3-nitrotyrosine and Hypoxia

"Live High-Train Low" (LHTL) training can alter oxidative status of athletes. This study compared prooxidant/antioxidant balance responses following two LHTL protocols of the same duration and at the same living altitude of 2250 m in either normobaric (NH) or hypobaric (HH) hypoxia. Twenty-four well-trained triathletes underwent the following two 18-day LHTL protocols in a cross-over and randomized manner: Living altitude (PIO2 = 111.9 ± 0.6 vs. 111.6 ± 0.6 mmHg in NH and HH, respectively); training "natural" altitude (~1000-1100 m) and training loads were precisely matched between both LHTL protocols. Plasma levels of oxidative stress [advanced oxidation protein products (AOPP) and nitrotyrosine] and antioxidant markers [ferric-reducing antioxidant power (FRAP), superoxide dismutase (SOD) and catalase], NO metabolism end-products (NOx) and uric acid (UA) were determined before (Pre) and after (Post) the LHTL. Cumulative hypoxic exposure was lower during the NH (229 ± 6 hrs.) compared to the HH (310 ± 4 hrs.; P<0.01) protocol. Following the LHTL, the concentration of AOPP decreased (-27%; P<0.01) and nitrotyrosine increased (+67%; P<0.05) in HH only. FRAP was decreased (-27%; P<0.05) after the NH while was SOD and UA were only increased following the HH (SOD: +54%; P<0.01 and UA: +15%; P<0.01). Catalase activity was increased in the NH only (+20%; P<0.05). These data suggest that 18-days of LHTL performed in either NH or HH differentially affect oxidative status of athletes. Higher oxidative stress levels following the HH LHTL might be explained by the higher overall hypoxic dose and different physiological responses between the NH and HH.

Topics: Adult; Advanced Oxidation Protein Products; Humans; Hypoxia; Male; Oxidative Stress; Physical Fitness; Superoxide Dismutase; Tyrosine; Uric Acid

Both acute hypoxia and physical exercise are known to increase oxidative stress. This randomized prospective trial investigated whether the addition of moderate exercise can alter oxidative stress induced by continuous hypoxic exposure.. Fourteen male participants were confined to 10-d continuous normobaric hypoxia (FIO2 = 0.139 ± 0.003, PIO2 = 88.2 ± 0.6 mm Hg, ∼4000-m simulated altitude) either with (HCE, n = 8, two training sessions per day at 50% of hypoxic maximal aerobic power) or without exercise (HCS, n = 6). Plasma levels of oxidative stress markers (advanced oxidation protein products [AOPP], nitrotyrosine, and malondialdehyde), antioxidant markers (ferric-reducing antioxidant power, superoxide dismutase, glutathione peroxidase, and catalase), nitric oxide end-products, and erythropoietin were measured before the exposure (Pre), after the first 24 h of exposure (D1), after the exposure (Post) and after the 24-h reoxygenation (Post + 1). In addition, graded exercise test in hypoxia was performed before and after the protocol.. Maximal aerobic power increased after the protocol in HCE only (+6.8%, P < 0.05). Compared with baseline, AOPP was higher at Post + 1 (+28%, P < 0.05) and nitrotyrosine at Post (+81%, P < 0.05) in HCS only. Superoxide dismutase (+30%, P < 0.05) and catalase (+53%, P < 0.05) increased at Post in HCE only. Higher levels of ferric-reducing antioxidant power (+41%, P < 0.05) at Post and lower levels of AOPP (-47%, P < 0.01) at Post + 1 were measured in HCE versus HCS. Glutathione peroxidase (+31%, P < 0.01) increased in both groups at Post + 1. Similar erythropoietin kinetics was noted in both groups with an increase at D1 (+143%, P < 0.01), a return to baseline at Post, and a decrease at Post + 1 (-56%, P < 0.05).. These data provide evidence that 2 h of moderate daily exercise training can attenuate the oxidative stress induced by continuous hypoxic exposure.

Topics: Adult; Advanced Oxidation Protein Products; Atmospheric Pressure; Catalase; Erythropoietin; Exercise; Exercise Test; Glutathione Peroxidase; Heart Rate; Humans; Hypoxia; Male; Malondialdehyde; Oxidative Stress; Oxygen; Physical Exertion; Superoxide Dismutase; Tyrosine; Young Adult

The aim of this study was to assess the role of the type 1 angiotensin II (AT(1)) receptor in the increase of oxidative stress and NO metabolism during a single 6 h exposure to intermittent hypoxia (IH). Nine healthy young men were exposed, while awake, to sham IH, IH with placebo medication, and IH with the AT(1) receptor antagonist, losartan, using a double-blind, placebo-controlled, randomized, crossover study design. In addition to blood pressure, oxidative stress, peroxynitrite activity, uric acid, global antioxidant status and the end-products of NO (NOx) metabolism were measured in plasma before and after 6 h of IH. Oxidative stress and peroxynitrite activity increased and NOx decreased during IH with placebo. In contrast, neither sham IH nor IH with losartan affected these parameters. With respect to each condition, blood pressure had the same profile as oxidative stress. These results demonstrate that blockade of AT(1) receptors prevented the increase in oxidative stress and peroxynitrite activity and the decrease in NO metabolism induced by IH. Finally, this study suggests that the renin-angiotensin system may participate in the overproduction of reactive oxygen species associated with IH by upregulation of the actions of angiotensin II.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Angiotensin II Type 1 Receptor Blockers; Cross-Over Studies; Deoxyguanosine; Double-Blind Method; Humans; Hypoxia; Losartan; Male; Nitric Oxide; Oxidative Stress; Tyrosine; Uric Acid

Indirect evidence implicates endothelial dysfunction in the pathogenesis of vascular diseases associated with obstructive sleep apnea (OSA). We investigated directly whether dysfunction and inflammation occur in vivo in the vascular endothelium of patients with OSA. The effects of continuous positive airway pressure (CPAP) therapy on endothelial function and repair capacity were assessed.. Thirty-two patients with newly diagnosed OSA and 15 control subjects were studied. Proteins that regulate basal endothelial nitric oxide (NO) production (endothelial NO synthase [eNOS] and phosphorylated eNOS) and inflammation (cyclooxygenase-2 and inducible NOS) and markers of oxidative stress (nitrotyrosine) were quantified by immunofluorescence in freshly harvested venous endothelial cells before and after 4 weeks of CPAP therapy. Vascular reactivity was measured by flow-mediated dilation. Circulating endothelial progenitor cell levels were quantified to assess endothelial repair capacity. Baseline endothelial expression of eNOS and phosphorylated eNOS was reduced by 59% and 94%, respectively, in patients with OSA compared with control subjects. Expression of both nitrotyrosine and cyclooxygenase-2 was 5-fold greater in patients with OSA than in control subjects, whereas inducible NOS expression was 56% greater. Expression of eNOS and phosphorylated eNOS significantly increased, whereas expression of nitrotyrosine, cyclooxygenase-2, and inducible NOS significantly decreased in patients who adhered to CPAP > or = 4 hours daily. Baseline flow-mediated dilation and endothelial progenitor cell levels were lower in patients than in control subjects, and both significantly increased in patients who adhered to CPAP > or = 4 hours daily.. OSA directly affects the vascular endothelium by promoting inflammation and oxidative stress while decreasing NO availability and repair capacity. Effective CPAP therapy is associated with the reversal of these alterations.

Topics: Adult; Biomarkers; Continuous Positive Airway Pressure; Cyclooxygenase 2; Endothelium, Vascular; Female; Humans; Hypoxia; Male; Middle Aged; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Severity of Illness Index; Sleep Apnea, Obstructive; Treatment Outcome; Tyrosine; Vasculitis; Vasodilation; Veins

Obstructive sleep apnea (OSA) in children is associated with cardiovascular morbidity such as systemic and pulmonary hypertension. However, it remains unclear whether endothelial dysfunction occurs in pediatric OSA and whether it is reversible on effective treatment of OSA.. Consecutive nonobese children (aged 6 to 11 years) who were diagnosed with OSA after overnight polysomnography and control children matched on the basis of age, gender, ethnicity, and body mass index underwent blood draw the next morning for soluble CD40 ligand, asymmetric dimethylarginine (ADMA), and nitrotyrosine levels, as well as 2 iterations of 60-second cuff-occlusion tests for assessment of endothelial function. These tests were repeated 4 to 6 months after adenotonsillectomy. OSA children showed blunted reperfusion kinetics after release of occlusion, which completely normalized in 20 of 26 patients after adenotonsillectomy. All 6 children in whom no improvements occurred had a strong family history of cardiovascular disease (versus 2 of the remaining 20 patients; P<0.04). Plasma nitrotyrosine and ADMA levels were similar in OSA and control children; however, soluble CD40 ligand levels were higher in OSA children and were reduced after treatment, particularly in those with normalized hyperemic responses.. Postocclusive hyperemia is consistently blunted in children with OSA, and such altered endothelial function is reversible 4 to 6 months after treatment, particularly if a family history of cardiovascular disease is not present. Although no evidence for either nitric oxide-dependent oxidative/nitrosative stress or for the increased presence of the circulating nitric oxide synthase inhibitor ADMA was found in children with OSA, soluble CD40 ligand levels were increased in OSA and reflected the changes in endothelial function after treatment.

Topics: Adenoidectomy; Arginine; Body Weight; CD40 Ligand; Child; Endothelium, Vascular; Female; Humans; Hyperemia; Hypoxia; Male; Nitric Oxide; Polysomnography; Sleep Apnea, Obstructive; Tonsillectomy; Tyrosine; Vasculitis

Endothelium-dependent hyperpolarization (EDH) plays important roles in the systemic circulation, whereas its role in the pulmonary circulation remains largely unknown. Furthermore, the underlying mechanisms of pulmonary hypertension (PH) also remain to be elucidated. We thus aimed to elucidate the role of EDH in pulmonary circulation in general and in PH in particular. In isolated perfused lung and using male wild-type mice, endothelium-dependent relaxation to bradykinin (BK) was significantly reduced in the presence of N

Topics: Animals; Biological Factors; Disease Models, Animal; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; Male; Membrane Potentials; Mice, Inbred C57BL; Microcirculation; Nitric Oxide; Pulmonary Artery; Pulmonary Circulation; Signal Transduction; Tyrosine; Vascular Remodeling; Vasodilation; Vasodilator Agents

Chronic intermittent hypoxia (CIH), main feature of obstructive sleep apnea, produces nitro-oxidative stress, which contributes to potentiate carotid body (CB) chemosensory discharges and sympathetic-adrenal-axis activity, leading to hypertension. The MnSOD enzymatic activity, a key enzyme on oxidative stress control, is reduced by superoxide-induced nitration. However, the effects of CIH-induced nitration on MnSOD enzymatic activity in the CB and adrenal gland are not known. We studied the effects of CIH on MnSOD protein and immunoreactive (MnSOD-ir) levels in the CB, adrenal gland and superior cervical ganglion (SCG), and on 3-nitrotyrosine (3-NT-ir), CuZnSOD (CuZnSOD-ir), MnSOD nitration, and its enzymatic activity in the CB and adrenal gland from male Sprague-Dawley rats exposed to CIH for 7 days. CIH increased 3-NT-ir in CB and adrenal gland, whereas MnSOD-ir increased in the CB and in adrenal cortex, but not in the whole adrenal medulla or SCG. CIH nitrated MnSOD in the CB and adrenal medulla, but its activity decreased in the adrenal gland. CuZnSOD-ir remained unchanged in both tissues. All changes observed were prevented by ascorbic acid treatment. Present results show that CIH for 7 days produced MnSOD nitration, but failed to reduce its activity in the CB, because of the increased protein level.

Topics: Adrenal Glands; Animals; Carotid Body; Hypoxia; Immunohistochemistry; Male; Nitrosative Stress; Oxidative Stress; Rats, Sprague-Dawley; Superoxide Dismutase; Tyrosine

Hyperglycemia-related oxidative stress and hypoxia are important mechanisms responsible for diabetes-induced embryopathy and other complications. High sucrose low copper diet (HSD), but not regular diet (RD), induces type 2 diabetes in the inbred Cohen diabetic sensitive (CDs) rats but not in the Sabra control rats. We recently demonstrated long-term changes of DNA methylation and gene expression in various groups of genes, including genes involved in oxidant-antioxidant activity in the liver of 2-4-week-old CDs offspring of diabetic dams. We now studied the postnatal effects of diabetes and/or HSD on several liver metabolic parameters in these offspring.. we studied lipid peroxidation, activity of the antioxidants enzymes superoxide dismutase (SOD) and Catalase (CAT). By immunohistochemistry: protein oxidation by nitrotyrosine staining, hypoxia inducing factor1α (HIF1α), apoptosis [caspase 3, bcl-2-like protein (BAX)], proliferation [proliferating cell nuclear antigen (PCNA)] and NF-κB.. In the Sabra rats fed HSD only few, early and transitional changes were observed in lipid peroxidation, SOD and CAT activity. In the CDs fed HSD more significant changes in lipid and protein oxidation, HIF1α, apoptosis and proliferation were observed, persisting for longer.. The changes in the Sabra rats HSD were attributed to the pro-oxidant effects of the diet and those in the diabetic CDs to the HSD and maternal diabetes. In light of the DNA methylation changes in the liver of the CDs HSD, we presume that changes in gene expression are responsible for our findings, and that similar changes may lead to the metabolic syndrome at adulthood.

Topics: Animals; Antioxidants; Apoptosis; Biomarkers; Catalase; Cell Proliferation; Copper; Diabetes Mellitus, Experimental; Diet; Female; Hypoxia; Liver; Malondialdehyde; NF-kappa B; Oxidative Stress; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Sucrose; Superoxide Dismutase; Tyrosine; Weight Gain

Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Chronic Disease; Enzyme Inhibitors; Free Radical Scavengers; Hypoxia; Lung; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Tyrosine; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

Nitric oxide (NO) is involved in neuronal modifications, and overproduction of NO contributes to memory deficits after acute hypobaric hypoxia-reoxygenation. This study investigated the ability of the iNOS inhibitor 1400W to counteract spatial memory deficits following acute hypobaric hypoxia-reoxygenation, and to affect expression of NOS, NO, 3-NT and MDA production, and apoptosis in rat cerebral cortex. We also used primary rat microglia to investigate the effect of 1400W on expression of NOS, NO, 3-NT and MDA production, and apoptosis. Acute hypobaric hypoxia-reoxygenation impaired spatial memory, and was accompanied by activated microglia, increased iNOS expression, NO, 3-NT and MDA production, and neuronal cell apoptosis in rat cerebral cortex one day post-reoxygenation. 1400W treatment inhibited iNOS expression without affecting nNOS or eNOS. 1400W also reduced NO, 3-NT and MDA production, and prevented neuronal cell apoptosis in cerebral cortex, in addition to reversing spatial memory impairment after acute hypobaric hypoxia-reoxygenation. Hypoxia-reoxygenation activated primary microglia, and increased iNOS and nNOS expression, NO, 3-NT, and MDA production, and apoptosis. Treatment with 1400W inhibited iNOS expression without affecting nNOS, reduced NO, 3-NT and MDA production, and prevented apoptosis in primary microglia. Based on the above findings, we concluded that the highly selective iNOS inhibitor 1400W inhibited iNOS induction in microglial cells, and reduced generation of NO, thereby mitigating oxidative stress and neuronal cell apoptosis in the rat cerebral cortex, and improving the spatial memory dysfunction caused by acute hypobaric hypoxia-reoxygenation.

Topics: Animals; Annexin A5; Apoptosis; Cells, Cultured; Cognition Disorders; Disease Models, Animal; Gene Expression Regulation; Hypoxia; Imines; Lipid Peroxidation; Malondialdehyde; Maze Learning; Microglia; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase Type II; Oxygen; Rats; Rats, Sprague-Dawley; Tyrosine

Independently, both inactivity and hypoxia augment oxidative stress. This study, part of the FemHab project, investigated the combined effects of bed rest-induced unloading and hypoxic exposure on oxidative stress and antioxidant status. Healthy, eumenorrheic women were randomly assigned to the following three 10-day experimental interventions: normoxic bed rest (NBR;n= 11; PiO2 = 133 mmHg), normobaric hypoxic bed rest (HBR;n= 12; PiO2 = 90 mmHg), and ambulatory hypoxic confinement (HAMB;n= 8: PiO2 = 90 mmHg). Plasma samples, obtained before (Pre), during (D2, D6), immediately after (Post) and 24 h after (Post+1) each intervention, were analyzed for oxidative stress markers [advanced oxidation protein products (AOPP), malondialdehyde (MDA), and nitrotyrosine], antioxidant status [superoxide dismutase (SOD), catalase, ferric-reducing antioxidant power (FRAP), glutathione peroxidase (GPX), and uric acid (UA)], NO metabolism end-products (NOx), and nitrites. Compared with baseline, AOPP increased in NBR and HBR on D2 (+14%; +12%;P< 0.05), D6 (+19%; +15%;P< 0.05), and Post (+22%; +21%;P< 0.05), respectively. MDA increased at Post+1 in NBR (+116%;P< 0.01) and D2 in HBR (+114%;P< 0.01) and HAMB (+95%;P< 0.05). Nitrotyrosine decreased (-45%;P< 0.05) and nitrites increased (+46%;P< 0.05) at Post+1 in HAMB only. Whereas SOD was higher at D6 (+82%) and Post+1 (+67%) in HAMB only, the catalase activity increased on D6 (128%) and Post (146%) in HBR and HAMB, respectively (P< 0.05). GPX was only reduced on D6 (-20%;P< 0.01) and Post (-18%;P< 0.05) in HBR. No differences were observed in FRAP and NOx. UA was higher at Post in HBR compared with HAMB (P< 0.05). These data indicate that exposure to combined inactivity and hypoxia impairs prooxidant/antioxidant balance in healthy women. Moreover, habitual activity levels, as opposed to inactivity, seem to blunt hypoxia-related oxidative stress via antioxidant system upregulation.

Topics: Adult; Antioxidants; Bed Rest; Biomarkers; Catalase; Female; Glutathione Peroxidase; Humans; Hypoxia; Malondialdehyde; Nitrites; Nitrogen Oxides; Oxidative Stress; Superoxide Dismutase; Tyrosine; Uric Acid; Women's Health

Oxidative stress is involved in the development of carotid body (CB) chemosensory potentiation and systemic hypertension induced by chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea. We tested whether peroxynitrite (ONOO(-)), a highly reactive nitrogen species, is involved in the enhanced CB oxygen chemosensitivity and the hypertension during CIH. Accordingly, we studied effects of Ebselen, an ONOO(-) scavenger, on 3-nitrotyrosine immunoreactivity (3-NT-ir) in the CB, the CB chemosensory discharge, and arterial blood pressure (BP) in rats exposed to CIH. Male Sprague-Dawley rats were exposed to CIH (5% O2, 12 times/h, 8 h/day) for 7 days. Ebselen (10 mg/kg/day) was administrated using osmotic minipumps and BP measured with radiotelemetry. Compared to the sham animals, CIH-treated rats showed increased 3-NT-ir within the CB, enhanced CB chemosensory responses to hypoxia, increased BP response to acute hypoxia, and hypertension. Rats treated with Ebselen and exposed to CIH displayed a significant reduction in 3-NT-ir levels (60.8 ± 14.9 versus 22.9 ± 4.2 a.u.), reduced CB chemosensory response to 5% O2 (266.5 ± 13.4 versus 168.6 ± 16.8 Hz), and decreased mean BP (116.9 ± 13.2 versus 82.1 ± 5.1 mmHg). Our results suggest that CIH-induced CB chemosensory potentiation and hypertension are critically dependent on ONOO(-) formation.

Topics: Animals; Azoles; Blood Pressure; Carotid Body; Diastole; Hypertension; Hypoxia; Isoindoles; Male; Neuronal Plasticity; Organoselenium Compounds; Peroxynitrous Acid; Rats, Sprague-Dawley; Systole; Tyrosine

Ischemia/reperfusion and the resulting oxidative/nitrative stress impair cerebral myogenic tone via actin depolymerization. While it is known that NADPH oxidase (Nox) family is a major source of vascular oxidative stress; the extent and mechanisms by which Nox activation contributes to actin depolymerization, and equally important, the relative role of Nox isoforms in this response is not clear.. To determine the role of Nox4 in hypoxia-mediated actin depolymerization and myogenic-tone impairment in cerebral vascular smooth muscle.. Control and Nox4 deficient (siRNA knock-down) human brain vascular smooth muscle cells (HBVSMC) were exposed to 30-min hypoxia/45-min reoxygenation. Nox2, Nox4, inducible and neuronal nitric oxide synthase (iNOS and nNOS) and nitrotyrosine levels as well as F:G actin were determined. Myogenic-tone was measured using pressurized arteriography in middle cerebral artery isolated from rats subjected to sham, 30-min ischemia/45-min reperfusion or ex-vivo oxygen glucose deprivation in the presence and absence of Nox inhibitors.. Nox4 and iNOS expression were significantly upregulated following hypoxia or ischemia/reperfusion. Hypoxia augmented nitrotyrosine levels while reducing F actin. These effects were nullified by inhibiting nitration with epicatechin or pharmacological or molecular inhibition of Nox4. Ischemia/reperfusion impaired myogenic-tone, which was restored by the selective inhibition of Nox4.. Nox4 activation in VSMCs contributes to actin depolymerization after hypoxia, which could be the underlying mechanism for myogenic-tone impairment following ischemia/reperfusion.

Topics: Actin Cytoskeleton; Actins; Animals; Cells, Cultured; Humans; Hypoxia; Membrane Glycoproteins; Middle Cerebral Artery; Muscle, Smooth, Vascular; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Reperfusion Injury; RNA, Small Interfering; Tyrosine; Up-Regulation

Chronic exposure to intermittent hypoxia (CIH) elicits plasticity of the carotid sinus and phrenic nerves via reactive oxygen species (ROS). To determine whether CIH-induced alterations in ventilation, metabolism, and heart rate are also dependent on ROS, we measured responses to acute hypoxia in conscious rats after 14 and 21 d of either CIH or normoxia (NORM), with or without concomitant administration of allopurinol (xanthine oxidase inhibitor), combined allopurinol plus losartan (angiotensin II type 1 receptor antagonist), or apocynin (NADPH oxidase inhibitor). Carotid body nitrotyrosine production was measured by immunohistochemistry. CIH produced an increase in the ventilatory response to acute hypoxia that was virtually eliminated by all three pharmacologic interventions. CIH caused a robust increase in carotid body nitrotyrosine production that was greatly attenuated by allopurinol plus losartan and by apocynin but unaffected by allopurinol. CIH caused a decrease in metabolic rate and a reduction in hypoxic bradycardia. Both of these effects were prevented by allopurinol, allopurinol plus losartan, and apocynin.

Topics: Acetophenones; Allopurinol; Analysis of Variance; Animals; Anti-Arrhythmia Agents; Antioxidants; Body Weight; Carbon Dioxide; Carotid Sinus; Catecholamines; Chemoreceptor Cells; Free Radical Scavengers; Heart Rate; Hypoxia; Losartan; Male; Oxidative Stress; Oxygen Consumption; Plethysmography; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Regression Analysis; Respiration; Tidal Volume; Time Factors; Tyrosine

It is well documented that the nitric oxide (NO) might be directly involved in brain response to hypobaric hypoxia, and could contribute to memory deficiencies. Recent studies have shown that melatonin could attenuate hypoxia or ischemia-induced nerve injuries by decreasing the production of free radicals. The present study, using immunohistochemical and immunoblot methods, aimed to explore whether melatonin treatment may affect the expression of nitric oxide system and protein nitration, and provide neuroprotection in the rat hippocampus injured by hypobaric hypoxia. Prior to hypoxic treatment, adult rats were pretreated with melatonin (100 mg/kg, i.p.) before they were exposed to the altitude chamber with 48 Torr of the partial oxygen concentration (pO2) for 7 h to mimic the ambience of being at 9000 m in height. They were then sacrificed after 0 h, 1, and 3 days of reoxygenation.. The results obtained from the immunohistochemical and immunoblotting analyses showed that the expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine (Ntyr) and Caspase 3 in the hypoxic hippocampus were increased from 0 h to 3 days of reoxygenation. Interestingly, the hypoxia-induced increase of nNOS, eNOS, iNOS, Ntyr and Caspase 3 protein expression was significantly depressed in the hypoxic rats treated with melatonin.. Activation of the nitric oxide system and protein nitration constitutes a hippocampal response to hypobaric hypoxia and administration of melatonin could provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaric hypoxia.

Topics: Altitude Sickness; Animals; Antioxidants; Caspase 3; Disease Models, Animal; Hippocampus; Hypoxia; Male; Melatonin; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Tyrosine

The present study was to explore the effect of metallothionein (MT) on intermittent hypoxia (IH) induced aortic pathogenic changes. Markers of oxidative damages, inflammation, and vascular remodeling were observed by immunohistochemical staining after 3 days and 1, 3, and 8 weeks after IH exposures. Endogenous MT was induced after 3 days of IH but was significantly decreased after 8 weeks of IH. Compared with the wild-type mice, MT knock-out mice exhibited earlier and more severe pathogenic changes of oxidative damages, inflammatory responses, and cellular apoptosis, as indicated by the significant accumulation of collagen, increased levels of connective tissue growth factor, transforming growth factor β1, tumor necrosis factor-alpha, vascular cell adhesion molecule 1,3-nitrotyrosine, and 4-hydroxy-2-nonenal in the aorta. These findings suggested that chronic IH may lead to aortic damages characterized by oxidative stress and inflammation, and MT may play a pivotal role in the above pathogenesis process.

Topics: Aldehydes; Animals; Aorta; Apoptosis; Connective Tissue Growth Factor; Hypoxia; Metallothionein; Mice; Mice, Knockout; NADPH Oxidases; Nitric Oxide Synthase Type III; Oxidative Stress; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

Topics: Animals; Antioxidants; Chronic Disease; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lipoproteins, LDL; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; NADPH Oxidases; Oxidative Stress; Pulmonary Artery; Reactive Oxygen Species; Scavenger Receptors, Class E; Signal Transduction; Tyrosine; Up-Regulation; Ventricular Function, Right; Ventricular Pressure

Hyperoxia causes oxidative stress. Breath-hold diving is associated with transient hyperoxia followed by hypoxia and a build-up of carbon dioxide (CO₂), chest-wall compression and significant haemodynamic changes. This study analyses variations in plasma oxidative stress markers after a series of repetitive breath-hold dives.. Thirteen breath-hold divers were asked to perform repetitive breath-hold dives to 20 metres' depth to a cumulative breath-hold time of approximately 20 minutes over an hour in the open sea. Plasma nitric oxide (NO), peroxinitrites (ONOO⁻) and thiols (R-SH) were measured before and after the dive sequence.. Circulating NO significantly increased after successive breath-hold dives (169.1 ± 58.26% of pre-dive values; P = 0.0002). Peroxinitrites doubled after the dives (207.2 ± 78.31% of pre-dive values; P = 0.0012). Thiols were significantly reduced (69.88 ± 19.23% of pre-dive values; P = 0.0002).. NO may be produced by physical effort during breath-hold diving. Physical exercise, the transient hyperoxia followed by hypoxia and CO₂ accumulation would all contribute to the increased levels of superoxide anions (O₂²⁻). Since interaction of O₂²⁻ with NO forms ONOO⁻, this reaction is favoured and the production of thiol groups is reduced. Oxidative stress is, thus, present in breath-hold diving.

Topics: Adult; Biomarkers; Breath Holding; Diving; Humans; Hyperoxia; Hypoxia; Male; Nitric Oxide; Nitrites; Oxidative Stress; Sulfhydryl Compounds; Time Factors; Tyrosine

Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2',7'-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.

Topics: Animals; Blood-Brain Barrier; Cell Membrane; Claudin-5; Endothelial Cells; Guinea Pigs; Hypoxia; Microvessels; Mitochondria; Oxidative Stress; Permeability; Reactive Oxygen Species; Reperfusion Injury; Tight Junction Proteins; Tight Junctions; Tyrosine; Zonula Occludens-1 Protein

Chronic liver diseases are commonly associated with tissue hypoxia that may cause inflammation, oxidative stress, liver cell injury and increased nuclear transcriptional regulation. The hepatic response to chronic hypoxia at the molecular level has not yet been clearly understood until now. The aim of this study is to investigate whether nuclear transcription factors [hypoxia-inducible factor-1 (HIF-1α), activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB)] exhibit activity changes during hepatic response to chronic hypoxia. Blood and liver samples were collected from adult Sprague-Dawley rats living in atmospheric air or 10% oxygen for four weeks. Levels of serum alanine aminotransferase (ALT), 8-isoprostane and nitrotyrosine were measured. The activities of nuclear transcription factors and the expression of downstream genes (iNOS, eNOS, ET-1 and VEGF) were measured using RT-PCR, Western blotting and Gel shift analysis. Results showed that serum ALT level, 8-isoprostane level and formation of nitrotyrosine were within normal range at all time-points. In the hypoxic liver, DNA-binding activities of HIF-1α, NF-κB and AP-1 increased significantly. Expression levels of iNOS, VEGF and ET-1 progressively increased from day 7 to day 28. eNOS was also elevated in the hypoxic liver. In conclusion, our study suggests that increased activity of HIF-1α, AP-1 and NF-κB may partly play a significant role in the hepatic response to oxidative stress and liver injury under chronic hypoxia. The increased expression of VEGF, ET-1, iNOS and eNOS may be partly due to the compensatory mechanism in the vascular beds of the liver in response to chronic hypoxia.

Topics: Alanine Transaminase; Animals; Biomarkers; Blotting, Western; Chronic Disease; Dinoprost; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Endothelin-1; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Liver; Male; NF-kappa B; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription Factor AP-1; Tyrosine; Vascular Endothelial Growth Factor A

The aim of this study was to assess the possible neuroprotective effect of the main nonsteroidal antiinflammatory drugs (NSAIDs) in an experimental model of hypoxia-reoxygenation in rat brain slices. After reoxygenation the increase in lactate dehydrogenase (LDH) efflux was inhibited by nimesulide, celecoxib and meloxicam with an IC(50) in the 10(-6)M range, by flurbiprofen, ibuprofen and diclofenac in the 10(-5)M range, and by salicylic acid, indomethacin, acetylsalicylic acid and mefenamic acid the 10(-4)M range. The effect of other NSAIDs was seen with an IC(50) greater than 10(-3)M. A statistically significant linear correlation between the values of LDH efflux and prostaglandin E(2) was found for NSAIDs whose IC(50) of cytoprotection (LDH efflux) was below 10(-4)M. The concentration of interleukin 10 was increased with nimesulide, celecoxib, meloxicam, flurbiprofen, ibuprofen and diclofenac. Flurbiprofen and diclofenac significantly inhibited the production of lipid peroxides. The increase in brain nitrite levels was significantly reduced with celecoxib, flurbiprofen, diclofenac and salicylic acid. Concentrations of 3-nitrotyrosine were significantly reduced with celecoxib, flurbiprofen, ibuprofen, salicylic acid and ketorolac. In conclusion, NSAIDs with the greatest cytoprotective effect (nimesulide, celecoxib and meloxicam) may exert their effect mainly through the blockade of cyclooxygenase-2 (COX-2) activity. Other compounds with neuroprotective activity may complement their lower anti-COX-2 effect with a slight increase in interleukin 10 and reduced oxidative and nitrosative stress in our model of hypoxia-reoxygenation in rat brain slices.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytoprotection; Dinoprostone; Glucose; Hypoxia; Interleukin-10; Interleukin-1beta; L-Lactate Dehydrogenase; Lipid Peroxidation; Lipid Peroxides; Male; Neuroprotective Agents; Nitrites; Nitrosation; Oxidative Stress; Oxygen; Rats; Rats, Wistar; Tyrosine

Chronic hypoxia increases the expression of inducible nitric oxide synthase (iNOS) mRNA and protein levels in fetal guinea pig heart ventricles. Excessive generation of nitric oxide (NO) can induce nitrosative stress leading to the formation of peroxynitrite, which can upregulate the expression of matrix metalloproteinases (MMPs). This study tested the hypothesis that maternal hypoxia increases fetal cardiac MMP9 and collagen through peroxynitrite generation in fetal hearts.. In heart ventricles, levels of malondialdehyde, 3-nitrotyrosine (3-NT), MMP9, and collagen were increased in hypoxic (HPX) vs. normoxic (NMX) fetal guinea pigs.. Thus, maternal hypoxia induces oxidative-nitrosative stress and alters protein expression of the extracellular matrix (ECM) through upregulation of the iNOS pathway in fetal heart ventricles. This identifies iNOS-derived NO as an important stimulus for initiating the adverse effects of peroxynitrite in HPX fetal hearts.. Pregnant guinea pigs were exposed to normoxia (room air) or hypoxia (10.5% O(2), 14 d) before term (term ≈ 65 d) and administered water, L-N6-(1-iminoethyl)-lysine (LNIL), an iNOS inhibitor, or N-acetylcysteine (NAC), an antioxidant.

Topics: Acetylcysteine; Animals; Body Weight; Collagen; Female; Fetal Heart; Guinea Pigs; Hypoxia; Lipid Peroxidation; Matrix Metalloproteinase 9; Nitric Oxide Synthase Type II; Organ Size; Peroxynitrous Acid; Pregnancy; Tyrosine

The effectiveness of sodium bicarbonate (SB) has recently been questioned although it is often used to correct metabolic acidosis of neonates. The aim of the present study was to examine its effect on hemodynamic changes and hydrogen peroxide (H(2)O(2)) generation in the resuscitation of hypoxic newborn animals with severe acidosis.. Newborn piglets were block-randomized into a sham-operated control group without hypoxia (n = 6) and two hypoxia-reoxygenation groups (2 h normocapnic alveolar hypoxia followed by 4 h room-air reoxygenation, n = 8/group). At 10 min after reoxygenation, piglets were given either i.v. SB (2 mEq/kg), or saline (hypoxia-reoxygenation controls) in a blinded, randomized fashion. Hemodynamic data and blood gas were collected at specific time points and cerebral cortical H(2)O(2) production was continuously monitored throughout experimental period. Plasma superoxide dismutase and catalase and brain tissue glutathione, superoxide dismutase, catalase, nitrotyrosine and lactate levels were assayed.. Two hours of normocapnic alveolar hypoxia caused cardiogenic shock with metabolic acidosis (PH: 6.99 ± 0.07, HCO(3)(-): 8.5 ± 1.6 mmol/L). Upon resuscitation, systemic hemodynamics immediately recovered and then gradually deteriorated with normalization of acid-base imbalance over 4 h of reoxygenation. SB administration significantly enhanced the recovery of both pH and HCO(3-) recovery within the first hour of reoxygenation but did not cause any significant effect in the acid-base at 4 h of reoxygenation and the temporal hemodynamic changes. SB administration significantly suppressed the increase in H(2)O(2) accumulation in the brain with inhibition of superoxide dismutase, but not catalase, activity during hypoxia-reoxygenation as compared to those of saline-treated controls.. Despite enhancing the normalization of acid-base imbalance, SB administration during resuscitation did not provide any beneficial effects on hemodynamic recovery in asphyxiated newborn piglets. SB treatment also reduced the H(2)O(2) accumulation in the cerebral cortex without significant effects on oxidative stress markers presumably by suppressing superoxide dismutase but not catalase activity.

Topics: Acid-Base Equilibrium; Acidosis; Animals; Animals, Newborn; Blood Gas Analysis; Catalase; Cerebral Cortex; Disease Models, Animal; Female; Glutathione; Hemodynamics; Hydrogen Peroxide; Hypoxia; Infusions, Intravenous; Lactic Acid; Male; Oxidation-Reduction; Oxidative Stress; Oxygen; Pulmonary Alveoli; Resuscitation; Sodium Bicarbonate; Superoxide Dismutase; Swine; Tyrosine

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension. IH causes oxidative stress that may limit bioavailability of the endothelium-derived vasodilator nitric oxide (NO) and thus contribute to this hypertensive response. We therefore hypothesized that increased vascular superoxide anion (O(2)(-)) generation reduces NO-dependent pulmonary vasodilation following IH. To test this hypothesis, we examined effects of the O(2)(-) scavenger tiron on vasodilatory responses to the endothelium-dependent vasodilator ionomycin and the NO donor S-nitroso-N-acetylpenicillamine in isolated lungs from hypocapnic-IH (H-IH; 3 min cycles of 5% O(2)/air flush, 7 h/day, 4 wk), eucapnic-IH (E-IH; cycles of 5% O(2), 5% CO(2)/air flush), and sham-treated (air/air cycled) rats. Next, we assessed effects of endogenous O(2)(-) on NO- and cGMP-dependent vasoreactivity and measured O(2)(-) levels using the fluorescent indicator dihydroethidium (DHE) in isolated, endothelium-disrupted small pulmonary arteries from each group. Both E-IH and H-IH augmented NO-dependent vasodilation; however, enhanced vascular smooth muscle (VSM) reactivity to NO following H-IH was masked by an effect of endogenous O(2)(-). Furthermore, H-IH and E-IH similarly increased VSM sensitivity to cGMP, but this response was independent of either O(2)(-) generation or altered arterial protein kinase G expression. Finally, both H-IH and E-IH increased arterial O(2)(-) levels, although this response was more pronounced following H-IH, and H-IH exposure resulted in greater protein tyrosine nitration indicative of increased NO scavenging by O(2)(-). We conclude that IH increases pulmonary VSM sensitivity to NO and cGMP. Furthermore, endogenous O(2)(-) limits NO-dependent vasodilation following H-IH through an apparent reduction in bioavailable NO.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium-Dependent Relaxing Factors; Free Radical Scavengers; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Lung; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Polycythemia; Pulmonary Artery; Rats; Rats, Wistar; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Superoxides; Tyrosine; Vasodilation

Diabetic retinopathy and retinopathy of prematurity are blinding disorders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (p7-p12) or (p7-p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vaso-obliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.

Topics: Acetylcysteine; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Catechin; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Glutathione; Hyperoxia; Hypoxia; Ischemia; Lipid Peroxidation; Metalloporphyrins; Mice; Mice, Inbred C57BL; Peroxynitrous Acid; Protective Agents; Retinal Neovascularization; Retinal Vessels; Tyrosine

We previously demonstrated that chronic exposure to intermittent hypoxia (CIH) impairs endothelium-dependent vasodilation in rats. To determine the time course of this response, rats were exposed to CIH for 3, 14, 28, or 56 days. Then, we measured acetylcholine- and nitroprusside-induced vasodilation in isolated gracilis arteries. Also, we measured endothelial and inducible nitric oxide synthase, nitrotyrosine, and collagen in the arterial wall and urinary isoprostanes. Endothelium-dependent vasodilation was impaired after 2 weeks of CIH. Three days of CIH was not sufficient to produce this impairment and longer exposures (i.e. 4 and 8 weeks) did not exacerbate it. Impaired vasodilation was accompanied by increased collagen deposition. CIH elevated urinary isoprostane excretion, whereas there was no consistent effect on either isoform of nitric oxide synthase or nitrotyrosine. Exposure to CIH produces functional and structural deficits in skeletal muscle resistance arteries. These impairments develop within 2 weeks after initiation of exposure and they are accompanied by systemic evidence of oxidant stress.

Topics: Acetylcholine; Analysis of Variance; Animals; Arteries; Chronic Disease; Collagen; Endothelium, Vascular; Hypoxia; Immunohistochemistry; Isoprostanes; Male; Muscle, Skeletal; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroprusside; Oxygen; Partial Pressure; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine; Vascular Resistance; Vasodilation; Vasodilator Agents

Intermittent hypoxia, a feature of obstructive sleep apnoea, potentiates ventilatory hypoxic responses, alters heart rate variability and produces hypertension, partially owing to an enhanced carotid body responsiveness to hypoxia. Since oxidative stress is a potential mediator of both chemosensory and cardiorespiratory alterations, we hypothesised that an antioxidant treatment may prevent these alterations. Accordingly, we studied the effects of ascorbic acid (1.25 g.L(-1) drinking water) on plasma lipid peroxidation, nitrotyrosine and inducible nitric oxide synthase (iNOS) immunoreactivity in the carotid body, ventilatory and carotid chemosensory responses to acute hypoxia, heart rate variability and arterial blood pressure in male Sprague-Dawley rats exposed to 5% O(2); 12 episodes.h(-1); 8 h.day(-1) or sham condition for 21 days. Intermittent hypoxia increased plasma lipid peroxidation, nitrotyrosine and iNOS expression in the carotid body, enhanced carotid chemosensory and ventilatory hypoxic responses, modified heart rate variability and produced hypertension. Ascorbic acid prevented the increased plasma lipid peroxidation and nitrotyrosine formation within the carotid body, and the enhanced carotid chemosensory and ventilatory responses to hypoxia, as well as heart rate variability alterations and hypertension. The present results support an essential role for oxidative stress in the generation of carotid body chemosensory potentiation and systemic cardiorespiratory alterations induced by intermittent hypoxia.

Topics: Animals; Antioxidants; Ascorbic Acid; Carotid Body; Chemoreceptor Cells; Heart Rate; Hypertension; Hypoxia; Lipid Peroxidation; Lipids; Male; Malondialdehyde; Nitric Oxide Synthase Type II; Nitrosamines; Oxidative Stress; Pulmonary Ventilation; Rats; Sleep Apnea, Obstructive; Tyrosine

To date, the role that NO derived from endothelial NO synthase (eNOS) plays in the development of the injuries occurring under hypoxia/reoxygenation (H/R) in the lung remains unknown and thus constitutes the subject of the present work. A follow-up study was conducted in Wistar rats submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 48 h and 5 days), with or without prior treatment using the eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis, protein nitration and NO production (NOx) were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose, implying that eNOS-derived NO may have a protective effect against the injuries occurring during H/R in the lung. These findings could open the possibility of future studies to design new therapies for this type of hypoxia based on NO-pharmacology.

Topics: Animals; Cytoprotection; Enzyme Inhibitors; Hypoxia; In Situ Nick-End Labeling; Lung; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Ornithine; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Tyrosine

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal approximately 4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Topics: Animals; Cell Respiration; Cytochrome P-450 CYP2E1; Dietary Fats; Fatty Liver; Hypoxia; Liver; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Mitochondrial Proteins; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Tyrosine

This study examined whether hypoxia causes free radical-mediated disruption of the blood-brain barrier (BBB) and impaired cerebral oxidative metabolism and whether this has any bearing on neurological symptoms ascribed to acute mountain sickness (AMS). Ten men provided internal jugular vein and radial artery blood samples during normoxia and 9-h passive exposure to hypoxia (12.9% O(2)). Cerebral blood flow was determined by the Kety-Schmidt technique with net exchange calculated by the Fick principle. AMS and headache were determined with clinically validated questionnaires. Electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence were employed for direct detection of spin-trapped free radicals and nitric oxide metabolites. Neuron-specific enolase (NSE), S100beta, and 3-nitrotyrosine (3-NT) were determined by ELISA. Hypoxia increased the arterio-jugular venous concentration difference (a-v(D)) and net cerebral output of lipid-derived alkoxyl-alkyl free radicals and lipid hydroperoxides (P < 0.05 vs. normoxia) that correlated with the increase in AMS/headache scores (r = -0.50 to -0.90, P < 0.05). This was associated with a reduction in a-v(D) and hence net cerebral uptake of plasma nitrite and increased cerebral output of 3-NT (P < 0.05 vs. normoxia) that also correlated against AMS/headache scores (r = 0.74-0.87, P < 0.05). In contrast, hypoxia did not alter the cerebral exchange of S100beta and both global cerebral oxidative metabolism (cerebral metabolic rate of oxygen) and neuronal integrity (NSE) were preserved (P > 0.05 vs. normoxia). These findings indicate that hypoxia stimulates cerebral oxidative-nitrative stress, which has broader implications for other clinical models of human disease characterized by hypoxemia. This may prove a risk factor for AMS by a mechanism that appears independent of impaired BBB function and cerebral oxidative metabolism.

Topics: Acute Disease; Adult; Altitude Sickness; Biomarkers; Blood-Brain Barrier; Brain; Free Radicals; Headache; Health Surveys; Humans; Hypoxia; Male; Nerve Growth Factors; Oxidative Stress; Oxygen; Phosphopyruvate Hydratase; Regional Blood Flow; Retrospective Studies; Risk Factors; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Tyrosine

Chronic lung hypoxia results in hypoxic pulmonary hypertension. Concomitant chronic hypercapnia partly inhibits the effect of hypoxia on pulmonary vasculature. Adult male rats exposed to 3 weeks hypoxia (Fi(02)=0.1) combined with hypercapnia (Fi(C02)=0.04-0.05) had lower pulmonary arterial blood pressure, increased weight of the right heart ventricle, and less pronounced structural remodeling of the peripheral pulmonary arteries compared with rats exposed only to chronic hypoxia (Fi(02)=0.1). According to our hypothesis, hypoxic pulmonary hypertension is triggered by hypoxic injury to the walls of the peripheral pulmonary arteries. Hypercapnia inhibits release of both oxygen radicals and nitric oxide at the beginning of exposure to the hypoxic environment. The plasma concentration of nitrotyrosine, the marker of peroxynitrite activity, is lower in hypoxic rats exposed to hypercapnia than in those exposed to hypoxia alone. Hypercapnia blunts hypoxia-induced collagenolysis in the walls of prealveolar pulmonary arteries. We conclude that hypercapnia inhibits the development of hypoxic pulmonary hypertension by the inhibition of radical injury to the walls of peripheral pulmonary arteries.

Topics: Animals; Blood Pressure; Chronic Disease; Disease Models, Animal; Hypercapnia; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung Injury; Male; Oxidative Stress; Pulmonary Artery; Rats; Rats, Wistar; Time Factors; Tyrosine

Obstructive sleep apnea is characterized by upper airway collapse, leading to intermittent hypoxia (IH). It has been postulated that IH-induced oxidative stress may contribute to several chronic diseases associated with obstructive sleep apnea. We hypothesize that IH induces systemic oxidative stress by upregulating NADPH oxidase, a superoxide-generating enzyme. NADPH oxidase is regulated by a cytosolic p47(phox) subunit, which becomes phosphorylated during enzyme activation. Male C57BL/6J mice were exposed to IH with an inspired O(2) fraction nadir of 5% 60 times/h during the 12-h light phase (9 AM-9 PM) for 1 or 4 wk. In the aorta and heart, IH did not affect lipid peroxidation [malondialdehyde (MDA) level], nitrotyrosine level, or p47(phox) expression and phosphorylation. In contrast, in the liver, exposure to IH for 1 wk resulted in a trend to an increase in MDA levels, whereas IH for 4 wk resulted in a 38% increase in MDA levels accompanied by upregulation of p47(phox) expression and phosphorylation. Administration of an NADPH oxidase inhibitor, apocynin, during IH exposure attenuated IH-induced increases in hepatic MDA. In p47(phox)-deficient mice, MDA levels were higher at baseline and, unexpectedly, decreased during IH. In conclusion, oxidative stress levels and pathways under IH conditions are organ and duration specific.

Topics: Acetophenones; Animals; Body Weight; Eating; Enzyme Inhibitors; Erythrocytes; Glutathione; Glutathione Disulfide; Hypoxia; Lipid Peroxidation; Lipoproteins, LDL; Liver; Male; Malondialdehyde; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; NADPH Oxidases; Oxidative Stress; Phosphorylation; Superoxide Dismutase; Tyrosine

The purpose of the study was to check whether hypoxia of corneal tissue increases the collagenolytic activity due to release of reactive oxygen and nitrogen species. Rats were exposed to hypoxia 10% O(2) for 4, 14, and 21 days. The radical tissue injury was measured by the level of nitrotyrosine and changes in the lipoperoxide-related fluorophores. Collagen protein composition was analyzed by slab gel electrophoresis. The activity of gelatinolytic enzymes was studied using the zymography. The vascularization of the corneas was measured. We found no differences in the corneal tissue in the gel electrophoretic profile of collagenous proteins and gelatinolytic activity between normoxic and hypoxic rats. We did not find any sign of radical tissue injury. There were no changes in the vascularization of corneas after exposition to hypoxia. The environmental 10% hypoxia does not induce radical tissue injury and an increase of collagenolytic activity in the rat cornea.

Topics: Analysis of Variance; Animals; Collagen; Cornea; Corneal Neovascularization; Extracellular Matrix; Eye Proteins; Free Radicals; Hypoxia; Male; Matrix Metalloproteinases; Oxidative Stress; Rats; Rats, Wistar; Time Factors; Tyrosine

Damage of the placenta resulting from ischemia-reperfusion is important to the pathophysiology of preeclampsia. Here we investigated whether low concentrations of glyceryl trinitrate (GTN), a nitric oxide mimetic with anti-apoptotic properties, inhibit hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of chorionic villous explants from human placentas. Compared with villi analyzed immediately after delivery or maintained under normoxic conditions, villi exposed to a 6-hour cycle of hypoxia/reoxygenation exhibited greater numbers of syncytiotrophoblasts with terminal dUTP nick-end labeling (TUNEL)-positive nuclei in the syncytiotrophoblast. This increased number of TUNEL-positive nuclei was paralleled by higher levels of 4-hydroxynonenal (marker of lipid peroxidation), nitrotyrosine residues, and active caspase-3 and polyADP-ribose polymerase expression. Morphological analysis of explants exposed to hypoxia/reoxygenation revealed apoptotic and aponecrotic features similar to those of chorionic villi from preeclamptic pregnancies. Treatment with GTN during the hy-poxia/reoxygenation cycle blocked the increases in the number of TUNEL-positive nuclei and in the levels of 4-hydroxynonenal, nitrotyrosine, and active caspase-3. Incubation with GTN also attenuated the hypoxia/reoxygenation-induced polyADP-ribose polymerase expression and the apoptotic and aponecrotic morphological alterations. These results suggest that small concentrations of nitric oxide protect chorionic villi from hypoxia/reoxygenation-induced damage and provide a rationale for the use of low doses of nitric oxide mimetics in the treatment and/or prevention of preeclampsia.

Topics: Aldehydes; Apoptosis; Blotting, Western; Caspase 3; Collagen Type XI; Female; Humans; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Microscopy, Electron, Transmission; Nitroglycerin; Organ Culture Techniques; Pre-Eclampsia; Pregnancy; Reperfusion Injury; Tocolytic Agents; Trophoblasts; Tyrosine

We previously reported that hypoxia attenuates cGMP-dependent protein kinase (PKG)-mediated relaxation in pulmonary vessels (Am J Physiol Lung Cell Mol Physiol 279: L611-L618, 2003). To determine whether hypoxia-induced reactive oxygen and nitrogen species (ROS and RNS, respectively) may be involved in the downregulation of PKG-mediated relaxation, ovine fetal intrapulmonary veins were exposed to 4 h of normoxia or hypoxia, with or without scavengers of ROS [N-acetylcysteine (NAC)] or peroxynitrite (quercetin and Trolox) and preconstricted with endothelin-1. Hypoxia decreased the relaxation response to 8-bromo-cGMP, PKG protein expression, and kinase activity and increased tyrosine nitration in PKG. However, ROS and RNS scavengers prevented these changes. To determine whether increased PKG nitration diminishes PKG activity, pulmonary vein smooth muscle cells (PVSMC) were exposed to shorter-term (30 min) hypoxia, which increased PKG nitration and decreased PKG activity but did not alter PKG protein expression. Increased dihydro-2,7-dichlorofluorescein diacetate (DCFH(2)-DA) fluorescence in PVSMC after 4 h or 30 min of hypoxia was not observed in the presence of NAC, quercetin, or Trolox, suggesting increased ROS and RNS production. Increased PKG nitration and the associated decrease in PKG activity in PVSMC after 30 min of hypoxia were also reversed on reoxygenation. The consequences of PKG nitration were assessed by exposure of purified PKG-Ialpha to peroxynitrite, which caused increased 3-nitrotyrosine immunoreactivity and inhibition of kinase activity. Our data suggest that, after 30 min of hypoxia, reversible covalent modification of PKG by hypoxia-induced reactive species may be an important mechanism by which the relaxation response to cGMP is regulated. However, after 4 h of hypoxia, PKG nitration and decreased PKG expression are involved.

Topics: Animals; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Fetus; Hypoxia; Isoenzymes; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitrates; Nitrites; Peroxynitrous Acid; Pulmonary Veins; Reactive Nitrogen Species; Reactive Oxygen Species; Sheep; Time Factors; Tyrosine; Vasodilation

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.

Topics: Animals; Enzyme Inhibitors; Hyperoxia; Hypoxia; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; omega-N-Methylarginine; Oxygen; Partial Pressure; Rats; Rats, Wistar; Reactive Oxygen Species; Tyrosine

Pathogenesis of hypoxic pulmonary hypertension is initiated by oxidative injury to the pulmonary vascular wall. Because nitric oxide (NO) can contribute to oxidative stress and because the inducible isoform of NO synthase (iNOS) is often upregulated in association with tissue injury, we hypothesized that iNOS-derived NO participates in the pulmonary vascular wall injury at the onset of hypoxic pulmonary hypertension. An effective and selective dose of an iNOS inhibitor, L-N6-(1-iminoethyl)lysine (L-NIL), for chronic peroral treatment was first determined (8 mg/l in drinking water) by measuring exhaled NO concentration and systemic arterial pressure after LPS injection under ketamine+xylazine anesthesia. A separate batch of rats was then exposed to hypoxia (10% O2) and given L-NIL or a nonselective inhibitor of all NO synthases, N(G)-nitro-L-arginine methyl ester (L-NAME, 500 mg/l), in drinking water. Both inhibitors, applied just before and during 1-wk hypoxia, equally reduced pulmonary arterial pressure (PAP) measured under ketamine+xylazine anesthesia. If hypoxia continued for 2 more wk after L-NIL treatment was discontinued, PAP was still lower than in untreated hypoxic controls. Immunostaining of lung vessels showed negligible iNOS presence in control rats, striking iNOS expression after 4 days of hypoxia, and return of iNOS immunostaining toward normally low levels after 20 days of hypoxia. Lung NO production, measured as NO concentration in exhaled air, was markedly elevated as early as on the first day of hypoxia. We conclude that transient iNOS induction in the pulmonary vascular wall at the beginning of chronic hypoxia participates in the pathogenesis of pulmonary hypertension.

Topics: Administration, Oral; Animals; Chronic Disease; Dose-Response Relationship, Drug; Enzyme Inhibitors; Exhalation; Hypertension, Pulmonary; Hypoxia; Lung; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Artery; Rats; Rats, Wistar; Time Factors; Tyrosine

To examine the effects of combined burn and smoke inhalation injury on hypoxic pulmonary vasoconstriction, 3-nitrotyrosine formation, and respiratory function in adult sheep.. Prospective, placebo-controlled, randomized, single-blinded trial.. University research laboratory.. Twelve chronically instrumented ewes.. Following a baseline measurement, sheep were randomly allocated to either healthy controls (sham) or the injury group, subjected to a 40%, third-degree body surface area burn and 48 breaths of cotton smoke according to an established protocol (n = 6 each). Hypoxic pulmonary vasoconstriction was assessed as changes in pulmonary arterial blood flow (corrected for changes in cardiac index) in response to left lung hypoxic challenges performed at baseline and at 24 and 48 hrs postinjury.. Combined burn and smoke inhalation was associated with increased expression of inducible nitric oxide (NO) synthase, elevated NO2/NO3 (NOx) plasma levels (12 hrs, sham, 6.2 +/- 0.6; injury, 16 +/- 1.6 micromol.L; p < .01) and increased peroxynitrite formation, as indicated by augmented lung tissue 3-nitrotyrosine content (30 +/- 3 vs. 216 +/- 8 nM; p < .001). These biochemical changes occurred in parallel with pulmonary shunting, progressive decreases in Pao2/Fio2 ratio, and a loss of hypoxic pulmonary vasoconstriction (48 hrs, -90.5% vs. baseline; p < .001). Histopathology revealed pulmonary edema and airway obstruction as the morphologic correlates of the deterioration in gas exchange and the increases in airway pressures.. This study provides evidence for a severe impairment of hypoxic pulmonary vasoconstriction following combined burn and smoke inhalation injury. In addition to airway obstruction, the loss of hypoxic pulmonary vasoconstriction may help to explain why blood gases are within physiologic ranges for a certain time postinjury and then suddenly deteriorate.

Topics: Animals; Burns; Female; Hypoxia; Lung; Multiple Trauma; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Pulmonary Gas Exchange; Random Allocation; Respiratory Distress Syndrome; Sheep; Single-Blind Method; Smoke Inhalation Injury; Survival Analysis; Tyrosine; Vasoconstriction

Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm increases peroxynitrite formation. In vitro force-frequency relationship, isotonic fatigability, and nitrotyrosine levels were assessed under hypoxic (Po(2) approximately 6.5 kPa) and hyperoxic (Po(2) approximately 88.2 kPa) control conditions and also in the presence of authentic peroxynitrite (60 min), ebselen (60 min), and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine acetate (L-NMMA) (90 min). A hypoxia-induced downward shift of the force-frequency relationship was associated with elevated nitrotyrosine level in the diaphragm. During hypoxia, both ebselen and L-NMMA decreased nitrotyrosine levels but did not affect force generation. Strenuous contractions impaired force generation but did not affect nitrotyrosine levels in the diaphragm during hypoxia. But under hyperoxic conditions, fatiguing contractions were associated with elevated diaphragm nitrotyrosine levels. Under hyperoxic conditions exogenous peroxynitrite impaired force generation and increased nitrotyrosine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relationship was found between diaphragm nitrotyrosine formation and in vitro force generation.

Topics: Animals; Azoles; Diaphragm; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Isoindoles; Lipid Peroxidation; Male; Muscle Contraction; Muscle Fatigue; omega-N-Methylarginine; Organoselenium Compounds; Peroxynitrous Acid; Rats; Rats, Wistar; Tyrosine

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.

Topics: Acute Disease; Anemia, Sickle Cell; Animals; Disease Models, Animal; Hemoglobin, Sickle; HSP90 Heat-Shock Proteins; Humans; Hypoxia; Lung Diseases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Tyrosine

Changes in the production system of nitric oxide (NO), a multifunctional biological messenger known to participate in blood-flow regulation, neuromodulation, and neuroprotection or neurotoxicity, were investigated in the caudate putamen of adult rats submitted to hypobaric hypoxia. Employing immunohistochemistry, Western blotting, enzymatic assay, and NADPH-diaphorase staining, we demonstrate that neuronal nitric oxide synthase (nNOS) expression and constitutive nitric oxide synthase (cNOS) activity were transiently activated by 7 h of exposure to a simulated altitude of 8325 m (27,000 ft). In addition, endothelial nitric oxide synthase (eNOS) immunoreactivity and blood vessel NADPH-diaphorase staining peaked immediately after the hypoxic stimulus, whereas inducible nitric oxide synthase (iNOS) expression and activity remained unaltered. Nitrotyrosine formation, a marker of protein nitration, was evaluated by immunohistochemistry and Western blotting, and was found to increase parallel to nitric oxide synthesis. We conclude that the nitric oxide system undergoes significant transient alterations in the caudate putamen of adult rats submitted to acute hypobaric hypoxia.

Topics: Altitude; Animals; Blood Vessels; Blotting, Western; Cell Count; Endothelium; Hypoxia; Immunohistochemistry; Male; NADPH Dehydrogenase; Neostriatum; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Time Factors; Tyrosine

Obstructive sleep apnea (OSA) has been increasingly linked to cardiovascular disease, endothelial dysfunction, and oxidative stress, generated by repetitive nocturnal hypoxemia and reperfusion. Circulating free nitrotyrosine has been reported as a novel biomarker of nitric oxide (NO)-induced oxidative/nitrosative stress. Nitrosative stress has been implicated as a possible mechanism for development of cardiovascular diseases. We tested the hypothesis that repetitive severe hypoxemia resulting from OSA would increase NO-mediated oxidative stress. We studied 10 men with newly diagnosed moderate to severe OSA who were free of other diseases, had never been treated for OSA, and were taking no medications. Nitrotyrosine measurements, performed by liquid chromatography-tandem mass spectrometry, were made before and after untreated apneic sleep. We compared free nitrotyrosine levels in these patients with those obtained at similar times in 10 healthy male control subjects without OSA, with similar age and body mass index. Evening baseline nitrotyrosine levels were similar before sleep in the control and OSA groups [0.16 +/- 0.01 and 0.15 +/- 0.01 ng/ml, respectively, P = not significant (NS)]. Neither normal nor disturbed apneic sleep led to significant changes of plasma nitrotyrosine (morning levels: control group 0.14 +/- 0.01 ng/ml; OSA group 0.15 +/- 0.01 ng/ml, P = NS). OSA was not accompanied by increased circulating free nitrotyrosine either at baseline or after sleep. This observation suggests that repetitive hypoxemia during OSA does not result in increased NO-mediated oxidative/nitrosative stress in otherwise healthy subjects with OSA.

Topics: Adult; Chromatography, Liquid; Humans; Hypoxia; Male; Mass Spectrometry; Middle Aged; Nitric Oxide; Sleep; Sleep Apnea, Obstructive; Tyrosine

Ischemic hypoxia provokes alterations in the production system of nitric oxide in the cerebellum. We hypothesize that the nitric oxide system may undergo modifications due to hypobaric hypoxia and that may play a role in high altitude pathophysiology. Therefore, changes in the nitric oxide system of the cerebellum of rats submitted to acute hypobaric hypoxia were investigated. Adult rats were exposed for 7 h to a simulated altitude of 8235 m (27000 ft.) and then killed after 0 h or 1, 3, 5 and 10 days of reoxygenation. Nitric oxide synthase calcium-dependent and -independent activity, immunoblotting and immunohistochemistry of neuronal, endothelial, and inducible nitric oxide synthase, and nitrotyrosine were evaluated. Immunoreactivity for neuronal nitric oxide synthase slightly increased in the baskets of the Purkinje cell layer and in the granule cells, after 0 h of reoxygenation, although no changes in neuronal nitric oxide synthase immunoblotting densitometry were detected. Calcium-dependent activity significantly rose after 0 h of reoxygenation, reaching control levels in the following points, and being coincident with a peak of eNOS expression. Nitrotyrosine formation showed significant increments after 0 h and 1 day of reoxygenation. Nitrotyrosine immunoreactivity showed an intracellular location change in the neurons of the cerebellar nuclei and in addition, an appearance of nitration in the soma of the Purkinje cells was detected. No changes in inducible nitric oxide synthase activity, immunoblotting or immunohistochemistry were detected. We conclude that at least part of the nitric oxide system is involved in cerebellum responses to hypobaric hypoxia.

Topics: Altitude; Animals; Cerebellum; Endothelium, Vascular; Hypoxia; Immunohistochemistry; Male; Nerve Tissue Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Tyrosine

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, and NO synthase (NOS) activity assays in adult rats submitted for 30 min to hypoxia, in a hypobaric chamber at a simulated altitude of 38,000 ft (11000 m) (154.9 mm Hg). The cerebral cortex was studied after different survival times, 0 and 24 h, 5, 8, 15, and 30 days of reoxygenation. This situation led to morphological alterations in the large type I interneurons, as well as immunoreactive changes in the appearance and number of the small neurons (type II), both containing neuronal NOS (nNOS). Some of these small neurons showed immunoreactive cytoplasm and short processes; others, the more numerous during all reoxygenation periods, contained the immunoreactive product mainly related to a perinuclear ring. Ultrastructurally, these small neurons exhibited changes in nuclear structures as in the shape of the nuclear membrane, in the distribution of heterochromatin, and in the nucleolar morphology. The reaction product for nitrotyrosine, as a marker of protein nitration, showed modifications in distribution of the immunoreactive product. No expression was found for inducible NOS (iNOS). All these modifications were accompanied by increased nNOS and nitrotyrosine production as demonstrated by Western blotting and calcium-dependent activity, returning to control conditions after 30 days of reoxygenation, suggesting a reversible NO mechanism of action.

Topics: Animals; Atmospheric Pressure; Brain Chemistry; Cerebral Cortex; Hypoxia; Immunohistochemistry; Male; Nitrergic Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Proteins; Rats; Rats, Wistar; Tyrosine

Changes in the nitric oxide system of the hippocampus from rats submitted to hypobaric hypoxia were investigated. Adult rats were exposed to a simulated altitude of 8,325 m (27,000 ft) for 7 h and killed after 0 h, 1, 3, 5, 10 and 20 days of reoxygenation. The number of neuronal nitric oxide synthase immunoreactive neurons and their dendritic plexus, as well as neuronal nitric oxide synthase immunoblotting densitometry and calcium-dependent activity increased from 0 h to 3 days of reoxygenation. In addition, endothelial nitric oxide synthase immunoreactivity peaked after 7 h of hypobaric hypoxia. Nitrotyrosine immunoreactivity showed an increase in the pyramidal cells of CA2-CA3 and in glial cells surrounding the blood vessels after 0 h, 1 and 3 days of reoxygenation. Immunoblotting densitometry of 1 of the 2 nitrotyrosine-immunoreactive bands detected also increased after 0 h and 1 day of reoxygenation. Inducible nitric oxide synthase immunoreactivity was found only in some blood vessels after 0 h, 1 and 3 days of reoxygenation, but no changes in inducible nitric oxide synthase activity or immunoblotting were detected. We conclude that transient activation of the nitric oxide system constitutes a hippocampal response to hypobaric hypoxia.

Topics: Animals; Blotting, Western; Cell Count; Disease Models, Animal; Endothelium, Vascular; Glial Fibrillary Acidic Protein; Hippocampus; Hypoxia; Immunohistochemistry; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Time Factors; Tyrosine

Thioredoxin (TRX) is a 13 kDa protein with antioxidant effect and redox regulating functions. Peroxynitrite is a strong oxidizing and nitrating agent which can react with all classes of biomolecules. In the present study, we focused on the association between TRX and nitrotyrosine, which served as a marker of peroxynitrite formation, in the neonatal hypoxia-ischemia (HI) rat brain. At 4-16 h after HI, the immunoreactivity for TRX was diminished in the injured region in the cortex and striatum, whereas nitrotyrosine immunoreactivity was enhanced. In contrast, around the injured region, TRX immunoreactivity was enhanced in survival neurons at 4-24 h after HI, while the immunoreactivity for nitrotyrosine was mostly not detected. Northern blot analysis showed increased TRX mRNA induction in the cerebral hemisphere ipsilateral to the carotid ligation from 4-24 h after HI but not in the contralateral hypoxic hemisphere. These findings suggest that production of peroxynitrite is involved in HI brain injury, and that induced TRX plays a neuroprotective role against oxidative stress resulting from HI.

Topics: Animals; Animals, Newborn; Blotting, Northern; Brain; Hypoxia; Models, Anatomic; Oxidants; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Thioredoxins; Time Factors; Tyrosine

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS-immunoreactive band and calcium-dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV-V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium-independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham-perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L-NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia-reperfusion contributes to cerebral injury.

Topics: Animals; Blotting, Western; Brain Ischemia; Calcium; Cerebral Cortex; Disease Models, Animal; Enzyme Inhibitors; Gene Expression; Glucose; Hypoxia; Immunohistochemistry; Magnetic Resonance Imaging; Male; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Peroxynitrous Acid; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Nitrotyrosine residues (NT), an index of oxidative stress arising from peroxynitrite formation and action, are found in placental vasculature of pregnancies complicated by pre-eclampsia (PE) or pregestational insulin-dependent diabetes mellitus (IDDM). This study correlates conventional placental pathology with NT immunostaining in 20 cases of perinatal mortality (13 stillbirths and seven cases of neonatal mortality) associated with PE, IDDM, amniotic fluid infection syndrome (AFIS), or from fetal/neonatal demise not related to these conditions (congenital anomalies) (n = five/group). Patients with PE have more decidual arteriolopathy and Tenney-Parker change, while patients with IDDM and ascending infection have more villous cytotrophoblastic hyperplasia. Archival paraffin-embedded placental sections were immunostained for NT for correlation with clinical features and H&E histological findings. The intensity of immunostaining for NT varied from absent (n = 7) to 1+ (n = 5) or 2+ (n = 8). All eight placentae with 2+ staining showed increased villous extracellular matrix (ECM), compared to none of five with 1+ staining and two of seven with no staining (chi2 = 14.3, P = 0.001). There was no statistically significant difference in the percentage of stem villi with luminal vascular abnormalities (5.7 vs 10 vs 35.7 per cent, F = 2.3, P = 0.1). Our data show that increased production of reactive oxygen species by placental tissue may be associated with increased extracellular matrix, itself produced by fibroblasts under the influence of oxygen. NT immunostaining may therefore help differentiate those cases of perinatal morbidity/mortality associated with post-placental hypoxia provided that the secondary impact of intrauterine fetal death can be excluded by future studies.

Topics: Adult; Diabetes Mellitus, Type 1; Embolism, Amniotic Fluid; Extracellular Matrix; Female; Fetal Death; Gestational Age; Humans; Hypoxia; Infant, Newborn; Placenta; Placental Circulation; Pre-Eclampsia; Pregnancy; Retrospective Studies; Tyrosine

Oxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2',7'-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and alpha-phenyl-N-tert-butylnitrone reduced levels of oxidative stress after reoxygenation. These changes are consistent with an ischemia-reperfusion injury, and mirror those seen in preeclampsia. Consequently, in vitro hypoxia/reoxygenation may represent a suitable model system for investigating the generation of placental oxidative stress in preeclampsia and other complications of pregnancy.

Topics: Aldehydes; Cyclic N-Oxides; Deferoxamine; Female; Fluorescent Antibody Technique; Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Hypoxia; Immunohistochemistry; Ischemia; Nitrogen Oxides; Oxidative Stress; Oxygen; Placenta; Pregnancy; Pregnancy Complications; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tissue Distribution; Tyrosine

Nitrotyrosine and eNOS were detected immunocytochemically using specific antibodies in paraffin sections of lung from rats subjected to hypoxia for 2, 7, or 14 days. The staining intensity for eNOS was enhanced in the endothelium of both resistance and conduit pulmonary arteries at 2 days. Staining intensity for eNOS remained elevated at 7 and 14 days in conduit arteries, whereas it progressively increased further in resistance arteries. Nitrotyrosine staining was elevated to a similar degree in endothelium and adjacent vascular smooth muscle. In resistance pulmonary arteries, there was a progressive increase in nitrotyrosine, which matched the increase in eNOS. In conduit pulmonary arteries, nitrotyrosine increased only after 14 days of hypoxia. The results suggest that in chronic hypoxia the up-regulation of eNOS leads to the formation of peroxynitrite which has access to both endothelium and vascular smooth muscle.

Topics: Actins; Animals; Arteries; Endothelium; Hypertrophy, Right Ventricular; Hypoxia; Immunohistochemistry; Lung; Nitric Oxide Synthase; Rats; Tyrosine; Vascular Resistance