Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays.
We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.
CEA, Service de Pharmacologie et d'Immunologie, DRIPP, Centre d'Etudes SACLAY, Gif-sur-Yvette, France.
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Créminon
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Habib
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Maclouf
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Pradelles
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Grassi
Y
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Frobert
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D004730:Endothelium, Vascular / Q000201:enzymology*; D004790:Enzyme Induction; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011451:Prostaglandin-Endoperoxide Synthases