Arachidonic acid metabolism in corneal epithelial cells: Identification of lipoxygenase isoforms
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
The metabolism of arachidonic acid in the human cornea was investigated and compared with the metabolism in cattle and primates. All three species showed significant arachidonic acid metabolism inthe epithelium, whereas the activity in the stroma and the endothelium was low or undetectable. Subcellular fractions of the human epithelium formed 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), whereas 12(S)- and 15(S)-HETEs were identified in the bovine and monkey epithelia.
Several human lipoxygenase isoforms were identified. The epithelium contained mRNAs of 15-lipoxygenase type 1 (15-LOX-l), 15LOX-2 and 5-LOX, as evaluated by reverse transcriptionpolymerase chain reaction (RT-PCR) and sequencing. Furthermore, mRNAs of leukotriene A4 (LTA4-hydrolase and cyclooxygenase-2 were detected by RT-PCR.
Two bovine lipoxygenases were cloned, sequenced and analysed by Northern blot. The deduced amino acid sequence of one lipoxygenase was 82% identical with human 15-LOX-2 and the other lipoxygenase was 87 % identical with human platelet type 12-LOX. In addition, mRNAs of 5-LOX, leukocyte type 12-LOX and LTA4-hydrolase were detected with RT-PCR and confirmed by Northern blot analysis.
To investigate the effect of inflammatory stimuli on the corneal lipoxygenase activities, an airinterface organ culture model of bovine cornea was developed. The lipoxygenase activities were maintained for at least 72 h in culture and 12-O-tetradecanoyl-phorbol-13-acetate increased the 12- and 15-LOX activities 40 % and 50 %, respectively, whereas the effect of lipopolysaccharide andultraviolet irradiation was small.
The biosynthesis of 12(S)- and 15(S)-HETE in the bovine cornea was quantified with liquidchromatography-mass Spectrometry. The average biosynthesis of 15(S)-HETE and 12(S)-HETE from 30 µM arachidonic acid was 38±8 and <3 ng/mg protein/30 min respectively, which increased more than 100 % in the presence of Ca2+. The metabolism of endogenous and exogenous arachidonic acid in bovine corneal epithelia differed. The endogenous substrate generated 12(S)-HETE as the main product, 50 ± 13 pg/mg tissue, whereas the amount of 15(S)-HETE was low or undetectable. Organ culture of cornea and subsequent LC-MS analysis of the cornea1 epithelial lipoxygenase products provide unique opportunities to study lipoxygenase isoforms in vitro.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2000. , 55 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 222
Pharmaceutical biosciences, Cornea, cDNA cloning, lipoxygenase, liquid chromatography-mass spectrometry, organ culture
Research subject Pharmaceutical Pharmacology
IdentifiersURN: urn:nbn:se:uu:diva-434ISBN: 91-554-4666-3OAI: oai:DiVA.org:uu-434DiVA: diva2:164855
2000-04-05, Sal B21, BMC, Uppsala universitet, Uppsala, 10:15