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Critical role of COX-1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone

Chiara Bolego^*,1, Carola Buccellati^*, Alberto Prada, Rosa Maria Gaion, Giancarlo Folco^* and Angelo Sala^*,2

^* Department of Pharmacological Sciences, University of Milan, Milan, Italy;

Rho Hospital, Rho, Italy; and

Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy

²Correspondence: Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: angelo.sala{at}unimi.it

We aimed at evaluating the relative contribution of cyclooxygenase (COX) -1 and COX-2 to the synthesis of prostacyclin in endothelial cells under static conditions in the presence or absence of exogenous arachidonic acid and/or altered intracellular redox balance. Selective inhibitors of either COX-1 (SC560 and FR122047) or COX-2 (SC236) concentration dependently (1–300 nM) reduced basal and interleukin (IL) -1β-induced prostacyclin production in human umbilical vein endothelial cells by 70% or more; compound selectivity was confirmed using a whole-blood assay (IC₅₀ COX-1/COX-2: 13 nM/930 nM for SC-560; 9 µM/457 nM for SC-236). The observed concomitant formation of isoprostane appeared to be associated with COX enzyme activity, while formation of COX-1/COX-2 heterodimers was detected by immunoprecipitation. In the presence of arachidonic acid and 12-hydroperoxy-eicosatetraenoic acid, either exogenous or provided by platelet activation, or after glutathione depletion, COX-1 inhibition but not COX-2 inhibition concentration dependently decreased prostacyclin production. Both isoforms appear to contribute to basal prostacyclin production by endothelial cells, with COX-2 providing the hydroperoxide tone required for COX-1 activity. Conversely, in the case of intracellular glutathione depletion or enhanced availability of arachidonic acid and hydroperoxides, selective COX-2 inhibition did not significantly affect the production of endothelial prostacyclin. These findings contribute to a better understanding of the effects of cyclooxygenase inhibitors on prostacyclin production.—Bolego, C., Buccellati, C., Prada, A., Gaion, R. M., Folco, G., Sala, A. Critical role of COX-1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone.

Key Words: selective cyclooxygenase inhibitors • prostaglandin • isoprostanes • COX-2 heterodimers • 12-HpETE