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).