title: Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.
abstract: Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.
authors.affiliation: Amersham Pharmacia Biotech UK Ltd., Cardiff, Forest Farm, CF4 7YT, United Kingdom.
authors.forename: J K
authors.initials: JK
authors.lastname: Horton
authors.forename: A S
authors.initials: AS
authors.lastname: Williams
authors.forename: Z
authors.initials: Z
authors.lastname: Smith-Phillips
authors.forename: R C
authors.initials: RC
authors.lastname: Martin
authors.forename: G
authors.initials: G
authors.lastname: O'Beirne
mesh_terms: D016475:3T3 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015232:Dinoprostone / Q000032:analysis* / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D016861:Cyclooxygenase Inhibitors; D005938:Glucocorticoids; D007476:Ionophores; D008070:Lipopolysaccharides; D011453:Prostaglandins; D005576:Colforsin; D000001:Calcimycin; D003907:Dexamethasone; D011451:Prostaglandin-Endoperoxide Synthases; D000262:Adenylyl Cyclases; D015232:Dinoprostone