FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044003876> ?p ?o ?g. }

• W2044003876 endingPage "34927" @default.
• W2044003876 startingPage "34918" @default.
• W2044003876 abstract "We have recently shown that two distinct prostaglandin (PG) E2 synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE2 biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE2 synthase, the perinuclear enzymes thromboxane synthase and PGI2 synthase generated their respective products via COX-2 in preference to COX-1 in both theA23187-induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD2 synthase preferentially used COX-1 and COX-2 in the A23187-induced immediate and interleukin-1-induced delayed PGD2-biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A2. Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA2s at a moment when PG production takes place. We have recently shown that two distinct prostaglandin (PG) E2 synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE2 biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE2 synthase, the perinuclear enzymes thromboxane synthase and PGI2 synthase generated their respective products via COX-2 in preference to COX-1 in both theA23187-induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD2 synthase preferentially used COX-1 and COX-2 in the A23187-induced immediate and interleukin-1-induced delayed PGD2-biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A2. Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA2s at a moment when PG production takes place. prostaglandin arachidonic acid cyclooxygenase phospholipase A2 cytosolic PLA2 secretory PLA2 group V sPLA2 group IID sPLA2 prostaglandin E2synthase cytosolic PGES membrane-bound PGES PGD2 synthase hematopoietic PGD2synthase prostaglandin I2 synthase thromboxane TX synthase fluorescein isocyanate human embryonic kidney interleukin-1 phosphate-buffered saline 10 mm Tris-HCl (pH 7.4) containing 150 mm NaCl and 0.1% Tween 20 5-lipoxygenase-activating protein endoplasmic reticulum human umbilical vein endothelial cells Biosynthesis of prostaglandins (PGs)1 through the cyclooxygenase (COX) pathway involves oxidation and subsequent isomerization of membrane-derived arachidonic acid (AA) via three sequential enzymatic reactions. The initial step of this metabolic pathway is the stimulus-induced liberation of AA from membrane glycerophospholipids by the action of phospholipase A2(PLA2) enzymes, including cytosolic PLA2α (cPLA2; group IVA) and several secretory PLA2(sPLA2) isozymes (groups IIA, IID, V, and X) (1Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar, 2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 8Hanasaki K. Ono T. Saiga A. Morita Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 10Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton W. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The released AA is sequentially metabolized to PGG2 and then to PGH2 by either COX-1 or COX-2. PGH2 is then converted to various bioactive PGs (thromboxane (TX) A2, PGD2, PGE2, PGF2α, and PGI2) by the respective terminal PG synthases, which have different structures and exhibit cell- and tissue-specific distributions. Segregated utilization of COX-1 and COX-2 in the PG biosynthetic events has been demonstrated by a number of cell biological, pharmacological, and genetic studies (3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 11Reddy S.T. Herschman H.R. J. Biol. Chem. 1994; 269: 15473-15480Abstract Full Text PDF PubMed Google Scholar, 12Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 13Harada Y. Hatanaka K. Kawamura M. Saito M. Ogino M. Majima M. Ohno T. Ogino K. Yamamoto K. Taketani Y. Yamamoto S. Katori M. Prostaglandins. 1996; 51: 19-33Crossref PubMed Scopus (97) Google Scholar, 14Langenbach R. Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1049) Google Scholar, 15Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Smithies O. Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1031) Google Scholar, 16Gross G.A. Imamura T. Luedke C. Vogt S.K. Olson L.M. Nelson D.M. Sadovsky Y. Muglia L.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11875-11879Crossref PubMed Scopus (213) Google Scholar, 17Lim H. Paria B.C. Das S.K. Dinchuk J.E. Langenbach R. Trzaskos J.M. Dey S.K. Cell. 1997; 91: 197-208Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar). Generally, the constitutive COX-1 is mainly utilized in the immediate PG biosynthesis, which occurs within several minutes after stimulation with Ca2+ mobilizers, whereas the inducible COX-2 is an absolute requirement for delayed PG biosynthesis, which lasts for several hours after proinflammatory stimuli. When cells are first treated with proinflammatory stimuli and subsequently exposed to Ca2+ mobilizers, the induced COX-2 can also promote the immediate response (priming response) (3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 18Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 19Chen Q-R. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5968Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 20Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 21Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 22Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). However, the precise molecular mechanisms underlying the functional segregation between the two COXs are still obscure. Although specific coupling between COXs and particular PLA2 subtypes have been proposed (23Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 24Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), subsequent coexpression studies have clearly demonstrated that both cPLA2 and several sPLA2isozymes (groups IIA, IID, V, and X) are capable of supplying AA to both COX-1 and COX-2 in the immediate responses and mainly to COX-2 in the delayed responses (3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Moreover, studies using cPLA2null mice have provided unequivocal evidence that cPLA2 is essential for both immediate and delayed phases of PG generation (25Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-621Crossref PubMed Scopus (645) Google Scholar,26Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Saporstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (760) Google Scholar). The actions of sPLA2s in either phase appear to be cell type-specific and depend on their temporal expression, secretion process, and sorting into particular membrane microdomains (4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). It has been suggested that the two COX isozymes are differently coupled with specific terminal PG synthases. For instance, rat peritoneal macrophages produce TXA2 and PGD2 through COX-1 in the A23187-induced immediate response and PGE2 and PGI2 through COX-2 in the lipopolysaccharide-induced delayed response (13Harada Y. Hatanaka K. Kawamura M. Saito M. Ogino M. Majima M. Ohno T. Ogino K. Yamamoto K. Taketani Y. Yamamoto S. Katori M. Prostaglandins. 1996; 51: 19-33Crossref PubMed Scopus (97) Google Scholar, 18Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 27Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1999; 274: 11660-11666Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). TXA2 generation by activated platelets depends entirely on COX-1 (14Langenbach R. Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1049) Google Scholar), whereas PGE2production by osteoblasts occurs predominantly through COX-2 irrespective of the co-presence of COX-1 (19Chen Q-R. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5968Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 22Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In the biphasic production of PGD2 by activated mouse cultured mast cells, only COX-1 is utilized in the immediate phase and only COX-2 in the delayed phase (12Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 28Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Murakami M. Bingham III, C.O. Matsumoto R. Austen K.F. Arm J.P. J. Immunol. 1995; 155: 4445-4453PubMed Google Scholar, 30Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In a rat inflammatory model, COX-2-selective inhibitors reduce the accumulation of PGE2but not of other PGs (18Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). Furthermore, the different enzyme kinetics of each terminal synthase could create a situation in which the ratio of the PG products follows a more complex pattern (31Penglis P.S. Cleland L.G. Demasi M. Caughey G.E. James M.J. J. Immunol. 2000; 165: 1605-1611Crossref PubMed Scopus (91) Google Scholar). Conversion of PGH2 to TXA2 and PGI2is catalyzed by TX synthase (TXS) and PGI2 synthase (PGIS), respectively, both of which belong to the cytochrome P-450 family and are reportedly localized in the endoplasmic reticulum (ER) and perinuclear membranes (32Ohashi K. Ruan K-H. Kulmacz R.J. Wu K.K. Wang L-H. J. Biol. Chem. 1992; 267: 789-793Abstract Full Text PDF PubMed Google Scholar, 33Hara S. Miyata A. Yokoyama C. Inoue H. Brugger R. Lottspeich F. Ullrich V. Tanabe T. J. Biol. Chem. 1994; 269: 19897-19903Abstract Full Text PDF PubMed Google Scholar, 34Lin Y-Z. Wu K-K. Ruan K-H. Arch. Biochem. Biophys. 1998; 352: 78-84Crossref PubMed Scopus (29) Google Scholar). PGD2 synthase (PGDS), which isomerizes PGH2 to PGD2, occurs in two distinct forms, the lipocalin-type PGDS (L-PGDS), a secretory enzyme known as β-trace that is abundantly present in the central nervous system (35Nagata A. Suzuki Y. Igarashi M. Eguchi N. Toh H. Urade Y. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4020-4024Crossref PubMed Scopus (175) Google Scholar), and hematopoietic PGDS (H-PGDS), which represents the ς class of the cytosolic glutathione S-transferase family (36Kanaoka Y. Ago H. Inagaki E. Nanayama T. Miyano M. Kikuno R. Fujii Y. Eguchi N. Toh H. Urade Y. Hayaishi O. Cell. 1997; 90: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The lung and liver types of PGF synthase are cytosolic proteins with high homology that belong to the aldo-keto reductase family (37Watanabe K. Yoshida R. Shimizu T. Hayaishi O. J. Biol. Chem. 1985; 260: 7035-7041Abstract Full Text PDF PubMed Google Scholar, 38Suzuki T. Fujii Y. Miyano M. Chen L.Y. Takahashi T. Watanabe K. J. Biol. Chem. 1999; 274: 241-248Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Several proteins that exhibit PGE2 synthase (PGES) activity have also been identified to date; they are the constitutive cytosolic PGES (cPGES), which is identical to p23 (39Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar), the inducible, perinuclear membrane-bound PGES (mPGES), which was originally designated MGST1-L1 (for membrane-boundGST1-like-1) (40Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 41Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (898) Google Scholar, 42Thoren S. Jakobsson P-J. Eur. J. Biochem. 2000; 267: 6428-6434Crossref PubMed Scopus (192) Google Scholar), and the cytosolic glutathione S-transferase isozymes μ2 and μ3 (43Beuckmann C.T. Fujimori K. Urade Y. Hayaishi O. Neurochem. Res. 2000; 25: 733-738Crossref PubMed Scopus (80) Google Scholar). The expression of mPGES is strongly induced by proinflammatory stimuliin vitro and at inflamed sites in vivo and is down-regulated by anti-inflammatory glucocorticoids (40Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 42Thoren S. Jakobsson P-J. Eur. J. Biochem. 2000; 267: 6428-6434Crossref PubMed Scopus (192) Google Scholar). More recently, we have demonstrated by coexpression and antisense experiments that cPGES and mPGES favor COX-1 and COX-2, respectively, over the other for conversion of exogenous and endogenous AA to PGE2 (39Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar, 40Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar). This finding has provided further support for the hypothesis that there is selective functional linkage between COX isozymes and terminal PG synthases. In this study, we have extended our coexpression approach to clarify diverse functional couplings between COXs and several other lineage-specific PG synthases (TXS, PGIS, and H-PGDS). Our results suggest that lineage-specific PG synthases are classified into three categories in terms of their localization and COX preference: (i) the perinuclear enzymes that prefer COX-2 (TXS, PGIS, and mPGES), (ii) the cytosolic enzyme that prefers COX-1 (cPGES), and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus (H-PGDS). Alterations in AA supply through exogenous and endogenous routes significantly affects the coupling between COXs and terminal PG synthases. The goat anti-human COX-2 and rabbit anti-human cPLA2 antibodies were purchased from Santa Cruz. The rabbit anti-mouse H-PGDS antibody was described previously (44Pinzar E. Miyano M. Kanaoka Y. Urade Y. Hayaishi O. J. Biol. Chem. 2000; 275: 31239-31244Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Rat TXS and mouse H-PGDS cDNAs were obtained by reverse-transcriptase polymerase chain reaction using mRNAs purified from rat platelets and mouse bone marrow-derived mast cells, respectively, as templates using 5′- and 3′-primers corresponding to the N- and C-terminal 23-base pair nucleotide sequences. The touchdown PCR condition was 94 °C for 30 s and then 30 cycles of 94 °C for 5 s and 68 °C for 4 min with Advantage cDNA polymerase mix (CLONTECH) using a DNA thermal cycler (PerkinElmer Life Sciences). Human endothelial cell-derived PGIS cDNA was derived previously (45Miyata A. Hara S. Yokoyama C. Inoue H. Ullrich V. Tanabe T. Biochem. Biophys. Res. Commun. 1994; 200: 1728-1734Crossref PubMed Scopus (118) Google Scholar). LipofectAMINE PLUS reagent, Opti-MEM medium, and TRIzol reagent were obtained from Life Technologies. Mouse anti-FLAG epitope monoclonal antibody was purchased from Sigma. Rabbit anti-human COX-1 antibody, AA, and the enzyme immunoassay kits for TXB2 and 6-keto-PGF1α were obtained from Cayman Chemical. Oligonucleotide primers were from Greiner. Geneticin, hygromycin, zeocin, and the mammalian expression vectors pCR3.1, pCDNA3.1/hyg(+), and pCDNA3.1/zeo(+) were obtained from Invitrogen. A23187 was purchased from Calbiochem. Human interleukin-1β (IL-1) was purchased from Genzyme. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, FITC-rabbit anti-goat IgG, FITC-goat anti-rabbit IgG antibodies, and horseradish peroxidase-conjugated anti-rabbit and anti-goat antibodies were purchased from Zymed Laboratories Inc. Other reagents were obtained from Wako Pure Chemical Industries. Human embryonic kidney (HEK) 293 cells were cultured in RPMI 1640 medium containing 10% (v/v) fetal calf serum as described previously (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). To obtain C-terminally FLAG-epitope-tagged TXS, PCR was performed using TXS/pCR3.1 as a template with a primer 5′-TTA CTT GTC ATC GTC GTC CTT GTA GTC GCG TGA CAC AAT CTT GAC-3′ (the FLAG epitope is underlined) in combination with a primer 5′-ATG GAA GTG TTG GGG CTT CTC-3′. To obtain C-terminally FLAG-tagged PGIS, PCR was performed using PGIS/pCDNA3.1/hyg (+) as a template with primers 5′-TTACTT GTC ATC GTC GTC CTT GTA GTC TGG GCG GAT GCG GTA GCG-3′ and 5′-ATG GCT TGG GCC GCG CTC-3′. The PCR reaction was carried out with 25 cycles of denaturation for 5 s at 94 °C and annealing and extension for 4 min at 68 °C. The fragments obtained were each subcloned into the pCR3.1 vector and sequenced. Establishment of HEK293 cells stably transfected with human COX-1 or COX-2 and those with rat group V or mouse group IID sPLA2s (sPLA2-V and -IID, respectively) were described previously (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). To establish 293 transfectants stably coexpressing COXs and specific PG synthases, the neomycin-resistant clones stably expressing either COX (3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) were subjected to the second transfection with each synthase cDNA that had been subcloned into pCDNA3.1/hyg(+). Transfection was performed using LipofectAMINE PLUS reagent as described previously (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The cells were cloned by limiting dilution in 96-well plates in culture medium supplemented with 70 μg/ml hygromycin. After culturing for 1 month, wells containing a single colony were chosen, and the expression of COXs and PG synthases was assessed by immunoblotting and/or RNA blotting, as described below. The double transfectants were subjected to the third transfection with cPLA2 cDNA subcloned into pCDNA3.1/zeo (+). After transfection, the cells were cloned by limiting dilution in the presence of 50 μg/ml zeocin. Expression of cPLA2 was assessed by immunoblotting. All procedures were described in our previous reports (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Briefly, HEK293 cells (5 × 104/ml) were seeded into each well of 24- or 48-well plates in 1 ml and 0.5 ml of culture medium, respectively. After culturing for 4 days, the cells were washed once with culture medium and then incubated with 250 μl (24-well plate) or 100 μl (48-well plate) of various concentrations of AA or 10 μmA23187 in medium containing 1% fetal calf serum for 30 min (immediate response) or 1 ng/ml IL-1 in medium containing 10% fetal calf serum for 4 h (delayed response). PGD2 released into the supernatants was quantified by a PGD2 radioimmunoassay kit (Amersham Pharmacia Biotech),and TXB2 and 6-keto-PGF1α(stable endoproducts of TXA2 and PGI2, respectively) were quantified by their respective enzyme immunoassay kits (Cayman Chemicals). To assess the transcellular action of sPLA2 (3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), cells expressing rat sPLA2-V or mouse sPLA2-IID and those coexpressing COX-1 or COX-2 and TXS or H-PGDS (2.5 × 104 cells/ml each) were cocultured for 4 days and then stimulated with 1 ng/ml IL-1 in medium containing 10% fetal calf serum for 4 h. TXB2 and PGD2 released into the supernatants were quantified. Generation of these prostanoids was also verified by thin layer chromatography, as required for the experiments. Cells prelabeled for 24 h with 0.1 μCi/ml [3H]AA (Amersham Pharmacia Biotech) were treated with AA, A23187, or IL-1 as noted above, and the products were extracted with diethyl ether, methanol, 1 mcitric acid (30/4/1, v/v/v). The extracts were separated on silica gel 60 plates (Roche Molecular Biochemicals) with a solvent system of ether/petroleum ether/acetic acid (90/2/5, v/v/v) at −20 °C. Distribution of radioactive products on the plates was detected by the BAS2000 imaging analyzer (Fujix). The Rf values of the products were compared with those of the authentic TXB2, 6-keto-PGF1α, and PGD2 standards (Cayman Chemical). The cells were lyzed in phosphate-buffered saline (PBS) containing 0.1% SDS, applied to SDS-polyacrylamide gels (10% for COXs and 12.5% for terminal PG synthases), and electrophoresed as previously reported (2Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kud" @default.
• W2044003876 created "2016-06-24" @default.
• W2044003876 creator A5014507709 @default.
• W2044003876 creator A5014565775 @default.
• W2044003876 creator A5030120964 @default.
• W2044003876 creator A5060772179 @default.
• W2044003876 creator A5069381935 @default.
• W2044003876 creator A5075092495 @default.
• W2044003876 creator A5077023503 @default.
• W2044003876 date "2001-09-01" @default.
• W2044003876 modified "2023-10-03" @default.
• W2044003876 title "Coupling between Cyclooxygenase, Terminal Prostanoid Synthase, and Phospholipase A2" @default.
• W2044003876 cites W1496876439 @default.
• W2044003876 cites W1500692723 @default.
• W2044003876 cites W1514194392 @default.
• W2044003876 cites W1524778875 @default.
• W2044003876 cites W1530740124 @default.
• W2044003876 cites W1532612328 @default.
• W2044003876 cites W1540849804 @default.
• W2044003876 cites W1544502281 @default.
• W2044003876 cites W1554644961 @default.
• W2044003876 cites W1630696756 @default.
• W2044003876 cites W1641540149 @default.
• W2044003876 cites W165922605 @default.
• W2044003876 cites W1771837556 @default.
• W2044003876 cites W1970898912 @default.
• W2044003876 cites W1975287199 @default.
• W2044003876 cites W1984062654 @default.
• W2044003876 cites W1991862371 @default.
• W2044003876 cites W1993507501 @default.
• W2044003876 cites W1993535520 @default.
• W2044003876 cites W1995533728 @default.
• W2044003876 cites W2000031384 @default.
• W2044003876 cites W2004016651 @default.
• W2044003876 cites W2004201056 @default.
• W2044003876 cites W2009676510 @default.
• W2044003876 cites W2014503546 @default.
• W2044003876 cites W2014768234 @default.
• W2044003876 cites W2015066288 @default.
• W2044003876 cites W2019328429 @default.
• W2044003876 cites W2019774786 @default.
• W2044003876 cites W2020563114 @default.
• W2044003876 cites W2021360223 @default.
• W2044003876 cites W2030109883 @default.
• W2044003876 cites W2031789588 @default.
• W2044003876 cites W2034904195 @default.
• W2044003876 cites W2040040765 @default.
• W2044003876 cites W2044959265 @default.
• W2044003876 cites W2049955643 @default.
• W2044003876 cites W2051737543 @default.
• W2044003876 cites W2051958752 @default.
• W2044003876 cites W2055687230 @default.
• W2044003876 cites W2059080791 @default.
• W2044003876 cites W2062818585 @default.
• W2044003876 cites W2065638310 @default.
• W2044003876 cites W2068990208 @default.
• W2044003876 cites W2072647758 @default.
• W2044003876 cites W2077047375 @default.
• W2044003876 cites W2077608143 @default.
• W2044003876 cites W2077641423 @default.
• W2044003876 cites W2081953970 @default.
• W2044003876 cites W2083651943 @default.
• W2044003876 cites W2085052924 @default.
• W2044003876 cites W2099431883 @default.
• W2044003876 cites W2108389970 @default.
• W2044003876 cites W2122117766 @default.
• W2044003876 cites W2126743548 @default.
• W2044003876 cites W2126835057 @default.
• W2044003876 cites W2138103319 @default.
• W2044003876 cites W2140182017 @default.
• W2044003876 cites W2142787693 @default.
• W2044003876 cites W2143930183 @default.
• W2044003876 cites W2145945997 @default.
• W2044003876 cites W2146388445 @default.
• W2044003876 cites W2146839586 @default.
• W2044003876 cites W2153465192 @default.
• W2044003876 cites W2167029659 @default.
• W2044003876 doi "https://doi.org/10.1074/jbc.m100429200" @default.
• W2044003876 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11418589" @default.
• W2044003876 hasPublicationYear "2001" @default.
• W2044003876 type Work @default.
• W2044003876 sameAs 2044003876 @default.
• W2044003876 citedByCount "191" @default.
• W2044003876 countsByYear W20440038762012 @default.
• W2044003876 countsByYear W20440038762013 @default.
• W2044003876 countsByYear W20440038762014 @default.
• W2044003876 countsByYear W20440038762015 @default.
• W2044003876 countsByYear W20440038762016 @default.
• W2044003876 countsByYear W20440038762017 @default.
• W2044003876 countsByYear W20440038762018 @default.
• W2044003876 countsByYear W20440038762019 @default.
• W2044003876 countsByYear W20440038762020 @default.
• W2044003876 countsByYear W20440038762021 @default.
• W2044003876 countsByYear W20440038762022 @default.
• W2044003876 countsByYear W20440038762023 @default.
• W2044003876 crossrefType "journal-article" @default.
• W2044003876 hasAuthorship W2044003876A5014507709 @default.
• W2044003876 hasAuthorship W2044003876A5014565775 @default.

• next