Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases.
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 micrograms/ml) for 0 to 24 hr at 37 degrees 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 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 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 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)
Patrignani
P
P
Division of Clinical Pharmacology, University of Chieti G. D'Annunzio, School of Medicine, Italy.
Panara
M R
MR
Greco
A
A
Fusco
O
O
Natoli
C
C
Iacobelli
S
S
Cipollone
F
F
Ganci
A
A
Créminon
C
C
Maclouf
J
J
D000328:Adult; D000595:Amino Acid Sequence; D015153:Blotting, Western; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000097:blood*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology; D011451:Prostaglandin-Endoperoxide Synthases / Q000097:blood*
D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D003907:Dexamethasone; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone