ABSTRACT
Active human cyclooxygenase-2 (Cox-2) was expressed at high levels in insect cells using a recombinant baculovirus. The specific activity of Cox-2 in the microsomes of infected cells was 0.51 mumol O2/min/mg and was comparable to that obtained for partially purified Cox-2 from ovine placenta (0.55 mumol O2/min/mg). The Cox-2 enzyme expressed in insect cells was glycosylated to varying extents and most of the cyclooxygenase activity was in the high-speed microsomal pellet. The insect-cell-expressed enzyme also showed characteristic 15-hydroxyeicosa-tetraenoic acid production after aspirin treatment and had typical inhibition profiles with a number of known nonsteroidal antiinflammatory drugs.
Subject(s)
Gene Expression , Prostaglandin-Endoperoxide Synthases/genetics , Spodoptera/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Baculoviridae/genetics , Base Sequence , Cell Line , Cloning, Molecular , Cyclooxygenase Inhibitors/pharmacology , DNA, Complementary/genetics , Glycoside Hydrolases/pharmacology , Glycosylation , Humans , Hydroxyeicosatetraenoic Acids/biosynthesis , Microsomes/enzymology , Molecular Sequence Data , Prostaglandin-Endoperoxide Synthases/metabolism , Recombinant Proteins , TransfectionABSTRACT
Rat prostaglandin endoperoxidase synthase-2 (PGHS-2) cDNA was cloned from rat calvarial osteoblasts total RNA by RT-PCR. The primary sequence of rat PGHS-2 had 98% and 92% identity to the mouse and human enzymes, respectively. Transfection of the rat PGHS-2 cDNA into COS 7 cells, followed by the addition of 20 microM arachidonic acid, resulted in a dramatic increase in PGE2 released from these cells. The amount of PGE2 produced was comparable to that obtained from cells similarly transfected with human PGHS-1 cDNA. In the rat paw carrageenin-oedema inflammatory model, the injected paw had elevated levels of PGHS-2 mRNA compared to the control paw. In a rat pyrexia model, injection of the pyrogen lipopolysaccharide, resulted in elevated levels of PGHS-2 mRNA in the brain. These results suggest that PGHS-2 expression plays a role both in inflammation and fever.
