Abstract
Nonsteroidal anti-inflammatory drugs inhibit the action of prostaglandin H synthase (PGH synthase), and this effect may constitute the basis for therapeutic and idiosyncratic responses to these agents. We found that aspirin treatment of cultured ovine tracheal epithelial cells blocked PGH synthase-catalyzed formation of PG as expected but also caused a dose-dependent increase in 15-hydroxyeicosatetraenoic acid (15-HETE) production from arachidonic acid. In contrast, aspirin caused only inhibition of PG production without enhancing 15-HETE formation in ovine seminal vesicle and other tissues. The 15-HETE formed by aspirin-treated ovine tracheal epithelial cells was generated by a PGH synthase-dependent mechanism because: (i) the 15-HETE forming activity was just as sensitive as PG forming activity to selective inhibition by indomethacin; (ii) both 15-HETE and PG forming activities were quantitatively immunoprecipitated (depleted from supernatants and recovered in immune complex pellets) by a specific anti-PGH synthase antiserum. Additional immunoprecipitation experiments indicated that anti-PGH synthase monoclonal antibodies (cyo-1 and cyo-5) raised against the aspirin-inhibited form of the enzyme (contained in seminal vesicle) did not recognize the aspirin-stimulated 15-HETE-forming PGH synthase (contained in cultured epithelial cells). Thus, sequential immunoprecipitation of cultured epithelial cell material first with excess cyo-1 followed by anti-PGH synthase antiserum indicated that two isoforms of PGH synthase were expressed in these cells. SDS-polyacrylamide gel electrophoresis of immunoprecipitated PGH synthase from cultured epithelial cells revealed distinct protein bands for each form of the enzyme (M(r) = 70,000 and 72,000). The identification of a distinct PGH synthase which may be modified by aspirin so that selective oxygenation of fatty acid substrate is enhanced (while PG formation is inhibited) indicates that isozymes of PGH synthase exist which are pharmacologically distinct.
Highlights
PG forming activity to selective inhibition by indo- (Ser530in ovine and murine PGH synthase) and other strucmethacin; (ii)both 15-HETE and PG forming activitietsural features of PGH synthase has been made possible by were quantitativeliymmunoprecipitate(ddepleted purification of the enzymefrombovine and ovine seminal from supernatants and recovered inimmune complex vesicle and by molecularcloning of ovine seminal vesicle, pellets) by a specific anti-PGHsynthaseantiserum. murine fibroblast, and human platelet cDNAs encoding the Additionailmmunoprecipitatioenxperimentisndicomplete protein [3,4,5,6]
Cated that anti-PGH synthase monoclonal antibodies Recent studiesfrom several laboratories indicate that there raised against the aspirin-inhibited maybe anadditional form of PGHsynthase mRNA and form of the enzyme did protein [7,8,9,10]
Previous work from our laboratory [11] not recognize the aspirin-stimulated 15-HETE-form- suggested theexistence of a PGHsynthasemRNA which ing PGH synthase(containedin culturedepithelial might be preferentially expressed in ovine tracheal epithelial cells).,sequentialimmunoprecipitation of cul- cells cultured under growth-stimulating conditions anmdight tured epithelial cell material first with excess c y o - l followed by anti-PGHsynthaseantiserumindicated that two isoforms of PGH synthase wereexpressed in these cells
Summary
HPLC Analysis of Oxygenation Products-Incubations were stopped by rapid cooling to 4 "C, and an aliquot of PGB, was added as an internal standard for productrecovery. Chiral-phase HPLC (on products first purified by wu '0 10 20 30 40 0 10 20 30 40 reverse-pbase HPLC) was performed with a pair of 4.6 X 250-mm columns in series which were packed with 5 ~ L Maminopropyl silica particles ionically bonded to dinitrobenzoylphenylglycineat a flow rate of 0.8 ml/min with two solvents (A and B) used in an isocratic program of 8%B where A was hexane and B was hexane/2-propanol (100:4). Products were extracted from cell supernatants and were analyzed by reverse-phase HPLC using an acetonitrile (ACN)/water/aceticacid solvent system. Tion were found in freshlyisolated tracheal epithelial cells This materialwas analyzed with monitoring of ions m/z 391 for authentic 15-HETE andm/z 399 for the *H8-labeled internal standard.For positive-ion electronimpactGC-MS,the reverse-phase (Table I). T o the methyl ester TMS derivative This materialwas analyzed In contrasttoresultswithculturedepithelial cells, we with selected monitoring of ions m/z 225, 316, and 335 for authentic 15-HETE and m/z 229, 323, and 343for the 'H8-labeled internal standard. Observed (asothers have reported)thataspirininvariably inhibitedPGHsynthase-catalyzedformation of 15-HETE (and11-HETE) in preparations of ovine seminal vesicle (Table 11).Additional experiments on culturedovine tracheal epithelial cells were performed to detect other as-
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