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Accumulating evidence suggests that the intrarenal renin-angiotensin system may be involved in the progression of diabetic nephropathy. Chymase is a potent local angiotensin II-forming enzyme in several species, including humans and hamsters. However, the pathophysiological role of chymase is not fully understood. Here, we report a causal role of chymase in diabetic nephropathy and the therapeutic effectiveness of chymase inhibition. In the present study, renal chymase expression was markedly upregulated in streptozotocin-induced diabetic hamsters. Oral administration of a specific chymase inhibitor, TEI-F00806, completely ameliorated proteinuria, the overexpression of transforming growth factor-[beta] and fibronectin in glomeruli, and renal mesangial expansion, by normalizing the increase in intrarenal angiotensin II levels in diabetic hamsters independently of blood pressure levels. In contrast, ramipril did not show such sufficient effects. These effects occurred in parallel with improvements in superoxide production and expression of NAD(P)H oxidase components [NAD(P)H oxidase 4 and [p22.sup.phox]] in glomeruli. This study showed for the first time that chymase inhibition may protect against elevated intrarenal angiotensin II levels, oxidative stress, and renal dysfunction in diabetes. These findings suggest that chymase offers a new therapeutic target for diabetic nephropathy. angiotensin II; NAD(P)H oxidase doi: 10.1152/ajprenal.00337.2010

Hindlimb suspension (HLS) elicits muscle atrophy, oxidative stress, and apoptosis in skeletal muscle. Increases in oxidative stress can have detrimental effects on muscle mass and function, and it can potentially lead to myonuclear apoptosis. Resveratrol is a naturally occurring polyphenol possessing both antioxidant and antiaging properties. To analyze the capacity of resveratrol to attenuate oxidative stress, apoptosis and muscle force loss were measured following 14 days of HLS. Young (6 mo) and old (34 mo) rats were administered either 12.5 mg x [kg.sup.-1] x [day.sup.-1] of trans-resveratrol, or 0.1% carboxymethylcellulose for 21 days, including 14 days of HLS. HLS induced a significant decrease in plantarflexor isometric force, but resveratrol blunted this loss in old animals. Resveratrol increased gastrocnemius catalase activity, MnSOD activity, and MnSOD protein content following HLS. Resveratrol reduced hydrogen peroxide and lipid peroxidation levels in muscles from old animals after HLS. Caspase 9 abundance was reduced and Bcl-2 was increased, but other apoptotic markers were not affected by resveratrol in the gastrocnemius muscle after HLS. The data indicate that resveratrol has a protective effect against oxidative stress and muscle force loss in old HLS animals; however, resveratrol was unable to attenuate apoptosis following HLS. These results suggest that resveratrol has the potential to be an effective therapeutic agent to treat muscle functional decrements via improving the redox status associated with disuse. oxidative stress; apoptosis; hindlimb suspension; aging doi: 10.1152/ajpregu.00489.2010.

Kallistatin is a regulator of vascular homeostasis capable of controlling a wide spectrum of biological actions in the cardiovascular and renal systems. We previously reported that kallistatin inhibited intracellular reactive oxygen species formation in cultured cardiac and renal cells. The present study was aimed to investigate the role and mechanisms of kallistatin in protection against oxidative stress-induced vascular injury and endothelial cell apoptosis. We found that kallistatin gene delivery significantly attenuated aortic superoxide formation and glomerular capillary loss in hypertensive DOCA-salt rats. In cultured endothelial cells, kallistatin suppressed TNF-[alpha]-induced cellular apoptosis, and the effect was blocked by the pharmacological inhibition of phosphatidylinositol 3-kinase and nitric oxide synthase (NOS) and by the knockdown of endothelial NOS (eNOS) expression. The transduction of endothelial cells with adenovirus expressing dominant-negative Akt abolished the protective effect of kallistatin on endothelial apoptosis and caspase activity. In addition, kallistatin inhibited TNF-[alpha]induced reactive oxygen species formation and NADPH oxidase activity, and these effects were attenuated by phosphatidylinositol 3-kinase and NOS inhibition. Kallistatin also prevented the induction of Bim protein and mRNA expression by oxidative stress. Moreover, the downregulation of forkhead box O 1 (FOXO1) and Bim expression suppressed TNF-[alpha]-mediated endothelial cell death. Furthermore, the antiapoptotic actions of kallistatin were accompanied by Akt-mediated FOXO1 and eNOS phosphorylation, as well as increased NOS activity. These findings indicate a novel role of kallistatin in the protection against vascular injury and oxidative stress-induced endothelial apoptosis via the activation of Akt-dependent eNOS signaling. endothelium; endothelial nitric oxide synthase superoxide; tumor necrosis factor-[alpha]; Bim; forkhead box O 1 doi: 10.1152/ajpheart.00591.2010.

Blood ketone body levels increase during starvation and untreated diabetes. Here we tested the hypothesis that ketone bodies directly inhibit insulin action in skeletal muscle. We investigated the effect of D,L-[beta]-hydroxybutyrate (BOH; the major ketone body in vivo) on insulin-mediated glucose uptake (2-deoxyglucose) in isolated mouse soleus (oxidative) and extensor digitorum longus (EDL; glycolytic) muscle. BOH inhibited insulin-mediated glucose uptake in soleus (but not in EDL) muscle in a time- and concentration-dependent manner. Following 19.5 h of exposure to 5 mM BOH, insulin-mediated (20 mU/ml) glucose uptake was inhibited by ~90% (substantial inhibition was also observed in 3-O-methylglucose transport). The inhibitory effect of BOH was reproduced with D- but not L-BOH. BOH did not significantly affect hypoxia- or AICAR-mediated (activates AMP-dependent protein kinase) glucose uptake. The BOH effect did not require the presence/utilization of glucose since it was also seen when glucose in the medium was substituted with pyruvate. To determine whether the BOH effect was mediated by oxidative stress, an exogenous antioxidant (1 mM tempol) was used; however, tempol did not reverse the BOH effect on insulin action. BOH did not alter the levels of total tissue GLUT4 protein or insulin-mediated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 but blocked insulin-mediated phosphorylation of protein kinase B by ~50%. These data demonstrate that BOH inhibits insulin-mediated glucose transport in oxidative muscle by inhibiting insulin signaling. Thus ketone bodies may be potent diabetogenic agents in vivo. ketone bodies; soleus; extensor digitorum longus; glucose transporter 4 doi: 10.1152/ajpendo.00142.2010.

Despite pathophysiological similarities to alcoholic liver disease, susceptibility to acetaminophen hepatotoxicity in metabolic syndrome-related nonalcoholic steatohepatitis (NASH) has not been well elucidated. In this study, therefore, we investigated acetaminophen-induced liver injury in KK-[A.sup.y] mice, an animal model of metabolic syndrome. Twelve-week-old male KK-[A.sup.y] and C57B1/6 mice were injected intraperitoneally with 300 or 600 mg/kg acetaminophen, and euthanized 6 h later. Liver histology was assessed, and hepatic expression of 4-hydroxy-2-nonenal was detected by immunohistochemistry. Levels of reduced glutathione were determined spectrophotometrically. Phosphorylation of c-Jun N[H.sub.2]-terminal kinase (JNK) was analyzed by Western blotting. Hepatocytes were isolated from both strains by collagenase perfusion, and cell death and oxidative stress were measured fluorometrically by use of propidium iodide and 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester, respectively. Acetaminophen induced more severe necrosis and apoptosis of hepatocytes in KK-[A.sup.y] mice than in C57B1/6 mice and significantly increased serum alanine aminotransferase levels in KK-[A.sup.y] mice. Acetaminophen-induction of 4-hydroxy-2-nonenal in the liver was potentiated, whereas the levels of reduced glutathione in liver were lower in KK-[A.sup.y] mice. Acetaminophen-induced phosphorylation of JNK in the liver was also enhanced in KK-[A.sup.y] mice. Exposure to 20 [micro]M tert-butyl hydroperoxide did not kill hepatocytes isolated from C57B1/6 mice but induced cell death and higher oxidative stress in hepatocytes from KK-[A.sup.y] mice. These results demonstrated that acetaminophen toxicity is increased in diabetic KK-[A.sup.y] mice mainly due to enhanced oxidative stress in hepatocytes, suggesting that metabolic syndrome-related steatohepatitis is an exacerbating factor for acetaminophen-induced liver injury. drug-induced liver injury; metabolic syndrome; steatohepatitis; oxidative stress; hepatotoxicity doi: 10.1152/ajpgi.00361.2009.

Epicardial adipose tissue (EAT) is an endocrine organ adjacent to coronary arteries and myocardium without anatomy barriers. Locally produced adipokines may reflect or affect to cardiovascular physiology and pathology. Our aim was to study the protein expression profiles of EAT and subcutaneous adipose tissue (SAT) to identify local candidate molecules characterizing EAT in patients with cardiovascular disease. EAT and SAT samples were collected from 55 patients undergoing heart surgery. Proteins from these tissues were separated by two-dimensional (2D) gel electrophoresis, and differences between them were identified by MALDI-TOF/TOF spectra. Differences in protein levels were further investigated by real-time RT-PCR and Western blots, and production of reactive oxygen species (ROS) in EAT and SAT was evaluated by nitroblue tetrazolium chloride assays. ROS production was higher in EAT than SAT. We have found mRNA differences for catalase, glutathione S-transferase P, and protein disulfide isomerase, and 2D Western blots additionally showed post-translational differences for phosphoglycerate mutase 1; all four are related to oxidative stress pathways. EAT suffers greater oxidative stress than SAT in patients with cardiovascular diseases and exhibits associated proteomic differences that suggest the possibility of its association with myocardial stress in these patients. epicardial adipose tisssue doi: 10.1152/ajpheart.00120.2010.

Thiamine is an important cofactor of metabolic enzymes, and its deficiency leads to cardiovascular dysfunction. First, we characterized the metabolic status measuring resting oxygen consumption rate and lactate blood concentration after 35 days of thiamine deficiency (TD). The results pointed to a decrease in resting oxygen consumption and a twofold increase in blood lactate. Confocal microscopy showed that intracellular superoxide (~40%) and [H.sub.2][O.sub.2] (2.5 times) contents had been increased. In addition, biochemical activities and protein expression of SOD, glutathione peroxidase, and catalase were evaluated in hearts isolated from rats submitted to thiamine deprivation. No difference in SOD activity was detected, but protein levels were found to be increased. Catalase activity increased 2.1 times in TD hearts. The observed gain in activity was attended by an increased catalase protein level. However, a marked decrease in glutathione peroxidase activity (control 435.3 [+ or -] 28.6 vs. TD 199.4 [+ or -] 30.2 nmol NADPH x [min.sup.-1] x [ml.sup.-l]) was paralleled by a diminution in the protein levels. Compared with control hearts, we did observe a greater proportion of apoptotic myocytes by TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 reactivity techniques. These results indicate that during TD, reactive oxygen species (ROS) production may be enhanced as a consequence of the installed acidosis. The perturbation in the cardiac myocytes redox balance was responsible for the increase in apoptosis. catalase; glutathione peroxidase; superoxide dismutase doi: 10.1152/ajpheart.00820.2009.

Studies from animal models suggest that myocardial mitochondrial damage contributes to cardiac dysfunction after burn injury. In this report, we used an ex vivo model of primary cardiomyocyte culture to investigate the mechanisms of burn-induced mitochondrial impairment. Briefly, blood serum was collected from Sprague-Dawley (SD) rats subjected to 40% total body surface area burn and added (10% vol/vol) to primary cardiomyocytes prepared from SD rats. The effect of the burn serum on mitochondrial function and membrane integrity in the myocytes was analyzed. Exposure of myocytes to burn serum doubled the mitochondrial membrane damage measured by two independent assays. This treatment also significantly elevated mitochondrial oxidative stress, indicated by a more than 30% increase in lipid oxidation. Downregulation of mitochondrial antioxidant defense was also evident since the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were reduced by about 30% and 50%, respectively. Burn serum also induced deficiency of mitochondrial metabolism, indicated by a 30% decrease in the activity of cytochrome e oxidase. These mitochondrial dysfunctions appear to be generated by oxidative stress because burn serum induced a significant increase of mitochondrial oxygen species (mtROS) in cardiomyocytes, and pretreatment of cardiomyocytes with the antioxidant N-acetyl-cysteine prevented the mitochondrial damages induced by burn serum. Remarkably, the increase in mtROS was abolished by an antibody-mediated blockade of CDI4. Furthermore, burn injury-induced mitochondrial damage in cardiomyocytes was prevented in CDI4 knockout mice. Taken together, these data suggested that burn injury produces CD 14-dependent mitochondrial damage via oxidative stress in myocardium. oxidative stress; mitochondrial metabolism; burn injury doi: 10.1152/ajpheart.00927.2009.

Mitochondrial dysfunction is involved in pathopysiology of ischemia-reperfusion-induced acute kidney injury (AM). The p66shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction, which might play a role in AM-induced renal tubular injury. Oxidative stress-mediated Serine36 phosphorylation of p66shc facilitates its transportation to the mitochondria where it oxidizes cytochrome c and generates excessive amount of reactive oxygen species (ROS). The consequence is mitochondrial depolarization and injury. Earlier we determined that p66shc plays an essential role in injury of cultured mouse renal proximal tubule cells during oxidative stress. Here, we studied the role of p66shc in ROS generation and consequent mitochondrial dysfunction during oxidative injury in renal proximal tubule cells. We employed p66shc knockdown renal proximal tubule cells and cells that overexpress wild-type, Serine phosphorylation (S36A), or cytochrome c-binding (W134F) mutants of p66shc. Inhibition of the mitochondrial electron transport chain or the mitochondrial permeability transition revealed that hydrogen peroxide-induced injury is mitochondrial ROS and consequent mitochondrial depolarization dependent. We also found that through Ser36 phosphorylation and mitochondria/cytochrome c binding, p66shc mediates those effects. We propose a similar mechanism in vivo as we demonstrated mitochondrial binding of p66shc as well as its association with cytochrome c in the postischemic kidneys of mice. Thus, manipulating p66shc might offer a new therapeutic modality to ameliorate renal ischemic injury. reactive oxygen species doi: 10.1152/ajprenal.00639.2009.

Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor fork-head box O 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahl salt-sensitive (DSS) rats, we found that high salt intake induced a time-dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, [H.sub.2][O.sub.2] dose-dependently lowered kallistatin mRNA and protein levels as determined by real-time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO1 expression with small-interfering RNA. [H.sub.2][O.sub.2] rapidly induced FOXO1 nuclear translocation, but the effect was blocked by c-Jun N[H.sub.2]-terminal kinase (JNK) inhibitor. Inhibition of JNK by pharmacological inhibitor or small-interfering RNA reversed [H.sub.2][O.sub.2]'s effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK-dependent FOXO1 activation in cultured endothelial cells. kallistatin; Forkhead box O 1; oxidative stress; c-Jun N[H.sub.2]-terminal kinase; blood vessel doi:10.1152/ajpheart.00826.2009

Pulmonary expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) is critically important for normal functional maturation of alveolar macrophages. We found previously that lung GMCSF is dramatically suppressed in mice exposed to hyperoxia. Alveolar epithelial cells (AEC) are a major source of GM-CSF in the peripheral lung, and in vivo hyperoxia resulted in greatly reduced expression of GM-CSF protein by AEC ex vivo. We now explore the mechanisms responsible for this effect, using primary cultures of murine AEC exposed to hyperoxia in vitro. Exposure of AEC to 80% oxygen/5% C[O.sub.2] for 48 h did not induce overt toxicity, but resulted in significantly decreased GM-CSF protein and mRNA expression compared with cells in normoxia. Similar effects were seen when AEC were stressed with serum deprivation, an alternative inducer of oxidative stress. The effects in AEC were opposite those in a murine lung epithelial cell line (MLE-12 cells), in which hyperoxia induced GM-CSF expression. Both hyperoxia and serum deprivation resulted in increased intracellular reactive oxygen species (ROS) in AEC. Hyperoxia and serum deprivation induced significantly accelerated turnover of GM-CSF mRNA. Treatment of AEC with catalase during oxidative stress preserved GM-CSF protein and mRNA and was associated with stabilization of GM-CSF mRNA. We conclude that hyperoxia-induced suppression of AEC GM-CSF expression is a function of ROS-induced destabilization of GM-CSF mRNA. We speculate that AEC oxidative stress results in significantly impaired pulmonary innate immune defense due to effects on local GM-CSF expression in the lung. lung; innate immunity; growth factors; oxidative stress doi:10.1152/ajplung.00161.2009.

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/[cm.sup.2] increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from [10.sup.-9] to [10.sup.-6] M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by [10.sup.-6]M ATP was significantly reduced by [10.sup.-5] M suramin. Suramin ([10.sup.-5] M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, [10.sup.-4] M apamin, [10.sup.-9] M iberiotoxin, [10.sup.-5] M 2-aminoethoxydephenyl borate, or [10.sup.-5]M xestospongin C, but not [10.sup.-5] M glybenclamide or [10.sup.-5] M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small--([SK.sub.Ca]) and big-conductance ([BK.sub.Ca]) [Ca.sup.2+]-activated [K.sup.+] channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphatemediated release of intracellular [Ca.sup.2+] ions and the activation of [Ca.sup.2+]-activated [K.sup.+] channels in LEC. lymph flow rate; [Ca.sup.2+]-activated [K.sup.+] channel; mRNA; 2-aminoethoxydephenyl borate; xestospongin C; apamin; iberiotoxin; pinacidil doi:10.1152/ajpcell.00249.2009

In sickle cell disease (SCD), the events originating from hemoglobin S polymerization and intravascular sickling lead to reperfusion injury, hemolysis, decreased nitric oxide (NO) bioavailability, and oxidative stress. Oxidative stress is implicated as a contributing factor to multiple organ damage in SCD. We hypothesize that inhibition of sickling by genetic manipulation to enhance antisickling fetal hemoglobin (HbF) expression will have an ameliorating effect on oxidative stress by decreasing intravascular sickling and hemolysis and enhancing NO bioavailability. We tested this hypothesis in BERK (Berkeley) mice expressing exclusively human [alpha]- and [[beta].sup.S]-globins and varying levels of HbF, i.e., BERK ( sickle cell disease; hemolysis; inflammation; reperfusion; multiple-organ damage doi: 10.1152/ajpregu.00611.2009

We have previously reported gender differences in ventricular remodeling and development of heart failure using the aortocaval fistula model of chronic volume overload in rats. In contrast to males, female rats exhibited no adverse ventricular remodeling and less mortality in response to volume overload. This gender-specific cardio-protection was lost following ovariectomy and was partially restored using estrogen replacement. However, it is not known if estrogen treatment would be as effective in males. The purpose of this study was to evaluate the structural and functional effects of estrogen in male rats subjected to chronic volume overload. Four groups of male rats were studied at 3 days and 8 wk postsurgery as follows: fistula and sham-operated controls, with and without estrogen treatment. Biochemical and histological studies were performed at 3 days postsurgery, with chronic structural and functional effects studied at 8 wk. Measurement of systolic and diastolic pressure-volume relationships was obtained using a blood-perfused isolated heart preparation. Both fistula groups developed significant ventricular hypertrophy after 8 wk of volume overload. Untreated rats with fistula exhibited extensive ventricular dilatation, which was coupled with a loss of systolic function. Estrogen attenuated left ventricular dilatation and maintained function in treated rats. Estrogen treatment was also associated with a reduction in oxidative stress and circulating endothelin-I levels, as well as prevention of matrix metalloproteinase-2 and -9 activation and breakdown of ventricular collagen in the early stage of remodeling. These data demonstrate that estrogen attenuates ventricular remodeling and disease progression in male rats subjected to chronic volume overload. cardiac function; heart failure; hypertrophy; aortocaval fistula; hormone; gender; contractility doi: 10.1152/ajpheart.00336.2009

Intracellular [Ca.sup.2+] overloading, coupled to induction of oxidative stress, is present at 4-wk aldosterone/salt treatment (ALDOST). This prooxidant reaction in cardiac myocytes and mitochondria accounts for necrotic cell death and subsequent myocardial scarring. It is intrinsically linked to increased intracellular zinc concentration ([[[Zn.sup.2+]].sub.i]) serving as an antioxidant. Herein, we addressed the temporal responses in coupled [Ca.sup.2+] and [Zn.sup.2+] dyshomeostasis, reflecting the prooxidant-antioxidant equilibrium, by examining preclinical (week 1) and pathological (week 4) stages of ALDOST to determine whether endogenous antioxidant defenses would be ultimately overwhelmed to account for this delay in cardiac remodeling. We compared responses in cardiomyocyte free [[[Ca.sup.2+]].sub.i] and [[[Zn.sup.2+]].sub.i] and mitochondrial total [[Ca.sup.2+]].sub.m] and [[[Zn.sup.2+]].sub.m], together with biomarkers of oxidative stress and antioxidant defenses, during 1- and 4-wk ALDOST. At week 1 and compared with controls, we found: 1) elevations in [[[Ca.sup.2+]].sub.i] and [[[Ca.sup.2+]].sub.m] were coupled with [[[Zn.sup.2+]].sub.i] and [[[Zn.sup.2+]].sub.m]; 2) increased mitochondrial [H.sub.2][O.sub.2] production, cardiomyocyte xanthine oxidase activity, and cardiac and mitochondrial 8-isoprostane levels, counterbalanced by increased activity of antioxidant proteins, enzymes, and the nonenzymatic antioxidants that can be considered as cumulative antioxidant capacity; some of these enzymes and proteins (e.g., metallothionein-1, Cu/Zn-superoxide, glutathione synthase) are regulated by metal-responsive transcription factor-1, and 3) although these augmented antioxidant defenses were sustained at week 4, they fell short in combating the persistent intracellular [Ca.sup.2+] overloading and marked rise in cardiac tissue 8-isoprostane and mitochondrial transition pore opening. Thus a coupled [Ca.sup.2+] and [Zn.sup.2+] dyshomeostasis occurs early during ALDOST in cardiac myocytes and mitochondria that regulate redox equilibrium until week 4 when ongoing intracellular [Ca.sup.2+] overloading and prooxidants overwhelm antioxidant defenses. aldosterone; oxidative stress; antioxidants doi: 10.1152/ajpheart.00593.2009

Impaired estrogen action is associated with the metabolic syndrome in humans. We sought to determine whether impaired estrogen action in female C57B16 mice, produced by whole body Esrl ablation, could recapitulate aspects of this syndrome, including inflammation, insulin resistance, and obesity. Indeed, we found that global knockout (KO) of the estrogen receptor (ER)[alpha] leads to reduced oxygen uptake and caloric expenditure compared with wild-type (WT) mice. In addition, fasting insulin, leptin, and PAL1 levels were markedly elevated, whereas adiponectin levels were reduced in normal chow-fed KO. Furthermore, ER[alpha]-KO mice exhibited impaired glucose tolerance and marked skeletal muscle insulin resistance that was accompanied by the accumulation of bioactive lipid intermediates, inflammation, and diminished PPAR[alpha], PPAR[delta], and UCP2 transcript levels. Although the relative glucose intolerance and insulin resistance phenotype in KO mice became more severe with high-fat feeding, WT mice were refractory to these dietary-induced effects, and this protection coincided with a marked increase in circulating adiponectin and heat shock protein 72 levels in muscle, liver, and fat. These data indicate that ER[alpha] is critical for the maintenance of whole body insulin action and protection against tissue inflammation during both normal chow and high-fat feeding. estrogen receptor-m; estrogen action; fatty acid metabolism; insulin action doi: 10.1152/ajpendo.00504.2009

Oxidative stress is one of the causative factors in progression and etiology of age-related cataract. Peroxiredoxin 6 (Prdx6), a savior for cells from internal or external environmental stresses, plays a role in cellular signaling by detoxifying reactive oxygen species (ROS) and thereby controlling gene regulation. Using targeted inactivation of the Prdx6 gene, we show that Prdx6-deficient lens epithelial cells (LECs) are more vulnerable to UV-triggered cell death, a major cause of skin disorders including cataractogenesis, and these cells display abnormal protein profiles. PRDX6-depleted LECs showed phenotypic changes and formed lentoid body, a characteristic of terminal cell differentiation and epithelial-mesenchymal transition. [Prdx6.sup.-/-] LECs exposed to UV-B showed higher ROS expression and were prone to apoptosis compared with wild-type LECs, underscoring a protective role for Prdx6. Comparative proteomic analysis using fluorescence-based difference gel electrophoresis along with mass spectrometry and database searching revealed a total of 13 proteins that were differentially expressed in [Prdx6.sup.-/-] cells. Six proteins were upregulated, whereas expression of seven proteins was decreased compared with [Prdx6.sup.+/+] LECs. Among the cytoskeleton-associated proteins that were highly expressed in Prdx6-deficient LECs was tropomyosin (Tm)2[beta]. Protein blot and real-time PCR validated dramatic increase of Tm2[beta] and Tm1[alpha] expression in these cells. Importantly, [Prdx6.sup.+/+] LECs showed a similar pattern of Tm2[beta] protein expression after transforming growth factor (TGF)-[beta] or [H.sub.2][O.sub.2] treatment. An extrinsic supply of PRDX6 could restore Tm2[beta] expression, demonstrating that PRDX6 may attenuate adverse signaling in cells and thereby maintain cellular homeostasis. Exploring redox-proteomics ([Prdx6.sup.-/-]) and characterization and identification of abnormally expressed proteins and their attenuation by PRDX6 delivery should provide a basis for development of novel therapeutic interventions to postpone ROS-mediated abnormal signaling deleterious to cells or tissues. peroxiredoxin; oxidative stress; proteomics; tropomyosin doi: 10.1152/ajpcell.00336.2009

Am J Physiol Renal Physiol 298: F125-F132, 2010. First published October 28, 2009; doi: 10.1152/ajprenal.00248.2009.--Glomerular endothelial cells (GEC) are strategically situated within the capillary loop and adjacent to the glomerular mesangium. GEC serve as targets of metabolic, biochemical, and hemodynamic signals that regulate the glomerular microcirculation. Unequivocally, hyperglycemia, hypertension, and the local renin-angiotensin system partake in the initiation and progression of diabetic nephropathy (DN). Whether free fatty acids (FFA) and reactive oxygen species (ROS) that have been associated with the endothelial dysfunction of diabetic macrovascular disease also contribute to DN is not known. Since endothelial cells from different organs and from different species may display different phenotypes, we employed human GEC to investigate the effect of high glucose (22.5 mmol/l), FFA (800 [micro]mol/l), and angiotensin II (ANG II; [10.sup.-7] mol/l) on the genesis of ROS and their effects on endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX-2), and the synthesis of prostaglandins (PGs). We demonstrated that high glucose but not high FFA increased the expression of a dysfunctional eNOS as well as increased ROS from NADPH oxidase (100%) and likely from uncoupled eNOS. ANG II also induced ROS from NADPH oxidase. High glucose and ANG II upregulated (100%) COX-2 via ROS and significantly increased the synthesis of prostacyclin ([PGI.sub.2]) by 300%. In contrast, FFA did not upregulate COX-2 but increased [PGI.sub.2] (500%). These novel studies are the first in human GEC that characterize the differential role of FFA, hyperglycemia, and ANG II on the genesis of ROS, COX-2, and PGs and their interplay in the early stages of hyperglcyemia. diabetes; prostaglandins; reactive oxygen species; nitric oxide

Am J Physiol Renal Physiol 298: F86-F94, 2010. First published November 11, 2009; doi: 10.1152/ajprenal.00351.2009.--Obesity and hypertension are risk factors for the development of chronic kidney disease. The mechanisms by which elevated blood pressure and fatty acids lead to the development of renal injury are incompletely understood. Here, we investigated the contributions of cholesterol and oxidative stress to renal endothelial dysfunction and glomerular injury in a model of obesity and hypertension. Male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were fed a normal diet, a high-fat diet, a high-fat diet with tempol, or a high-fat diet with simvastatin for up to 10 wk. Blood pressure was not altered by a high-fat diet or treatments. After 3 wk, renal afferent dilatory responses to acetylcholine were impaired in WKY rats and SHR fed a high-fat diet. Tempol treatment prevented this vascular dysfunction in both strains; however, simvastatin treatment demonstrated greater beneficial effects in the SHR. Albuminuria was observed in the SHR and was exacerbated by a high-fat diet. Tempol and simvastatin treatment significantly ameliorated albuminuria in the SHR fed a high-fat diet. Ten weeks on a high-fat resulted in an increase in urinary 8-isoprostane in WKY rats and SHR, and tempol and simvastatin treatment prevented this increase, indicating a reduction in renal oxidative stress. Monocyte chemoattractant protein-1 (MCP-1) excretion was significantly elevated by a high-fat diet in both strains, and tempol prevented this increase. Interestingly, simvastatin treatment had no effect on MCP-1 levels. These data indicate that tempol and simvastatin treatment via a reduction in oxidative stress improve renal endothelial function and decrease glomerular injury in a model of obesity and hypertension. kidney; oxidative stress; metabolic syndrome; inflammation

Am J Physiol Heart Circ Physiol 298: H194-H201, 2010. First published November 6, 2009; doi: 10.1152/ajpheart.00767.2009.--The intracellular redox state is stringently maintained by thiol-based antioxidants to establish a balance for the physiological and pathophysiological roles of reactive oxygen species. The relative contributions of the thioredoxin (Trx) and glutathione/glutaredoxin systems to intracellular redox balance are incompletely understood, as are the consequences of altered thiol metabolism on endothelial nitric oxide (NO) synthase (eNOS) and NO-dependent pathways in the endothelium. We designed duplex small interfering RNA (siRNA) constructs to specifically 'knock down' the expression of three key thiol-metabolizing enzymes in cultured aortic endothelial cells. Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR 1), or mitochondrial Trx reductase (TrxR2) significantly decreased the intracellular reduced glutathione-to-oxidized glutathione ratio, siRNA-mediated knockdown of either GR, TrxR1, or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor) and also blocked eNOS enzyme activity (using the [[sup.3]H]arginine/[[sup.3]H]citrulline assay). Pretreatment of endothelial cells with N,N'-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production, siRNA-mediated TrxR2 knockdown led to a marked increase in hydrogen peroxide ([H.sub.2][O.sub.2]) production in endothelial cells. In contrast, knockdown of GR or TrxR1 only slightly increased [H.sub.2][O.sub.2] production. Supplementation of endothelial cells with tetrahydrobiopterin prevented the increase in [H.sub.2][O.sub.2] generation seen with siRNA-mediated knockdown of GR. These studies show that the differential regulation of thiol-metabolizing proteins leads to critical changes in oxidative and nitrosative stress pathways. Greater understanding of the differential regulation of thiol-metabolizing proteins may lead to the development of new pharmacological targets for diseases associated with oxidative stress in the vascular wall. oxidative stress; endothelial function; thioredoxin; glutathione; nitric oxide; tetrahydrobiopterin

Wu J, Mei C, Vlassara H, Striker GE, Zheng F. Oxidative stress-induced JNK activation contributes to proinflammatory phenotype of aging diabetic mesangial cells. Am J Physiol Renal Physiol 297: F1622-F 1631, 2009. First published September 23, 2009; doi: 10.1152/ajprenal.00078.2009.--Chronic inflammation and increased oxidative stress (OS) play an important role in diabetic nephropathy progression. Herein, we show that mesangial cells from streptozotocin-induced aging diabetic mice, a model of progressive diabetic nephropathy, exhibited increased OS and a proinflammatory phenotype characterized by elevated chemokines and ICAM-1 expression. This phenotypic change was consistent with the extensive inflammatory lesions present in aging diabetic kidneys and was not found in mesangial cells from old and young controls or young diabetic mice. Activation of the c-Jun N[H.sub.2]-terminal kinase (JNK) pathway was a likely contributor to the proinflammatory phenotype of aging diabetic mesangial cells since 1) phosphorylated JNK levels and JNK kinase activity were increased in these cells, 2) suppression of JNK significantly decreased monocyte chemoattractant protein-1 (MCP-1) production in these cells, and 3) activation of JNK in normal mesangial cells induced inflammation. Elevated OS in aging diabetic mesangial cells may be a cause of JNK activation and inflammation, because antioxidant treatment decreased JNK phosphorylation and MCP-1 production. Additionally, decreased expression of mitogen-activated protein kinase phosphatase 5 (MKP5) may also contribute to increased JNK and inflammation in aging diabetic mesangial cells since overexpression of MKP5 in these cells normalized phosphorylated JNK levels and reversed the proinflammatory phenotype. Moreover, knocking down of MKP5 expression in old control mesangial cells resulted in JNK activation and MCP-1 production, a phenotype seen in aging diabetic mesangial cells. Interestingly, MKP5 phosphatase activity was diminished by free radicals in vitro. Thus, OS may induce inflammation in mesangial cells by activating JNK through either a direct activation of JNK or indirectly by suppression of MKP5 activity. Proinflammatory phenotype of mesangial cells may contribute to chronic inflammatory lesions and disease progression of aging diabetic mice. diabetic nephropathy; mice doi: 10.1152/ajprenal.00078.2009

Raab EL, Vuguin PM, Stoffers DA, Simmons RA. Neonatal exendin-4 treatment reduces oxidative stress and prevents hepatic insulin resistance in intrauterine growth-retarded rats. Am J Physiol Regul Integr Comp Physiol 297: R1785-R1794, 2009. First published October 21, 2009; doi: 10.1152/ajpregu.00519.2009.--Intrauterine growth retardation (IUGR) has been linked to the development of Type 2 diabetes in adulthood. We have developed an IUGR model in the rat whereby the animals develop diabetes later in life. Previous studies demonstrate that administration of the long-acting glucagons-like- peptide -1 agonist, Exendin-4, during the neonatal period prevents the development of diabetes in IUGR rats. IUGR animals exhibit hepatic insulin resistance early in life (prior to the onset of hyperglycemia), characterized by blunted suppression of hepatic glucose production (HGP) in response to insulin. Basal HGP is also significantly higher in IUGR rats. We hypothesized that neonatal administration of Exendin-4 would prevent the development of hepatic insulin resistance. IUGR and control rats were given Exendin-4 on days 1-6 of life. Hyperinsulinemic-euglycemic clamp studies showed that Ex-4 significantly reduced basal HGP by 20% and normalized insulin suppression of HGP in IUGR rats. While Ex-4 decreased body weight and fat content in both Control and IUGR animals, these differences were only statistically significant in Controls. Exendin-4 prevented development of oxidative stress in liver and reversed insulin-signaling defects in vivo, thereby preventing the development of hepatic insulin resistance. Defects in glucose disposal and suppression of hepatic glucose production in response to insulin were reversed. Similar results were obtained in isolated Ex-4-treated neonatal hepatocytes. These results indicate that exposure to Exendin-4 in the newborn period reverses the adverse consequences of fetal programming and prevents the development of hepatic insulin resistance. intrauterine growth retardation; diabetes; liver; insulin resistance doi: 10.1152/ajpregu.00519.2009

Kato K, Yamanouchi D, Esbona K, Kamiya K, Zhang F, Kent KC, Liu B. Caspase-mediated protein kinase C-5 cleavage is necessary for apoptosis of vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 297: H2253-H2261, 2009. First published October 16, 2009; doi: 10.1152/ajpheart.00274.2009.--Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C-8 (PKC-[delta]) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC-[delta] proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC-[delta] occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-[delta], we were able to prevent the cleavage of PKC-[delta], as well as apoptosis. Inhibition of PKC-[delta] with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (AD-Pribose) polymerase, cleavage of PKC-[delta], and DNA fragmentation, confirming the previously reported role of PKC-[delta] in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-[delta], and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC-[delta] cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC-[delta] acts both upstream and downstream of caspase-3. oxidative stress; remodeling; phosphorylation; necrosis doi: 10.1152/ajpheart.00274.2009.

Lustgarten MS, Jang YC, Liu Y, Muller FL, Qi W, Steinhelper M, Brooks SV, Larkin L, Shimizu T, Shirasawa T, McManus LM, Bhattacharya A, Richardson A, Van Remmen H. Conditional knockout of Mn-SOD targeted to type IIB skeletal muscle fibers increases oxidative stress and is sufficient to alter aerobic exercise capacity. Am J Physiol Cell Physiol 297: C 1520-C1532, 2009. First published September 23, 2009; doi: 10.1152/ajpcell.00372.2009.--In vitro studies of isolated skeletal muscle have shown that oxidative stress is limiting with respect to contractile function. Mitochondria are a potential source of muscle function-limiting oxidants. To test the hypothesis that skeletal muscle-specific mitochondrial oxidative stress is sufficient to limit muscle function, we bred mice expressing Cre recombinase driven by the promoter for the inhibitory subunit of troponin (TnIFastiCre) with mice containing a floxed Sod2 (Sod[2.sup.fl/fl]) allele. Mn-SOD activity was reduced by 82% in glycolytic (mainly type II) muscle fiber homogenates from young TnIFastCreSod[2.sup.fl/fl] mice. Furthermore, Mn-SOD content was reduced by 70% only in type IIB muscle fibers. Aconitase activity was decreased by 56%, which suggests an increase in mitochondrial matrix superoxide. Mitochondrial superoxide release was elevated more than twofold by mitochondria isolated from glycolytic skeletal muscle in TnIFastCreSod[2.sup.fl/fl] mice. In contrast, the rate of mitochondrial [H.sub.2][O.sub.2] production was reduced by 33%, and only during respiration with complex II substrate. [F.sub.2]-isoprostanes were increased by 36% in tibialis anterior muscles isolated from TnIFastCreSod[2.sup.fl/fl] mice. Elevated glycolytic muscle-specific mitochondrial oxidative stress and damage in TnIFastCreSod[2.sup.fl/fl] mice were associated with a decreased ability of the extensor digitorum longus and gastrocnemius muscles to produce contractile force as a function of time, whereas force production by the soleus muscle was unaffected. TnIFastCreSod.[2.sup.fl/fl] mice ran 55% less distance on a treadmill than wild-type mice. Collectively, these data suggest that elevated mitochondrial oxidative stress and damage in glycolytic muscle fibers are sufficient to reduce contractile muscle function and aerobic exercise capacity. muscle function; contractile function; oxidative damage; free radical doi: 10.1152/ajpcell.00372.2009

Sustained activation of the c-Jun N[H.sub.2]-terminal kinase (JNK) signaling pathway mediates the development and progression of experimental diet-induced nonalcoholic fatty liver disease (NAFLD). Delineating the mechanism of JNK overactivation in the setting of a fatty liver is therefore essential to understanding the pathophysiology of NAFLD. Both human and experimental NAFLD are associated with oxidative stress and resultant lipid peroxidation, which have been proposed to mediate the progression of this disease from simple steatosis to steatohepatitis. The ability of oxidants and the lipid peroxidation product 4-hydroxynonenal (HNE) to activate JNK signaling suggested that these two factors may act synergistically to trigger JNK overactivation. The effect of HNE on hepatocyte injury and JNK activation was therefore examined in cells under chronic oxidant stress from overexpression of the prooxidant enzyme cytochrome P450 2E1 (CYP2E1), which occurs in NAFLD. CYP2E1-generated oxidant stress sensitized a rat hepatocyte cell line to death from normally nontoxic concentrations of HNE. CYP2E1-overexpressing cells underwent a more profound depletion of glutathione (GSH) in response to HNE secondary to decreased [gamma]-glutamylcysteine synthetase activity. GSH depletion led to overactivation of JNK/c-Jun signaling at the level of mitogenactivated protein kinase kinase 4 that induced cell death. Oxidant stress and the lipid peroxidation product HNE cause synergistic overactivation of the JNK/c-Jun signaling pathway in hepatocytes, demonstrating that HNE may not be just a passive biomarker of hepatic oxidant stress but rather an active mediator of hepatocellular injury through effects on JNK signaling. nonalcoholic fatty liver disease; glutathione; lipid peroxidation doi: 10.1152/ajpgi.00151.2009.

We have previously reported that age-associated oxidative stress via protein kinase C (PKC) increases D1 receptor (D1R) phosphorylation and causes D1R-G protein uncoupling in renal proximal tubules (RPTs) of old Fischer 344 rats. This results in reduced ability of D1R agonist SKF-38393 to inhibit [Na.sup.+]-[K.sup.+]-ATPase in RPTs of old rats. Here, we studied the effect of treadmill exercise on markers of oxidative stress, PKC, D1R phosphorylation, D1R-G protein coupling, and [Na.sup.+]-[K.sup.+]-ATPase activity in RPTs of adult and old rats. We found increased levels of malondialdehyde, a marker of oxidative stress, in RPTs of old rats, which decreased during exercise. Nuclear levels of nuclear erythroid-related factor (Nrf)-2 and nuclear factor (NF)-[kappa]B in RPTs, transcription factors involved in antioxidant enzyme gene transcription, increased in exercised old rats. This was accompanied by an increase in the activity and expression of antioxidant enzymes, superoxide dismutase and heme oxygenase-1. Age-related decrease in the levels of D1R mRNAs and proteins was attenuated during exercise. Furthermore, exercise in old rats decreased PKC activity and D1R phosphorylation and increased SKF-38393-mediated [[sup.35]S]guanosine 5'-O-(3-thiotriphosphate) binding (an index of D1R-G protein coupling). SKF-38393 also caused inhibition of [Na.sub.+]-[K.sup.+]-ATPase in these animals. Also, exercise caused a decrease in proteinuria and increase in phosphaturia in old rats. These results suggest beneficial effects of exercise in terms of increasing antioxidant defenses, decreasing oxidative stress, and improving kidney function in general and D1R function in particular in aging. Both Nrf-2 and NF-[kappa]B seem to play key role in this phenomenon. exercise; aging; [D.sub.1] receptor; nuclear erythroid-related factor-2; nuclear factor-[kappa]B doi: 10.1152/ajprenal.00397.2009.

Induction of heme oxygenase-1 (HO-1) in the renal medulla increases carbon monoxide and bilirubin production and decreases ANG II-mediated superoxide production. The goal of this study was to determine the importance of increases in bilirubin to the antioxidant effects of HO-1 induction in cultured mouse thick ascending loop of Henle (TALH) and inner medullary collecting duct (IMCD3) cells. Bilirubin levels were decreased by using small interfering RNAs (siRNAs) targeted to biliverdin reductase (BVR), which is the cellular enzyme responsible for the conversion of biliverdin to bilirubin. Treatment of cultured TALH or IMCD-3 cells with BVR siRNA (50 or 100 nM) resulted in an 80% decrease in the level of BVR protein and decreased cellular bilirubin levels from 46 [+ or -] 5 to 23 [+ or -] 4 nM (n = 4). We then determined the effects of inhibition of BVR on ANG II-mediated superoxide production. Superoxide production induced by ANG II ([10.sup.-9] M) significantly increased in both TALH and IMCD-3 cells. Treatment of TALH cells with BVR siRNA resulted in a significant increase in ouabain-sensitive rubidium uptake from 95 [+ or -] 6 to 122 [+ or -] 5% control (n = 4, P < 0.05). Lastly, inhibition of BVR with siRNA did not prevent the decrease in superoxide levels observed in cells pretreated with the HO-1 inducer, hemin. We conclude that decreased levels of cellular bilirubin increase ANG II-mediated superoxide production and sodium transport; however, increases in bilirubin are not necessary for HO-1 induction to attenuate ANG II-mediated superoxide production. bilirubin; renal medulla; heme oxygenase; oxidative stress; small interfering RNA; sodium transport; rubidium uptake doi: 10.1152/ajpregu.90933.2008.

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT 1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted [H.sub.2][O.sub.2]-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular [H.sub.2][O.sub.2] levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. vasoprotection; histone deacetylase; sirtuin 1 doi: 10.1152/ajpheart.00375.2009.

We sought to examine the potential role of oxidative stress on skeletal muscle function with advancing age. Nuclear magnetic resonance (NMR) was employed to simultaneously assess muscle perfusion (arterial spin labeling) and energetics ([sup.31]P NMR spectroscopy) in the lower leg of young (26 [+ or -] 5 yr, n = 6) and older (70 [+ or -] 5 yr, n = 6) healthy volunteers following the consumption of either placebo (PL) or an oral antioxidant (AO) cocktail (vitamins C and E and [alpha]-lipoic acid), previously documented to decrease plasma free radical concentration. NMR measurements were made during and after 5 min of moderate intensity ([approximately equal to] 5 W) plantar flexion exercise. AO administration significantly improved end-exercise perfusion (AO, 50 [+ or -] 5, and PL, 43 [+ or -] 4 ml x 100 [g.sup.-1] x [min.sup.-1]) and postexercise perfusion area under the curve (AO, 1,286 [+ or -] 236, and PL, 866 [+ or -] 144 ml/100 g) in older subjects, whereas AO administration did not alter hemodynamics in the young group. Concomitantly, muscle oxidative capacity (time constant of phosphocreatine recovery, [tau]) was improved following AO in the older (AO, 43 [+ or -] 1, and PL, 51 [+ or] 7 s) but not the young (AO, 54 [+ or -] 5, and PL, 48 [+ or -] 7 s) group. These findings support the concept that oxidative stress may be partially responsible for the age-related decline in skeletal muscle perfusion during physical activity and reveal a muscle metabolic reserve capacity in the elderly that is accessible under conditions of improved perfusion. leg blood flow; oxidative capacity; exercise; nuclear magnetic resonance doi: 10.1152/ajpheart.00709.2009.

Angiotensin type 1 receptors ([AT.sub.1]Rs) play a critical role in a variety of physiological functions and pathophysiological states. They have been strongly implicated in the modulation of sympathetic outflow in the brain. An understanding of the mechanisms by which [AT.sub.1]Rs are regulated in a variety of disease states that are characterized by sympathoexcitation is pivotal in development of new strategies for the treatment of these disorders. This review concentrates on several aspects of [AT.sub.1]R regulation in the setting of chronic heart failure (CHF). There is now good evidence that [AT.sub.1]R expression in neurons is mediated by activation of the transcription factor activator protein 1 (AP-1). This transcription factor and its component proteins are upregulated in the rostral ventrolateral medulla of animals with CHF. Because the increase in [AT.sub.1]R expression and transcription factor activation can be blocked by the [AT.sub.1]R antagonist losartan, a positive feedback mechanism of [AT.sub.1]R expression in CHF is suggested. Oxidative stress has also been implicated in the regulation of receptor expression. Recent data suggest that the newly discovered catabolic enzyme angiotensin-converting enzyme 2 (ACE2) may play a role in the modulation of [AT.sub.1]R expression by altering the balance between the octapeptide ANG II and ANG(1-7). Finally, exercise training reduces both central oxidative stress and [AT.sub.1]R expression in animals with CHF. These data strongly suggest that multiple central and peripheral influences dynamically alter [AT.sub.1]R expression in CHF. sympathetic nerve activity; neuronal signaling; oxidative stress doi: 10.1152/ajpheart.00073.2009.

A number of type 1 receptor cytokine family members protect the heart from acute and chronic oxidative stress. This protection involves activation of two intracellular signaling cascades: the reperfusion injury salvage kinase (RISK) pathway, which entails activation of phosphatidylinositol 3-kinase (PI3-kinase) and ERK1/2, and JAK-STAT signaling, which involves activation of transcription factor signal transducer and activator of transcription 3 (STAT3). Obligatory for activation of both RISK and STAT3 by nearly all of these cytokines are the kinases JAK1 and JAK2. Yet surprisingly little is known about how JAK1 and JAK2 are regulated in the heart or how they couple to PI3-kinase activation. Although the JAKs are linked to antioxidative stress programs in the heart, we recently reported that these kinases are inhibited by oxidative stress in cardiac myocytes. In contrast, others have reported that cardiac JAK2 is activated by acute oxidative stress by an undefined process. Here we summarize recent insights into the regulation of JAK1 and JAK2. Besides oxidative stress, inhibitory regulation involves phosphorylation, nitration, and intramolecular restraints. Stimulatory regulation involves phosphorylation and adaptor proteins. The net effect of stress on JAK activity in the heart likely represents the sum of both inhibitory and stimulatory processes, along with their dynamic interaction. Thus the regulation of JAKs in the heart, once touted as the paragon of simplicity, is proving rather complicated indeed, requiring a second look. It is our contention that a better understanding of the regulation of this kinase family that is implicated in cardiac protection could translate into effective therapeutic strategies for preventing myocardial damage or repairing the injured heart. Janus kinase; oxidative stress; redox; cardiac remodeling; cytokine; cardiac myocyte doi: 10.1152/ajpheart.00032.2009.

Significant reduction of renal mass initiates a series of hemodynamic and nonhemodynamic events which lead to proteinuria, glomerulosclerosis, tubulointerstitial injury, and end-stage renal failure. Lipid mediators derived from fatty acids participate in regulation of renal hemodynamic and nonhemodynamic processes that influence progression of renal disease. Composition of cellular fatty acids and hence related signaling responses are influenced by their dietary contents. Consumption of omega-3 fatty acids (O-3FA) has proven effective in mitigating atherosclerosis. We tested the hypothesis that O-3FA supplementation may retard progression and attenuate upregulation of pathways involved in oxidative stress, inflammation, and fibrosis in rats with renal mass reduction. Sprague-Dawley rats were subjected to 5/6 nephrectomy [chronic renal failure (CRF)] and randomly assigned to the untreated and O-3FA-treated (0.3 g x [kg.sup.-1] x [day.sup.-1] by gastric gavage for 12 wk) groups. Sham-operated rats served as controls. The untreated CRF rats exhibited proteinuria, hypertension, azotemia, upregulations of renal tissue NAD(P)H oxidase, MCP-1, COX-2, PAI-1, TGF-[beta], Smad2, [alpha]-smooth muscle actin, fihronectin, and hepatocyte growth factor, activation of ERK1/2 and NF-[kappa]B, downregulation of Smad7, intense mononuclear leukocyte infiltration, tubulointerstifial fibrosis, and glomerulosclerosis. O-3FA supplementation significantly lowered COX-2, NAD(P)H oxidase (NOX-4, [gp91.sup.phox], [p47.sup.phox], [p22.sup.phox]), PAI-1, TGF-[beta], connective tissue growth factor, [alpha]-smooth muscle actin, fibronectin, Smad2, and MCP-1, raised Smad7, and attenuated ERK1/2 and NF-[kappa]B activation, tubulointerstitial fibrosis, and inflammation. Thus, long-term O-3FA supplementation can reduce or reverse upregulation of prooxidant, proinflanunatory, and profibrotic pathways and attenuate tubulointerstitial fibrosis in the remnant kidney. chronic kidney disease; NAD(P)H oxidase; TGF-[beta]; Smad; epithelialmesenchymal transition; MAP Kinase doi: 10.1152/ajprenal.00217.2009

This study investigated the extent to which the increase in torpor expression in the grey mouse lemur, due to graded food restriction, is modulated by a trade-off between a whole body sparing of polyunsaturated dietary fatty acids and the related oxidative stress generated during daily torpor. We measured changes in torpor frequency, total energy expenditure (TEE), linoleate (polyunsaturated fatty acid) and palmitate (saturated fatty acid) oxidation, hexanoyl-lysine (HEL; the product of linoleate peroxidation), and 8-hydroxydeoxyguanosine (8OHdG; a marker of DNA damage). Animals under summer-acclimated long days (LD) or winter-acclimated short days (SD) were exposed to a 40% (LD40 and SD40) and 80% (LD80 and SD80) 35-day calorie restriction (CR). During CR, all groups reduced their body mass, but LD80 animals reached survival-threatened levels at day 22 and were then excluded from the CR trial. Only SD mouse lemurs increased their torpor frequency with CR and displayed a decrease in their TEE adjusted for fat-free mass. After CR, SD40 mouse lemurs shifted the dietary fatty acid oxidation toward palmitate and spared linoleate. Such a shift was not observed in LD animals and during severe CR, during which oxidation of both dietary fatty acids was increased. Concomitantly, HEL increased in both LD40 and SD80 groups, whereas DNA damage was only seen in SD80 food-restricted animals. HEL correlated positively with linoleate oxidation confirming in vivo the substrate/product relationship demonstrated in vitro, and negatively with TEE adjusted for fat-free mass, suggesting higher oxidative stress associated with increased torpor expression. This suggests a seasonal-dependant, cost-benefit trade-off between maximizing torpor propensity and minimizing oxidative stress that is associated with a shift toward sparing of dietary polyunsaturated fatty acids that is dependent upon the expression of a winter phenotype. polyunsaturated fatty acid; energy savings; Microcebus murinus doi: 10.1152/ajpregu.00214.2009.

Low-voltage-activated calcium channels are reexpressed in ventricular myocytes in pathological conditions associated with hypoxic episodes, but a direct relation between oxidative stress and T-type channel function and regulation in cardiomyocytes has not been established. We aimed to investigate low-voltage-activated channel regulation under oxidative stress in neonatal rat ventricular myocytes. RT-PCR measurements of voltage-gated [Ca.sup.2+] ([Ca.sub.v])3.1 and [Ca.sub.v]3.2 mRNA levels in oxidative stress were compared with whole cell patch-clamp recordings of T-type calcium current. The results indicate that hypoxia reduces T-type current density at -30 mV (the hallmark of this channel) based on the shift of the voltage dependence of activation to more depolarized values and downregulation of [Ca.sub.v]3.1 at the mRNA level. Upon reoxygenation, both [Ca.sub.v]3.1 mRNA levels and the voltage dependence of total T-type current are restored, although differently for activation and inactivation. Using [Ni.sup.2+], we distinguished different effects of hypoxia/reoxygenation on the two current components. Long-term incubation in the presence of 100 [micro]M CO[Cl.sub.2] reproduced the effects of hypoxia on T-type current activation and inactivation, indicating that the chemically induced oxidative state is sufficient to alter T-type calcium current activity, and that hypoxia-inducible factor-1[alpha] is involved in [Ca.sub.v]3.1 downregulation. Our results demonstrate that [Ca.sub.v]3.1 and [Ca.sub.v]3.2 T-type calcium channels are differentially regulated by hypoxia/reoxygenation injury, and, therefore, they may serve different functions in the myocyte in response to hypoxic injury. low-voltage-activated calcium channel; oxidative stress: cardiomyocytes doi: 10.1152/ajpheart.00528.2009

We have recently reported that transgenic (mRen2)27 rats (Ren2 rats) exhibit pulmonary arterial hypertension (PAH), which is, in part, mediated by oxidative stress. Since 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exhibit beneficial vascular effects independent of cholesterol synthesis, we hypothesized that rosuvastatin (RSV) treatment ameliorates PAH and pulmonary vascular remodeling in Ren2 rats. in part, by reducing oxidative stress. Six-week-old male Reh2 and Sprague-Dawley rats received RSV (10 mg x [kg.sup.-1] x [day.sup.-1] ip) or vehicle for 3 wk. After treatment, right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. To evaluate treatment effects on pulmonary arteriole remodeling, morphometric analyses were performed to quantitate medial thickening and cell proliferation, whereas whole lung samples were used to quantitate the levels of 3-nitro-tyrosine, superoxide, stable nitric oxide (NO)metabolites [nitrates and nitrites N[O.sub.x], and expression of NO synthase isoforms. In the Ren2 rat, RVSP is normal at 5 wk of agc, PAH dexelops between 5 and 7 wk of age, and the elevated pressure is maintained with little variation through 13 wk. At 8 wk of age, left ventricular function and blood gases were normal in the Reh2 rat. Ren2 rats exhibited elevations in medial hypertrophy due to smooth muscle cell proliferation, 3-nitro-tyrosine, NO, NADPH oxidase activity, and endothelial NO synthase expression compared with Sprague-Dawley rats. RSV significantly blunted the increase in RVSP but did not reduce MAP in the Ren2 rat; additionally, RSV significantly attenuated the elevated parameters examined in the Ren2 rat. These data suggest that statins may be a clinically viable adjunct treatment of PAH through reducing peroxy-nitrite formation. oxidative stress; NADPH oxidase

We have previously shown that avian uncoupling protein (avUCP) is downregulated on exposure to acute heat stress, stimulating mitochondrial reactive oxygen species (ROS) production and oxidative damage. In this study, we investigated whether upregulation of avUCP could attenuate oxidative damage caused by acute heat stress. Broiler chickens (Gallus gallus) were fed either a control diet or an olive oil-supplemented diet (6.7%), which has been shown to increase the expression of UCP3 in mammals, for 8 days and then exposed either to heat stress (34[degree]C, 12 h) or kept at a thermoneutral temperature (25[degrees]C). Skeletal muscle mitochondrial ROS (measured as [H.sub.2][O.sub.2]) production, avUCP expression, oxidative damage, mitochondrial membrane potential, and oxygen consumption were studied. We confirmed that heat stress increased mitochondrial ROS production and malondialdehyde levels and decreased the amount of avUCP. As expected, feeding birds an olive oil-supplemented diet increased the expression of avUCP in skeletal muscle mitochondria and decreased ROS production and oxidative damage. Studies on mitochondrial function showed that heat stress increased membrane potential in state 4, which was reversed by feeding birds an olive oil-supplemented diet, although no differences in basal proton leak were observed between control and heat-stressed groups. These results show that under heat stress, mitochondrial ROS production and olive oil-induced reduction of ROS production may occur due to changes in respiratory chain activity as well as avUCP expression in skeletal muscle mitochondria. mitochondria; reactive oxygen species; membrane potential; uncoupling protein: proton leak

Insulin resistance or diabetes is associated with limited exercise capacity, which can be caused by the abnormal energy metabolism in skeletal muscle. Oxidative stress is involved in mitochondrial dysfunction in diabetes. We hypothesized that increased oxidative stress could cause mitochondrial dysfunction in skeletal muscle and make contribution to exercise intolerance in diabetes. C57/BL6J mice were fed on normal diet or high fat diet (HFD) for 8 wk to induce obesity with insulin resistance and diabetes. Treadmill tests with expired gas analysis were performed to determine the exercise capacity and whole body oxygen uptake ([Vo.sub.2). The work (vertical distance x body weight) to exhaustion was reduced in the HFD mice by 36%, accompanied by a 16% decrease of peak [Vo.sub.2]. Mitochondrial ADP-stimulated respiration, electron transport chain complex I and III activities, and mitochondrial content in skeletal muscle were decreased in the HFD mice. Furthermore, superoxide production and NAD(P)H oxidase activity in skeletal muscle were significantly increased in the HFD mice. Intriguingly, the treatment of HFD-fed mice with apocynin [10 mmol/1; an inhibitor of NAD(P)H oxidase activation] improved exercise intolerance and mitochondrial dysfunction in skeletal muscle without affecting glucose metabolism itself. The exercise capacity and mitochondrial function in skeletal muscle were impaired in type 2 diabetes, which might be due to enhanced oxidative stress. Therapies designed to regulate oxidative stress and maintain mitochondrial function could be beneficial to improve the exercise capacity in type 2 diabetes. exercise intolerance; insulin resistance; mitochondrial function; oxygen uptake; superoxide

The p38 mitogen-activated protein kinase (MAPK) is activated during heart diseases that might be associated with myocardial damage and cardiac remodeling process. Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. The purpose of this study was to investigate the role of p38[alpha] MAPK after experimental diabetes by using transgenic (TG) mice with cardiac-specific expression of a dominant-negative mutant form of p38[alpha] MAPK. The elevation of blood glucose was comparable between the nontransgenic (NTG) and TG mice. The expression of phospho-p38 MAPK and phosphoMAPK-activated protein kinase 2 levels were significantly suppressed in TG mice heart than in NTG mice after diabetes induction. Left ventricular (LV) dimension in systole was smaller, and the percent fractional shortening was higher in diabetic TG mice compared with diabetic NTG mice. In addition, diabetic TG mice had reduced cardiac myocyte diameter, content of cardiac fibrosis, LV tissue expressions of atrial natriuretic peptide, transforming growth factor [beta]1, and collagen III compared with diabetic NTG mice. Moreover, LV expression of NADPH oxidase subunits, [p22.sup.thox], [p67.sup.phox], [gp91.sup.phox], and Nox4, reactive oxygen species and lipid peroxidation levels were significantly increased in diabetic NTG mice, but not in diabetic TG mice. Furthermore, myocardial apoptosis, the number of caspase-3-positive cells, and the downregulation of antiapoptotic protein Bcl-[X.sub.L] were less in diabetic TG mice compared with diabetic NTG mice. In conclusion, our data establish that p38[alpha] MAPK activity is required for cardiac remodeling after diabetes induction and suggest that p38[alpha] MAPK may promote cardiomyocyte apoptosis by downregulation of Bcl-[X.sub.L]. p38 mitogen-activated protein kinase; oxidative stress

In human Schwann cells, the role of taurine in regulating glucose-induced changes in antioxidant defense systems has been examined. Treatment with high glucose for 7 days induced reactive oxygen species, increased 4-hydroxynoneal adducts (20 [+ or -] 5%, P < 0.05) and poly(ADP-ribosyl)ated proteins (40 [+ or -] 13%, P < 0.05). Increases in these markers of oxidative stress were reversed by simultaneous incubation in 0.25 mM taurine. Both high glucose and taurine independently increased superoxide dismutase and catalase activity and decreased glutathione levels, but their effects were not additive. Glucose reduced taurine transporter (TauT) mRNA and protein in a dose-dependent manner with maximal decreases of 66 [+ or -] 6 and 63 [+ or -] 12%, respectively (P < 0.05 both). The [V.sub.max] for taurine uptake was decreased in 30 mM glucose from 61 [+ or -] 5 to 42 [+ or -] 3 pmol x [min.sup.-] x mg [protein.sup.-1] (P < 0.001). Glucose-induced TauT downregulation could be reversed by inhibition of aldose reductase, a pathway that depletes NADPH and increases osmotic stress and protein glycation. TauT protein was increased more than threefold, and the [V.sub.max] for taurine uptake doubled (P < 0.05 both) by prooxidants. TauT downregulation was reversed both by treatment with the antioxidant [alpha]-lipoic acid, which increased TauT mRNA by 60% and [V.sub.max] by 50% (P < 0.05 both), and by the aldose reductase inhibitor sorbinil, which increased Taut mRNA 380% and [V.sub.max] by 98% (P < 0.01 both). These data highlight the potential therapeutic benefits of taurine supplementation in diabetic complications and provide mechanisms whereby taurine restoration could be achieved in order to prevent or reverse diabetic complications.

Although most attention has been focused on mitochondrial ATP production and transfer in failing hearts, less has been focused on the nonfailing hypertensive heart. Here, energetic complications are less obvious, yet they may provide insight into disease ontogeny. We studied hearts from 12-mo-old spontaneously hypertensive rats (SHR) relative to normotensive Wistar-Kyoto (WKY) rats. The ex vivo working-heart model of SHR showed reduced compliance and impaired responses to increasing preloads. High-resolution respirometry showed higher state 3 (with excess ADP) respiration in SHR left ventricle fibers with complex I substrates and maximal uncoupled respiration with complex I + complex II substrates. Respiration with ATP was depressed 15% in SHR fibers relative to WKY fibers, suggesting impaired ATP hydrolysis. This finding was consistent with a 50% depression of actomyosin ATPase activities. Superoxide production from SHR fibers was similar to that from WKY fibers respiring with ADP; however, it was increased by 15% with ATP. In addition, the apparent Km for ADP was 54% higher for SHR fibers, and assays conducted after ex vivo work showed a 28% depression of complex I in SHR, but not WKY, fibers. Transmission electron microscopy showed similar mitochondrial volumes but a decrease in the number of cristae in SHR mitochondria. Tissue lipid peroxidation was also 15% greater in SHR left ventricle. Overall, these data suggest that although cardiac mitochondria from nonfailing SHR hearts function marginally better than those from WKY hearts, they show dysfunction after intense work. Impaired ATP turnover in hard-working SHR hearts may starve cardiac mitochondria of ADP and elevate superoxide. adenylate control; saponin-permeabilized fibers; hypertensive heart; oxidative stress; complex I dysfunction; respiration

Oxidative stress contributes significantly to the migration of vascular smooth muscle cells (VSMCs), the major pathogenic process of vascular diseases, but the mechanism remains unclear. In the present study, we explored the role of peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] (PGC-1[alpha]), a major regulator of mitochondrial biogenesis and energy balance, in VSMC migration in vitro and in vivo. Overexpression of PGC-l[alpha] in cultured VSMCs led to a 74.5% reduction of migration activity and mitochondrial ROS generation by the increased expression of antioxidative proteins such as SOD-2 in the mitochondria. The knockdown of PGC-1[alpha] by specific small interfering (si)RNA markedly augmented VSMC migration activity and greatly reduced mitochondrial antioxidative protein expression. Furthermore, knockdown of SOD-2 expression by siRNA greatly reversed the inhibitory effect of PGC-l[alpha] overexpression on VSMC migration. In a rat carotid balloon injury model, adenovirus-mediated overexpression of PGC-1[alpha] greatly reduced neointimal formation (ratio of intima to media: 0.78 [+ or -] 0.09 vs. 1.45 [+ or -] 0.18 in the adenovims + green fluorescent protein gene-transfected group). Moreover, the expression of SOD-2 was significantly increased in vivo in local vessels after injury in the PGC-l[alpha]overexpressing group. These data strongly suggest that PGC-1[alpha] inhibits VSMC migration and neointimal formation after vascular injury in rats, mainly by upregulating the expression of the mitochondrial antioxidant enzyme SOD-2. restenosis; antioxidants; smooth muscle cells; metabolic syndrome; peroxisome proliferator-activated receptor-[gamma], coactivator-1[alpha]

Recently, kidney fibrosis following transplantation has become recognized as a main contributor of chronic allograft nephropathy. In transplantation, transient ischemia is an inescapable event. Reactive oxygen species (ROS) play a critical role in ischemia and reperfusion (I/R)-induced acute kidney injury, as well as progression of fibrosis in various diseases such as hypertension, diabetes, and ureteral obstruction. However, a role of ROS/ oxidative stress in chronic kidney fibrosis following I/R injury remains to be defined. In this study, we investigated the involvement of ROS/oxidative stress in kidney fibrosis following kidney I/R in mice. Mice were subjected to 30 min of bilateral kidney ischemia followed by reperfusion on day 0 and then administered with either manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, 5 mg/kg body wt ip), a cell permeable superoxide dismutase (SOD) mimetic, or 0.9% saline (vehicle) beginning at 48 h after I/R for 14 days. I/R significantly increased interstitial extension, collagen deposition, apoptosis of tubular epithelial cells, nitrotyrosine expression, hydrogen peroxide production, and lipid peroxidation and decreased copper-zinc SOD, manganese SOD, and glucose 6-phosphate dehydrogenase activities in the kidneys 16 days after the procedure. MnTMPyP administration minimized these postischemic changes. In addition, MnTMPyP administration significantly attenuated the increases of [alpha]-smooth muscle actin, PCNA, S100A4, CD68, and heat shock protein 47 expression following I/R. We concluded that kidney fibrosis develops chronically following I/R injury, and this process is associated with the increase of ROS/ oxidative stress. interstitial fibrosis; ischemia-reperfusion; superoxide; s-smooth muscle actin; manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin

The nucleus tractus solitarius (NTS), a major hindbrain area involved in cardiovascular regulation, receives primary afferent fibers from peripheral baroreceptors and chemoreceptors. Hydrogen peroxide ([H.sub.2][O.sub.2]) is a relatively stable and diffusible reactive oxygen species (ROS), which acting centrally, may affect neural mechanisms. In the present study, we investigated effects of [H.sub.2][O.sub.2] alone or combined with the glutamatergic antagonist kynurenate into the NTS on mean arterial pressure (MAP) and heart rate (HR). Conscious or anesthetized (urethane and [alpha]-chloralose) male Holtzman rats (280-320 g) were used. Injections of [H.sub.2][O.sub.2] (125 to 1500 pmol/40 nl) into the intermediate NTS of anesthetized rats evoked dose-dependent and transient hypotension (- 18 [+ or -] 3 to -55 [+ or -] 11 mmHg) and bradycardia (- 16 [+ or -] 5 to - 116 [+ or -] 40 bpm). Injection of the catalase inhibitor 3-amino-l,2,4-triazole (100 nmol/40 nl) into the NTS also produced hypotension and bradycardia. Previous injection of the ionotropic L-glutamate receptor antagonist kynurenate (7 nmol/40 nl) attenuated by 48% the bradycardic response, without changing the hypotension evoked by [H.sub.2][O.sub.2] (500 pmol/40 nl) in anesthetized rats. The antioxidant L-ascorbate (600 pmol/80 nl) injected into the NTS attenuated the bradycardic (42%) and hypotensive (67%) responses to [H.sub.2][O.sub.2] (500 pmol/40 nl) into the NTS. In conscious rats, injection of [H.sub.2][O.sub.2] (50 nmol/100 nl) into the NTS also evoked intense bradycardia (-207 [+ or -] 8 bpm) and hypotension (-54 [+ or -] 6 mmHg) that were abolished by prior injection of kynurenate (7 nmol/100 nl). The results show that [H.sub.2][O.sub.2] into the NTS induces hypotension and bradycardia probably due to activation of glutamatergic mechanisms. reactive oxygen species; catalase inhibition; kynurenic acid; blood pressure; heart rate

In human thyroid, caveolin-1 is localized at the apex of thyrocytes, but its role there remains unknown. Using immunohistochemistry, [sup.127]I imaging, transmission electron microscopy, immunogold electron microscopy, and quantification of [H.sub.2][O.sub.2], we found that in caveolin-1 knockout mice thyroid cell homeostasis was disrupted, with evidence of oxidative stress, cell damage, and apoptosis. An even more striking phenotype was the absence of thyroglobulin and iodine in one-half of the follicular lumina and their presence in the cytosol, suggesting that the iodide organification and binding to thyroglobukin were intracellular rather than at the apical membrane/ extracellular colloid interface. The latter abnormality may be secondary to the observed mislocalization of the thyroid hormone synthesis machinery (dual oxidases, thyroperoxidase) in the cytosol. Nevertheless, the overall uptake of radioiodide, its organification, and secretion as thyroid hormones were comparable to those of wild-type mice, suggesting adequate compensation by the normal TSH retrocontrol. Accordingly, the levels of free thyroxine and TSH were normal. Only the levels of free triiodothyronine showed a slight decrease in caveolin-1 knockout mice. However, when TSH levels were increased through low-iodine chow and sodium perchlorate, the induced goiter was more prominent in caveolin-1 knockout mice. We conclude that caveolin-1 plays a role in proper thyroid hormone synthesis as well as in cell number homeostasis. Our study demonstrates for the first time a physiological function of caveolin-1 in the thyroid gland. Because the expression and subcellular localization of caveolin-1 were similar between normal human and murine thyroids, our findings in caveolin-1 knockout mice may have direct relevance to the human counterpart. dual oxidase; thyroperoxidase; iodination; thyrocytes; hydrogen peroxide; oxidative stress; thyroid hormone synthesis; iodide

Adiponectin (APN) exerts its metabolic regulation largely through AMP-dependent protein kinase (AMPK). However, the role of AMPK in APN's antiapoptotic effect in ischemic-reperfused (I/R) adult cardiomyocytes remains incompletely understood. The present study was designed to determine the involvement of AMPK in the antiapoptotic signaling of APN. Cardiomyocytes from adult male mice overexpressing a dominant-negative [[alpha].sub.2]-subunit of AMPK (AMPK-DN) or wild-type (WT) littermates were subjected to simulated I/R (SI/R) and pretreated with 2 [micro]g/ml globular domain of APN (gAPN) or vehicle. SI/R-induced cardiomyocyte apoptosis was modestly increased in AMPK-DN cardiomyocytes (P < 0.05). Treatment with gAPN significantly reduced SI/R-induced apoptosis in WT cardiomyocytes as well as in AMPK-DN cardiomyocytes, indicating that the antiapoptotic effect of gAPN is partially AMPK independent. Furthermore, gAPN-induced endothelial nitric oxide synthase (eNOS) phosphorylation was significantly reduced in AMPK-DN cardiomyocytes, suggesting that the APN-eNOS signaling axis is impaired in AMPK-DN cardiomyocytes. Additional experiments demonstrated that treatment of AMPK-DN cardiomyocytes with gAPN reduced SI/R-induced NADPH oxidase overexpression, decreased superoxide generation, and blocked peroxynitrite formation to the same extent as that observed in WT cardiomyocytes. Collectively, our present study demonstrated that although the metabolic and eNOS activation effect of APN is largely mediated by AMPK, the superoxide-suppressing effect of APN is not mediated by AMPK, and this AMPK-independent antioxidant property of APN increased nitric oxide bioavailability and exerted significant antiapoptotic effect. apoptosis; oxidative stress; nitric oxide; adipocytokine

The tra2[beta] gene encoding an alternative splicing regulator, transformer 2-[beta] (Tra2[beta]), generates five alternative splice variant transcripts (tra2[beta]1-5). Functionally active, full-length Tra2[beta] is encoded by tra2[beta]1 isoform. Expression and physiological significance of the other isoforms, particularly tra2[beta]4, are not fully understood. Rat gastric mucosa constitutively expressed tra2[beta]1 isoform and specifically generated tra2[beta]4 isoform that includes premature termination codon-containing exon 2, when exposed to restraint and water immersion stress. Treatment of a gastric cancer cell line (AGS) with arsenite (100 [micro]M) preferentially generated tra2[beta]4 isoform and caused translocation of Tra2[beta] from the nucleus to the cytoplasm in association with enhanced phosphorylation during the initial 4-6 h (acute phase). Following the acute phase, AGS cells continued upregulated tra2[beta]1 mRNA expression, and higher amounts of Tra2[beta] were reaccumulated in their nuclei. Treatment with small interference RNAs targeting up-frameshift-1 or transfection of a plasmid containing tra2[beta]1 cDNA did not induce tra2[beta]4 isoform expression and did not modify the arsenite-induced expression of this isoform, suggesting that neither the nonsense-mediated mRNA decay nor the autoregulatory control by excess amounts of Tra2[beta] participated in the tra2[beta]4 isoform generation. Knockdown of Tra2[beta] facilitated skipping of the central variable region of the CD44 gene and suppressed cell growth. In contrast, overexpression of Tra2[beta] stimulated combinatorial inclusion of multiple variable exons in the region and cell growth. The similar skipping and inclusion of the variable region were observed in arsenite-treated cells. Our results suggest that Tra2[beta] may regulate cellular oxidative response by changing alternative splicing of distinct genes including CD44. arsenite; splicing regulator; tra2[beta]4 isoform; premature termination codon; cell growth

In prosthetic loosening, bone resorption is induced by wear debris particles generated from the artificial joint articulation. Our prior work showed that synovial-like fibroblasts respond to titanium particles by producing receptor activator of NF-[kappa]B ligand (RANKL), a critical activator of osteoclastogenesis. While this effect occurs through a cyclooxygenase-2 (COX-2)-dependent pathway, the mechanism of COX-2 stimulation by titanium particles is not clear. Here we show that titanium particles induce COX-2 gene expression by activating NF-[kappa]B signaling. Inhibitor of NF-[kappa]B (I[kappa]B[alpha]) is degraded following particle treatment, permitting active NF-[kappa]B to translocate to the nucleus where it interacts with the COX-2 promoter and drives transcription. NF-[kappa]B activation is dependent on reactive oxygen species since antioxidants block the NF-[kappa]B signaling induced by particles. Surprisingly, I[kappa]B[alpha] degradation is independent of IKK (I[kappa]B kinase) and the 26S proteasome. Instead, calpain inhibitor can block the I[kappa]B[alpha] degradation induced by particles. Furthermore, the calpain-targeted COOH-terminal PEST sequence of I[kappa]B[alpha] is necessary for phosphorylation and degradation, consistent with a proteasome-independent mechanism of catabolism. Altogether, the data demonstrate a signaling pathway by which titanium particles induce oxidative stress, stimulate calpain-mediated NF-[kappa]B activation, and activate target gene expression, including COX-2. These findings define important targets for osteolysis but may also have importance in other diseases where fibroblasts respond to environmental particles, including pulmonary diseases. cyclooxygenase-2; osteolysis; inflammation; cell signaling; reactive oxygen species

Exercise-induced oxidative stress is instrumental in achieving the health benefits from regular exercise. Therefore, inappropriate use of fruit-derived products (commonly applied as prophalytic antioxidants) may counteract the positive effects of exercise. Using human exercise and cellular models we found that 1) blackcurrant supplementation suppressed exercise-induced oxidative stress, e.g., plasma carbonyls (0.9 [+ or -] 0.1 vs. 0.6 [+ or -] 0.1 nmol/mg protein, placebo vs. blackcurrant), and 2) preincubation of THP-1 cells with an anthocyanin-rich blackcurrant extract inhibited LPS-stimulated cytokine secretion [TNF-[alpha] (16,453 [+ or -] 322 vs. 10,941 [+ or -] 82 pg/ml, control vs. extract, P < 0.05) and IL-6 (476 [+ or -] 14 vs. 326 [+ or -] 32 pg/ml, control vs. extract, P < 0.05)] and NF-[kappa]B activation. In addition to its antioxidant and anti-inflammatory properties, we found that postexercise plasma collected after blackcurrant supplementation enhanced the differential temporal LPS-stimulated inflammatory response in THP-1 cells, resulting in an early suppression of TNF-[alpha] (1,741 [+ or -] 32 vs. 1,312 [+ or -] 42 pg/ml, placebo vs. blackcurrant, P < 0.05) and IL-6 (44 [+ or -] 5 vs. 36 [+ or -] 3 pg/ml, placebo vs. blackcurrant, P < 0.05) secretion after 24 h. Furthermore, by using an oxidative stress cell model, we found that preincubation of THP- 1 cells with hydrogen peroxide (H202) prior to extract exposure caused a greater suppression of LPS-stimulated cytokine secretion after 24 h, which was not evident when cells were simultaneously incubated with [H.sub.2] [O.sub.2] and the extract. In summary, our findings support the concept that consumption of blackcurrant anthocyanins alleviate oxidative stress, and may, if given at the appropriate amount and time, complement the ability of exercise to enhance immune responsiveness to potential pathogens. acute inflammation; fruit anthocyanins; moderate exercise

Vascular dysfunction occurs with aging. We hypothesized that oxidative stress and ANG II [acting via ANG II type 1 ([AT.sub.1] receptors] promotes cerebral vascular dysfunction with aging. We studied young (5-6 mo), old (17-19 mo), and very old (23 [+ or -] 1 mo) mice. In basilar arteries in vitro, acetylcholine (an endothelium-dependent agonist) produced dilation in young wild-type mice that was reduced by ~60 and 90% (P < 0.05) in old and very old mice, respectively. Similar effects were seen using A23187, a second endothelium-dependent agonist. The vascular response to acetylcholine in very old mice was almost completely restored with tempol (a scavenger of superoxide) and partly restored by PJ34, an inhibitor of poly(ADP-ribose) polymerase (PARP). We used mice deficient in Mn-SOD (Mn-[SOD.sup.+/-]) to test whether this form of SOD protected during aging but found that age-induced endothelial dysfunction was not altered by Mn-SOD deficiency. Cerebral vascular responses were similar in young mice lacking AT; receptors ([AT.sub.1.sup.-/-]) and wild-type mice. Vascular responses to acetylcholine and A23187 were reduced by ~50% in old wild-type mice (P < 0.05) but were normal in old [AT.sub.1]-deficient mice. Thus, aging produces marked endothelial dysfunction in the cerebral artery that is mediated by ROS, may involve the activation of PARP, but was not enhanced by Mn-SOD deficiency. Our findings suggest a novel and fundamental role for ANG II and [AT.sub.1] receptors in age-induced vascular dysfunction. basilar artery; endothelium, genetically altered mice; acetylcholine; angiotensin II; A23187

We have recently shown that the inability of repetitive ischemia (RI) to activate p38 MAPK (p38) and Akt in metabolic syndrome [JCR:LA-cp (JCR)] rats was associated with impaired coronary collateral growth (CCG). Furthermore, Akt and p38 activation correlated with optimal [O.sup.-.sub.2]. levels and were altered in JCR rats, and redox-sensitive p38 activation was required for CCG. Here, we determined whether the activation of Src, a possible upstream regulator, was altered in JCR rats and whether redox-dependent Src and Akt activation were required for CCG. CCG was assessed by myocardial blood flow (microspheres) and kinase activation was assessed by Western blot analysis in the normal zone and collateral-dependent zone (CZ). RI induced Src activation (~3-fold) in healthy [Wistar-Kyoto (WKY)] animals but not in JCR animals. Akt inhibition decreased (~50%), and Src inhibition blocked RI-induced CCG in WKY rats. Src inhibition decreased p38 and Akt activation. Myocardial oxidative stress ([O.sub.2] x and oxidized/reduced thiols) was measured quantitatively (X-band electron paramagnetic resonance). An antioxidant, apocynin, reduced RI-induced oxidative stress in JCR rats to levels induced by RI in WKY rats versus the reduction in WKY rats to very low levels. This resulted in a significant restoration of p38 (~80%), Akt (~65%), and Src (~90%) activation in JCR rats but decreased the activation in WKY rats (p38: ~45%, Akt: ~65%, and Src: ~100%), correlating with reduced CZ flow in WKY rats (~70%), but significantly restored CZ flow in JCR rats (~75%). We conclude that 1) Akt and Src are required for CCG, 2) Src is a redox-sensitive upstream regulator of RI-induced p38 and Akt activation, and 3) optimal oxidative stress levels are required for RI-induced p38, Akt, and Src activation and CCG. signal transduction; oxidative stress; coronary artery disease; syndrome X