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) direct ed against a specific carboxyterminal sequence of human cyclooxygenase -2 (residues 580-598). A rabbit polyclonal antiserum was also raised a gainst another sequence of 10 amino acids (residues 570-581) not prese nt in human constitutive cyclooxygenase-1. Affinity-purified polyclona l antibodies, coated on microtiter plates, were used as capture antibo dies in a two-site immunometric assay, with an mAb-acetylcholinesteras e conjugate used as tracer. The detection limit was 500 fmol/ml of pep tide C3-COX2 (residues 570-595). The assay was specific for the cycloo xygenase-2 (COX-2) isoform, since no immunoreactivity could be detecte d in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). I n contrast, extracts from cultured human umbilical vein endothelial ce lls challenged with 20 nM phorbol myristate acetate (PMA) showed an in crease in COX-2 immunoreactivity related both to the increase in enzym e activity and the Variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immu nometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by meas uring the immunoreactivity of the fractions obtained after molecular s ieve chromatography of control and stimulated cell extracts, and corro borated the marked enhancement of COX-2 by comparison with COX-1. Trea tment of PMA-activated cells with II-7 or actinomycin D totally abolis hed the COX-2 signal and had little effect on COX-1. No significant va riation in COX-2 immunoreactivity was observed using the inactive isom er 4 alpha-PMA, even at 100 nM. These assays constitute the first quan titative analysis of constitutive COX-1 and of inducible COX-2 in nucl eated cells at the protein level.