Studies of lipoxygenases in the epithelium of cultured bovine cornea using an air interface model.
Epithelial lipoxygenases of bovine cornea were investigated in organ culture models. Subcellular fractions of the epithelium were incubated with(14)C-labelled arachidonate and the metabolites were analysed. Bovine corneal epithelial cells contain 15-lipoxygenase type 2 and 12-lipoxygenases of the leukocyte and the platelet types. The 15-lipoxygenase activity was prominent in the cytosolic fraction. Twelve- and 15-lipoxygenases occurred in the microsomal fraction, where the 15-lipoxygenase activity appeared to be favoured by low protein levels. The lipoxygenase activities strongly declined within 24 hr when the cornea was covered with cell culture medium, but were maintained with high activity in an air interface organ culture model for at least 72 hr. Cultured corneas were studied in pairs in the air interface model under influence of inflammatory stimuli. The epithelial 15- and 12-lipoxygenase activities were only slightly augmented by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (10 microM, 8-72 hr), and remained unchanged after treatment with lipopolysaccharide (1-100 microgram ml(-1), 8-72 hr) or UV irradiation (301 nm, 0.17 J cm(-2); 8-24 hr). In some experiments, 5-lipoxygenase activity was detectable, as judged from liquid chromatography-mass spectrometry and chiral chromatography. Reverse transcription-polymerase chain reaction and Northern blot analysis were therefore used to identify mRNA of 5-lipoxygenase and related enzymes in bovine epithelium. 5-Lipoxygenase was detected as an amplicon of 695 bp, which had 91% nucleotide sequence identity with human 5-lipoxygenase and by Northern blot as a 3.0 kb mRNA. Leukotriene A(4)hydrolase was detected with the same techniques. The amino acid sequence of a 612 bp fragment was 90% identical with human leukotriene A(4)hydrolase and the size of the mRNA was 2.7 kb. The two enzymes were also detected in human corneal epithelium by reverse transcription-polymerase chain reaction.
Liminga
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Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, SE-751 24, Sweden. Maria.Liminga@farmbio.uu.se
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D000388:Air; D000818:Animals; D015152:Blotting, Northern; D002273:Carcinogens / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D017572:Leukotriene A4 / Q000032:analysis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008084:Lipoxygenase / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014466:Ultraviolet Rays
D002273:Carcinogens; D017572:Leukotriene A4; D008070:Lipopolysaccharides; D008084:Lipoxygenase; D013755:Tetradecanoylphorbol Acetate