Studies of lipoxygenases in the epithelium of cultured bovine cornea usingan air interface model

Epithelial lipoxygenases of bovine cornea were investigated in organ cultur e models. Subcellular fractions of the epithelium were incubated with C-14- labelled arachidonate and the metabolites were analysed. Bovine corneal epi thelial cells contain 15-lipoxygenase type 2 and 12-lipoxygenases of the le ukocyte and the platelet types. The 15-lipoxygenase activity was prominent in the cytosolic fraction. Twelve- and 15-lipoxygenases occurred in the mic rosomal fraction, where the 15-lipoxygenase activity appeared to be favoure d by low protein levels. The lipoxygenase activities strongly declined with in 24 hr when the cornea was covered with cell culture medium, but were mai ntained with high activity in an air interface organ culture model for at l east, 72 hr, Cultured corneas were studied in pairs in the air interface mo del under influence of inflammatory stimuli. The epithelial 15- and 12-lipo xygenase activities were only slightly augmented by treatment with 12-O-tet radecanoyl-phorbol-13-acetate (10 mu M, 8-72 hr), and remained unchanged af ter treatment with lipopolysaccharide (1-100 mu g ml-l. 8-72 hr) or UV irra diation (301 nm, 0.17 J cm(-2); 8-24 hr). In some experiments, 5-lipoxygena se activity was detectable, as judged from liquid chromatography-mass spect rometry and chiral chromatography. Reverse transcription-polymerase chain r eaction 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 iden tity with human 5-lipoxygenase and by Northern blot as a 3.0 bp mRNA. Leuko triene 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 c hain reaction. (C) 2000 Academic Press.