This study was undertaken to investigate the enzymatic regulation of the biosynthesis of vasoconstrictor prostanoids by resting and interleukin (IL)-1(beta)stimulated human umbilical vein endothelial cells (HUVECs). Biosynthesis of eicosanoids in response to IL-1beta, exogenous labeled arachidonic acid (AA), or histamine, as well as their spontaneous release, was evaluated by means of HPLC and RIA. HUVECs exposed to IL-1beta produced prostaglandin (PG) I2 for no longer than 30 seconds after the substrate was added irrespective of the cyclooxygenase (COX) activity, whereas the time course of PGE2 and PGD2 formation was parallel to the COX activity. The ratio of PGE2 to PGD2 produced by HUVECs was similar to that obtained by purified COX-1 and COX-2. Production of PGF2alpha from exogenous AA was limited and similar in both resting and IL-1beta-treated cells. PGF2alpha was the main prostanoid released into the medium during exposure to IL-1beta, whereas when HUVECs treated with IL-1beta were stimulated with histamine or exogenous AA, PGE2 was released in a higher quantity than PGF2alpha. PGF2alpha released into the medium during treatment with IL-1beta and the biosynthesis of PGE2 and PGD2 in response to exogenous AA or histamine increased with COX-2 expression, whereas this did not occur in the case of PGI2. We observed that PGI synthase (PGIS) mRNA levels were not modified by the exposure to IL-1beta, but the enzyme was partially inactivated. When SnCl2 was added to the incubation medium, the transformation of exogenous AA-derived PGH2 into PGE2 and PGD2 was totally diverted toward PGF2alpha. Overall, these results support the conclusions that PGE2 and PGD2 (and also probably PGF2alpha) were nonenzymatically derived from PGH2 in HUVECs. The concept that a high ratio of PGH2 was released by the IL-1beta-treated HUVECs and isomerized outside the cell into PGE2 and PGD2 was supported by the biosynthesis of thromboxane B2 by COX-inactivated platelets, indicating the uptake by platelets of HUVEC-derived PGH2. The IL-1beta-induced increase in the release of PGH2 by HUVECs was suppressed by the COX-2-selective inhibitor SC-58125 and correlated with both COX-2 expression and PGIS inactivation. An approach to the mechanism of inactivation of PGIS by the exposure to IL-1beta was performed by using labeled endoperoxides as substrate. The involvement of HO. in the PGIS inactivation was supported by the fact that deferoxamine, pyrrolidinedithiocarbamate, DMSO, mannitol, and captopril antagonized the effect of IL-1beta on PGIS to different degrees. The NO synthase inhibitor NG-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1beta, indicating that NO. was also involved. NO. reacts with O2-. to form peroxynitrite, which has been reported to inactivate PGIS. Homolytic fission of the O-O bond of peroxynitrite yields NO2. and HO.. The fact that 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which reacts with NO. to form NO2., dramatically potentiated the IL-1beta effect sugg...

1 Cyclo-oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ), exists in two forms, termed COX-1 and COX-2 which are encoded by di erent genes. COX-1 is expressed constitutively and is known to be the site of action of aspirin and other nonsteroidal anti-in¯ammatory drugs. COX-2 may be induced by a series of pro-in¯ammatory stimuli and its role in the development of in¯ammation 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 ®nite 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 di erent stimuli, phorbol 12-myristate 13-acetate (PMA) and interleukin-1b (IL-1b). 4 Both PMA and IL-1b produced dose-and time-dependent increases of the synthesis of the COXderived 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 PGE 2 . 5 The increase of COX activity by PMA or IL-1b correlated with an increase of the enzyme's apparent V max , whilst the a nity for the substrate, measured as apparent K m , remained una ected. 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 re¯ected 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 IC 50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX-1 selective assay. Classical non-steroidal anti-in¯ammatory 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.

This study was focused on the characterization of the metabolism of linoleic acid by human dermal fibroblasts and the effect of interleukin-1 on the biosynthesis of octadecanoids. Dermal fibroblasts untreated and treated with recombinant IL-1beta were incubated with exogenous labeled linoleic acid. A combination of high performance liquid chromatography and gas chromatography-mass spectrometry was used as the analytic technique. We found that dermal fibroblasts convert linoleic acid mainly into 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) and 9-hydroxy-10-trans,12-cis-octadecadienoic acid (9-HODE), 13(S)-HODE and 9(R)-HODE being the predominant enantiomers. IL-1beta increased the formation of both 13-HODE and 9-HODE in a concentration-dependent manner with similar EC50 values as for prostanoid formation. This effect of IL-1beta on HODEs formation was concomitant with the expression of prostaglandin H-synthase-2. Formation of octadecanoids was inhibited in a concentration-dependent manner by acetylsalicylic acid and indomethacin. Dexamethasone, actinomycin D, and cycloheximide abolished the effect of IL-1beta on HODEs biosynthesis. Octadecanoid biosynthetic activity was associated with the microsomal fraction. Dermal fibroblasts incorporated [14C]-9-HODE and [14C]-13-HODE into phospholipids, mainly into phosphatidylcholine. IL-1beta increased significantly the esterification of 13-HODE in all glycerophospholipids, the major increase being observed in phosphatidylinositol. These results indicate that prostaglandin H-synthase-2 is the enzyme responsible for the increase in the ability to form HODEs of dermal fibroblasts stimulated with IL-1beta.