8-11-14-eicosatrienoic-acid and 12-hydroxy-5-8-14-eicosatrienoic-acid

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cattle; Cornea; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Eye; Hydroxyeicosatetraenoic Acids; Isoenzymes; Oxygenases; Sodium-Potassium-Exchanging ATPase

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal (14)C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Animals, Newborn; Arachidonic Acid; Aryl Hydrocarbon Hydroxylases; Chromatography, Liquid; Cytochrome P450 Family 2; Lipoxygenase; Mass Spectrometry; Mice; Microsomes; Mixed Function Oxygenases; NADP; Skin; Stereoisomerism

Injury to the ocular surface induces the production of the corneal epithelial-derived 12-hydroxyeicosatetrienoic acid (12-HETrE), which exhibits stereospecific potent inflammatory and angiogenic properties and is formed by a cytochrome P450 (P450) enzyme, CYP4B1. We have cloned the rabbit corneal CYP4B1 into the expression plasmid pIRES2-enhanced green fluorescent protein (EGFP) and examined the effect of CYP4B1 overexpression on corneal inflammation in vivo and limbal vessel sprouting ex vivo. Cultured rabbit corneal epithelial cells transfected with pIRES2-EGFP-CYP4B1 metabolized arachidonic acid to 12-HETrE at a rate five times higher than that of pIRES2-EGFP-transfected cells (3.53 +/- 0.08 versus 0.62 +/- 0.10 nmol/h/10(6) cells; mean +/- S.E.M., n = 6, p < 0.05), indicating a functional expression of the CYP4B1. Injection of either plasmid into the rabbit cornea resulted in EGFP fluorescence in the corneal epithelium. However, corneal neovascularization, as measured by the length of penetrating blood vessels, was significantly greater in the corneas of eyes transfected with the pIRES2-CYP4B1 compared with pIRES2-EGFP. Corneal-limbal explants from eyes transfected with pIRES2-CYP4B1 showed a marked angiogenic activity (46 +/- 10 versus 12 +/- 3 mm capillary length, n = 6, p < 0.05), which correlated with increased levels of 12-HETrE, the CYP4B1-derived angiogenic 12-hydroxyeicosanoid (0.93 +/- 0.18 versus 0.15 +/- 0.02 pmol/explant, n = 6, p < 0.05), and was inhibited (76 +/- 5%) by the P450 inhibitor 17-octadecynoic acid. The results further implicate the corneal CYP4B1 as a component of the inflammatory and angiogenic cascade initiated by injury to the ocular surface and raise the possibility of a new therapeutic target for preventing corneal neovascularization, namely, the CYP4B1-12-HETrE system.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aryl Hydrocarbon Hydroxylases; Cornea; Corneal Neovascularization; Male; Rabbits; Transfection

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Burns; Contact Lenses; Cytochrome P-450 Enzyme System; Disease Models, Animal; Eicosanoids; Eye Injuries; Inflammation; Neovascularization, Physiologic; Rabbits; Wound Healing

Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate.. Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection.. In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE.. Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Blotting, Western; Chromatography, High Pressure Liquid; Culture Media; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Epithelium, Corneal; Gas Chromatography-Mass Spectrometry; Hypoxia; Immunoenzyme Techniques; Isoenzymes; Organ Culture Techniques; Prostaglandin-Endoperoxide Synthases; Rabbits

The corneal epithelium metabolizes arachidonic acid by a cytochrome P450-(CYP) mediated pathway to 12(R)hydroxy-5,8,10,14-eicosatrienoic acid [12(R)-HETE] and 12(R)hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE]. Both metabolites possess potent inflammatory properties with 12(R)-HETrE being a powerful angiogenic factor and assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea, in vivo. We developed an in vitro model of corneal organ culture to characterize the biochemical and molecular events involved in the increased synthesis of these metabolites. These cultured corneas exhibit epithelial cytochrome P450 CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis as indicated by chiral analysis and by the ability of CYP enzyme inhibitors to repress their synthesis. Hypoxia greatly and selectively stimulated the synthesis of 12(R)-HETE (7-fold over control normoxic conditions) and 12(R)-HETrE. The bacterial endotoxin, lipopolysaccharide, also increased the synthesis of these eicosanoids, substantiating the notion that this activity may function as an inflammatory pathway. These metabolites were detected in the culture medium by gas chromatography/mass spectroscopy (GC/MS) analysis and their levels significantly increased in hypoxia-treated corneas, further indicating their endogenous formation in response to injury. This in vitro model provides an excellent preparation for studying factors regulating the synthesis of these inflammatory eicosanoids and for isolating, identifying and characterizing the CYP protein responsible for their synthesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Cell Hypoxia; Culture Media; Cytochrome P-450 Enzyme System; Epithelium, Corneal; Inflammation; Lipopolysaccharides; Organ Culture Techniques; Rabbits

Delayed-type hypersensitivity (DTH) reactions are initiated by sensitized T cells. Their progression is dependent upon the local release of various autacoids, including cytokines and eicosanoids, by T cells, infiltrating inflammatory cells, and resident tissue cells. 12(R)-hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE], an eicosanoid produced by skin and cornea, possesses potent proinflammatory properties at picomolar concentrations including vasodilation, increase in membrane permeability, neutrophil chemotaxis, and angiogenesis. Because DTH reactions are associated with many of these same phenomena, we examined the effect of 12(R)-HETrE and related 12-hydroxyeicosanoids on the expression of DTH to purified protein derivative of tuberculin in sensitized guinea pigs. In the absence of purified protein derivative of tuberculin, none of the eicosanoids evoked erythema or edema after intradermal injection at doses up to 100 pmol. When injected together with purified protein derivative of tuberculin, 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], but not its enantiomer 12(S)-HETE, significantly inhibited macroscopic expression of delayed reactivity (erythema) only at the highest dose tested, 10 pmol. In contrast, 12(R)-HETrE significantly enhanced expression of DTH at doses between 1 fmol and 1 pmol (50% and 30% increases above control, respectively). Its stereoisomer, 12(S)-HETrE, did not enhance DTH at any tested dose, but was able to block the activity of 12(R)-HETrE when injected simultaneously. Enhancement or inhibition of visible skin responses was not associated with qualitative or quantitative changes in cellular infiltrates at the reaction site. 12(R)-HETrE had no effect on the nonimmunologic inflammatory skin reaction induced by phorbol myristate acetate, suggesting selectivity toward DTH. We conclude that 12(R)-HETrE enhances DTH via a yet to be determined mechanism and that its stereoisomer, 12(S)-HETrE, may be a useful antagonist for studying the inflammatory actions of this eicosanoid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Capillary Permeability; Dermatitis; Erythema; Guinea Pigs; Hypersensitivity, Delayed; Male; Skin; Tetradecanoylphorbol Acetate; Tuberculin

The corneal epithelium of several species, has the capacity to metabolize arachidonic acid (arachidonic acid) via an NADPH-dependent cytochrome P450 mechanism. The major metabolites are 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), both of which exist in stereoisomeric configurations. However, the R enantiomers are predominantly produced by this enzyme system and exhibit potent biological activities. 12(R)-HETE inhibits Na-K-ATPase, increases corneal thickness and reduces intraocular pressure. 12(R)-HETrE causes vasodilation, neutrophil chemoattraction and angiogenesis. The formation of these metabolites is unaffected by cyclooxygenase and lipoxygenase inhibitors (indomethacin, diclofenac and BW755C) but inhibited by cytochrome P450 enzyme inhibitors such as carbon monoxide, SKF-525A and clotrimazole. The capacity of the normal corneal epithelium to metabolize arachidonic acid via cytochrome P450 is very low although under certain conditions this enzymatic pathway may become greatly induced. Corneal epithelial hypoxia in response to contact lens wear results in the time-dependent formation of NADPH-cytochrome P450-dependent arachidonate metabolites, 12(R)-HETE and 12(R)-HETrE. Under this condition, metabolite production correlates strongly with the in situ inflammatory response and inhibition of their formation significantly attenuates inflammation. It is evident that the cytochrome P450 arachidonate metabolites should be added to the realm of cyclooxygenase and lipoxygenase-derived eicosanoids as possible inflammatory mediators. Therefore, studies to evaluate eicosanoid involvement in inflammation should examine inhibitors of this pathway in addition to the classically studied non-steroidal antiinflammatory drugs (NSAIDs).

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cattle; Chromatography, High Pressure Liquid; Clotrimazole; Cornea; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Epithelium; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Microsomes; Proadifen; Rabbits

The R and S enantiomers of 12-hydroxyeicosatetraenoic acid (12-HETE) exhibit different biological activities. Although they appear to be produced by different enzymatic pathways, cytochrome P-450 monooxygenase and lipoxygenase, respectively, they display similar metabolism in both corneal epithelium and neutrophils. In corneal epithelial microsomes, both enantiomers are subject to oxidation and keto reduction reactions to form the dihydro metabolite, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), via a keto intermediate. The apparent Km for the formation of 12-HETrE was 17.9 and 20 microM for 12(R)-HETE and 12(S)-HETE, respectively, and the apparent Vmax of the reaction was 17.4 and 8.2 pmol/mg per min, respectively. Chiral analysis of the dihydro metabolite demonstrated a product enantiospecificity. Arachidonic acid, 12(R)-HETE, 12(S)-HETE and the intermediate of this reaction, 12-oxo-ETrE, were metabolized predominantly to 12(R)-HETrE in a ratio [12(R)-HETrE: 12(S)-HETrE] of 7.3:1, 4.3:1, 1.5:1 and 2.3:1, respectively. 12(R)-HETrE is a potent vasodilator, chemotactic and angiogenic factor whose synthesis is induced in inflamed tissues; 12(S)HETrE is devoid of these properties. 12(R)-HETE, derived from NADPH-dependent cytochrome P-450 monooxygenases, and 12(S)-HETE, derived from 12-lipoxygenase, may both play an important role in regulating the inflammatory response by serving as substrates for the local synthesis of 12(R)-HETrE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Cattle; Cornea; Cytochrome P-450 Enzyme System; Epithelium; Hydroxyeicosatetraenoic Acids; Microsomes; NADP; Oxidation-Reduction; Stereoisomerism

Two biologically active cytochrome P-450 arachidonate metabolites previously were characterized: 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) and 12(R)-hydroxy-5,8,14-eicosatrienoic acid (12(R)-DH-HETE), which are endogenously formed in the corneal epithelium. The functional activity of these novel metabolites mimics changes observed in hypoxic corneas. Therefore, the effect of hypoxic stress was examined on metabolite formation in rabbits fitted with polymethylmethacrylate contact lenses. Although applied lenses fit tightly to the rabbit cornea, mechanical irritation also may contribute to the ocular response. Contact lens-induced hypoxic stress stimulated endogenous formation of both 12(R)-HETE (a sodium, potassium adenosine triphosphatase inhibitor) and 12(R)-DH-HETE (a vasodilatory, chemotactic, and angiogenic factor) in a time-dependent manner. After 4 hr of contact lens wear, a 21-fold increase in endogenous 12(R)-HETE formation concomitant with an increase in corneal thickness was observed. After prolonged contact lens wear (144 hr), a 23-fold increase in endogenous 12(R)-DH-HETE formation was found, corresponding with the appearance of a marked conjunctival inflammation characterized by corneal neovascularization. The increased formation of these compounds was associated with time-dependent changes in corneal endothelial morphology. The ability of 12(R)-HETE and 12(R)-DH-HETE to mediate the clinical signs of corneal hypoxia suggest these metabolites may be potential mediators of contact lens complications that followed conditions of hypoxic stress and possibly mechanical irritation in this model.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Cell Count; Chromatography, High Pressure Liquid; Conjunctivitis; Contact Lenses; Cornea; Corneal Neovascularization; Cytochrome P-450 Enzyme System; Endothelium, Corneal; Epithelium; Hydroxyeicosatetraenoic Acids; Methylmethacrylates; Oxygen Consumption; Rabbits; Time Factors