Generation of 8-epi-prostaglandin F(2alpha) in isolated rat kidney glomeruli by a radical-independent mechanism.
Isoprostanes comprise a group of free radical-catalyzed products of arachidonic acid. However, there is recent evidence pointing towards an enzyme-dependent formation of isoprostanes. With the use of isolated rat glomeruli we addressed the mechanisms of isoprostane generation. Synthesis of prostanoids and isoprostanes, including 8-epi-PGF(2alpha), was studied under conditions favouring radical formation. Cultured glomeruli formed different prostanoids including 8-epi-PGF(2alpha). Upon LPS challenge cyclo-oxygenase (COX)-2 expression was enhanced, and this was paralleled by a 2 - 9-fold increase in prostanoid formation, including isoprostanes. Addition of COX-isoform unselective inhibitors (diclofenac, indomethacin) or a selective inhibitor (NS-398) suppressed the synthesis of prostanoids, 8-epi-PGF(2alpha) and total isoprostane fraction; however, inhibition of the latter was less pronounced. Antioxidants such as butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), or dimethylurea exhibited an only minimal inhibitory effect on 8-epi-PGF(2alpha) synthesis. Moreover, ROS-generating drugs (menadione, methylviologen) or NADPH-driven radical formation were unable to cause the generation of significant amounts of 8-epi-PGF(2alpha) by rat glomeruli. In contrast, the total isoprostane fraction could be increased by menadione addition. These data provide further evidence for a radical-independent, but COX-dependent formation of 8-epi-PGF(2alpha) in renal tissue. Regarding the other isoprostanes, both radicals and COX enzymes contribute to their formation. Based on our data we assume that elevated release of vasoactive 8-epi-PGF(2alpha) has to be expected under conditions when the prostanoid system in the kidney is stimulated, e.g. under inflammatory conditions. Regarding renal oxidative injuries, the usefulness of 8-epi-PGF(2alpha) as a representative marker molecule of oxidative stress has to be questioned.
Klein
T
T
Department of Pediatrics, Philipps-University of Marburg, D-35033 Marburg, Germany.
Neuhaus
K
K
Reutter
F
F
Nüsing
R M
RM
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D016718:Arachidonic Acid / Q000737:chemistry / Q000494:pharmacology; D015153:Blotting, Western; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004008:Diclofenac / Q000494:pharmacology; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D011464:Epoprostenol / Q000235:genetics / Q000378:metabolism; D004952:Esters; D028441:F2-Isoprostanes; D005260:Female; D005609:Free Radicals / Q000378:metabolism*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007678:Kidney Glomerulus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D011453:Prostaglandins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013930:Thromboxane-A Synthase / Q000235:genetics / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000975:Antioxidants; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004952:Esters; D028441:F2-Isoprostanes; D005609:Free Radicals; D007527:Isoenzymes; D008565:Membrane Proteins; D009578:Nitrobenzenes; D011453:Prostaglandins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D004008:Diclofenac; C075750:8-epi-prostaglandin F2alpha; D016718:Arachidonic Acid; D003907:Dexamethasone; D015237:Dinoprost; D011464:Epoprostenol; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C496513:Ptgs1 protein, rat; D013930:Thromboxane-A Synthase