Regulation of cerebral microvascular endothelial cell cyclooxygenase-2 message and activity by blood derived vasoactive agents

We have investigated the effects of prolonged treatment of cerebral microvascular endothelial cells with vasoconstrictor products of blood clot hemolysis on prostanoid production and cyclooxygenase (COX)/prostacyclin synthase activity and message. Confluent primary cultures of endothelial cells derived from piglet cerebral microvessels were incubated with endothelin-1 (ET-1; 10 nM) or thromboxane A2 analog U-46619 (1 μM), alone or combined, and COX/prostacyclin synthase activity determined following exposure of treated cells to arachidonic acid (10 μM) for 30 min. 6-KetoPGF1α and PGE2 levels in the medium were determined using radioimmunoassay. Effect of treatments on COX-2 message was determined by RNAse Protection Assay. Combined treatment with ET-1 (10 nM) and U-46619 (1 μM) for 24 h significantly reduced 6-ketoPGF1α and PGE2 levels in the media by 57% and 33%. Treatment of cells with U-46619 alone increased both 6-ketoPGF1α and PGE2 level in the media by 170% and 42%. Incubation of control cells with arachidonic acid (10 μM) for 30 min increased 6-ketoPGF1α and PGE2 production by 163% and 567%. Pretreatment with ET-1 or U-46619 alone for 24 h had no significant effect on 6-ketoPGF1α produced from exogenous arachidonic acid. However, PGE2 production from exogenous arachidonic acid by cells pretreated with ET-1 but not with U-46619 was attenuated by 35%. Combined treatment with ET-1 and U-46619 reduced both PGE2 and 6-ketoPGF1α production from arachidonic acid by 14% and 40%, respectively. Acute incubation of cells with ET-1 or U-46619 did not have any significant effects on COX-2 mRNA. In conclusion, combined ET-1 and U-46619 reduced prostanoid production. The reduction cannot be fully explained by changes in COX/prostacyclin synthase activity and/or message, but the changes could be due to reduced availability of free arachidonic acid potentially resulting from inhibition of endothelial phospholipase A2.