title: Regulation of prostaglandin synthesis in Madin Darby canine kidney cells: role of prostaglandin G/H synthase and secreted phospholipase A2.
abstract: The renal epithelial cell line MDCK (Madin Darby canine kidney) was used as a model system to investigate the contribution of the secreted phospholipase A2 type II(sPLA2) and cyclooxygenases to prostaglandin E2 (PGE2) synthesis. Activation of protein kinase C by the phorbol ester TPA led to an enhanced PGE2 synthesis within 1 hour, which continued for more than 20 hours. Treatment of the cells with TPA increased the activities of sPLA2 and cyclooxygenase. Activation of cyclooxygenase was reflected by an increase in cyclooxygenase-2 mRNA. Coincubation of the cells with TPA and a specific sPLA2 inhibitor (BM 16.2224) almost completely inhibited sPLA2 activity in the cell culture supernatants. TPA-induced PGE2 synthesis was reduced by the inhibitor to about 50%. The inhibitor had no effect on cyclooxygenase activity or expression, indicating an involvement of sPLA2 in PGE2 synthesis. These experiments show that in resting cells, even in the presence of exogenous arachidonic acid, PGE2 synthesis was limited by the low abundance of cyclooxygenase. Enhanced expression and activity of cyclooxygenase, however, was not sufficient for increased prostaglandin synthesis. Availability of the precursor arachidonic acid seemed to be rate limiting in prostaglandin synthesis in stimulated MDCK cells.
authors.affiliation: Medizinische Klinik IV, Universität Erlangen-Nürnberg, Germany.
authors.forename: H J
authors.initials: HJ
authors.lastname: Schaefers
authors.forename: J
authors.initials: J
authors.lastname: Haselmann
authors.forename: M
authors.initials: M
authors.lastname: Goppelt-Struebe
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D003990:Dibenzoxepins / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007668:Kidney; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: C100191:BM 16.2224; D017931:DNA Primers; D003990:Dibenzoxepins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate