Selective induction of cyclo-oxygenase-2 activity in the permanent human endothelial cell line HUV-EC-C: biochemical and pharmacological characterization.
1. Cyclo-oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX-1 and COX-2 which are encoded by different genes. COX-1 is expressed constitutively and is known to be the site of action of aspirin and other nonsteroidal anti-inflammatory drugs. COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the development of inflammation has been claimed. 2. Endothelial cells are an important physiological source of prostanoids and the selective induction of COX-2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines. 3. The HUV-EC-C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12-myristate 13-acetate (PMA) and interleukin-1 beta (IL-1 beta). 4. Both PMA and IL-1 beta produced dose- and time-dependent increases of the synthesis of the COX-derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2. 5. The increase of COX activity by PMA or IL-1 beta correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected. 6. Treatment of the cells with PMA induced a time-dependent increase in the expression of both COX-1 and COX-2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX-2 isoenzyme at the protein level. 7. The enzymatic activity of the PMA-induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX-1 selective assay. Classical non-steroidal anti-inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX-2 inhibitors (SC-58125, NS-398 and nimesulide) showed higher potency towards the COX of PMA-treated HUV-EC-C. 8. Overall, it appears that the stimulation of the HUV-EC-C line with PMA selectively induces the COX-2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin.
Miralpeix
M
M
Laboratory of Inflammation Mediators, Santa Creu i Sant Pau Hospital, Barcelona, Spain.
Camacho
M
M
López-Belmonte
J
J
Canalías
F
F
Beleta
J
J
Palacios
J M
JM
Vila
L
L
D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000187:drug effects*; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007375:Interleukin-1; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate; D007213:Indomethacin