Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression and prostaglandin I synthase activity

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-1 beta, 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-1 beta produced prostaglandin (PG) I-2 for no longer than 30 seconds after the substrate was added irrespective of the cyclooxygenase (COX) activity, whereas the time course of PGE(2) and PGD(2) formation was parallel to the COX activity. The ratio of PGE(2) to PGD(2) produced by HUVECs was similar to that obtained by purified COX-1 and COX-2. Production of PGF(2 alpha) from exogenous AA was limited and similar in both resting and IL-1 beta-treated cells. PGF(2 alpha) was the main prostanoid released into the medium during exposure to IL-1 beta, whereas when HUVECs treated with IL-1 beta were stimulated with histamine or exogenous AA, PGE(2) was released in a higher quantity than PGF(2 alpha). PGF(2 alpha) released into the medium during treatment with IL-1 beta and the biosynthesis of PGE(2) and PGD(2) in response to exogenous AA or histamine increased with COX-2 expression, whereas this did not occur in the case of PGI(2). We observed that PGI synthase (PGIS) mRNA levels were not modified by the exposure to IL-1 beta, but the enzyme was partially inactivated. When SnCl2 was added to the incubation medium, the transformation of exogenous AA-derived PGH(2) into PGE(2) and PGD(2) was totally diverted toward PGF(2 alpha). Overall, these results support the conclusions that PGE(2) and PGD(2) (and also probably PGF(2 alpha)) were nonenzymatically derived from PGH(2) in HUVECs. The concept that a high ratio of PGH(2) was released by the LL-1 beta-treated HUVECs and isomerized outside the cell into PGE(2) and PGD(2) was supported by the biosynthesis of thromboxane B-2 by COX-inactivated platelets, indicating the uptake by platelets of HUVEC-derived PGH(2). The IL-1 beta-induced increase in the release of PGH(2) 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-1 beta 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-1 beta on PGIS to different degrees. The NO synthase inhibitor N-G-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1 beta, indicating that NO was also involved. NO reacts with O-2(-.) 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-1 beta effect suggests that NO2. could be a species implicated in the inactivation of PGIS. Cooperation of HO was supported by the fact that DMSO partially antagonized the effect of carboxy-PTIO. Although our results on the exact mechanism of the inactivation of PGIS caused by IL-1 beta were not conclusive, they strongly suggest that both NO and HO were involved.