title: Regulation of cerebral microvascular endothelial cell cyclooxygenase-2 message and activity by blood derived vasoactive agents.
abstract: 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).
authors.affiliation: Vascular Biology Unit, Center for Cardiovascular Diseases, College of Pharmacy and Health Sciences, Texas Southern University, 3100 Cleburne Avenue, Houston, TX 77004, USA. yakubu_ma@tsu.edu
authors.forename: Momoh A
authors.initials: MA
authors.lastname: Yakubu
authors.forename: Charles W
authors.initials: CW
authors.lastname: Leffler
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D016718:Arachidonic Acid / Q000494:pharmacology; D015152:Blotting, Northern; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D004338:Drug Combinations; D004347:Drug Interactions; D019332:Endothelin-1 / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D011506:Proteins / Q000378:metabolism; D011863:Radioimmunoassay / Q000379:methods; D013552:Swine; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: D004338:Drug Combinations; D019332:Endothelin-1; D011506:Proteins; D014662:Vasoconstrictor Agents; C103637:mesoderm specific transcript protein; D016718:Arachidonic Acid; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D051546:Cyclooxygenase 2; D015232:Dinoprostone