Mitochondrial reactive oxygen species contribute to high NaCl-induced activation - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:44 |只看该作者 |倒序浏览 |打印

作者：Xiaoming Zhou, Joan D. Ferraris, and Maurice B. Burg作者单位：1 Division of Nephrology, Department of Medicine, Uniformed Services University of the Health Sciences, and 2 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
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      【摘要】: |+ O2 h. U# T  a) W+ r1 y' N
   Hypertonicity activates the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), resulting in increased expression of genes involved in osmoprotective accumulation of organic osmolytes, including glycine betaine, and in increased expression of osmoprotective heat shock proteins. Our previous studies showed that high NaCl increases reactive oxygen species (ROS), which contribute to activation of TonEBP/OREBP. Mitochondria are a major source of ROS. The purpose of the present study was to examine whether mitochondria produce the ROS that contribute to activation of TonEBP/OREBP. We inhibited mitochondrial ROS production in HEK293 cells with rotenone and myxothiazol, which inhibit mitochondrial complexes I and III, respectively. Rotenone (250 nM) and myxothiazol (12 nM) reduce high NaCl-induced ROS over 40%, whereas apocynin (100 µM), an inhibitor of NADPH oxidase, and allopurinol (100 µM), an inhibitor of xanthine oxidase, have no significant effect. Rotenone and myxothiazol reduce high NaCl-induced increases in TonEBP/OREBP transcriptional activity (ORE/TonE reporter assay) and BGT1 (betaine transporter) mRNA abundance ranging from 53 to 69%. They inhibit high NaCl-induced TonEBP/OREBP transactivating activity, but not its nuclear translocation. Release of ATP into the medium on hypertonic stress has been proposed to be a signal that triggers cellular osmotic responses. However, we do not detect release of ATP into the medium or inhibition of high NaCl-induced ORE/TonE reporter activity by an ATPase, apyrase (20 U/ml), indicating that high NaCl-induced activation of TonEBP/OREBP is not mediated by release of ATP. We conclude that high NaCl increases mitochondrial ROS production, which contributes to the activation of TonEBP/OREBP by increasing its transactivating activity. : k" [" S. w8 S; _/ A4 \/ w
      【关键词】 superoxides rotenone myxothiazol transactivation nuclear translocation ATP release
   THE TRANSCRIPTION FACTOR tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), when activated by high NaCl and other hypertonic stresses, increases transcription of genes involved in osmoprotective accumulation of organic osmolytes, including glycine betaine and heat shock proteins ( 26, 34, 48 ). In the kidney, TonEBP/OREBP also controls expression of a urea transporter (UT-A) ( 36 ) and of aquaporin-2 ( 22, 26 ), thus regulating the urinary concentrating mechanism. Hypertonicity, induced by high NaCl, increases reactive oxygen species (ROS) in mIMCD3 ( 50 ), mIMCD-K2 ( 49 ), HEK293 cells ( 51 ), and, presumably, also in the renal medulla, where interstitial NaCl normally is high and there is oxidative stress that results in protein carbonylation ( 50 ). We previously found that high NaCl-induced ROS contribute to activation of TonEBP/OREBP ( 51 ). The purpose of the present study was to identify the source within cells of the ROS that are produced in response to high NaCl and whether it is necessary for full activation of TonEBP/OREBP.4 l- o7 X& ]9 s  z5 l

ROS are a by-product of mitochondrial generation of ATP through oxidative phosphorylation. This is true even in the renal papilla, where the oxygen tension is low ( 9 ). Under physiological conditions, 0.2-2% of the oxygen taken up by cells is converted by mitochondria to ROS, initially through production of superoxide that is subsequently dismutated to hydrogen peroxide ( 17 ). There are two main sites of superoxide generation in the inner mitochondrial membrane: NADH dehydrogenase at complex I, and the interface between ubiquinone and complex III ( 30 ).
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The number of mitochondria in renal medullary cells apparently is affected by osmolality. Brattleboro rats have hereditary diabetes insipidus, which reduces their medullary interstitial osmolality because of lack of vasopressin. Their renal papillas have a reduced number of mitochondria ( 29 ). Infusion of vasopressin into the rats, which increases osmolality in the renal medullary interstitium, increases the number of mitochondria ( 4 ). Similarly, low renal medullary osmolality in aquaporin-1 null mice ( 27 ) is associated with reduced expression of several mitochondrial genes ( 31 ). The increased mitochondrial capacity associated with hyperosmolality presumably reflects increased energy demand. Furthermore, mitochondria are major sources of ROS in renal medullas where the urine is concentrated ( 52 ).

In the present study, we examined whether high NaCl-induced ROS were produced by mitochondria and whether the ROS originating from mitochondria contributed to activation of TonEBP/OREBP.
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MATERIALS AND METHODS
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Cells, cell culture, and chemicals. Human embryonic kidney 293 (HEK293) cells, purchased from American Type Culture Collection (Manassas, VA) and grown as a monolayer, were incubated in Eagle's minimal essential medium plus 10% fetal bovine serum in 5% CO 2 -95% air at 37°C. Cells were used below passage 50. The osmolality of the control, "isotonic," medium was 290 mosmol/kgH 2 O. Dihydroethidium was purchased from Molecular Probes (Eugene, OR), and all other chemicals were purchased from Sigma (St. Louis, MO). All inhibitors and probes were freshly prepared. All controls, except in Fig. 6, contained 0.1% DMSO in the medium.4 ^4 s3 ]3 \, [9 Y4 V, w+ n. R

Fig. 6. A : high NaCl does not cause detectable release of ATP from HEK293 cells. Osmolality was increased to 500 mosmol/kgH 2 O by addition of NaCl in the presence of suramin (200 µM) to prevent loss of ATP. B : apyrase (20 U/ml) has no significant effect on high NaCl-induced ORE reporter activity. HEK293 cells were incubated with apyrase for 30 min, and then osmolality was increased to 500 mosmol/kgH 2 O by addition of NaCl for 16 h. C : apyrase remains active after 16 h at 500 mosmol/kgH 2 O. An aliquot of medium, taken at the end of experiments in B, was incubated with an ATP standard solution at 37°C for 30 min, and then ATP was measured ( n = 3).! d$ G2 V8 v$ I2 ]$ A7 t
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Measurement of ROS. ROS were measured as previously described ( 51 ). Briefly, 0.8 to 1 x 10 6 cells were grown overnight on the optical glass bottom of a dish (MatTek, Ashland, MA) and preincubated with 30 µM dihydroethidium in phenol red-free culture medium for 30 min. Then, the medium was replaced with an otherwise identical one at 290 or 450 mosmol/kgH 2 O (NaCl added) for 45 min. The fluorophore was excited at 488 nm. Fluorescent emission at 525 nm was recorded by confocal microscopy (LSM510 Meta, Zeiss, Thornwood, NY) and analyzed with MetaMorph software.

Measurement of ATP abundance. An ATP assay kit from Calbiochem (San Diego, CA) was used according to the manufacturer's instructions. This assay is based on oxidation of luciferin, which is proportional to the amount of ATP present in the reaction buffer. For measuring cellular ATP, 2-3 x 10 4 cells/well were seeded in 96-well white view plates (Packard, Wellesley, MA) and incubated for 24 h at 290 mosmol/kgH 2 O. Then, the medium was changed for 16 h to one still at 290 mosmol/kgH 2 O or the osmolality was elevated to 500 mosmol/kgH 2 O by the addition of NaCl. The cells were lysed, the protein concentrations were estimated by the BCA reagent (Pierce, Rockford, IL), and ATP/mg cell protein was measured. For measuring ATP release to medium, the cells were seeded at 2-3 x 10 4 cells/well in a 96-well plastic plate in phenol red-free cell culture medium. Osmolality was increased to 500 mosmol/kgH 2 O (NaCl added) in the presence of 200 µM suramin, which prevents the degradation of any ATP that might be released ( 8, 25 ). In experiments involving depletion of medium ATP by apyrase, apyrase activity was measured at the end of the experiment by incubating 10 µl medium with a standard ATP solution to be sure that it had remained active.
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Measurement of TonEBP/OREBP transcriptional activity. The osmotic responsive element (ORE) reporter, -1233/-1105 IL2min-GL3, was constructed by inserting nucleotides -1233 to -1105 of the 5'-flanking region of the human aldose reductase gene into Mlu I/ Nhe I sites upstream of the human IL-2 minimal promoter, as previously described ( 51 ). An otherwise identical reporter to which binding of TonEBP/OREBP is prevented by mutation of the potentiating and ORE elements and the adjacent AP-1 site was used to control for specificity for TonEBP/OREBP. Mutations were performed using a QuikChange site mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Accuracy of the mutations was confirmed by sequencing ( 51 ).* J* t$ F, `  O* |
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Stable reporter cell lines were established by transfection with Effectene (Qiagen, Valencia, CA), blasticidin selection, and screening for luciferase activity to select clones with the highest expression. Next, 2-3 x 10 4 cells/well were seeded in 96-well white view plates (Packard) and incubated for 24 h at 290 mosmol/kgH 2 O; then, the medium was changed for 16 h to one still at 290 mosmol/kgH 2 O or the osmolality was elevated to 500 mosmol/kgH 2 O by the addition of NaCl. Luciferase activity was measured with Bright-Glo substrate (Promega, Madison, WI) in a Victor luminometer (PerkinElmer, Wellesley, MA). Cell proteins were determined with the BCA reagent (Pierce).
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Measurement of BGT1 mRNA expression. As previously described ( 13 ), 2.0 x 10 5 cells/well were plated in a six-well dish, then incubated at the indicated osmolality for 16 h. Total RNA was isolated using an RNeasy Mini Kit (Qiagen) and reverse transcribed with Taq man RT reagents (Applied Biosystems, Foster City, CA). Amplicons were detected with the ABI Prism 7900HT detection system (Applied Biosystems). Primers for the human BGT1 mRNA are 5'-TGTTCAGCTCCTTCACCTCTGA-3' and 5'-GCAATGCTCTGTGTTCCAAAAG-3'. The 6-carboxyfluorescein (FAM)-labeled probe is 5'-CTGCCCTGGACGACCTGCAACAA-3'. As a control for RT efficiency and loading, 18S RNA was measured at the same time, using primers and a probe from Applied Biosystems. The fold-difference in RNA abundance between conditions ( F ) was calculated, as F = E (Ct1-Ct2). E is the efficiency of the reaction determined from the results of reactions containing 8 or 80 ng of cDNA template. The efficiency was (means ± SE) 1.98 ± 0.056 in 18 experiments. Ct 1 and Ct 2 are the numbers of cycles required to reach the threshold of amplicon abundance in respective experimental conditions ( 13 ).

Measurement of TonEBP/OREBP transactivating activity. A yeast binary GAL4 reporter assay system was used, as previously described ( 13 ). Briefly, the assay system comprises an expression vector containing the TonEBP/OREBP transactivation domain (TAD), GAL4dbd-TAD, and a reporter plasmid (GAL4UAS-GL3) that are cotransfected. GAL4dbd-TAD contains in-frame insertion of cDNA coding for amino acids 548-1531 of TonEBP/OREBP in a vector containing the neomycin resistance gene (pFA-CMV, Stratagene). An otherwise identical construct, in which the GAL4dbd construct does not contain a TAD, was used as a control for nonspecific effects. GAL4UAS-GL3 contains five tandem repeats of the yeast GAL4 binding site (upstream activating sequence) and a minimal promoter (TATATA) derived from pFR-Luc (Stratagene) and inserted into the Nhe I/ Hin dIII sites of pGL3 (Promega) upstream of the Photinus pyralis luciferase gene GAL4UAS-GL3. It was further modified for blasticidin resistance as described before ( 13 ). Stable reporter cell lines were established by blasticidin and neomycin selection and used as described above for assays of TonEBP/OREBP transcriptional activity.$ @% Q6 j+ x8 ]. K
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Measurement of TonEBP/OREBP nuclear translocation. TonEBP/OREBP nuclear translocation was measured by Western blot analysis. Briefly, after 6 x 10 6 cells/10-cm dish were seeded and incubated for 24 h, the medium was changed for 30 min to one still at 290 mosmol/kgH 2 O or osmolality was elevated to 500 mosmol/kgH 2 O by the addition of NaCl. The cytoplasmic and nuclear proteins were separated with a NE-PER kit (Pierce) with a protease inhibitor cocktail tablet (Roche, Indianapolis, IN), 2.5 mM NaF and 2.5 mM Na 3 VO 4 added. The protein concentrations were determined with the BCA reagent (Pierce). Thirty micrograms cytoplasmic proteins/lane or 20 µg nuclear proteins/lane were loaded into a 15-well precast 4-12% gradient polyacrylamide gel (Invitrogen, Carlsbad, CA). The nitrocellulose membrane was probed with TonEBP/OREBP antibody (Affinity BioReagents, Golden, CO) overnight at 4°C, followed by incubation with Alexa fluophor-conjugated secondary antibody (Molecular Probes) for 60 min at 37°C. Display and quantification were by infrared imaging (Odyssey, Li-Cor, Lincoln, NE)./ w8 a5 M5 X# g5 h- v/ K
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Statistical analysis. Data are expressed as means ± SE. Statistical analyses were performed by paired t -test or repeated ANOVA, as appropriate. Post hoc comparison was made by Dunnett's test. P

RESULTS
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High NaCl-induced ROS come from mitochondria. We previously showed that high NaCl increases ROS in HEK293 cells ( 51 ), renal epithelial cells of human origin ( 40 ). Rotenone inhibits mitochondria complex I ( 42 ). Myxothiazol inhibits mitochondria complex III ( 10 ). To examine whether hypertonicity-induced ROS are produced by mitochondria, we examined the effects of rotenone and myxothiazol on ROS activity by confocal microscopy, using dihydroethidium as a probe. Apocynin inhibits NADPH oxidase ( 41 ). Allopurinol inhibits xanthine oxidase ( 45 ). To examine whether NADPH oxidase and xanthine oxidase contribute to high NaCl-induced ROS, we also examined the effect of apocynin and allopurinol on high NaCl-induced ROS. Rotenone and myxothiazol reduce high NaCl-induced ROS by 44 and 47%, respectively, whereas apocynin and allopurinol have no significant effect ( Fig. 1 A ). These results point to a mitochondrial source of the high NaCl-induced increase in ROS.

Fig. 1. A : rotenone (250 nM) and myxothiazol (12 nM), but not apocynin (100 µM) or allopurinol (100 µM), significantly inhibit high NaCl-induced reactive oxygen species (ROS) activity in HEK293 cells. ROS activity was estimated by confocal microscopy, using dihydroethidium (DHE) as a probe. Osmolality of medium bathing the cells was increased from 290 to 450 mosmol/kgH 2 O (NaCl added) for 45 min. The dye and inhibitors were added, as indicated, 30 min before the increase in NaCl. Results are expressed relative to control at 290 mosmol/kgH 2 O (* P : S6 E5 [$ T; _; P- k
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Mitochondrial ROS contribute to high NaCl-induced activation of TonEBP/OREBP. We next examined the influence of mitochondrial ROS on high NaCl-induced activation of TonEBP/OREBP by measuring the effect of rotenone and myxothiazol on ORE/TonE luciferase reporter activity. High NaCl increases ORE/TonE reporter activity by 58-fold ( Fig. 2 A ). Rotenone (250 nM) and myxothiazol (12 nM) attenuate the effect of high NaCl by 62 and 53%, respectively. As a control for specificity to TonEBP/OREBP, we used an otherwise identical reporter in which the ORE/TonE sites are mutated. This prevents the binding of TonEBP/OREBP and thus inhibits high NaCl-induced increase in reporter activity. Rotenone and myxothiazol have no significant effect on activity of the mutated ORE reporter ( Fig. 2 B ), excluding nonspecific effects.

Fig. 2. A : rotenone (250 nM) and myxothiazol (12 nM), but not allopurinol (100 µM), inhibit high NaCl-induced osmotic responsive element (ORE) luciferase reporter activity in HEK293 cells. B : rotenone (250 nM) and myxothiazol (12 nM) have no significant effect with mutated ORE luciferase reporter, used as a control for specificity. C : rotenone (250 nM) and myxothiazol (12 nM), but not allopurinol (100 µM), inhibit high NaCl-induced increase in betaine transporter (BGT1) mRNA expression. HEK293 cells were incubated with inhibitors for 30 min before osmolality was increased to 500 mosmol/kgH 2 O by addition of NaCl for 16 h. Results are expressed relative to control at 290 mosmol/kgH 2 O (* P

As an additional test, we measured the mRNA abundance of BGT1, a transcriptional target of TonEBP/OREBP ( 16 ). Rotenone and myxothiazol inhibit the high NaCl-induced increase of BGT1 mRNA expression by 69% ( Fig. 2 C ), further evidence of a mitochondrial origin of the ROS that contribute to activation of TonEBP/OREBP.
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Rotenone and myxothiazol reduce cellular ATP. However, rotenone and myxothiazol can reduce cellular ATP, which complicates the interpretation. In fact, each greatly reduces ATP in HEK293 cells ( Fig. 1 B ). Therefore, we performed additional experiments in an attempt to reduce mitochondria-derived ROS without decreasing ATP. Oligomycin at high concentrations inhibits mitochondrial respiration and reduces ATP synthesis ( 7 ). However, at low concentrations, oligomycin inhibits ROS without a reduction in ATP abundance ( 19 ). Oligomycin (5 ng/ml) inhibits high NaCl-induced increase in ROS ( Fig. 3 A ) without significantly affecting cellular ATP abundance ( Fig. 3 B ). It also inhibits high NaCl-induced ORE reporter activity by 54% ( Fig. 3 C ), similar to the effects of rotenone and myxothiazol. As a control for specificity, oligomycin does not significantly affect activity measured with the mutated ORE reporter ( Fig. 3 D ). Combined with our previous investigation showing that antioxidants N -acetylcysteine (NAC) and manganese (III) tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP) inhibit high NaCl-induced activation of TonEBP ( 51 ), we conclude that a reduction in mitochondrial-derived ROS can decrease high NaCl-induced activation of TonEBP/OREBP independently of any decrease in cellular ATP.

Fig. 3. A : oligomycin (5 ng/ml) inhibits high NaCl-induced ROS in HEK293 cells. ROS were measured by confocal microscopy, using DHE as a probe. Osmolality of medium bathing the cells was increased from 290 to 450 mosmol/kgH 2 O by adding NaCl for 45 min. The dye and inhibitor were added 30 min before exposure of cells to high NaCl. Results are expressed relative to control at 290 mosmol/kgH 2 O (* P
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Mitochondrial ROS contribute to high NaCl-induced activation of TonEBP/OREBP by increasing its transactivation. TonEBP/OREBP contains a TAD, whose activity is increased by high NaCl ( 12, 13, 23 ). To test whether mitochondrial ROS contribute to high NaCl-induced transactivating activity of TonEBP/OREBP, we examined the effect of rotenone and myxothiazol in HEK293 cells stably transfected with a binary GAL4 transactivation reporter system. Raising osmolality from 290 to 500 mosmol/kgH 2 O by adding NaCl increases transactivating activity by 11.8-fold ( Fig. 4 A ). Rotenone and myxothiazol, but not allopurinol, reduce high NaCl-induced transactivating activity by 66 and 46%, respectively ( Fig. 4 A ). We used an otherwise identical reporter that does not contain the TAD of TonEBP/OREBP to exclude a general effect on transactivation. Rotenone and myxothiazol have no significant effect on the control reporter activity ( Fig. 4 B ). We conclude that ROS contribute to activation of TonEBP/OREBP by transactivating it.- }3 w: x0 ^5 C7 w6 ~; X% b

Fig. 4. A : rotenone (250 nM) and myxothiazol (12 nM), but not allopurinol (100 µM), inhibit high NaCl-induced increase in tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP) transactivating activity. B : rotenone (250 nM), myxothiazol (12 nM), and allopurinol (100 µM) do not significantly affect transactivation when the reporter contains no TonEBP/OREBP transactivation domain (TAD). HEK293 cells were incubated with chemicals for 30 min before osmolality was increased to 500 mosmol/kgH 2 O by addition of NaCl for 16 h. Results are expressed relative to control at 290 mosmol/kgH 2 O (* P

Contribution of mitochondrial ROS to high NaCl-induced activation of TonEBP/OREBP does not involve nuclear translocation. At 300 mosmol/kgH 2 O, TonEBP/OREBP distributes between the cytosol and nucleus. On hypertonic stress, TonEBP/OREBP translocates into the nucleus within 30 min ( 5, 32 ). We tested whether mitochondrial ROS contribute to TonEBP/OREBP nuclear translocation by examining the effect of rotenone and myxothiazol on TonEBP/OREBP protein abundance in cytoplasm and nucleus. Rotenone and myxothiazol do not significantly affect high NaCl-induced nuclear translocation of TonEBP/OREBP, nor does the antioxidant NAC (15 mM) ( Fig. 5 ). We conclude that high NaCl-induced TonEBP/OREBP nuclear translocation is independent of ROS in general and of mitochondrial ROS in particular.

Fig. 5. Rotenone (Rot; 250 nM; A and B ), myxothiazol (Myx; 12 nM; A and B ), and N -acetylcysteine (NAC; C and D ) do not significantly affect high NaCl-induced TonEBP/OREBP nuclear translocation in HEK293 cells. The cells were incubated with each chemical for 30 min, and then osmolality was increased to 500 mosmol/kgH 2 O by addition of NaCl for an additional 30 min. TonEBP/OREBP was measured in cytoplasmic and nuclear extracts by Western blot analysis ( n = 3).# c3 o4 x. q8 n" w, r
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High NaCl does not induce release of cellular ATP. In Jurkat T and neutrophil cells, hypertonicity induces release of cellular ATP, which triggers cellular osmotic responses ( 8, 25 ). To determine whether this is also true of HEK293 cells, we measured ATP in the medium. High NaCl does not induce release of HEK293 cellular ATP ( Fig. 6 A ). To exclude the possibility that some ATP might be released, but maintain measurable levels only at the surface of the cells, where it could still act as a signal, we added the ATPase apyrase (20 U/ml) to eliminate any traces of ATP. Apyrase does not reduce high NaCl-induced ORE reporter activity ( Fig. 6 B ), excluding a role for extracellular ATP. We confirmed that apyrase remains active throughout the 16-h incubation at 500 mosmol/kgH 2 O by directly measuring the activity at the end of the experiment ( Fig. 6 C ). We conclude that high NaCl-induced activation of TonEBP/OREBP does not depend on release of ATP from the cells.

DISCUSSION
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Rotenone and myxothiazol have been widely used to inhibit mitochondrial production of ROS, despite the fact that they have other effects as well. Rotenone and myxothiazol inhibit high NaCl-induced increase in ROS ( Fig. 1 A ) and increase in TonEBP/OREBP transcriptional activity ( Fig. 2 ). They also attenuate the high NaCl-induced increase in TonEBP/OREBP transactivating activity ( Fig. 4 ) but do not affect high NaCl-induced TonEBP/OREBP nuclear translocation ( Fig. 5 ). Thus the ROS that are induced by high NaCl and contribute to the activation of TonEBP/OREBP come from mitochondria, and they mediate high NaCl-induced activation of TonEBP/OREBP by increasing its transactivating activity, not its translocation to the nucleus." |# t* ]# Q4 y% L

In the renal cortex, ROS are primarily generated by NADH oxidase ( 52 ). However, this differs in the renal medulla, associated with higher interstitial osmolality. In the outer portions of the renal medulla, NADH oxidase and mitochondria are both major sources of ROS, but in the renal papilla mitochondria are the only major source ( 52 ), which we attribute to the higher interstitial NaCl concentration. An increased contribution of mitochondria to overall cellular production of ROS when NaCl is high can explain why rotenone and myxothiazol reduce ROS at 500 but not at 290 mosmol/kgH 2 O ( Fig. 1 A ). Previously, hypoxia and mechanical stretch were also found to increase mitochondrial production of ROS ( 1, 6 ), and increased ROS were found to trigger cellular responses ( 1, 6 ), analogous to the responses found in the present studies.; X' O& O3 }5 R2 K( I1 [. [
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In Jurkat T and neutrophil cells, hypertonicity activates NADPH oxidase to produce ROS. This follows release of cellular ATP and phosphorylation of p38 kinase through a putative osmoreceptor system ( 8, 25 ). This mechanism must be cell type specific, however, because we do not observe any high NaCl-induced release of ATP ( Fig. 6 ), nor do we find effects of apocynin in HEK293 cells, which express NADPH oxidase ( 39 ) ( Fig. 1 A ). Along the same line, the high NaCl-induced increase in ROS is not reduced when either gp91 phox or p47 phox, which are critical NADPH oxidase components, is inactivated in primary cultures of inner medullary collecting duct cells ( 49 ). In contrast, rotenone and other inhibitors of mitochondrial ROS production block high NaCl-induced ROS in these cells, consistent with a mitochondrial source of the ROS ( 49 ).

Rotenone and myxothiazol inhibit ROS activity by only 44-47%, and higher concentrations of the inhibitors do not increase the effect (data not shown), implying that there may be other sources of high NaCl-induced ROS besides mitochondria. Other possible sources besides mitochondria and NADPH oxidase include xanthine oxidase ( 46 ) and nitric oxide synthase ( 44 ). However, N G -monomethyl- L -arginine (a general inhibitor of nitric oxide synthases) ( 2, 51 ), apocynin, and allopurinol ( Fig. 1 A ) do not inhibit high NaCl-induced ROS production, leaving the question as to what the other sources of ROS might be.8 X/ p( l6 R7 {  ]2 D9 D. ]
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How does hypertonicity increase mitochondrial ROS production? One possibility follows from the fact that mitochondria are anchored to the cytoskeleton via actin binding complexes in the outer membrane ( 3, 18, 24 ). This transmits forces associated with cytoskeleton restructuring to the outer membrane, leading to altered respiratory chain activity and superoxide generation. Examples include increased mitochondrial ROS production in response to actin cytoskeleton remodeling triggered by cyclic strain ( 1 ) or antibody binding ( 47 ). Hypertonicity also induces actin cytoskeleton reorganization ( 11, 43 ), which could produce the same effect. A direct effect of cellular hyperosmolality is another possibility. Acute hypertonic stress results in inhibition of substrate oxidation and decrease in ATP synthesis ( 15 ). Studies with isolated mitochondria show that hypertonicity does not inhibit each electron transport complex per se. Instead, hypertonicity compresses voids and decreases the availability of voids in the membrane phase for quinone diffusion ( 28 ). This could lead to an increase in the life of electron transport intermediates such as ubisemiquinone, which generates a major component of ROS, superoxide, by transferring one electron to one oxygen molecule.

At this point, we can only speculate about how ROS contribute to high NaCl-induced activation of TonEBP/OREBP. High NaCl is known to induce a MAPK cascade that phosphorylates p38, increasing its kinase activity ( 43 ). This activation of p38 contributes to high NaCl-induced increase in TonEBP/OREBP transcriptional activity ( 20, 35 ) by transactivating TonEBP/OREBP ( 20 ). Phosphorylation of p38 is a dynamic and reversible process that is dictated by the balance of activities of MAPK kinases like MKK3/6 and protein phosphatases, including protein tyrosine phosphatase (PTP)s. ROS inhibit PTPs through either direct oxidation of the thiol group in the active cysteine residue or glutathionylation of it due to an increased oxidized glutathione level ( 14 ). Hypertonicity inhibits protein phosphatase activity, which can be prevented by the antioxidant NAC ( 37 ). Thus the high NaCl-induced increase in mitochondrial ROS production might contribute to activation of p38 by inactivating a PTP that restricts p38 phosphorylation and activity. Increased p38 activity could then transactivate TonEBP/OREBP. The process could be expedited by the presence of both PTPs ( 38 ) and p38 ( 21 ) in mitochondria. A previous example of ROS affecting transactivation via phosphorylation by a MAPK is provided by the effect of PDGF. Activation of PDGF increases ROS, which contribute to activity of the transcription factor AP-1 ( 33 ), mediated by ERK, which increases phosphorylation of a TAD located at the COOH terminal of c-Fos, a partner in AP-1 heterodimers ( 33 ).
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ACKNOWLEDGMENTS$ E4 k4 G5 j6 m6 p
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The authors thank Drs. Chris Combs and Daniela A. Malide for expert help with confocal microscopy." y7 ^0 p- M; m( W
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