Detection of endogenous 12-hydroxyeicosatrienoic acid in human tear film.

PURPOSE: Increased production of 12-hydroxyeicosatetraenoic acid [12(R)-HETE] and 12-hydroxyeicosatrienoic acid [12(R)-HETrE] positively correlates with the in vivo progression of ocular surface inflammation in rabbits. Tear film was collected from human subjects with inflamed eyes to determine whether these eicosanoids could be detected from endogenous sources. METHODS: Control and inflamed eyes were assessed and assigned a subjective inflammatory score. Tears were collected and extracted with an internal standard. Single-ion-monitoring gas chromatography-mass spectrometry (SIM-GC-MS) was performed to quantitate endogenous levels of 12-HETE and 12-HETrE. RESULTS: 12-HETrE was detected in the tear film of both control and inflamed eyes, with the mean level being seven times higher in inflamed tears. 12-HETE was not detected in control tears and was detected in only 6 of 38 inflamed-eye tear samples. CONCLUSIONS: The current findings demonstrate that the human eye produces detectable amounts of 12-HETrE, which is released into the tear flow. The increased levels of 12-HETrE associated with ocular surface inflammation suggest that this eicosanoid may contribute to inflammation of the ocular surface in humans.

The effect of hypoxia on endogenous corneal epithelial eicosanoids.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Regulation of cyclooxygenase-2 by hypoxia and peroxisome proliferators in the corneal epithelium.

Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR alpha and beta but not gamma mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E(2) accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation.

Hypoxia stimulates the synthesis of cytochrome P450-derived inflammatory eicosanoids in rabbit corneal epithelium.

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.

pH profiles indicative of rate-limiting nucleophilic displacement in thioltransferase catalysis.

The apparent pKa for the active site thiol of human thioltransferase (TTase) is about 3.5, but the pH dependence of TTase-catalyzed rates of glutathione (GSH)-dependent reduction of disulfide substrates displays an inflection point near pH 8.5. The similarity of the pH-rate profile with the titration of the GSH thiol moiety suggested rate-limiting nucleophilic attack by the glutathionyl thiolate species to regenerate reduced TTase from the TTase-SSG intermediate. To test this hypothesis pH-rate profiles for TTase-catalyzed dethiolation of the glutathionyl mixed disulfide of bovine serum albumin ([35S]BSA-SSG) were measured according to release of radiolabeled GS-equivalents. Various thiol compounds, whose thiol pKa values range on both sides of the pKa of GSH (pKa = 8.7), were used as reducing substrates, e.g., trifluoroethanethiol (pKa = 7.5) and 3-mercaptopropionic acid (pKa = 10.3). The pH-rate profiles paralleled the titration of the respective thiol groups of the reducing substrates, consistent with the hypothesis. In addition, second-order rate constants (k) were determined for the nonenzymatic and TTase-catalyzed reactions of the various thiols with BSA-SSG. A simple linear free energy relationship (log k vs pKa) was displayed for the nonenzymatic reactions. In contrast, the relationship for the enzymatic reactions revealed GSH to be different from the other thiol substrates, i.e., GSH gave a second-order rate constant greater than expected for its thiol pKa. This result suggests a special interaction of GSH with the TTase enzyme in the transition state that enhances the nucleophilicity of GSH.