Hypertonic induction of COX-2 in collecting duct cells by reactive oxygen species of mitochondrial origin.
Our previous studies have documented MAPK mediation of the hypertonicity-induced stimulation of COX-2 expression in cultured renal medullary epithelial cells. The present study extends this observation by examining the role of reactive oxygen species (ROSs). ROS levels, determined using dichlorodihydrofluorescence diacetate and cytochrome c, were rapidly and significantly increased following exposure of mIMCD-K2 cells to media made hypertonic by adding NaCl. Hypertonic treatment (550 mosmol/kg) for 16 h induced a 5.6-fold increase in COX-2 protein levels and comparable increases in prostaglandin E(2) release, both of which were completely abolished by the NADPH oxidase inhibitor diphenyleneiodonium (25-50 microM). The general antioxidant N-acetyl-l-cysteine (6 mM), and the superoxide dismutase mimetic TEMPO (2.0 mm) reduced COX-2 levels by 75.6 and 79.8%, respectively. Exposure of mIMCD-K2 cells to exogenous O(2)(-.) generated by the xanthine/xanthine oxidase system mimicked the effect of hypertonicity on COX-2 expression and prostaglandin E(2) release. The increases in phosphorylation of ERK1/2 and p38 were detected 20 min following the hypertonic treatment and were both prevented by N-acetyl-l-cysteine. The increases in ROSs in response to hypertonic treatment were completely blocked by any one of the mitochondrial inhibitors tested, such as rotenone, thenoyltrifluoroacetone, or carbonyl cyanide m-chlorophenylhydrazone, associated with remarkable inhibition of COX-2 expression. In contrast, the increases in ROSs were not significantly altered in IMCD cells deficient in either gp91(phox) or p47(phox), nor were the increases in COX-2 expression. We conclude that ROSs derived from mitochondria, but not NADPH oxidase, mediate the hypertonicity-induced phosphorylation of MAPK and the stimulation of COX-2 expression.
Yang
Tianxin
T
Department of Internal Medicine, University of Utah and Veterans Affairs Medical Center, Salt Lake City, Utah 84148, USA. tianxin.yang@hsc.utah.edu
Zhang
Aihua
A
Honeggar
Matthew
M
Kohan
Donald E
DE
Mizel
Diane
D
Sanders
Karl
K
Hoidal
John R
JR
Briggs
Josephine P
JP
Schnermann
Jurgen B
JB
D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D005452:Fluoresceins / Q000494:pharmacology; D005799:Genes, Dominant; D006835:Hydrazones / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D009995:Osmosis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology; D013997:Time Factors; D019820:Xanthine / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C110400:2',7'-dichlorodihydrofluorescein diacetate; D000975:Antioxidants; D003497:Cyclic N-Oxides; D005452:Fluoresceins; D006835:Hydrazones; D008562:Membrane Glycoproteins; D009861:Onium Compounds; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D012402:Rotenone; C070053:carbonyl 3-chlorophenylhydrazone; D019820:Xanthine; D013804:Thenoyltrifluoroacetone; C007517:diphenyleneiodonium; D045304:Cytochromes c; D051546:Cyclooxygenase 2; D014969:Xanthine Oxidase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone; C003959:TEMPO; D000111:Acetylcysteine