Rate of Vasoconstrictor Prostanoids Released by Endothelial Cells Depends on Cyclooxygenase-2 Expression and Prostaglandin I Synthase Activity

From the Laboratory of Inflammation Mediators, Institute of Research of Santa Creu i Sant Pau Hospital, Barcelona, Spain.

Abstract—This study was undertaken to investigate the enzymatic regulation of the biosynthesis of vasoconstrictor prostanoids by resting and interleukin (IL)-1ß–stimulated human umbilical vein endothelial cells (HUVECs). Biosynthesis of eicosanoids in response to IL-1ß, 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-1ß 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 PGF₂ from exogenous AA was limited and similar in both resting and IL-1ß–treated cells. PGF₂ was the main prostanoid released into the medium during exposure to IL-1ß, whereas when HUVECs treated with IL-1ß were stimulated with histamine or exogenous AA, PGE₂ was released in a higher quantity than PGF₂. PGF₂ released into the medium during treatment with IL-1ß 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-1ß, 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 PGF₂. Overall, these results support the conclusions that PGE₂ and PGD₂ (and also probably PGF₂) were nonenzymatically derived from PGH₂ in HUVECs. The concept that a high ratio of PGH₂ was released by the IL-1ß–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-1ß–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-1ß 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-1ß on PGIS to different degrees. The NO synthase inhibitor N^G-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1ß, 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-1ß 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-1ß were not conclusive, they strongly suggest that both NO· and HO· were involved.

Key Words: cyclooxygenase • prostanoid • endothelial cell

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