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1.
Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases. 相似文献
2.
Wei S Brittin JJ Falck JR Anjaiah S Nithipatikom K Cui L Campbell WB Capdevila JH 《Analytical biochemistry》2006,352(1):129-134
An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase. 相似文献
3.
Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney. 总被引:4,自引:0,他引:4
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M L Schwartzman N G Abraham M A Carroll R D Levere J C McGiff 《The Biochemical journal》1986,238(1):283-290
Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla. 相似文献
4.
Omega- and (omega-1)-hydroxylation of lauric acid and arachidonic acid by rat renal cytochrome P-450 总被引:1,自引:0,他引:1
We resolved four cytochrome P-450s, designated as P450 K-2, K-3, K-4, and K-5, from the renal microsomes of untreated male rats by high-performance liquid chromatography (HPLC) and investigated the lauric acid and arachidonic acid hydroxylation activities of these fractions. P450 K-4 and K-5 had high omega- and (omega-1)-hydroxylation activities toward lauric acid. The ratio of the omega-/(omega-1)-hydroxylation activity of P450 K-4 and K-5 was 3 and 6, respectively. Also, P450 K-4 and K-5 effectively catalyzed the omega- and (omega-1)-hydroxylation of arachidonic acid. P450 K-3 was not efficient in the hydroxylation of either lauric acid or arachidonic acid. P450 K-2 had low omega- and (omega-1)-hydroxylation activities toward arachidonic acid, and efficiently catalyzed the hydroxylation of lauric acid at the (omega-1)-position only, not at the omega-position. 相似文献
5.
Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P450 总被引:1,自引:0,他引:1
Cytochrome P450-dependent oxidation of arachidonic acid was studied in liver microsomes from normal fed, protein-energy malnourished, and refed rats. The overall rate of arachidonic acid oxidation was very similar in microsomes from the three groups, but microsomes from malnourished rats showed a higher turnover rate than microsomes from normal fed and refed rats. The regiospecificity of cytochrome P450 oxidation of arachidonic acid was drastically altered by the animal nutritional status. Thus, protein-energy malnutrition results in a clear stimulation of total omega and omega-1 hydroxylation, concomitant with a marked decrease in olefin epoxidation and allyllic oxidations. These changes, as well as the documented biological activity of some of the cytochrome P450 arachidonate metabolites, suggest that protein-energy deficiency might help to select P450 isozymes which are probably involved in key monooxygenation reactions of physiological substrates. 相似文献
6.
Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase 总被引:16,自引:0,他引:16
The demonstration of in vivo arachidonic acid epoxidation and omega-hydroxylation established the cytochrome P450 epoxygenase and omega/omega-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and omega/omega-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products. 相似文献
7.
Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid 总被引:1,自引:0,他引:1
M L Schwartzman P J Pagano J C McGiff N G Abraham 《Archives of biochemistry and biophysics》1987,252(2):635-645
We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid. 相似文献
8.
A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways. 相似文献
9.
Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains 总被引:2,自引:0,他引:2
cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytochrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NADPH-P450 reductases were expressed in Escherichia coli and characterized enzymatically. Measurements of NADPH cytochrome c reductase activity showed fusion-dependent increases in the rates of cytochrome c reduction by the bacterial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes). Neither the bacterial flavoprotein nor the truncated rat reductase supported arachidonic acid metabolism by P450 2C11. In contrast, fusion of P450 2C11 to either reductase yielded proteins that metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P450 2C11 and native rat P450 reductase. Addition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11. These increases occurred with preservation of the regioselectivity of arachidonic acid metabolism. The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H(2)O(2) formation [73 +/- 10 and 10 +/- 1 nmol of H(2)O(2) formed min(-)(1) (nmol of P450)(-)(1) for the reconstituted and fused protein systems, respectively]. These studies demonstrate that (a) a self-sufficient, catalytically active arachidonate epoxygenase can be constructed by fusing P450 2C11 to mammalian or bacterial P450 reductases and (b) the P450 BM3 reductase interacts efficiently with mammalian P450 2C11 and catalyzes the reduction of the heme iron. However, fusion is required for metabolism and product formation. 相似文献
10.
Chen JK Chen J Imig JD Wei S Hachey DL Guthi JS Falck JR Capdevila JH Harris RC 《The Journal of biological chemistry》2008,283(36):24514-24524
Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system. 相似文献
11.
Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family. 相似文献
12.
Sánchez-Mendoza A López-Sánchez P Vázquez-Cruz B Rios A Martínez-Ayala S Escalante B 《Biochemical and biophysical research communications》2000,272(2):423-430
To assess the effect of angiotensin II on ion transport in rat isolated proximal tubules and establish the arachidonic acid cytochrome P450 metabolites' role mediating angiotensin II effect and to analyze whether corticosteroids play a role modulating this effect, we studied the effect of low (10 and 100 pM) and high (0.1-1 microM) angiotensin II concentrations on proximal tubule ion transport, measured as (86)Rb uptake. Low angiotensin II produced a stimulation on the (86)Rb uptake (195.79 +/- 35, 377.9 +/- 81, and 300 +/- 49 pg (86)Rb/microg protein/2 min, for control and 10 and 100 pM angiotensin II, respectively). High angiotensin II concentration inhibited ion transport (0.1 microM, 57.9 +/- 5 and 1 microM, 47.3 +/- 4 pg (86)Rb/microg protein/2 min), this effect was prevented by 17-ODYA and by losartan, while indomethacin had no effect. Dexamethasone treatment increased angiotensin II-induced (86)Rb uptake inhibition and arachidonic acid metabolism (19-, 20-HETE and 12-HETE), while adrenalectomy partly prevented angiotensin II-induced inhibition and decreased cytochrome P450-dependent arachidonic acid metabolism. In conclusion, high doses of angiotensin II produce inhibition of ion transport in rat isolated proximal tubules; this effect is mediated by AT(1) receptors, involves cytochrome P450-dependent arachidonic acid metabolites, and is upregulated by corticosteroids. 相似文献
13.
Gottlieb RA 《Archives of biochemistry and biophysics》2003,420(2):262-267
While attention has historically focused on mitochondria as the primary source of ROS in myocardial ischemia/reperfusion injury, recent evidence has implicated cytochrome P450 monooxygenases (CYPs) as a significant factor. CYPs represent a large family of enzymes that catalyze the oxidation of endogenous and exogenous compounds. They catalyze arachidonic acid oxidation to a variety of biologically active eicosanoids that regulate ion channels and protein kinases, with effects on vasomotor tone and cardiac inotropy. They also represent a significant source of reactive oxygen species that may target cellular homeostatic mechanisms and mitochondria. In this review, we will consider the contribution of cytochrome P450 enzymes to reperfusion injury and will speculate on whether the mechanism of injury is due to CYP-mediated ROS production or arachidonic acid metabolites. 相似文献
14.
Platelet microsomes were shown to contain cytochromes P-450 and b5 and their respective reductases, NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase. Metyrapone and carbon monoxide (CO), two inhibitors of cytochrome P-450, inhibited both the arachidonic acid-induced platelet aggregation and the formation of aggregating factors from arachidonic acid by isolated microsomes. In addition metyrapone produced a type II spectral change with platelet microsomal cytochrome P-450. The data suggest that cytochrome P-450 may play a role in the complex enzyme systems which convert arachidonic acid to the platelet aggregating factors, cyclic endoperoxides and thromboxane A2. 相似文献
15.
5,6-Epoxyeicosatrienoic acid stimulates growth hormone release in rat anterior pituitary cells 总被引:1,自引:0,他引:1
J R Cashman 《Life sciences》1989,44(19):1387-1393
The effect of arachidonic acid and some of its metabolites have been examined in rat anterior pituitary cells for their ability to release growth hormone. The cytochrome P-450 metabolite, 5,6-epoxyeicosatrienoic acid is a much more effective growth-hormone releasing agent than 15-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid methyl ester, 5-hydroxyeicosatetraenoic acid or arachidonic acid. The release of growth hormone is rapid, dose-dependent and reaches an apparent saturation after eight minutes. These studies described herein provide evidence that lipoxygenase and cyclooxygenase products of arachidonic acid are less potent while cytochrome P-450 products are more potent in the release of growth hormone from anterior pituitary cells. 相似文献
16.
Approaches to the chemical synthesis of cytochrome P-450-dependent metabolites of arachidonic acid and the biological role of novel metabolites in the arachidonic acid cascade are discussed. 相似文献
17.
Cytochrome P-450 enzyme-specific control of the regio- and enantiofacial selectivity of the microsomal arachidonic acid epoxygenase 总被引:5,自引:0,他引:5
J H Capdevila A Karara D J Waxman M V Martin J R Falck F P Guenguerich 《The Journal of biological chemistry》1990,265(19):10865-10871
Chiral analysis of the rat liver microsomal arachidonic acid epoxygenase metabolites shows enantioselective formation of 8,9-, 11,12-, and 14,15-cis-epoxyeicosatrienoic acids in an approximately 2:1, 4:1, and 2:1 ratio of antipodes, respectively. Animal treatment with the cytochrome P-450 inducer phenobarbital increased the overall enantiofacial selectivity of the microsomal epoxygenase and caused a concomitant inversion in the absolute configurations of its metabolites. These effects of phenobarbital were time-dependent and temporally linked to increases in the concentration of microsomal cytochrome P-450 enzymes. Reconstitution of the epoxygenase reaction utilizing several purified cytochrome P-450 demonstrated that the asymmetry of epoxidation is under cytochrome P-450 enzyme control. These results established that the chirality of the hepatic arachidonic acid epoxygenase is under regulatory control and confirm cytochromes P-450 IIB1 and IIB2 as two of the endogenous epoxygenases induced in vivo by phenobarbital. 相似文献
18.
Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver 总被引:3,自引:0,他引:3
M Laniado-Schwartzman K L Davis J C McGiff R D Levere N G Abraham 《The Journal of biological chemistry》1988,263(5):2536-2542
A novel human liver cytochrome P-450 isozyme (P-450-AA), which catalyzes arachidonic acid epoxidation, has been purified to electrophoretic homogeneity from human liver. As judged spectrally, the newly described isozyme is low spin in the oxidized state, with a soret band at 415 nm and an increased maximum at 451 nm in the CO-difference spectrum. Cytochrome P-450-AA appeared homogeneous as judged by the appearance of a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 53,100. Although cytochrome P-450-AA had a relatively low specific content of 10.8 nmol/mg, it possessed a high activity of arachidonic acid epoxidation. The P-450-AA oxidized arachidonic acid in a reconstituted system into the four regioisomeric epoxyeicosatrienoic acids (EETs) (5, 6-, 8, 9-, 11, 12-, 14, 15-EETs) at a rate of 2,010 pmol/nmol/min, a rate which is 37-fold higher than that observed with the crude microsomal preparation. Moreover, the purified cytochrome P-450-AA catalyzed the de-ethylation of 7-ethoxyresorufin at the rate of 2970 pmol/nmol/min, whereas other cytochrome P-450-dependent reactions were carried out at 23-2,000-fold lower rates and ranged between 0.3-130 pmol/nmol/min. The amino acid composition is different from that of other cytochrome P-450 isozymes. The NH2-terminal sequence of 20-amino acid residues was compared to that of LM2 and PB2-B2, the phenobarbital-induced forms in rabbit and rats, respectively. Comparison was also made with two forms of human cytochrome P-450, HLc and HLd. There were 7/20 identical residues for P-450-AA and LM2 and 4/20 for P-450-AA and PB2-B2. There were 2/20 identical residues for P-450-AA and HLd, and no identical residues were found for HLc. We conclude that the biologically active EETs, are formed by a distinct and unique P-450 isozyme from human liver and that arachidonic acid can serve as a screen for detection of the novel P-450 isozyme. 相似文献
19.
Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis 总被引:5,自引:0,他引:5
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The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H(2)O(2), etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptotic effects of 14,15-EET were blocked by two mechanistically and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitors, wortmannin and LY294002, but not by the specific mitogen-activated protein kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in vitro kinase assay revealed activation of Akt kinase within 10 min after 14,15-EET addition, which was completely abolished by either wortmannin or LY294002 pretreatment. In summary, the present studies demonstrated that 14,15-EET inhibits apoptosis by activation of a PI-3 kinase-Akt signaling pathway. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preventing activation of the neutral sphingomyelinase pathway and proapoptotic ceramide formation. 相似文献
20.
M E Addy 《Prostaglandins, leukotrienes, and essential fatty acids》1992,47(1):85-91
The effects of three chemically different groups of compounds, (triterpenoid saponins, beta-phenylethylamines and tetrahydroisoquinolines), known to be present in Desmodium adscendens, on plasma membrane ion channel, cytochrome P450 NADPH-dependent oxygenation of arachidonic acid, and production of prostaglandins by the cyclooxygenase enzyme system, are described. The very high-conductance calcium-activated potassium ion channel, which is responsible for the maintenance of tone in smooth muscles, was activated by the saponins. The cytochrome P450 NADPH-dependent monooxygenase reaction, which produces epoxy- and hydroxylated eicosanoids from arachidonic acid metabolism, was inhibited by an analogue of the tetrahydroisoquinoline present in the plant. This analogue also acted as a reductant in the prostaglandin synthesizing system using microsomes from ram seminal vesicles. The same system was activated by the beta-phenylethylamines found in the plant material, with the formation of more prostaglandins, the type being dependent on the amount of cyclooxygenase enzyme used and the presence or absence of coenzyme. 相似文献