- Research Paper
- Open Access
Antioxidative defense and mitochondrial thermogenic response in brown adipose tissue
© Springer-Verlag 2009
- Received: 26 June 2009
- Accepted: 19 November 2009
- Published: 13 December 2009
Cold-exposure activates interscapular brown adipose tissue (IBAT) non-shivering thermogenesis that relies primarily on intensification of metabolic rate and uncoupling. During cold-acclimation, uncoupling in IBAT decreases superoxide (O2 ·−) production and as an adaptive response the activities of manganese and copper, zinc superoxide dismutase (Mn- and CuZn-SOD, respectively) are decreased, as well. However, molecular mechanisms governing this SODs adaptive response are still unsolved. Besides, knowing that NO reinforces IBAT uncoupling, we wondered whether nitric oxide (NO) is taking part in SODs regulation? Mn- and CuZn-SOD mRNA and protein expression, uncoupling protein 1 (UCP1), nitrotyrosine and nuclear factor-kappa B (NF-κB) immunolabeling, as well as total SOD (tSOD) activity in IBAT of rats subjected to cold (4 ± 1°C) for 1, 3, 7, 12, 21 and 45 days and treated by l-arginine or N ω-nitro-l-arginine-methyl ester (l-NAME) were examined. Cold increased UCP1 immunopositivity and decreased tSOD activity during entire cold-acclimation and transiently, (day 3), activated NF-κB and increased Mn and CuZn-SOD mRNA expression and nitrotyrosine labeling, suggesting NO involvement in this signaling. However, SODs mRNA expression was decreasing from day 12 till the end of cold-acclimation. l-arginine augmented and prolonged cold-induced UCP1 and nitrotyrosine immunopositivity, NF-κB activation and SODs mRNA expression increase, while l-NAME expressed an opposite effect. Related to cold, l-arginine decreased, while l-NAME increased Mn-SOD protein expression. In contrast, neither low temperature nor both treatments applied affected CuZn-SOD protein expression. The results showed that adaptive decrease in SODs activity on uncoupling-decreased O2 ·− production was achieved already at the level of gene transcription and that NO takes part in the regulation of IBAT SOD isoforms.
- Brown adipose tissue
Interscapular brown adipose tissue (IBAT) is the main site of non-shivering thermogenesis . The thermogenic capacity of IBAT relies on its high mitochondrial density and unique presence of uncoupling protein 1 (UCP1) that uncouples phosphorylation from respiration and dissipates proton gradient as heat [2, 3]. Exposure of animals to cold activates IBAT thermogenic program , a complex process consisting of a series of molecular, biochemical and structural tissue changes that recruit a large capacity for tissue oxidative metabolism increase and heat production. However, increase in tissue metabolic activity unavoidably leads to generation of reactive oxygen and nitrogen species (ROS and RNS, respectively), well known mediators of several forms of tissue damage. Cellular homeostasis in these conditions is maintained by an almost perfect match in the tissue antioxidative defense (AD) [5–10]. In addition, we have recently shown that the changes in AD occurring during cold-acclimation should be observed as a part of newly established IBAT homeostasis that characterizes intensive oxidative metabolism, uncoupling, lipolysis and decreased rate of apoptosis [9–11]. Therefore, shown decrease in manganese and copper, zinc superoxide dismutase (Mn- and CuZn-SOD, respectively, EC 126.96.36.199) activity in rats kept at cold for 45 days, accompanied by an increased UCP1 expression, was explained as adaptive response of enzymatic activities on decreased superoxide (O2 ·−) production by uncoupling . However, with the exception of the data on acute cold-exposure , the results on molecular mechanisms involving this SODs adaptation are still lacking. Precisely, it is unknown how SODs expression, in terms of protein and mRNA level, changes during various periods of acclimation to cold.
On the other hand, it has been shown that reactive species first of all nitric oxide (NO) and O2 ·− also participate in numerous redox-sensitive pathways that regulate different IBAT functions [9, 11, 13–15]. NO has been reported to regulate tissue blood flow , proliferation and differentiation of brown adipocytes, apoptosis , capillary remodeling  and mitochondriogenesis [13, 15]. It regulates some of these pathways directly, but the targeting of some NO effects in IBAT has been found to be mediated by O2 ·− and glutathione [10, 13, 14]. Thus, the enzymes involved in reactive species metabolism became an essential component of redox signaling, tightly regulating and targeting ROS and RNS production, which is crucial for their effects.
Nevertheless, despite the vast knowledge on the role NO plays in IBAT thermogenesis, there is still no data on possible effects of NO on both Mn- and CuZn-SOD mRNA and protein expression, especially during transition to the new tissue homeostatic state, i.e. in multiple stages of acclimation to cold.
Because of all aforementioned facts, we performed this time-course study to gain a better insight into the regulatory molecular mechanisms underlying SODs activity adaptation during cold-exposure by examining Mn- and CuZn-SOD mRNA and protein content in IBAT of rats kept at cold for different time periods. A special attention has been paid to a possible influence of NO on cold-induced expressional profiles of IBAT SOD isoforms. For this purpose, adult male rats kept at room temperature, or exposed to cold for 1, 3, 7, 12, 21 and 45 days and receiving NO-manipulating agents l-arginine or N ω-nitro-l-arginine methyl ester (l-NAME) as drinking liquids were used. Changes in Mn- and CuZn-SOD mRNA and protein level, total SOD (tSOD) activity, UCP1 and nitrotyrosine immunopositivity and activation of nuclear factor-κB (NF-κB), a potential mediator of NO effects, were assessed.
The experimental protocol was approved by the Ethical Committee for the Treatment of Experimental Animals of the Institute for Biological Research, Belgrade. Mill Hill hybrid hooded, 4-month-old rat (Rattus norvegicus Berkenhout 1769) males were divided into two main groups: a control group, kept at room temperature (22 ± 1°C) for the duration of the experiment, and the second group, maintained at cold (4 ± 1°C). Cold-acclimated group was divided into three subgroups: (1) untreated; (2) l-arginine-treated and (3) l-NAME-treated. Drugs were administered as drinking liquids, containing 2.25% solution of l-arginine·HCl, i.e. 0.01% l-NAME·HCl in tap water, as described earlier [9, 11]. The rats were housed in individual plastic cages with drinking liquids and food ad libitum. The duration of cold-exposure ranged from 1 to 45 days (1, 3, 7, 12, 21 and 45 days), with six animals per experimental group.
The animals were killed at each experimental point by decapitation, and the IBAT was harvested and quickly cut into halves on an ice bath. Approximately one half of the tissue was snap-frozen in liquid nitrogen and stored at −80°C until subsequent RNA extraction and western blotting. The remaining IBAT part was used for immunohistochemistry and total SOD activity determination. For immunohistochemical examination, fresh tissue was immediately fixed in neutral buffered formalin and subjected to light microscopy. For SOD activity determination, tissue samples were homogenized (a Janke and Kunkel Ka/Werke Ultra/Turrax homogenizer) at 0–4°C in 0.25 M sucrose, 0.1 mM EDTA and 50 mM Tris buffer, pH 7.4 and sonicated.
RNA extraction and semi-quantitative RT–PCR
Total RNA was prepared from 100 mg of isolated IBAT using TRIzol (Invitrogen, Life Technologies, CA, USA) and one microgram of total RNA was reverse transcribed to cDNA using an iScript™ cDNA synthesis kit (Bio Rad Laboratories, USA) according to manufacturer’s instructions.
Primers sequences and cycling conditions
Product size (bp)
60″ at 95°C, 60″ at 57°C, 90″ at 72°C
60″ at 95°C, 60″ at 53°C, 90″ at 72°C
30″ at 95°C, 30″ at 56°C, 30″ at 72°C
SDS–PAGE and western blotting
Western blot was done as described previously [9, 11] using mouse monoclonal antibody against Mn-SOD (1:1000, Chemicon International Inc., Temecula, CA, USA) and rabbit polyclonal antibody against CuZn-SOD (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentrations are expressed relative to the control acclimated to room temperature taken as 100%. Quantitative analysis of immunoreactive bands was done by ImageQuant software. Volume is the sum of all the pixel intensities within a band; 1 pixel = 0.007744 mm2.
Five-μm-thick sections were immunostained with the avidin–biotin-peroxidase method, as described previously . Primary antibodies were anti-nitrotyrosine, anti-UCP1 (1:200 and 1:300 Abcam, Cambridge, UK, respectively) and anti-NF-κB (1:200, v/v Upstate, USA). The specificity of the immune reaction was tested by replacing primary antibody with a non-immune rabbit serum or by incubating the sections with the secondary antibody alone.
Activity of superoxide dismutase
Total SOD activity was examined  and enzymatic activity expressed in U mg−1 protein. SOD units were defined as the amount of the enzyme inhibiting epinephrine oxidation by 50% under the appropriate reaction conditions.
Other assays and statistics
Protein content was estimated using bovine serum albumin as a reference . Analysis of variance (ANOVA) was used for within-group comparison of the data. If the F test showed an overall difference, Tukey’s test was applied to evaluate significance of the differences. Statistical significance was accepted at P < 0.05.
The data obtained throughout the present work showed that cold, as an indispensable part of IBAT thermogenic program, increased UCP1 immunopositivity in untreated rats during the entire 45-day-acclimation period. In line with this and the fact that uncoupling acts decreasing production of O2 ·− in IBAT mitochondria [20, 21], decreased tSOD activity during the entire examined acclimation period was observed. Similarly, one-day-cold- exposure decreased both Mn- and CuZn-SOD mRNA expression. This is in line with Niakao et al.  who have shown that acute cold exposure rapidly decreased mRNA level of all SOD isoforms. However, on day 3 of cold-acclimation, both Mn- and CuZn-SOD transcripts were higher than the control and this was followed by restitution of their expression at control level on day 7 and a decrease below control until the end of experimental period. This SODs transcriptional activation is in accordance with our recent data demonstrating maximal transcriptional activation of IBAT thermogenic genes, leading to tissue metabolic adaptation on day 3 of cold-acclimation . These results clearly reflect intensification of IBAT synthetic processes and metabolic rate oriented to preserve thermal homeostasis. During that time, IBAT O2 consumption can increase tremendously, which could transiently increase ROS production and activate AD. Accordingly, activation of NF-κB was recorded here on day 3 of cold-acclimation, clearly confirming above hypothesized tissue redox perturbations. Namely, NF-κB is the major redox sensitive factor shown to activate gene expression of a number of antioxidative enzymes . Therefore, it could be involved in the activation of SODs transcription observed in the present study.
Besides, we have shown recently that NO participates in the activation of IBAT thermogenic gene expression during cold-acclimation . Thus, we have examined here IBAT appearance of nitrotyrosine, a redox signaling component that mediates a variety of NO actions [25, 26]. The data showed that time-course of SODs transcriptional activation correlates well with nitrotyrosine immunopositivity increase, suggesting NO involvement in this signaling. However, it has been shown that during the time of exposure to cold, the rate of uncoupling increases and consequently, mitochondrial production of O2 ·− decreases [21, 27]. Accordingly, after transient activation of SODs transcription on day 3, their mRNA expression was decreased and remained below control value until the end of cold-acclimation. Thus, a prolonged decrease in the abundance of Mn- and CuZn-SOD transcripts observed here strongly indicates that previously shown adaptive decrease in SODs activities on uncoupling-decreased O2 ·− production [10, 11] is achieved already at the level of their transcription.
On the other hand, in most tissues examined, cold-induced changes in the SOD isoenzyme mRNA levels appeared not to be concomitant with those in the protein levels as reported by Niakao et al. . These authors emphasized that this is particularly accurate in IBAT in which acute cold-exposure rapidly decreased expression of all SOD isoforms without any alteration in the amount of the enzymatic proteins. This is in accordance with our results on the changes of CuZn-SOD expression demonstrating that neither low temperature nor either of the applied treatments affected protein expression of this enzyme. However, changes in Mn-SOD protein expression were in line with the changes in mRNA expression of untreated rats during acclimation to cold. These data support the fact that CuZn-SOD is a highly stable protein  and the data revealing Mn-SOD more inducible than CuZn-SOD in conditions of metabolic turnover [28, 29].
The observed different response of Mn- and CuZn-SOD mRNA and protein levels during cold-acclimation appears to depend on their different subcellular localization. Namely, Mn-SOD is compartmentalized in the mitochondria, whereas CuZn-SOD mainly occurs in the cytosol . Thus, the changes in Mn-SOD protein expression reflect the state in these compartments and the observed decrease could be explained by adaptive response of the enzyme on decreased production of O2 ·− by an intense uncoupling.
Compared with cold, treatment with l-arginine, a substrate for NO synthesis, augmented and prolonged period of increased Mn- and CuZn-SOD mRNA expression, as well as UCP1 immunolabeling, while l-NAME markedly attenuated mRNA expression of both SOD isoforms and decreased UCP1 immunopositivity. Such opposite effects of l-arginine and l-NAME strongly indicate that stimulatory effects of l-arginine, could be, at least in part, mediated by NO. Further support of this hypothesis is provided by the results on immunohistochemical staining of nitrotyrosine that was higher than the control on day 3 and day 7 of cold-acclimation. Stimulatory effect of NO on UCP1 appearance in IBAT is in line with our previous data showing induction of UCP1 protein expression by NO . On the other hand, induction of Mn- and CuZn-SOD transcription by l-arginine strongly suggests that NO activates IBAT SODs transcription and agrees well with the results of other authors obtained both in vitro  and in vivo [31, 32] and showing that both SOD isoforms contain NO-binding sites and that their transcription could be up-regulated by NO. In addition, Mn-SOD gene promoter contains NF-κB binding site and represents one of key targets for NF-κB signaling . Accordingly, nuclear localization of P65 NF-κB subunit, which signals its activation, was detected in the present study from day 3 to day 7 of l-arginine treatment. Considering that NO can up-regulate NF-κB [34, 35], it could be supposed that the observed stimulatory effect of NO on Mn-SOD expression might also be mediated by NF-κB activation. However, precise mechanisms involved in the regulation of IBAT SODs mRNA expression by NO remain to be elucidated.
As already stated, in accordance with tissue active-, i.e. thermogenic-state characterized by an intensive uncoupling, Mn-SOD protein expression was generally decreased in all groups during the acclimation to cold, except on day 3 in untreated and day one in l-NAME-treated animals. The same holds true for tSOD activity decrease and UCP1 labeling increase observed throughout this work during the entire period of acclimation to cold. However, the values of Mn-SOD protein expression were lower in l-arginine-treated group in each time-point of cold-acclimation, in relation to those detected in untreated rats. This result is in line with our previous data showing stimulatory effect of NO on cold-induced increase of UCP1 protein expression  and mitochondriogenesis . Thus, this might reflect enzymatic response at protein level on NO-induced increase of uncoupling and thermogenic capacity. In addition, an assumed decrease in O2 ·− availability could be the consequence of its interaction with NO. These molecules are rapidly reacting with the threefold higher rate constant than that of dismutation reaction catalyzed by SOD . In this context, NO may act as O2 ·− scavenger, thus decreasing its concentration. Interaction of these redox molecules leads to the formation of peroxynitrite , RNS that takes part in NO cGMP-independent signaling and mediates variety of NO actions, such as induction of Mn-SOD gene expression without concomitant response in the protein level [22, 38]. Precisely, alongside with stimulatory effect of peroxynitrite on Mn-SOD transcription, it inactivates Mn-SOD via nitration causing either a decrease of its activity or its protein level . In vivo effects of peroxynitrite are possible to observe by nitrotyrosine formation [39–41]. So, nitrotyrosine appearance detected here in l-arginine-treated group on day 3 and 7 of cold-acclimation, accompanied by Mn-SOD mRNA expression increase and its protein expression decrease, indicate that peroxynitrite could play a role in the regulation of IBAT Mn-SOD mRNA and protein expression.
In general, the results of the present study corroborate and extend our earlier findings demonstrating that adaptive decrease in SODs activity on uncoupling-decreased O2 ·− production is achieved already at the level of gene transcription. Our data also clearly demonstrated that besides regulating various IBAT functions, NO is implicated in the regulation of IBAT SOD isoforms, while differentially affecting mRNA, protein content and SODs activities. Furthermore, NO increases tissue thermogenic capacity in thermogenically active IBAT, through increased UCP1 expression and uncoupling. Thus, it seems likely that primary effect of NO on IBAT during cold-acclimation is to reinforce tissue thermogenic capacity and that during this acting NO indirectly modulates the response of IBAT SOD isoforms.
This work was supported by the Ministry of Science and Technological Development of the Republic of Serbia, Grant No 143050 and by the COST FA0602 Action.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
- Himms-Hagen J (1990) Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB J 4:2890–2898PubMedGoogle Scholar
- Nicholls DG, Locke RM (1984) Thermogenic mechanisms in brown fat. Physiol Rev 64:1–64PubMedGoogle Scholar
- Cannon B, Nedergaard J (1986) Brown adipose tissue thermogenesis in neonatal and cold-adapted animals. Biochem Soc Trans 14:233–236PubMedGoogle Scholar
- Suter ER (1969) The fine structure of brown adipose tissue. II. Perinatal development in the rat. Lab Invest 21:246–258PubMedGoogle Scholar
- Halliwell B, Gutteridge JM (1990) The antioxidants of human extracellular fluids. Arch Biochem Biophys 280:1–8View ArticlePubMedGoogle Scholar
- Buzadžić B, Blagojević D, Korać B, Saičić ZS, Spasić MB, Petrović VM (1997) Seasonal variation in the antioxidant defense system of the brain of the ground squirrel (Citellus citellus) and response to low temperature compared with rat. Comp Biochem Physiol C 117:141–149View ArticlePubMedGoogle Scholar
- Buzadžić B, Korać B, Petrović VM (1999) The effect of adaptation to cold and re-adaptation to room temperature on the level of glutathione in rat tissues. J Therm Biol 24:373–377View ArticleGoogle Scholar
- Korać B, Buzadžić B (2001) Doxorubicin toxicity to the skin: possibility of protection with antioxidants enriched yeast. J Dermatol Sci 25:45–52View ArticlePubMedGoogle Scholar
- Petrović V, Korać A, Buzadžić B, Korać B (2005) The effects of l-arginine and l-NAME supplementation on redox-regulation and thermogenesis in interscapular brown adipose tissue. J Exp Biol 208:4263–4271View ArticlePubMedGoogle Scholar
- Petrović V, Buzadžić B, Korać A, Vasilijević A, Janković A, Korać B (2006) Free radical equilibrium in interscapular brown adipose tissue: relationship between metabolic profile and antioxidative defense. Comp Biochem Physiol C 142:60–65Google Scholar
- Korać A, Buzadžić B, Petrović V, Vasilijević A, Janković A, Mićunović K, Korać B (2008) The role of nitric oxide in remodeling of capillary network in rat interscapular brown adipose tissue after long-term cold-acclimation. Histol Histopathol 23:441–450PubMedGoogle Scholar
- Niakao C, Ookawara T, Kizaki T, Suzuki K, Haga S, Sato Y, Ohno H (1999) Effects of acute cold stress on mRNA expression and immunoreactivity of three superoxide dismutase isoenzymes in genetically obese mice. Res Commun Mol Pathol Pharmacol 106:47–61PubMedGoogle Scholar
- Petrović V, Korać A, Buzadžić B, Vasilijević A, Janković A, Mićunović K, Korać B (2008) Nitric oxide regulates mitochondrial re-modelling in interscapular brown adipose tissue: ultrastructural and morphometric-stereologic studies. J Microsc 232:542–548View ArticlePubMedGoogle Scholar
- Nisoli E, Clementi E, Tonello C, Sciorati C, Briscini L, Carruba MO (1998) Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures. Br J Pharmacol 125:888–894View ArticlePubMedGoogle Scholar
- Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, Bracale R, Valerio A, Francolini M, Moncada S, Carruba MO (2003) Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science 299:896–899View ArticlePubMedGoogle Scholar
- Kikuchi-Utsumi K, Gao B, Ohinata H, Hashimoto M, Yamamoto N, Kuroshima A (2002) Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold-exposure. Am J Physiol Regul Integr Comp Physiol 282:623–626Google Scholar
- Petrović V, Buzadžić B, Korać A, Vasilijević A, Janković A, Korać B (2009) l-Arginine supplementation induces glutathione synthesis in interscapular brown adipose tissue through activation of glutamate-cysteine ligase expression: the role of nitric oxide. Chem Biol Interact 182:204–212View ArticlePubMedGoogle Scholar
- Misra HP, Fridovich I (1972) The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175PubMedGoogle Scholar
- Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
- Cino M, Del Maestro RF (1989) Generation of hydrogen peroxide by brain mitochondria: the effect of reoxygenation following postdecapitative ischemia. Arch Biochem Biophys 269:623–638View ArticlePubMedGoogle Scholar
- Skulachev VP (1994) Decrease in the intracellular concentration of O2 as a special function of cellular respiratory system. Biokhimiia 59:1910–1912PubMedGoogle Scholar
- Jackson RM, Parish G, Helton ES (1998) Peroxynitrite modulates Mn-SOD gene expression in lung epithelial cells. Free Radic Biol Med 25:463–472View ArticlePubMedGoogle Scholar
- Ji LL (2007) Modulation of skeletal muscle antioxidative defense by exercise: role of redox signaling. Free Radic Biol Med 44:142–152View ArticlePubMedGoogle Scholar
- Petrović V (2008) Influence of l-arginine and l-NAME on enzymes involved in metabolism of nitric oxide, carbon monoxide and superoxide anion radical, during cold-induced IBAT hyperplasia. Dissertation, University of BelgradeGoogle Scholar
- Foster MW, McMahon TJ, Stamler JS (2003) S-nitrosylation in health and disease. Trends in Mol Med 9:160–168View ArticleGoogle Scholar
- Stamler JS, Lamas S, Fang FC (2001) Nitrosylation: the prototypic redox-based signaling mechanism. Cell 106:675–683View ArticlePubMedGoogle Scholar
- Boveris A, Oshino N, Chance B (1972) The cellular production of hydrogen peroxide. Biochem J 128:617–630PubMedGoogle Scholar
- Petrović VM, Saičić ZS, Radojičić R, Buzadžić B, Spasić BM (1989) Difference in the inducibility between copper zinc containing superoxide dismutase and manganese containing superoxide dismutase activity in the brown adipose tissue of the rat exposed or adapted to cold; correlation with tissue hyperplasia. Iugosl Physiol Pharmacol Acta 25:33–38Google Scholar
- Perera CS, St Clair DK, McClain CJ (1995) Differential regulation of manganese superoxide dismutase activity by alcohol and TNF in human hepatoma cells. Arch Biochem Biophys 323:471–476View ArticlePubMedGoogle Scholar
- Sano H, Hirai M, Saito H, Nakashima I, Isobe KI (1997) A nitric oxide-releasing reagent, S-nitroso-N-acetylpenicillamine, enhances the expression of superoxide dismutases mRNA in the murine macrophage cell line RAW264–7. Immunology 92:118–122View ArticlePubMedGoogle Scholar
- Frank S, Zacharowski K, Wray GM, Thiemermann C, Pfeilschifter J (1999) Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats. FASEB J 13:869–882PubMedGoogle Scholar
- Keller T, Plesková M, McDonald MC, Thiemermann C, Pfeilschifter J, Beck KF (2003) Identification of manganese superoxide dismutase as a NO-regulated gene in rat glomerular mesangial cells by 2D gel electrophoresis. Nitric Oxide 9:183–193View ArticlePubMedGoogle Scholar
- Guo G, Yan-Sanders Y, Lyn-Cook BD, Wang T, Tamae D, Ogi J, Khaletskiy A, Li Z, Weydert C, Longmate JA, Huang TT, Spitz DR, Oberley LW, Li JJ (2003) Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses. Mol Cell Biol 23:2362–2378View ArticlePubMedGoogle Scholar
- Connelly L, Palacios-Callender M, Ameixa C, Moncada S, Hobbs AJ (2001) Biphasic regulation of NF-κB activity underlies the pro- and anti-inflammatory actions of nitric oxide. J Immunol 166:3873–3881PubMedGoogle Scholar
- Connelly L, Jacobs AT, Palacios-Callender M, Moncada S, Hobbs AJ (2003) Macrophage endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory protein expression. J Biol Chem 278:26480–26487View ArticlePubMedGoogle Scholar
- Huie RE, Padmaja S (1993) The reaction of NO with superoxide. Free Radic Res Commun 18:195–199View ArticlePubMedGoogle Scholar
- Patel RP, Darley-Usmar VM (1996) Using peroxynitrite as oxidant with low-density lipoprotein. Methods Enzymol 269:375–384View ArticlePubMedGoogle Scholar
- Nilakantan V, Halligan NL, Nguyen TK, Hilton G, Khanna AK, Roza AM, Johnson CP, Adams MB, Griffith OW, Pieper GM (2005) Post-translational modification of manganese superoxide dismutase in acutely rejecting cardiac transplants: role of inducible nitric oxide synthase. J Heart Lung Transplant 24:1591–1599View ArticlePubMedGoogle Scholar
- Govindaraju K, Shan J, Levesque K, Hussain SN, Powell WS, Eidelman DH (2008) Nitration of respiratory epithelial cells by myeloperoxidase depends on extracellular nitrite. Nitric Oxide 18:184–194View ArticlePubMedGoogle Scholar
- Lavi S, Yang EH, Prasad A, Mathew V, Barsness GW, Rihal CS, Lerman LO, Lerman A (2008) The interaction between coronary endothelial dysfunction, local oxidative stress, and endogenous nitric oxide in humans. Hypertension 51:127–133View ArticlePubMedGoogle Scholar
- Hu J, Ng YK, Chin CM, Ling EA (2007) Effects of l-arginine and N(G)-nitro-l-arginine methyl ester treatments on expression of neuronal nitric oxide synthase in the guinea-pig bladder after partial bladder outlet obstruction. Neuroscience 151:680–691View ArticlePubMedGoogle Scholar