Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases
Patrignani, P.; Panara, M.R.; Greco, A.; Fusco, O.; Natoli, C.; Iacobelli, S.; Cipollone, F.; Ganci, A.; Créminon, C.; Maclouf, J.
Journal of Pharmacology and Experimental Therapeutics 271(3): 1705-1712
1994
ISSN/ISBN: 0022-3565 PMID: 7996488 Document Number: 429031
The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 mu-g/ml) for 0 to 24 hr at 37 degree C, The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 mu-g/ml) in vitro at time 0. PGE-2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 mu-g/ml LPS, PGE-2 production averaged 12.1 +- 6.2 ng/ml (mean +- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 mu-M) inhibited LPS-induced PGG-2 production by 96 +- 4% (mean +- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGHS-2 and thrombin-stimulated platelet PGHS-1. IC-50 values (mu-M) for inhibition of PGHS-1 and PGHS-2 were, indomethacin, 0.70 +- 0.20 vs. 0.36 +- 0.10 (P lt .05); S-indobufen, 0.64 +- 0.22 vs. 14.9 +- 8 (P lt .05). R-indobufen, 38 +- 18 vs. 230 +- 68 (P lt .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +- 96 vs. 187 +- 96. Nabumetone (500 and 1000 mg for 7 days) was equipotent in suppressing PGHS-1 and PGHS-2 activities in human whole blood ex vivo. We conclude that 1) LPS-induced biosynthesis of PGHS-2 in circulating monocytes accounts for the time-dependent expression of low-dose aspirin-insensitive cyclooxygenase activity in human whole blood; and 2) this technique allows assessing pharmacological inhibition of human PGHS-2 both in vitro and ex vivo.