis (5-30 microM) and had no measurable effect on cyclooxygenase metabolism (1-10 microM) i.e., 6-keto-prostaglandin F1 alpha and prostaglandin E2 synthesis. Addition of exogenous 15-HETE produced similar effects. These results suggest that the manipulation of macrophage 15-OH-20:3n-6 levels may provide a measure of cellular control over 20:4n-6 metabolism, specifically, leukotriene production. 相似文献
9.
The present study was conducted to determine whether protein kinase C was involved in angiotensin II-mediated release of 12-hydroxyeicosatetraenoic acid (12-HETE) from bovine adrenal glomerulosa cells. Activators of protein kinase C, 12-O-tetradecanoylphorbol 4-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG), significantly increased release of 12-HETE. The effect of OAG was potentiated by BAYK8644, a stimulator of calcium entry. Sphingosine, H-7 and staurosporine, which inhibited the activity of protein kinase C in vitro, almost completely blocked 12-HETE release induced by TPA. These agents also significantly reduced angiotensin II-mediated 12-HETE release. When time course of the liberation of 12-HETE was measured, angiotensin II elicited sustained release of 12-HETE, which was inhibited by staurosporine. These results indicate that angiotensin II induces sustained release of 12-HETE, a feed forward regulator of aldosterone secretion, and that protein kinase C may be involved in this process. 相似文献
10.
Shoko Ueki Junichi Takagi Yuichi Kobayashi Fumie Sato Yuji Saito 《Journal of cellular physiology》1999,178(1):63-68
We report here that the 12-lipoxygenase metabolite of arachidonic acid, 12-hydroxy-5Z, 8Z, 10E, 14Z, eicosatetraenoic acid (12-HETE), stimulates cAMP production in human fibroblasts among various cultured cell lines tested. Although 12-HETE seemed to stimulate the phospholipase C (PLC)-protein kinase C (PKC) system, inhibitors against PLC and PKC did not reduce the cAMP production induced by 12-HETE, indicating that the activation of PLC-PKC system is not positively coupled with the stimulation of cAMP production. On the other hand, the cAMP production induced by 12-HETE was dependent on the Ca2+/calmodulin system in the cells. The results suggest that 12-HETE specifically stimulates Ca2+/calmodulin-dependent adenylyl cyclase to increase cAMP level in the fibroblasts. J Cell Physiol 178:63–68, 1999. © 1999 Wiley-Liss, Inc. 相似文献
11.
The metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated in mouse peritoneal macrophages enriched in cholesterol by incubation with acetylated low density lipoproteins. After incubating with labeled arachidonic acid, cholesterol-rich cells released more 12-HETE into the medium than unmodified macrophages. With time, however, 12-HETE decreased in the medium of both cell preparations suggesting re-uptake of this monohydroxyfatty acid and perhaps further metabolism. When control macrophages were incubated with radiolabeled 12-HETE for 2 hr, almost 70% of the cell-associated 12-HETE label was incorporated into phospholipids. In contrast, in cholesterol-rich cells, only 31% of the 12-HETE label was incorporated into phospholipids. Bee venom phospholipase completely hydrolyzed the label, suggesting that the monohydroxyfatty acid was esterified at the sn-2 position of the phospholipid. In cholesterol-rich cells, 69% of the 12-HETE was diverted into neutral lipids. Two major neutral lipids were identified in cholesterol-rich macrophages. One neutral lipid band which migrated with an Rf value of 0.34 contained the hydroxylated fatty acid esterified to a glyceride. The other neutral lipid band having an Rf value of 0.49 contained cholesterol and by further analysis was found to contain predominantly cholesteryl-12-HETE. The labeled fatty acids in these two neutral lipids were mostly oxidized products of 12-HETE in contrast to the native 12-HETE observed in the phospholipids. Cholesterol-rich macrophages released 25% more products of 12-HETE metabolism than control macrophages. Two major products were observed in the medium which eluted in the area of a standard di-HETE, LTB4, on high performance liquid chromatography (HPLC) analysis. We propose that the reincorporation of 12-HETE into these neutral lipids and the increased capacity for further metabolism of this biologically potent hydroxyfatty acid could be a mechanism by which the cholesterol-rich macrophage maintains its membrane function, and regulates the amount of 12-HETE in the pericellular space. 相似文献
12.
T. Radeau P. Godard C. Chavis A. Crastes de Paulet M. Damon 《Prostaglandins & other lipid mediators》1992,43(6)
Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory porperties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min , total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 ± 3 % versus 47 ± 2 % of the total synthesis, p < 0.005), whereas intracellular free 5-HETE was decreased (13 ± 3 % versus 37 ± 4 %, p < 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process. 相似文献
13.
J.E. Graeber B.M. Glaser B.N.Y. Setty J.A. Jerdan R.W. Walenga M.J. Stuart 《Prostaglandins & other lipid mediators》1990,39(6)
We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10−7 M) sitmulated human retinal microvessel endothelial cell migration by 42 ± 10% (mean ± S.E.M., p<0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization
. Concentrations at which the
effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration
and neovascularization
suggests that it may play a role in vasoproliferative disorders. 相似文献
14.
Mark W. Musch Robert W. Bryant Corazon Coscolluela Robin F. Myers Marvin I. Siegel 《Prostaglandins & other lipid mediators》1985,29(3):405-430
A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 uM ionophore, MC9 cells produce 270 ± 43 pmoles 5-HETE, 74 ± 40 pmoles 5,12 di HETEs and 65 ± 31 pmoles LTC4/106 cells from 37 uM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-di HETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 ± 2.4 and 5.4 ± 1.2 pmoles/106 cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 ± 3% cellular histamine) and continued for 30 minutes (37 ± 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release. 相似文献
15.
Conjugated linoleic acid (CLA) is a dietary fatty acid that has been shown to reduce tumorigenesis and metastasis in breast, prostate and colon cancer in animals. However, the mechanism of its action has not been clarified. The goal of this study was to determine whether CLA altered mouse mammary tumor cell growth and whether specific metabolites of the lipoxygenase pathway were involved in CLA action. Both t10, c12-CLA and a lipoxygenase inhibitor, but not c9, t11-CLA or linoleic acid (LA), reduced mouse mammary tumor cell viability and growth by inducing apoptosis and reducing cell proliferation. t10, c12-CLA reduced the production of the 5-lipoxygenase metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE). That effect was not seen with c9, t11-CLA or LA. Adding 5-HETE back to tumor cells reduced the t10, c12-CLA effect on both apoptosis and cell proliferation. These data suggest that t10, c12-CLA reduction of tumor cell growth may involve the suppression of the 5-lipoxygenase metabolite, 5-HETE, with subsequent effects on apoptosis and cell proliferation. 相似文献
16.
Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells 总被引:1,自引:0,他引:1
Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides. 相似文献
17.
Eva Arbogast Wolfgang Schfer Hans Peter Zahradnik 《Prostaglandins & other lipid mediators》1996,51(2):125-137
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-PGF1α 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
18.
The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 μM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-14Carachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB4 were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB4. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed. 相似文献
19.
Cheng J Ou JS Singh H Falck JR Narsimhaswamy D Pritchard KA Schwartzman ML 《American journal of physiology. Heart and circulatory physiology》2008,294(2):H1018-H1026
Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by N(G)-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction. 相似文献
20.
Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus. 相似文献