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

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 A(2) analog U-46619 (1 microM), alone or combined, and COX/prostacyclin synthase activity determined following exposure of treated cells to arachidonic acid (10 microM) for 30 min. 6-KetoPGF(1)alpha and PGE(2) 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 microM) for 24h significantly reduced 6-ketoPGF(1)alpha and PGE(2) levels in the media by 57% and 33%. Treatment of cells with U-46619 alone increased both 6-ketoPGF(1)alpha and PGE(2) level in the media by 170% and 42%. Incubation of control cells with arachidonic acid (10 microM) for 30 min increased 6-ketoPGF(1)alpha and PGE(2) production by 163% and 567%. Pretreatment with ET-1 or U-46619 alone for 24h had no significant effect on 6-ketoPGF(1)alpha produced from exogenous arachidonic acid. However, PGE(2) 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 PGE(2) and 6-ketoPGF(1)alpha 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 A(2).