Nitric oxide stimulates cyclooxygenase-2 in cultured cTAL cells through a p38-dependent pathway.
To examine the interaction of nitric oxide (NO) and cyclooxygenase (COX-2) and the signaling pathway involved, primary cultured rabbit cortical thick ascending limb (cTAL) were used. In these cells, immunoreactive COX-2 and vasodilatory prostaglandins were increased by a NO donor, S-nitros-N-acetylpenicillamine (SNAP; 2.5 +/- 0.3-fold control, n = 6, P < 0.01). SNAP increased expression of phosphorylated p38 (pp38; 2.4 +/- 0.3-fold control; n = 5; P < 0.01), which was inhibited by the p38 inhibitor SB-203580 (1.3 +/- 0.1-fold control, n = 5, P < 0.01). SB-203580 inhibited SNAP-induced COX-2 expression [1.4 +/- 0.2-fold control, n = 6, not significant (NS) vs. control] and levels of PGE2 significantly. In cTAL cells transfected with a luciferase reporter driven by the wild-type mouse COX-2 promoter, SNAP stimulated luciferase activity, which was reversed by SB-203580 (control vs. SNAP vs. SNAP + SB-203580: 1.4 +/- 0.2-, 8.3 +/- 1.4-, and 0.4 +/- 0.1-fold control, respectively, n = 4, P < 0.01). Electrophoretic mobility shift assay indicated that SNAP stimulated nuclear factor (NF)-kappaB binding activity in cTAL that was also inhibited by the p38 inhibitor. SNAP was not able to stimulate a mutant COX-2 promoter construct that is not activated by NF-kappaB (0.9 +/- 0.1, 1.2 +/- 0.1, and 1.0 +/- 0.2 respectively, n = 4, NS). Low chloride increased COX-2 expression (2.7 +/- 0.4-fold control, n = 6, P < 0.01) and pp38 expression (2.8 +/- 0.3-fold; n = 5, P < 0.01), which were reversed by the specific NO synthase (NOS) inhibitor 7-nitroindazole. Administration of a low-salt diet increased immunoreactive COX-2 and neuronal NOS (nNOS) in the macula densa and surrounding cTAL of kidneys of wild-type mice but did not significantly elevate COX-2 expression in nNOS-/- mice. In summary, these studies indicate that, in cTAL, NO can increase COX-2 expression in cTAL and macula densa through p38-dependent signaling pathways via activation of NF-kappaB.
Cheng
Hui-Fang
HF
George M. O'Brien Kidney and Urologic Diseases Center and Division of Nephrology, Vanderbilt Univ. School of Medicine, and Nashville Veterans Affairs Hospital, Nashville, TN 37232, USA.
Zhang
Ming-Zhi
MZ
Harris
Raymond C
RC
D000818:Animals; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D004039:Diet, Sodium-Restricted; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D008138:Loop of Henle / Q000187:drug effects / Q000201:enzymology*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000172:deficiency; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D016328:NF-kappa B; D020030:Nitric Oxide Donors; D011993:Recombinant Fusion Proteins; C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; D008156:Luciferases; D052248:Nitric Oxide Synthase Type I; D051546:Cyclooxygenase 2; D048051:p38 Mitogen-Activated Protein Kinases; D010396:Penicillamine