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The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide--cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells. 10.1126/science.1192912

Significant reduction of renal mass causes progressive deterioration of renal function and structure which is mediated by systemic and glomerular hypertension, hyperfiltration, oxidative stress, inflammation, and dyslipidemia. Niacin is known to improve lipid metabolism and exert antioxidant/anti-inflammatory actions. Therefore, we considered that niacin supplementation may attenuate oxidative stress, inflammation, and tissue injury in the remnant kidney. To this end, 5/6 nephrectomized [chronic kidney disease (CKD)] rats were randomly assigned to niacin-treated (50 mg x [kg.sup.-1] x [day.sup.-1] in the drinking water for 12 wk) and untreated groups. Sham-operated rats served as controls. The untreated CKD rats exhibited azotemia, hypertension, hypertriglyceridemia, proteinuria, glomerulosclerosis, tubulointerstitial damage, upregulation of MCP-1, plasminogen activator inhibitor-1 (PAI-1), transforming growth factor (TGF)-[beta], cyclooxygenase (COX)-1, COX-2, and NAD(P)H oxidase (NOX-4, gpg[1.sup.phox], p4[7.sup.phox] and p2[2.sup.phox] subunits) and activation of NF-[kappa]B (I[kappa]B phosphorylation). Niacin administration reduced MCP-1, PAI- 1, TGF-[beta], p4[7.sup.phox], p2[2.sup.phox], COX-I, and NF-[kappa]B activation, ameliorated hypertension, proteinuria, glomerulosclerosis, and tubulointersfitial injury. Although niacin lowered serum creatinine and raised creafinine clearance, the differences did not reach statistical significance. Thus niacin supplementation helps to attenuate histological injury and mitigate upregulafion of oxidative and inflammatory systems in the remnant kidney. malnutrition; glomerulosclerosis; fibrosis; TGF; CTGF; NAD(P)H oxidase; cyclooxygenase; NF-[kappa]B; lipid disorder; atherosclerosis

Prolonged stress results in elevation of glucocorticoid (GC) hormones, which can have deleterious effects in the brain. The hippocampus, which has a high concentration of glucocorticoid receptors, is especially vulnerable to increasing levels of GCs. GCs have been suggested to endanger hippocampal neurons by exacerbating the excitotoxic glutamatecalcium--reactive oxygen species (ROS) cascade. In an effort to reveal the mechanisms underlying GC-mediated hippocampal neurotoxicity, we aimed to clarify the molecular pathway of GC-induced ROS increase by using organotypic hippocampal slice cultures. Assays for ROS, using 2',7'-dichlorodihydrofluorescein diacetate fluorescence, showed that treatment of synthetic GC, dexamethasone (DEX) significantly enhanced ROS levels. Time course and dose response analyses indicated that peak amount of ROS was generated at 4 h after treatment with 50 µmol/L DEX. By contrast, other steroid hormones, progesterone and estradiol did not influence ROS production. N-acetyl-L-cysteine completely suppressed ROS produced by DEX. Propidium iodide staining exhibited prominent cell death in the hippocampal layer after 96 h of DEX treatment. RU486, a GC receptor antagonist, almost completely blocked the effect of DEX on ROS production and cell death, indicating that DEX-induced ROS overproduction and hippocampal death are mediated via GC receptors. Real-time reverse transcriptase PCR analysis demonstrated that after DEX treatment the level of glutathione peroxidase mRNA was decreased whereas that of NADPH oxidase mRNA was significantly enhanced. These findings suggest that excess GCs cause hippocampal damage by regulating genes involved in ROS generation. Key words: glucocorticoid, hippocampus, neurotoxicity, organotypic hippocampal slice culture, reactive oxygen species, stress. Un stress prolonge entraine une augmentation d'hormones glucocorticoides (GC), ce qui peut avoir des effets nefastes dans le cerveau. L'hippocampe, qui contient une grande concentration de recepteurs aux glucocorticoides, est particulierement vulnerable a l'augmentation des taux de GC. On a suggere que les GC compromettent la survie des neurones hippocampiques en exacerbant la cascade excitotoxique glutamate-calcium-especes reactives de l'oxygene (ROS). Dans le but d'identifier les mecanismes sous-jacents a la neurotoxicite hippocampique vehiculee par les GC, notre avons utilise des cultures organotypiques de tranches d'hippocampe pour examiner la voie moleculaire de l'augmentation des ROS induite par les CG. Les essais, effectues en utilisant la fluorescence du 1',7'-dichlorodihydrofluoresceine diacetate, ont montre qu'un traitement a la GC synthetique, dexamethasone (DEX), a augmente significativement les taux de ROS. Les analyses de l'evolution temporelle et de la relation dose-reponse ont indique que la production maximale de ROS a eu lieu 4 h apres un traitement avec 50 µ M de DEX. Toutefois, d'autres hormones steroidiennes, progesterone et oestradiol, n'ont pas influe sur la production des ROS. La N-acetyl-L-cysteine a totalement supprime les ROS produites par la DEX. La coloration a l'iodure de propidium a montre une mort cellulaire significative dans la couche hippocampique apres 96 h de traitement a la DEX. RU486, un antagoniste des recepteurs aux GC, a presque completement bloque la production de ROS et la mort des cellules induites par le traitement a la DEX, ce qui indique que la surproduction de ROS et la mort hippocampique induites par la DEX sont vehiculees par les recepteurs aux GC. La technique de RT-PCR en temps reel a demontre que le taux d'ARNm de la glutathion peroxydase a diminue, alors que le taux d'ARNm oxydase de la NADPH a augmente significativement apres le traitement a la DEX. Ces resultats donnent a penser qu'un taux tres eleve de GC provoque une degradation de l'hippocampe en regulant les genes impliques dans la production des ROS. Mots-cles : glucocorticoide, hippocampe, neurotoxicite, culture organotypique de tranche d'hippocampe, especes reactives de l'oxygene, stress. [Traduit par la Redaction], Introduction The glucocorticoid (GC) steroid hormones released from the adrenal cortex are the principal effectors in the stress response (Sapolsky et al. 2000). Although short-term elevation of GCs can be [...]

Cytosolic [NADP.sup.+]-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/ (GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-[PK.sub.1] cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R. reduced nicotinamide adenine dinucleotide phosphate; glutathione; proximal tubular cell; acute kidney injury; oxidative stress; ROS

Insulin activation of the phosphatidylinositol 3-kinase (PI3K) pathway stimulates glucose uptake in peripheral tissues and synthesis of nitric oxide (NO) in the endothelium. Insulin resistance (IR) and hypertension frequently coexist, particularly among individuals with salt-sensitive hypertension. The mechanisms underlying this association are poorly understood. We investigated these mechanisms in a model of salt-sensitive hypertension in which we have previously shown that endothelial dysfunction is mediated by superoxide anion ([O.sup.-.sub.2]) linked to local ANG II. Dahl salt-sensitive rats were fed, for 6 wk, a normal salt diet (NS; 0.5% NaCl), high-salt diet (HS; 4% NaCl), HS plus the ANG II type 1 receptor (A[T.sub.1]R) blocker (ARB) candesartan (10 mg x [kg.sup.-1] x [day.sup.-1]), or HS plus the antioxidant tempol (172 mg/l in drinking water). Hypertensive (mean arterial pressure: 145 [+ or -] 4 vs. 102 [+ or -] 5 mmHg in NS, P < 0.05) rats manifested increased aortic A[T.sub.1]R mRNA (210%) and protein (101%) expression and [O.sub.2] production (104%) and impaired endothelium-dependent relaxation (EDR) to acetylcholine [maximal response ([E.sub.max]): 68 [+ or -] 9 vs. 91 [+ or -] 8% in NS, P < 0.05]. ARB or tempol normalized [O.sup.-.sub.2] and EDR despite that they did not normalize mean arterial pressure, which was reduced only 25%. Hypertensive rats manifested metabolic IR (36% reduction in the glucose infusion rate by insulin clamp), impaired NO-mediated insulin-induced EDR ([E.sub.max] : 12 [+ or -] 5 vs. 32 [+ or -] 4% in NS, P < 0.05), and impaired insulin activation of PI3K/endothelial NO synthase. ARB or tempol improved insulin-mediated EDR, PI3K, Akt/endothelial NO synthase phosphorylation, and metabolic IR (all P < 0.05). This study provides insight into the mechanisms that underlie the association between metabolic and hypertensive cardiovascular diseases and support the notion that [O.sup.-.sub.2] overproduction linked to tissue ANG II interferes with shared insulin signaling pathways in metabolic and cardiovascular tissues. endothelial function; salt sensitivity

Hypercholesterolemia is associated with phenotypic changes in endothelial cell function that lead to a proinflammatory and prothrombogenic state in different segments of the microvasculature. CD40 ligand (CD40L) and its receptor CD40 are ubiquitously expressed and mediate inflammatory responses and platelet activation. The objective of this study was to determine whether CD40/CD40L, in particular T-cell CD40L, contributes to microvascular dysfunction induced by hypercholesterolemia. Intravital microscopy was used to quantify blood cell adhesion in cremasteric postcapillary venules, endothelium-dependent vasodilation responses in arterioles, and microvascular oxidative stress in wild-type (WT) C57BL/6, CD40-deficient (-/-), [CD40L.sup.-/-], or severe combined immune deficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 wk. WT-HC mice exhibited an exaggerated leukocyte and platelet recruitment in venules and impaired vasodilation responses in arterioles compared with ND counterparts. A deficiency of CD40, CD40L, or lymphocytes attenuated these responses to HC. The HC phenotype was rescued in [CD40L.sup.-/-] and SCID mice by a transfer of WT T cells. Bone marrow chimeras revealed roles for both vascular- and blood cell-derived CD40 and CD40L in the HC-induced vascular responses. Hypercholesterolemia induced an oxidative stress in both arterioles and venules of WT mice, which was abrogated by either CD40 or CD40L deficiency. The transfer of WT T cells into [CD40L.sup.-/-] mice restored the oxidative stress. These results implicate CD40/CD40L interactions between circulating cells and the vascular wall in both the arteriolar and venular dysfunction elicited by hypercholesterolemia and identify T-cell-associated CD40L as a key mediator of these responses. T lymphocytes; platelets; oxidative stress; arteriolar vasodilation; leukocyte adhesion

A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced acute lung injury (ALI) has been shown; however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species and has been implicated in endothelial injury in ALI. Pulmonary microvascular endothelial cells (PMVEC) monolayers were grown on Transwell inserts and incubated with wild-type and NADPH oxidase-deficient AM in the presence or absence of cytomix (equimolar TNF-[alpha], IL-1[beta], and IFN-[gamma]). Injury to the mono-layers was assessed by trans-PMVEC Evans blue (EB)-labeled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers, and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC cocultures, we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NADPH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute to PMVEC injury in our studies. From our findings we have concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability. alveolar macrophages; endothelial cells; oxidative stress

The protective effects of nitric oxide (NO), a physiological activator of soluble guanylyl cyclase (sGC), have been reported in ischemia-reperfusion (I/R) syndrome of the lung. Therefore, we studied the effects of BAY 41-2272, a novel sGC stimulator, on I/R injury of the lung in an isolated intact organ model. Lung injury was assessed by measuring weight gain and microvascular permeability (capillary filtration coefficient, [K.sub.fc]). Release of reactive oxygen species (ROS) into the perfusate was measured during early reperfusion by electron spin resonance (ESR) spectroscopy. Rabbit lungs were treated with BAY 41-2272, [N.sup.G]-monomethyl-L-arginine (L-NMMA), or NO to evaluate the effects on I/R-induced lung injury. In untreated lungs, a dramatic rise in [K.sub.fc] values and weight gain during reperfusion were observed, and these results were associated with increased ROS production. Both, BAY 41-2272 and L-NMMA significantly attenuated vascular leakage and suppressed ROS release. Additional experiments showed that BAY 41-2272 diminished PMA-induced ROS production by NADPH oxidase. A pharmacological inhibition of the enzyme with consequent reduction in ROS levels decreased I/R injury. NO had only marginal effect on I/R injury. Thus BAY 41-2272 protects against I/R-induced lung injury by interfering with the activation of NADPH oxidases. acute lung injury; nitric oxide; oxidative stress; reactive oxygen species; guanosine 3',5'-cyclic monophosphate

Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-Jun N[H.sub.2]terminal kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma alanine aminotransferase activities increased from 46 [+ or -] 4 U/l in controls to 955 [+ or -] 90 U/l at 6 h after diquat treatment. Hematoxylin and eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6 h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP-600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury, but, in combination with phorone and FeS[O.sub.4], induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation. diquat; galactosamine; endotoxin; menadione; superoxide; mechanisms of cell death; caspases

Bacterial endotoxin lipopolysaccharide (LPS) is responsible for the multiorgan dysfunction that characterizes septic shock and is causal in the myocardial depression that is a common feature of endotoxemia in patients. In this setting the myocardial dysfunction appears to be due, in part, to the production of proinflammatory cytokines. A line of evidence also indicates that LPS stimulates autophagy in cardiomyocytes. However, the signal transduction pathway leading to autophagy and its role in the heart are incompletely characterized. In this work, we wished to determine the effect of LPS on autophagy and the physiological significance of the autophagic response. Autophagy was monitored morphologically and biochemically in HL-1 cardiomyocytes, neonatal rat cardiomyocytes, and transgenic mouse hearts after the administration of bacterial LPS or TNF-[alpha]. We observed that autophagy was increased after exposure to LPS or TNF-[alpha], which is induced by LPS. The inhibition of TNF-[alpha] production by AG126 significantly reduced the accumulation of autophagosomes both in cell culture and in vivo. The inhibition of p38 MAPK or nitric oxide synthase by pharmacological inhibitors also reduced autophagy. Nitric oxide or H202 induced autophagy in cardiomyocytes, whereas Nacetyl-cysteine, a potent antioxidant, suppressed autophagy. LPS resulted in increased reactive oxygen species (ROS) production and decreased total glutathione. To test the hypothesis that autophagy might serve as a damage control mechanism to limit further ROS production, we induced autophagy with rapamycin before LPS exposure. The activation of autophagy by rapamycin suppressed LPSmediated ROS production and protected cells against LPS toxicity. These findings support the notion that autophagy is a cytoprotective response to LPS-induced cardiomyocyte injury; additional studies are needed to determine the therapeutic implications. lipopolysaccharide; HL-1 cardiac myocyte; green fluorescent proteinmicrotubule-associated protein light chain 3; oxidative stress

Glucose-mediated oxidative stress and the upregulation of cyclooxygenase (COX)-2 pathway activity have been implicated in the pathogenesis of several vascular complications of diabetes including diabetic neuropathy. However, in nondiabetic subjects, the cardiovascular safety of selective COX-2 inhibition is controversial. The aim of this study was to explore the links between hyperglycemia, oxidative stress, activation of the COX-2 pathway, cardiac sympathetic integrity, and the development of left ventricular (LV) dysfunction in experimental diabetes. R wave-to-R wave interval (R-R interval) and parameters of LV function measured by echocardiography using 1% isoflurane, LV sympathetic nerve fiber density, LV collagen content, and markers of myocardial oxidative stress, inflammation, and PG content were assessed after 6 mo in control and diabetic COX-2-deficient ([COX2.sup.-/-]) and littermate, wild-type ([COX-2.sup.+/+]) mice. There were no differences in blood glucose, LV echocardiographic measures, collagen content, sympathetic nerve fiber density, and markers of oxidative stress and inflammation between nondiabetic (ND) [COX-2.sup.+/+] and [COX-2.sup.- /-] mice at baseline and thereafter. After 6 too, diabetic [COX-2.sup.+/+] mice developed significant deteriorations in the R-R interval and signs of LV dysfunction. These were associated with a loss of LV sympathetic nerve fiber density, increased LV collagen content, and a significant increase in myocardial oxidative stress and inflammation compared with those of ND mice. Diabetic [COX-2.sup.-/-] mice were protected against all these biochemical, structural, and functional deficits. These data suggest that in experimental diabetes, selective COX-2 inactivation confers protection against sympathetic denervation and LV dysfunction by reducing intramyocardial oxidative stress, inflammation, and myocardial fibrosis. sympathetic function; oxidative stress; inflammation

Malhotra A, Vashistha H, Yadav VS, Dube MG, Kalra SP, Abdellatif M, Meggs LG. Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis. Am J Physiol Heart Circ Physiol 296: H380-H388, 2009. First published December 5, 2008; doi:10.1152/ajpheart.00225.2008. Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcAJcytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification. diabetes mellitus; reactive oxygen species

Mitochondrial (mt)DNA mutations contribute to various disease states characterized by low ATP production. In contrast, thyroid hormone [3,3',5-triiodothyronine ([T.sub.3])] induces mitochondrial biogenesis and enhances ATP generation within cells. To evaluate the role of T3-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leigh's syndrome and one with hypertrophic cardiomyopathy. Compared with control cells, patient fibroblasts displayed similar levels of mitochondrial mass, peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] (PGC1[alpha]), mitochondrial transcription factor A (Tram), and uncoupling protein 2 (UCP2) protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold elevation in mitochondrial membrane potential, and 30% less complex V activity compared with control cells. Patient cells also displayed 20-25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with control cells. After [T.sub.3] treatment of patient cells, ROS production was decreased by 40%, cytoplasmic [Ca.sup.2+] was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1[alpha], Tfam, or UCP2 in either [T.sub.3]-treated patient or control cells. However, [T.sub.3] restored the mitochondrial membrane potential, complex V activity, and levels of MnSOD to normal values in patient cells and elevated MnSOD levels by 21% in control cells. These results suggest that [T.sub.3] acts to reduce cellular oxidative stress, which may help attenuate ROSmediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects. reactive oxygen species; mitochondrial biogenesis; cytoplasmic calcium; mitochondrial disease; 3,3',5-triiodothyronine; mitochondrial DNA

Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidantinduced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 [micro]M [H.sub.2][O.sub.2]) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to [H.sub.2][O.sub.2] significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), [alpha]-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate [alpha]II-spectrin following 4 h and 24 h of [H.sub.2][O.sub.2] treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 [micro]M). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for [H.sub.2][O.sub.2]-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress. skeletal muscle; protease; oxidative stress

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (A/S) and parkinsonism-dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of [Ca.sup.2+] and [Mg.sup.2+], coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, [TRPM2.sup.P1018L], produces a missense change in the channel protein whereby proline 1018 ([Pro.sup.1018]) is replaced by leucine ([Leu.sup.1018]). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states. calcium | neurodegeneration | oxidative stress | channelopathy | gene environment

We previously showed that long-term consumption of a soy protein diet (SoyP) reduces renal damage in obese Zucker (ObeseZ) rats by restoring urinary NO2 and NO3 excretion ([UNO.sub.2]/[NO.sub.3]V), suggesting that nitric oxide (NO) deficiency may contribute to the renal progression observed in this model. In addition, there is compelling evidence that hyperleptinemia produced deleterious effects on the kidney through its interaction with the short leptin receptor (ObRa). This study was designed to evaluate the contribution of the NO/endothelial NO synthase (eNOS) system, renal oxidative stress, and ObRa expression to the renoprotection conferred by the consumption of a SoyP in ObeseZ rats. Ten lean and ten male ObeseZ rats were included. One-half of each group was fed with a 20% SoyP and the other half with a 20% casein protein diet (CasP) over the course of 160 days. eNOS protein levels and phosphorylation, renal lipoperoxidation (rLPO), and antioxidant enzyme activity were assessed. In addition, renal ObRa, TGF-[beta], and kidney injury molecule (Kim-1) mRNA levels, as well as urinary Kim-1 levels, were measured. Renal injury observed in ObeseZ rats fed with CasP was not associated with changes in eNOS expression or phosphorylation. However, this group did present with increased rLPO, reduced catalase activity, and upregulation of ObRa, TGF-[beta]1, and Kim- 1. In contrast, ObeseZ rats fed with a SoyP exhibited a reduction in NOS-Thr495 phosphorylation and rLPO, as well as an enhanced catalase activity. These findings were associated with a significant reduction of ObRa, TGF-[beta]1, and Kim-1 mRNA levels and urinary Kim-1 protein. Our results show that renoprotection by SoyP in ObeseZ rats is in part mediated by increased NO availability secondary to a reduction in eNOS-T495 phosphorylation and oxidative stress, together with a significant reduction in ObRa and TGF-[beta] expression. oxidative stress; nitric oxide; endothelial nitric oxide synthase phosphorylation; short leptin receptor

Endothelin (ET) type B receptors ([ET.sub.B]R) are expressed in multiple tissues and perform different functions depending on their location. [ET.sub.B]R mediate endothelium-dependent vasodilation, clearance of circulating ET, and diuretic effects; all of these should produce a fall in arterial blood pressure. However, we recently showed that chronic activation of [ET.sub.B]R in rats with the selective agonist sarafotoxin 6c (S6c) causes sustained hypertension. We have proposed that one mechanism of this effect is constriction of capacitance vessels. The current study was performed to determine whether S6c hypertension is caused by increased generation of reactive oxygen species (ROS) and/or activation of the sympathetic nervous system. The model used was continuous 5-day infusion of S6c into male Sprague-Dawley rats. No changes in superoxide anion levels in arteries and veins were found in hypertensive S6c-treated rats. However, superoxide levels were increased in sympathetic ganglia from S6c-treated rats. In addition, superoxide levels in ganglia increased progressively the longer the animals received S6c. Treatment with the antioxidant tempol impaired S6c-induced hypertension and decreased superoxide levels in ganglia. Acute ganglion blockade lowered blood pressure more in S6c-treated rats than in vehicle-treated rats. Although plasma norepinephrine levels were not increased in S6c hypertension, surgical ablation of the celiac ganglion plexus, which provides most of the sympathetic innervation to the splanchnic organs, significantly attenuated hypertension development. The results suggest that S6c-induced hypertension is partially mediated by sympathoexcitation to the splanchnic organs driven by increased oxidative stress in prevertebral sympathetic ganglia. endothelin type B receptor; oxidative stress; neuronal regulation

Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) is a member of the Bcl-2 homology domain 3-only subfamily of proapoptotic Bcl-2 proteins and is associated with cell death in the myocardium. In this study, we investigated the potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT-sensitive homodimer that increased after myocardial ischemia-reperfusion (I/R). The presence of the antioxidant N-acetylcysteine reduced I/R-induced homodimerization of Bnip3. Overexpression of Bnip3 in cells revealed that most of exogenous Bnip3 exists as a DTT-sensitive homodimer that correlated with increased cell death. In contrast, endogenous Bnip3 existed mainly as a monomer under normal conditions in the heart. Screening of the Bnip3 protein sequence revealed a single conserved cysteine residue at position 64. Mutation of this cysteine to alanine (Bnip3C64A) or deletion of the N[H.sub.2]-terminus (amino acids 1-64) resulted in reduced cell death activity of Bnip3. Moreover, mutation of a histidine residue in the COOH-terminal transmembrane domain to alanine (Bnip3H173A) almost completely inhibited the cell death activity of Bnip3. Bnip3C64A had a reduced ability to interact with Bnip3, whereas Bnip3H173A was completely unable to interact with Bnip3, suggesting that homodimerization is important for Bnip3 function. A consequence of I/R is the production of reactive oxygen species and oxidation of proteins, which promotes the formation of disulfide bonds between proteins. Thus, these experiments suggest that Bnip3 functions as a redox sensor where increased oxidative stress induces homodimerization and activation of Bnip3 via cooperation of the N[H.sub.2]-terminal cysteine residue and the COOH-terminal transmembrane domain. Bcl-2 homology domain 3 proteins; apoptosis; reactive oxygen species; cysteine residues

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-[alpha] expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser l 5 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-[alpha], which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-[alpha] blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity. oxidative stress; mitochondrial death pathway

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial [O.sub.2.sup.*-] and [H.sub.2][O.sub.2] production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial [O.sub.2.sup.x-] and [H.sub.2][O.sub.2] production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular [O.sub.2.sup.*-] and [H.sub.2][O.sub.2] production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress. senescence; vascular disease; atherosclerosis; [Prop1.sup.df/df] mice

Impairment of the normal spatiotemporal pattern of intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) signaling, and in particular, the transition to an irreversible '[Ca.sup.2+] overload' response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this [Ca.sup.2+] overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide ([H.sub.2][O.sub.2]) evokes a [Ca.sup.2+] overload response and inhibition of plasma membrane [Ca.sup.2+]-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [[Ca.sup.2+]]I clearance assay in which mitochondrial [Ca.sup.2+] uptake was blocked with Ru-360, [H.sub.2][O.sub.2] (50[micro]M-1 mM) markedly inhibited the PMCA activity. This [H.sub.2][O.sub.2]-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The [H.sub.2][O.sub.2]-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial [Ca.sup.2+] handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the [Ca.sup.2+] overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states. calcium overload; oxidative stress; pancreatitis

Earlier we reported defects in D1 receptor function in renal proximal tubules (RPTs) of aged Fischer 344 (F344) and obese Zucker rats. However, the defects in the receptor function in RPTs of obese Zucker rats do not pass onto primary cultures of RPTs from these animals. Here, we determined whether the defects in D1 receptor function in RPTs of aged F344 rats pass onto the primary cultures. RPTs from aged (24-mo) and adult (6-mo) F344 rats were grown into primary cultures. The microscopic studies showed that ceils in cultures from adult and old rats were healthy as determined by the shape and size of the cells and nuclei. D1 receptor agonist SKF-38393 produced inhibition of [sup.86]Rb (rubidium) uptake, index of Na-K-ATPase activity, in ceils from adult rats, but this was reduced in old rats. Also, SKF-38393 increased the [[sup.35]S]GTP3[gamma]S binding, index of receptor activation, in the membranes of cells from adult rats but to a lesser extent from old rats. Furthermore, there was a downward trend in the levels of D 1 receptor numbers and in the receptor proteins in old rats. Interestingly, [gp.sup.91phox] subunit of NADPH oxidase and cellular protein carbonyl levels (oxidative stress marker) were higher in cultures from old rats. These results show that RPTs from adult and old F344 rats grow into epithelial cells in cultures. Furthermore, cells in cultures from old rats are at a higher level of oxidative stress, which may be contributing to the reduced D1 receptor function in the cells from old compared with adult rats. dopamine; NADPH oxidase; G protein-coupled receptor; aging

Humans and most other animals use 2 different genetic codes to translate their hereditary information: the standard code for nuclear-encoded proteins and a modern variant of this code in mitochondria. Despite the pivotal role of the genetic code for cell biology, the functional significance of the deviant mitochondrial code has remained enigmatic since its first description in 1979. Here, we show that profound and functionally beneficial alterations on the encoded protein level were causative for the AUA codon reassignment from isoleucine to methionine observed in most mitochondrial lineages. We demonstrate that this codon reassignment leads to a massive accumulation of the easily oxidized amino acid methionine in the highly oxidative inner mitochondrial membrane. This apparently paradoxical outcome can yet be smoothly settled if the antioxidant surface chemistry of methionine is taken into account, and we present direct experimental evidence that intramembrane accumulation of methionine exhibits antioxidant and cytoprotective properties in living cells. Our results unveil that methionine is an evolutionarily selected antioxidant building block of respiratory chain complexes. Collective protein alterations can thus constitute the selective advantage behind codon reassignments, which authenticates the 'ambiguous decoding' hypothesis of genetic code evolution. Oxidative stress has shaped the mitochondrial genetic code. evolution | methionine sulfoxide | nonstandard genetic code | protein oxidation | oxidative stress

Endothelial dysfunction is a characteristic feature during the renal damage induced by mild hyperuricemia. The mechanism by which uric acid reduces the bioavailability of intrarenal nitric oxide is not known. We tested the hypothesis that oxidative stress might contribute to the endothelial dysfunction and glomerular hemodynamic changes that occur with hyperuricemia. Hyperuricemia was induced in Sprague-Dawley rats by administration of the uricase inhibitor, oxonic acid (750 mg/kg per day). The superoxide scavenger, tempol (15 mg/kg per day), or placebo was administered simultaneously with the oxonic acid. All groups were evaluated throughout a 5-wk period. Kidneys were fixed by perfusion and afferent arteriole morphology, and tubulointerstitial 3-nitrotyrosine, 4-hydroxynonenal, NOX-4 subunit of renal NADPH-oxidase, and angiotensin II were quantified. Hyperuricemia induced intrarenal oxidative stress, increased expression of NOX-4 and angiotensin II, and decreased nitric oxide bioavailability, systemic hypertension, renal vasoconstriction, and afferent arteriolopathy. Tempol treatment reversed the systemic and renal alterations induced by hyperuricemia despite equivalent hyperuricemia. Moreover, because tempol prevented the development of preglomerular damage and decreased blood pressure, glomerular pressure was maintained at normal values as well. Mild hyperuricemia induced by uricase inhibition causes intrarenal oxidative stress, which contributes to the development of the systemic hypertension and the renal abnormalities induced by increased uric acid. Scavenging of the superoxide anion in this setting attenuates the adverse effects induced by hyperuricemia. superoxide; glomerular hypertension; arteriolopathy; tempol; uric acid

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and [H.sub.2][O.sub.2]. Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70. arterial injury; oxidative stress; extracellular signal-regulated kinase

Many vascular diseases are characterized by increased levels of ROS that destroy the biological activity of nitric oxide and limit cGMP formation. In the present study, we investigated the cGMP-forming ability of HMR-1766 in cells exposed to oxidative stress. Pretreatment of smooth muscle cells with [H.sub.2][O.sub.2] reduced cGMP production stimulated by sodium nitroprusside (SNP) or BAY 41-2272. However, pretreatment with [H.sub.2][O.sub.2] significantly increased HMR-1766 responses. Similar results were obtained with SIN-l, menadione, and rotenone. In addition, HMR-1766 was more effective in stimulating heme-free sGC compared with the wild-type enzyme. Interestingly, in cells expressing heme-free sGC, [H.sub.2][O.sub.2] inhibited instead of potentiated HMR-1766 responses, suggesting that the ROS-induced enhancement of cGMP formation was heme dependent. Moreover, using truncated forms of sGC, we observed that the N[H.sub.2]-terminus of the [[beta].sub.1]-subunit is required for the action of HMR-1766. Finally, to study tolerance development to HMR-1766, cells were pretreated with this sGC activator and reexposed to HMR-1766 or SNP. Results from these experiments demonstrated lack of tolerance development to HMR-1766 as well as lack of cross-tolerance with SNP. We conclude that HMR-1766 is an improved sGC activator as it has the ability to activate oxidized/heme-free sGC and is resistant to the development of tolerance; these observations make HMR-1766 a promising agent for treating diseases associated with increased vascular tone combined with enhanced ROS production. reactive oxygen species; cGMP; nitric oxide; soluble guanylyl cyclase

Multiparity is associated with increased risk of cardiovascular disease. We tested whether multiparity induces oxidative stress in rat vascular tissue. Coronary arteries and thoracic aorta were isolated from multiparous and age-matched virgin rats. Relaxation to ACh and sodium nitroprusside (SNP) was measured by wire myography. We also tested the effect of the superoxide dismutase mimetic MnTE2PyP (30 [micro]M), the NADPH oxidase inhibitor apocynin (10 [micro]M), and the peroxynitrite scavenger FeTPPs (10 [micro]M) on ACh-mediated relaxation in coronary arteries. Vascular superoxide anion was measured using the luminol derivative L-012 and nitric oxide (NO) generation by the Griess reaction. Multiparity reduced maximal response and sensitivity to ACh in coronary arteries [maximal relaxation ([E.sub.max]: multiparous 49 [+ or -] 3% vs. virgins 95% [+ or -] 3%; ECso: multiparous 135 [+ or -] 1 nM vs. virgins 60 [+ or -] 1 nM], and in aortic rings ([E.sub.max]: multiparous 38 [+ or -] 3% vs. virgins 79 [+ or -] 4%; [EC.sub.50]: multiparous 160 [+ or -] 2 nM vs. virgins 90 [+ or -] 3 nM). Coronary arteries from the two groups relaxed similarly to SNP. Superoxide anions formation was significantly higher in both coronary arteries (2.8-fold increase) and aorta (4.1-fold increase) from multiparous rats compared with virgins. In multiparous rats, incubation with MnTE2PyP, apocynin, and FeTPPs improved maximal relaxation to ACh (MnTE2PyP: 74 [+ or -] 5%; vehicle: 41 [+ or -] 5%; apocynin: 73 [+ or -] 3% vs. vehicle: 41 [+ or -] 3%: FeTPPs: 72 [+ or -] 3% vs. vehicle: 46 [+ or -] 3%) and increased sensitivity ([EC.sub.50]: MnTE2PyP: 61 [+ or -] 0.5 nM vs. vehicle: 91 [+ or -] 1 nM; apocynin: 45 [+ or -] 3 nM vs. vehicle: 91 [+ or -] 6 nM; FeTPP: 131 [+ or -] 2 nM vs. vehicle: 185 [+ or -] 1 nM). Multiparity also reduced total nitrate/nitrite levels (multiparous: 2.5 [+ or -] 2 [micro]mol/mg protein vs. virgins: 7 [+ or -] 1 [micro]mol/mg protein) and endothelial nitric oxide synthase protein levels (multiparous: 0.53 [+ or -] 0.1 protein/actin vs. virgins: 1.0 [+ or -] 0.14 protein/actin). These data suggest that multiparity induces endothelial dysfunction through decreased NO bioavailability and increased reactive oxygen species formation. nitric oxide synthase; sodium nitroprusside; [N.sup.G]-nitro-L-arginine methyl ester

Hyperlcplincmia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide ([O.sup.-.sub.2]), and peroxynitrite ([ONOO.sup.-]) nanosensors were placed near the surface (5 [+ or -] 2 [micro]m) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI)-stimulated NO, [O.sup.-.sub.2], [ONOO.sup.-] were recorded. Endothelial NO synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did not directly stimulate NO, [O.sup.-.sub.2], or [ONOO.sup.-] release from HUVEC. However, a 12-h exposure of HUVEC to leptin increased eNOS expression and Cal-stimulated NO (625 [+ or -] 30 vs. 500 [+ or -] 24 nmol/l control) and dramatically increased cytotoxic [O.sup.-.sub.2] and [ONOO.sup.-] levels. The [NO]-to-[[ONOO.sup.-]] ratio ([NO]/[[ONOO.sup.-]) decreased from 2.0 [+ or -] 0.1 in normal to 1.30 [+ or -] 0.1 in leptin-induced dysfunctional endothelium. In obese mice, a 2.5-fold increase in leptin concentration coincided with 100% increase in eNOS and about 30% decrease in intracellular L-arginine. The increased eNOS expression and a reduced L-arginine content led to eNOS uncoupling, a reduction in bioavailable NO (250 [+ or -] 10 vs. 420 [+ or -] 12 mnol/l control), and an elevated concentration of [O.sub.2] (240%) and [ONOO.sup.-] (70%). L-Arginine and sepiapterin supplementation reversed eNOS uncoupling and partially restored [NO]/[[ONOO.sup.-]] balance in obese mice. In obesity, leptin increases eNOS expression and decreases intracellular L-arginine, resulting in eNOS an uncoupling and depletion of endothelial NO and an increase of cytotoxic [ONOO.sup.-]. Hyperleptinemia triggers an endothelial NO/[ONOO.sup.-] imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e., atherosclerosis and diabetes. nitric oxide; endothelium; leptin; nitroxidative stress

Regulation of coronary function in diabetic hearts is an important component in preventing ischemic cardiac events but remains poorly studied. Exercise is recommended in the management of diabetes, but its effects on diabetic coronary function are relatively unknown. We investigated coronary artery myogenic tone and endothelial function, essential elements in maintaining vascular fluid dynamics in the myocardium. We hypothesized that exercise reduces pressure-induced myogenic constriction of coronary arteries while improving endothelial function in db/db mice, a model of type 2 diabetes. We used pressurized mouse coronary arteries isolated from hearts of control and db/db mice that were sedentary or exercised for 1 h/day on a motorized exercise-wheel system (set at 5.2 m/day, 5 days/wk). Exercise caused a ~10% weight loss in db/db mice and decreased whole body oxidative stress, as measured by plasma 8-isoprostane levels, but failed to improve hyperglycemia or plasma insulin levels. Exercise did not alter myogenic regulation of arterial diameter stimulated by increased transmural pressure, nor did it alter smooth muscle responses to U-46619 (a thromboxane agonist) or sodium nitroprusside (an endothelium-independent dilator). Moderate levels of exercise restored ACh-simulated, endothelium-dependent coronary artery vasodilation in db/db mice and increased expression of Mn SOD and decreased nitrotyrosine levels in hearts of db/db mice. We conclude that the vascular benefits of moderate levels of exercise were independent of changes in myogenic tone or hyperglycemic status and primarily involved increased nitric oxide bioavailability in the coronary microcirculation. coronary artery; manganese superoxide dismutase; diabetes; endothelium; oxidative stress

Fibroblasts are key structural cells that can be damaged by cigarette smoke. Cigarette smoke contains many components capable of eliciting oxidative stress, which may induce heine oxygenase (HO)-1, a cytoprotective enzyme. There are no data on HO-1 expression in primary human lung fibroblasts after cigarette smoke extract (CSE) exposure. We hypothesized that human lung fibroblasts exposed to cigarette smoke would increase HO-I though changes in intracellular glutathione (GSH). Primary human lung fibroblasts were exposed to CSE, and changes in HO-1 expression and GSH levels were assessed. CSE induced a time- and dose-dependent increase in expression of HO-1, but not HO-2 or biliverdin reductase, in two different primary human lung fibroblast strains, a novel finding. This induction of HO-1 paralleled a decrease in intracellular GSH, and a sustained reduction in GSH resulted in a dramatic increase in HO-1. Treatment with the antioxidants N-acetyl-L-cysteine or GSH reduced the expression of HO-I induced by CSE. We also examined the signal transduction mechanism responsible for HO-1 induction. Nuclear factor erythroid-derived 2, like 2 (Nrf2) was not involved in HO-1 induction by CSE. Activator protein-1 (AP-1) is a redox-sensitive transcription factor shown in other systems to regulate HO-1 expression. CSE exposure resulted in nuclear accumulation of c-Fos and c-Jun, two key AP-1 components. Reduction of c-Fos and c-Jun nuclear translocation by SP-600125 attenuated the CSE-induced expression of HO-1. These data support the concept that changes in the cellular redox status brought on by cigarette smoke induce HO-1 in fibroblasts. This increase in HO-1 may help protect against cigarette smoke-induced inflammation and/or cell death. oxidative stress; activator protein-l; biliverdin reductase; chronic obstructive pulmonary disease; nuclear factor erythroid-derived 2, like 2

Cellular localization determines whether the serine protease HtrA2 exerts pro- or antiapoptotic functions. In contrast to the well-characterized proapoptotic function of cytosolic HtrA2, mechanisms underlying the mitochondrial protective role are poorly understood. Mpv17l is a transmembrane protein previously implicated in peroxisomal reactive oxygen species metabolism and a close homolog of the inner mitochondrial membrane protein Mpv17. Here we demonstrate a previously undescribed direct interaction between Mpv17l and HtrA2 in mitochondria. The interaction is mediated by a PDZ domain and induces protease activation of HtrA2. HtrA2 inhibits mitochondrial superoxide generation, stabilizes mitochondrial membrane potential, and prevents apoptosis at baseline and in response to extracellular inducers of mitochondrial stress. The physiological role of Mpv17l is underscored by the finding that oxidative stress-induced down-regulation of Mpv17l is a consistent feature in renal injury models. Our findings identify Mpv17l as a unique interacting protein and regulator of HtrA2 protease mediating antioxidant and anti-apoptotic function in mitochondria. reactive oxygen species | proximal tubular epithelial cells | kidney injury | transforming growth factor beta | hypoxia

Major life history traits, such as fecundity and survival, have been consistently demonstrated to covary positively in nature, some individuals having more resources than others to allocate to all aspects of their life history. Yet, little is known about which resources (or state variables) may account for such covariation. Reactive oxygen species (ROS) are natural by-products of metabolism and, when ROS production exceeds antioxidant defenses, organisms are exposed to oxidative stress that can have deleterious effects on their fecundity and survival. Using a wild, long-lived bird, the Alpine Swift (Apus melba), we examined whether individual red cell resistance to oxidative stress covaried with fecundity and survival. We found that males that survived to the next breeding season tended to be more resistant to oxidative stress, and females with higher resistance to oxidative stress laid larger clutches. Furthermore, the eggs of females with low resistance to oxidative stress were less likely to hatch than those of females with high resistance to oxidative stress. By swapping entire clutches at clutch completion, we then demonstrated that hatching failure was related to the production of low-quality eggs by females with low resistance to oxidative stress, rather than to inadequate parental care during incubation. Although male and female resistance to oxidative stress covaried with age, the relationships among oxidative stress, survival, and fecundity occurred independently of chronological age. Overall, our study suggests that oxidative stress may play a significant role in shaping fecundity and survival in the wild. It further suggests that the nature of the covariation between resistance to oxidative stress and life history traits is sex specific, high resistance to oxidative stress covarying primarily with fecundity in females and with survival in males. Key words: Alpine Swift; annual survival; antioxidant defenses; Apus melba; fecundity; fitness traits; life history; oxidative theory of aging; sex-specific effect.

Both short-term and long-term nitric oxide (NO) blockade were shown to cause an increase in [O.sup.-.sub.2] activity. To assess the contribution of such enhanced [O.sup.-.sub.2] activity in the kidney, responses to administration of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 200 [micro]g x [min.sup.-1] x kg body [wt.sup.-1]) were assessed in knockout mice the lacking NAD(P)H oxidase subunit [gp91.sup.phox] (KO; n = 10) and in wild-type (WT; n = 10) mice. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearances, respectively. Baseline RBF was higher in KO compared with WT mice (5.8 [+ or -] 0.5 vs. 4.5 [+ or -] 0.3 ml x [min.sup.-1] x [g.sup.-1]; p < 0.04) without significant differences in GFR (0.62 [+ or -] 0.04 vs. 0.73 [+ or -] 0.05 ml x [min.sup.-1] x [g.sup.-1]) and in mean arterial pressure (MAP; 91 [+ or -] 6 vs. 88 [+ or -] 4 mmHg). L-NAME infusion for 60 min caused similar increases in MAP (114 [+ or -] 6 vs. 113 [+ or -] 3 mmHg) in both groups but resulted in a lesser degree of reduction in RBF in KO compared with WT mice (-7 [+ or -] 3 vs. -17 [+ or -] 3%; P < 0.02), although GFR remained unchanged in both groups. The natriuretic response to systemic L-NAME infusion was attenuated in KO compared with WT mice ([DELTA]: 3.1 [+ or -] 0.7 vs. 5.2 [+ or -] 0.6 [micro]mol x [min.sup.-1] x [g.sup.-1]). L-NAME increased urinary 8-isoprostane excretion rate in WT (5.9 [+ or -] 1 to 7.7 [+ or -] 1 pg x [min.sup.-1] x [g.sup.-1]; P < 0.02) but not in KO mice (5.6 [+ or -] 1 to 4.9 [+ or -] 0.3 pg x [min.sup.-1] x [g.sup.-1]). In contrast, responses to another vasoconstrictor, norepinephrine, were similar in both strains of mice. These data indicate that activation of NAD(P)H oxidase results in the enhancement of [O.sup.-.sub.2] activity that influences renal hemodynamics and excretory function in the condition of NO deficiency. superoxide; oxidative stress; renal blood flow; sodium excretion; 8-isoprostane excretion

Reactive oxygen species have emerged as important molecules in cardiovascular dysfunction such as diabetes and hypertension. Recent work has shown that oxidative stress and angiotensin II signaling mutually regulate each other by multiple mechanisms and contribute to the development of hypertension. Most of the known biological actions of angiotensin II can be attributed to AT1 receptors. The present study was carried out to investigate the role of renal AT1 receptor signaling in oxidative stress-mediated hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol (an antioxidant) for 2 wk. Compared with control rats, BSO-treated rats exhibited increased oxidative stress and reduced antioxidant levels and developed hypertension. BSO treatment also caused increased renal proximal tubular AT1 receptor protein abundance, message levels, and ligand binding. In these rats, angiotensin II caused significantly higher accumulation of inositol trisphosphate (IP3) and phospholipase C (PLC) activation which was sensitive to blockade by AT1 but not to AT2 antagonist. Also, angiotensin II-mediated, ATl-dependent MAP kinase, Na-K-ATPase, and Na/H exchanger 3 activation was higher in BSO-treated rats than in control rats. Tempol supplementation of BSO-treated rats restored redox status, normalized AT1 receptor expression, and decreased blood pressure. Tempol also normalized the angiotensin II-mediated, AT1-dependent IP3 accumulation and PLC, MAP kinase, Na-K-ATPase, and Na/H exchanger 3 stimulation. These data suggest that oxidative stress leads to AT1 receptor upregulation, which in turn causes overstimulation of sodium transporters and subsequently contributes to sodium retention and hypertension. Tempol, while reducing oxidative stress, normalizes AT1 receptor signaling and decreases blood pressure. L-buthionine sulfoximine; MAP kinase; Na-K-ATPase; Na/H exchanger; tempol

The objective of this study was to determine whether patients with chronic obstructive lung disease (COPD) display differences in organization of the metabolic pathways and segments involved in energy supply compared with healthy control subjects. Metabolic pathway potential, based on the measurement of the maximal activity ([V.sub.max]) of representative enzymes, was assessed in tissue extracted from the vastus lateralis in seven patients with COPD (age 67 [+ or -] 4 yr; [FEV.sub.1]/FVC = 44 [+ or -] 3%, where [FEV.sub.1] is forced expiratory volume in 1 s and FVC is forced vital capacity; means [+ or -] SE) and nine healthy age-matched controls (age 68 [+ or -] 2 yr; [FEV.sub.1]/FVC = 75 [+ or -] 2%). Compared with control, the COPD patients displayed lower (P < 0.05) [V.sub.max] (mol x kg [protein.sup.-1] x [h.sup.-1]) for cytochrome c oxidase (COX; 21.2 [+ or -] 2.0 vs. 28.7 [+ or -] 2.2) and 3-hydroxyacyl-CoA dehydrogenase (HADH; 2.54 [+ or -] 0.14 vs. 3.74 [+ or -] 0.12) but not citrate synthase (CS; 2.20 [+ or -] 0.16 vs. 3.19 [+ or -] 0.5). While no differences between groups were observed in [V.sup.max] for creatine phosphokinase, phosphorylase (PHOSPH), phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase, hexokinase (HEX) was elevated in COPD (P < 0.05). Enzyme activity ratios were higher (P < 0.05) for HEX/CS, HEX/COX, PHOSPH/HADH and PFK/ HADH in COPD compared with control. It is concluded that COPD patients exhibit a reduced potential for both the electron transport system and fat oxidation and an increased potential for glucose phosphorylation while the potential for glycogenolysis and glycolysis remains normal. A comparison of enzyme ratios indicated greater potentials for glucose phosphorylation relative to the citric acid cycle and the electron transport chain and glycogenolysis and glycolysis relative to [beta]-oxidation. lung disease; skeletal muscle; enzymes; oxidative; glycolytic.

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis. vascular endothelial function; oxidative stress

It has been shown that reactive oxygen species (ROS) contribute to the central effect of ANG II on blood pressure (BP). Recent studies have implicated an antihypertensive action of estrogen in ANG II-infused female mice. The present study used in vivo telemetry recording and in vitro living mouse brain slices to test the hypothesis that the central activation of estrogen receptors in male mice inhibits ANG II-induced hypertension via the modulation of the central ROS production. In male wild-type mice, the systemic infusion of ANG II induced a significant increase in BP ([DELTA]30.1 [+ or -] 2.5 mmHg). Either central infusion of Tempol or 17[beta]-estradiol ([E.sub.2]) attenuated the pressor effect of ANG II ([DELTA]10.9 [+ or -] 2.3 and [DELTA]4.5 [+ or -] 1.4 mmHg), and the protective effect of [E.sub.2] was prevented by the coadministration of an estrogen receptor, antagonist ICI-182780 ([DELTA]23.6 [+ or -] 3.1 mmHg). Moreover, the ganglionic blockade on day 7 after the start of ANG II infusions resulted in a smaller reduction of BP in central Tempol- and in central [E.sub.2]-treated males, suggesting that estrogen inhibits the central ANG II-induced increases in sympathetic outflow. In subfornical organ slices, the application of ANG II resulted in a 21.5 [+ or -] 2.5% increase in ROS production. The coadministration of irbesartan, an ANG II type 1 receptor antagonist, or the preincubation of brain slices with Tempol blocked ANG II-induced increases in ROS production (-1.8 [+ or -] 1.6% and -1.0 [+ or -] 1.8%). The ROS response to ANG II was also blocked by [E.sub.2] (-3.2 [+ or -] 2.4%). The results suggest that the central actions of [E.sub.2] are involved in the protection from ANG II-induced hypertension and that estrogen modulation of the ANG II-induced effects may involve interactions with ROS production. sex hormone; blood pressure; oxidative stress; subfornical organ

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and [H.sub.2][O.sub.2] were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from ~50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and [H.sub.2][O.sub.2] were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and [H.sub.2][O.sub.2] concentrations (2,572 [+ or -] 241 vs. 1,059 [+ or -] 160 nM, P < 0.01; and 26 [+ or -] 7 vs. 7 [+ or -] 1 [micro]M, P < 0.05, respectively). With flow elevation, [H.sub.2][O.sub.2] but not NO increased in SHR; NO but not [H.sub.2][O.sub.2] was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and [H.sub.2][O.sub.2] to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR [H.sub.2][O.sub.2] to WKY levels. Addition of topical [H.sub.2][O.sub.2] to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR. hypertension; oxidative stress; flow mediated; NAD(P)H oxidase inhibition; spontaneously hypertensive rats

Heat shock proteins (Hsps) are protective in models of transplantation, yet practical strategies to upregulate them remain elusive. The heat shock protein 90-binding agent (HBA) geldanamycin and its analogs (17-AAG and 17-DMAG) are known to upregulate Hsps and confer cellular protection but have not been investigated in a model relevant to transplantation. We examined the ability of HBAs to upregulate Hsp expression and confer protection in renal adenocarcinoma (ACHN) cells in vitro and in a mouse model of kidney ischemiareperfusion (I/R) injury. Hsp70 gene expression was increased 30-40 times in ACHN cells treated with HBAs, and trimerization and DNA binding of heat shock transcription factor-I (HSF1) were demonstrated. A three- and twofold increase in Hsp70 and Hsp27 protein expression, respectively, was found in ACHN cells treated with HBAs. HBAs protected ACHN cells from an [H.sub.2][O.sub.2]-mediated oxidative stress, and HSF1 short interfering RNA was found to abrogate HBA-mediated Hsp induction and protection. In vivo, Hsp70 was upregulated in the kidneys, liver, lungs, and heart of HBA-treated mice. This was associated with a functional and morphological renal protection from I/R injury. Therefore, HBAs mediate upregulation of protective Hsps in mouse kidneys which are associated with reduced I/R injury and may be useful in reducing transplant-associated kidney injury. kidney transplantation; ischemia-reperfusion injury; oxidative stress; geldanamycin

Hyperoxaluric kidneys show an impaired diuretic response to acute infusion of L-arginine. In this study, we examined the chronic effect of L-arginine supplementation on CaOx crystal formation in hyperoxaluric rat kidneys. Eight groups were tested: control (received drinking water), L group (received L-arginine, 0.6%), LN group [received [N.sup.G]-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg)], L + LN group (received L-arginine + L-NAME), HP group [received hydroxyl-L-proline (HP, 5%) mixed with chow to induce hyperoxaluria], L + HP group (received HP + t.-arginine), HP + LN group, and L + HP + LN group. The duration was 42 days, and each group had eight animals. Urinary biochemistry and renal CaOx amounts were measured, as well as renal expressions of nitric oxide synthase (NOS) isoforms and NAD(P)H oxidase. The distribution of inducible NOS (iNOS), NAD(P)H oxidase, ED1-positive cells, and nitrotyrosine was examined by immunohistochemical and immunofluorescence studies, whereas superoxide production from the kidneys was examined by fluorescence spectrometric assay. Compared with the HP group, the L + HP group had excessive CaOx crystal accumulation and enhanced endothelial NOS (eNOS), iNOS, and NAD(P)H oxidase protein expression in the kidney. Urinary excretion of nitrotyrosine was markedly increased. Increased superoxide formation in the L + HP kidney was derived from NAD(P)H oxidase and uncoupled eNOS, and increased nitrotyrosine formation might derive from iNOS and EDl-positive cells that gathered around the CaOx crystals. L-NAME cotreatment (L + HP + LN group) reduced renal oxidative nitrosative stress and tubular damage, which were induced by L + HP. The results showed that chronic L-arginine treatment to the hyperoxaluric kidney with massive CaOx crystal deposition may have a toxic effect by enhancing intrarenal oxidative and nitrosative stress. hyperoxaluria; nephrolithiasis; NAD(P)H oxidase; nitric oxide synthase

Previously, we found that high intraluminal pressure leads to production of reactive oxygen species (ROS) and also upregulates several components of the renin-angiotensin system in the wall of small arteries. We hypothesized that acute exposure of arterioles to high intraluminal pressure in vitro via increasing ROS production enhances the functional availability of type 1 angiotensin II (Ang II) receptors (AT1 receptors), resulting in sustained constrictions. In arterioles (~180 [micro]m) isolated from rat skeletal muscle, Ang II elicited dose-dependent constrictions, which decreased significantly by the second application [maximum (max.): from 59% [+ or -] 4% to 26% [+ or -] 5% at [10.sup.-8] M; P < 0.05] in the presence of 80 mmHg of intraluminal pressure. In contrast, if the arterioles were exposed to high intraluminal pressure (160 mmHg for 30 min), Ang II-induced constrictions remained substantial on the second application (max.: 51% [+ or -] 3% at [10.sup.-8] M). In the presence of Tiron and polyethylene glycol (PEG)-catalase, known to reduce the level of superoxide anion and hydrogen peroxide ([H.sub.2][O.sub.2]), second applications of Ang II evoked similarly reduced constrictions, even after high-pressure exposure (29% [+ or -] 4% at [10.sup.-8] M). Furthermore, when arterioles were exposed to [H.sub.2][O.sub.2] (for 30 min, [10.sup.-7] M, at normal 80 mmHg pressure), Ang II-induced constrictions remained substantial on second applications (59% [+ or -] 5% at [10.sup.-8] M). These findings suggest that high pressure, likely via inducing [H.sub.2][O.sub.2] production, increases the functional availability of AT1 receptors and thus enhances Ang II-induced arteriolar constrictions. We propose that in hypertension-regardless of etiology-high intraluminal pressure, via oxidative stress, enhances the functional availability of AT1 receptors augmenting Ang II-induced constrictions. hypertension; oxidative stress; hydrogen peroxide

There is increasing evidence that TGF-[beta] family member cytokine bone morphogenetic protein (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to 10 ng/ml) in CAECs increased [[O.sub.2].sup.*-] and [H.sub.2][O.sub.2] generation, induced NF-[kappa]B activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased [[O.sub.2].sup.*-] production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation. systemic; oxidative stress; endothelial dysfunction; pulmonary

Lead is a ubiquitous environmental toxin that is capable of causing numerous acute and chronic illnesses. Population studies have demonstrated a link between lead exposure and subsequent development of hypertension (HTN) and cardiovascular disease. In vivo and in vitro studies have shown that chronic lead exposure causes HTN and cardiovascular disease by promoting oxidative stress, limiting nitric oxide availability, impairing nitric oxide signaling, augmenting adrenergic activity, increasing endothelin production, altering the renin-angiotensin system, raising vasoconstrictor prostaglandins, lowering vasodilator prostaglandins, promoting inflammation, disturbing vascular smooth muscle [Ca.sup.2+] signaling, diminishing endothelium-dependent vasorelaxation, and modifying the vascular response to vasoactive agonists. Moreover, lead has been shown to cause endothelial injury, impede endothelial repair, inhibit angiogenesis, reduce endothelial cell growth, suppress proteoglycan production, stimulate vascular smooth muscle cell proliferation and phenotypic transformation, reduce tissue plasminogen activator, and raise plasminogen activator inhibitor-1 production. Via these and other actions, lead exposure causes HTN and promotes arteriosclerosis, atherosclerosis, thrombosis, and cardiovascular disease. In conclusion, studies performed in experimental animals, isolated tissues, and cultured cells have provided compelling evidence that chronic exposure to low levels of lead can cause HTN, endothelial injury/dysfunction, arteriosclerosis, and cardiovascular disease. More importantly, these studies have elucidated the cellular and molecular mechanisms of lead's action on cardiovascular/renal systems, a task that is impossible to accomplish using clinical and epidemiological investigations alone. oxidative stress; renin-angiotensin-aldosterone system; endothelin; catecholamines; adrenergic system; nitric oxide; endothelial dysfunction; endothelial cell; vascular smooth muscle; heart; atherosclerosis; heparan sulfate proteoglycans; thrombosis; angiogenesis; calcium signaling; antioxidant system; cGMP

In general, blood pressure is higher in normotensive men than in age-matched women, and the prevalence of hypertension in men is also higher until after menopause, when the prevalence of hypertension increases for women. It is likely then that the mechanisms by which blood pressure increases in men and women with aging may be different. Although clinical trials to reduce blood pressure with antioxidants have typically not been successful in human cohorts, studies in male rats suggest that oxidative stress plays an important role in mediating hypertension. The exact mechanisms by which oxidative stress increases blood pressure have not been completely elucidated. There may be several reasons for the discrepancies between clinical and animal studies. In this review, the data obtained in selected clinical and animal studies are discussed, and the hypothesis is put forward that oxidative stress may not be as important in mediating hypertension in females as has been shown previously in male rats. Furthermore, it is likely that differences in genetics, age, length of time with hypertension, endothelial dysfunction, and sex are all factored in to modulate the responses to antioxidants in humans. As such, future clinical trials should be designed and powered to evaluate the effects of oxidative stress on blood pressure separately in men and women. [F.sub.2]-isoprostanes

Previously, we have reported that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in pulmonary arterial endothelial cells (PAECs) in response to hydrogen peroxide ([H.sub.2][O.sub.2]). Thus the objective of this study was to identify the cis-element(s) and transcription factor(s) responsible for oxidant-mediated downregulation of the eNOS gene. Initial promoter experiments in PAECs treated with [H.sub.2][O.sub.2] revealed a significant decrease in activity of a promoter fragment containing 840 bp of upstream sequence of the human eNOS gene fused to a luciferase reporter. However, a promoter construct containing only 640 bp of upstream sequence had a significantly attenuated response to [H.sub.2][O.sub.2] challenge. As the 840-bp promoter construct had a putative binding site for the transcription factor activator protein-1 (AP-1) that was lacking in the 640-bp construct, we evaluated the effect of [H.sub.2][O.sub.2] on promoter activity after mutation of the AP-l binding sequence (TGAGTCA at -661 to TGAGTtg in the 840-bp construct). Similar to the results seen with the 640 bp, the AP-1 mutant promoter had a significantly attenuated response to [H.sub.2][O.sub.2]. EMSA revealed decreased binding of AP-1 during [H.sub.2][O.sub.2] treatment. Supershift analysis indicated that the AP-I complex consisted of a c-Jun and FosB heterodimer. Furthermore, in vitro EMSA analysis indicated the c-Jun binding was significantly decreased after [H.sub.2][O.sub.2] exposure. Using chromatin immunoprecipitation analysis, we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to [H.sub.2][O.sub.2]. These data suggest a role of decreased AP-1 binding likely through c-Jun in the [H.sub.2][O.sub.2]mediated decrease in eNOS promoter activity. oxidative stress; gene regulation; pulmonary hypertension

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. D J-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of D J-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that D J-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that D J-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that D J-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of D J-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner. gene expression | oxidative stress | Parkinson's disease | translation

We have recently shown that aorta from streptozotocin (STZ)-induced diabetic rats and A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited decreased levels of inhibitory guanine nucleotide regulatory protein ([G.sub.i])[alpha] proteins. In the present studies, we investigated the implication of oxidative stress in the hyperglycemia/diabetes-induced decreased expression of the [G.sub.i][alpha] protein and adenylyl cyclase signaling in VSMCs by using antioxidants. The levels of [G.sub.i][alpha] proteins were significantly decreased in A10 VSMCs exposed to high glucose and in aortic VSMCs from STZ-diabetic rats compared with control cells and were restored to control levels by antioxidants. In addition, [.sup.111]Mn-tetralis(benzoic acid porphyrin) and uric acid, scavengers of peroxynitrite, and [N.sup.G]-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase but not catalase, also restored the high glucose-induced decreased expression of [G.sub.i][alpha] proteins to the control levels in A10 VSMCs. Furthermore, the enhanced production of superoxide anion (O[) and increased activity of NADPH oxidase in these cells were also restored to control levels by diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, the diminished inhibition of adenylyl cyclase activity by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentrations of GTP[gamma]S as well as the enhanced stimulation of adenylyl cyclase by stimulatory agonists in hyperglycemic cells were restored to control levels by antioxidant treatments. These results suggest that high glucose-induced decreased levels of [G.sub.i][alpha] proteins and associated signaling in A 10 VSMCs may be attributed to the enhanced oxidative stress due to augmented levels of peroxynitrite and not to [H.sub.2][O.sub.2]. G proteins; antioxidants; inhibitory guanine nucleotide regulatory protein

The transgenic (mRen2)27 (Ren2) rat overexpresses mouse renin in extrarenal tissues, causing increased local synthesis of ANG II, oxidative stress, and hypertension. However, little is known about the role of oxidative stress induced by the tissue renin-angiotensin system (RAS) as a contributing factor in puhnonary hypertension (PH). Using male Ren2 rats, we test the hypothesis that lung tissue RAS overexpression and resultant oxidative stress contribute to PH and pulmonary vascular remodeling. Mean arterial pressure (MAP), right ventricular systolic pressure (RVSP), and wall thickness of small pulmonary arteries (PA), as well as intrapulmonary NADPH oxidase activity and subunit protein expression and reactive oxygen species (ROS), were compared in age-matched Ren2 and Sprague-Dawley (SD) rats pretreated with the SOD/catalase mimetic tempol for 21 days. In placebo-treated Ren2 rats, MAP and RVSP, as well as intrapulmonary NADPH oxidase activity and subunits (Nox2, [p22.sup.phox], and Rac- 1) and ROS, were elevated compared with placebo-treated SD rats (P < 0.05). Tempol decreased RVSP (P < 0.05), but not MAP, in Ren2 rats. Tempol also reduced intrapulmonary NADPH oxidase activity, Nox2, [p22.sup.phox], and Rac-1 protein expression, and ROS in Ren2 rats (P < 0.05). Compared with SD rats, the cross-sectional surface area of small PA was 38% greater (P < 0.001) and luminal surface area was 54% less (P < 0.001) in Ren2 rats. Wall surface area was reduced and luminal area was increased in tempol-treated SD and Ren2 rats compared with untreated controls (P < 0.05). Collectively, the results of this investigation support a seminal role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling. renin; angiotensin II; NADPH oxidase

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient ([bcl-2.sup.-/-]) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from [bcl-2.sup.+/+] and [bcl.-2.sup.-/-] mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The [bcl-2.sup.-/-] retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from [bcl-2.sup.-/-] mice compared with [bcl-2.sup.+/+] mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in [bcl-2.sup.-/-] retinal EC. Mechanistically, [bcl-2.sup.-/-] cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from [bcl-2.sup.-/-] mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis. angiogenesis; apoptosis; endothelial nitric oxide; oxidative stress