Assay of cyclooxygenase-1 and 2 in human monocytes.
OBJECTIVE
There is frequently poor correlation between in vitro methods for calculated cyclooxygenase (COX)-1/COX-2 selectivities of inflammatory agents. Therefore, we have examined the use of a single stimulus in a single cell type containing both COX isoforms, for determining the selectivities of COX-inhibitory agents.


METHODS
Fresh human monocytes were stimulated with arachidonic acid (AA; 10 microM) for 15 min and prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production were used as a measure of COX-1 activity. To measure COX-2 activity, cells were transiently pre-treated with aspirin to irreversibly inhibit constitutive COX-1, treated with lipopolysaccharide (LPS) to induce COX-2 and then stimulated with AA.


RESULTS
Eicosanoid production in resting monocytes was predominantly COX-1 derived since it was not inhibited by NS-398 and also, COX-2 was not detectable. In LPS treated monocytes pre-treated transiently with aspirin, neither the level of induced COX-2 nor the activity was affected. Using the mean of the results for PGE2 and TXB2 inhibition, the COX-1/COX-2 ratios of the IC50 values for aspirin and NS-398 are < 0.1 and > 130, respectively.


CONCLUSIONS
This study has provided a system for investigating inhibition of COX isotypes without the potentially confounding effects of using different cell types with different stimuli for each isotype as seen in other published systems. Dose responses to aspirin and NS-398 which are COX- and COX-2 selective inhibitors respectively, confirmed the utility of this system.
Demasi
M
M
Rheumatology Unit, Royal Adelaide Hospital, South Australia, Australia.
Caughey
G E
GE
James
M J
MJ
Cleland
L G
LG
D001241:Aspirin / Q000494:pharmacology; D015153:Blotting, Western; D002469:Cell Separation; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D015777:Eicosanoids / Q000096:biosynthesis; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins; D009000:Monocytes / Q000201:enzymology*; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013929:Thromboxane B2 / Q000494:pharmacology
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D015777:Eicosanoids; D007527:Isoenzymes; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D009578:Nitrobenzenes; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D013929:Thromboxane B2; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D001241:Aspirin