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The airway epithelium is a broad interface with the environment, mandating well-orchestrated responses to properly modulate inflammation. Classically, autophagy is a homeostatic pathway triggered in response to external cellular stresses, and is elevated in chronic airway diseases. Recent findings highlight the additional role of autophagy in vesicle trafficking and protein secretion, implicating autophagy pathways in complex cellular responses in disease. Th2 cytokines, IL-13 and IL-4, are increased in asthma and other airway diseases contributing to chronic inflammation. Previously, we observed that IL-13 increases reactive oxygen species (ROS) in airway epithelial cells in an autophagy-dependent fashion. Here, we tested our hypothesis that autophagy is required for IL-13-mediated superoxide production via the NADPH oxidase DUOX1. Using a mouse model of Th2-mediated inflammation induced by OVA-allergen, we observed elevated lung amounts of IL-13 and IL-4 accompanied by increased autophagosome levels, determined by LC3BII protein levels and immunostaining. ROS levels were elevated and DUOX1 expression was increased 70-fold in OVA-challenged lungs. To address the role of autophagy and ROS in the airway epithelium, we treated primary human tracheobronchial epithelial cells with IL-13 or IL-4. Prolonged, 7-day treatment increased autophagosome formation and degradation, while brief activation had no effect. Under parallel culture conditions, IL-13 and IL-4 increased intracellular superoxide levels as determined by electron paramagnetic resonance (EPR) spectroscopy. Prolonged IL-13 activation increased DUOX1, localized at the apical membrane. Silencing DUOX1 by siRNA attenuated IL-13-mediated increases in superoxide, but did not reduce autophagy activities. Notably, depletion of autophagy regulatory protein ATG5 significantly reduced superoxide without diminishing total DUOX1 levels. Depletion of ATG5, however, diminished DUOX1 localization at the apical membrane. The findings suggest non-canonical autophagy activity regulates DUOX1-dependent localization required for intracellular superoxide production during Th2 inflammation. Thus, in chronic Th2 inflammatory airway disease, autophagy proteins may be responsible for persistent intracellular superoxide production., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Methotrexate (MTX) is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD). Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA) and acetaldehyde (AA), known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA) or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Angiotensin II (AngII) elicits the production of superoxide (O 2 •- ) from mitochondria in numerous cell types within peripheral organs and in the brain suggesting a role for mitochondrial-produced O 2 •- in the pathogenesis of hypertension. However, it remains unclear if mitochondrial O 2 •- is causal in the development of AngII-induced hypertension, or if mitochondrial O 2 •- in the absence of elevated AngII is sufficient to increase blood pressure. Further, the tissue specific (i.e. central versus peripheral) redox regulation of AngII hypertension remains elusive. Herein, we hypothesized that increased mitochondrial O 2 •- in the absence of pro-hypertensive stimuli, such as AngII, elevates baseline systemic mean arterial pressure (MAP), and that AngII-mediated hypertension is exacerbated in animals with increased mitochondrial O 2 •- levels. To address this hypothesis, we generated novel inducible knock-down mouse models of manganese superoxide dismutase (MnSOD), the O 2 •- scavenging antioxidant enzyme specifically localized to mitochondria, targeted to either the brain subfornical organ (SFO) or peripheral tissues. Contrary to our hypothesis, knock-down of MnSOD either in the SFO or in peripheral tissues was not sufficient to alter baseline systemic MAP. Interestingly, when mice were challenged with chronic, peripheral infusion of AngII, only the MnSOD knock-down confined to the SFO, and not the periphery, demonstrated an increased sensitization and potentiated hypertension. In complementary experiments, over-expressing MnSOD in the SFO significantly decreased blood pressure in response to chronic AngII. Overall, these studies indicate that mitochondrial O 2 •- in the brain SFO works in concert with other AngII-dependent factors to drive an increase in MAP, as elevated mitochondrial O 2 •- alone, either in the SFO or peripheral tissues, failed to raise baseline blood pressure., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Norepinephrine (NE) produces multifaceted regulatory patterns in T-lymphocytes. Recently, we have shown that NE utilizes redox signaling as evidenced by increased superoxide (O2●-) causally linked to the observed changes in these cells; however, the source of this reactive oxygen species (ROS) remains elusive. Herein, we hypothesized that the source of increased O2●- in NE-stimulated T-lymphocytes is due to disruption of mitochondrial bioenergetics. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T-lymphocytes stimulated with NE and assessed O2●- levels, mitochondrial metabolism, cellular proliferation, and cytokine profiles. We demonstrate that the increase in O2●- levels in response to NE is time-dependent and occurs at later points of T-lymphocyte activation. Moreover, the source of O2●- was indeed the mitochondria as evidenced by enhanced MitoSOX Red oxidation as well as abrogation of this signal by the addition of the mitochondrial-targeted O2●--scavenging antioxidant MitoTempol. NE-stimulated T-lymphocytes also demonstrated decreased mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2●-. The effects of NE in regards to redox signaling appear to be adrenergic receptor-dependent as specific receptor antagonists could reverse the increase in O2●-; however, differential receptors regulating these processes were observed in CD4+ versus CD8+ T-lymphocytes. Finally, mitochondrial O2●- was shown to be mechanistic to the NE-mediated T-lymphocyte phenotype as supplementation of MitoTempol could reverse specific changes in cytokine expression observed with NE treatment. Overall, these studies indicate that mitochondrial metabolism and O2●--mediated redox signaling play a regulatory role in the T-lymphocyte response to NE., Competing Interests: The authors have declared that no competing interests exist.

Angiotensin II (AngII) is the main effector peptide of the renin-angiotensin system (RAS), and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2 (•-)). Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2 (•-). We have previously reported that over-expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix-localized O2 (•-) scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD), which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2 (•-). Using a neuronal cell culture model (CATH.a neurons), we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD) are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2 (•-) levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2 (•-), and inhibits AngII intra-neuronal signaling.

The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone, and 1-methyl-4-phenylpyridinium (MPP(+)) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2(•-)), oxidative stress, and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation were determined in the cytosol, intermembrane (IMS), and mitochondrial matrix compartments, using dihydroethidine derivatives and the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and the mitochondrial matrix prior to cell death. MPP(+) and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on alterations in ROS steady-state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm), and dopaminergic cell death induced by MPP(+) or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP(+) or rotenone, induced the transcriptional activation of the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2(•-) in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP(+) does not depend directly on mitochondrial O2(•-) formation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

Fullerene, the third allotrope of carbon, has been referred to as a "radical sponge" because of its powerful radical scavenging activities. However, the hydrophobicity and toxicity associated with fullerene limits its application as a therapeutic antioxidant. In the present study, we sought to overcome these limitations by generating water-soluble nanoformulations of fullerene (C(60)). Fullerene (C(60)) was formulated with poly(N-vinyl pyrrolidine) (PVP) or poly(2-alkyl-2-oxazoline)s (POx) homopolymer and random copolymer to form nano-complexes. These C(60)-polymer complexes were characterized by UV-vis spectroscopy, infrared spectroscopy (IR), dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). Cellular uptake and intracellular distribution of the selected formulations in catecholaminergic (CATH.a) neurons were examined by UV-vis spectroscopy, immunofluorescence and immunogold labeling. Electron paramagnetic resonance (EPR) spectroscopy was used to determine the ability of these C(60)-polymer complexes to scavenge superoxide. Their cytotoxicity was evaluated in three different cell lines. C(60)-POx and C(60)-PVP complexes exhibited similar physicochemical properties and antioxidant activities. C(60)-poly(2-ethyl-2-oxazoline) (PEtOx) complex, but not C(60)-PVP complex, were efficiently taken up by CATH.a neurons and attenuated the increase in intra-neuronal superoxide induced by angiotensin II (Ang II) stimulation. These results show that C(60)-POx complexes are non-toxic, neuronal cell permeable, superoxide scavenging antioxidants that might be promising candidates for the treatment of brain-related diseases associated with increased levels of superoxide., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Overexpressing superoxide dismutase 1 (SOD1; also called Cu/ZnSOD), an intracellular superoxide (O(2)(*-))-scavenging enzyme, in central neurons inhibits angiotensin II (AngII) intraneuronal signaling and normalizes cardiovascular dysfunction in diseases associated with enhanced AngII signaling in the brain, including hypertension and heart failure. However, the blood-brain barrier and neuronal cell membranes impose a tremendous impediment for the delivery of SOD1 to central neurons, which hinders the potential therapeutic impact of SOD1 treatment on these diseases. To address this, we developed conjugates of SOD1 with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer (Pluronic) (SOD1-P85 and SOD1-L81), which retained significant SOD1 enzymatic activity. The modified SOD1 effectively scavenged xanthine oxidase/hypoxanthine-derived O(2)(*-), as determined by HPLC and the measurement of 2-hydroxyethidium. Using catecholaminergic neurons, we observed an increase in neuronal uptake of SOD1-Pluronic after 1, 6, or 24h, compared to neurons treated with pure SOD1 or PEG-SOD1. Importantly, without inducing neuronal toxicity, SOD1-Pluronic conjugates significantly inhibited AngII-induced increases in intraneuronal O(2)(*-) levels. These data indicate that SOD1-Pluronic conjugates penetrate neuronal cell membranes, which results in elevated intracellular levels of functional SOD1. Pluronic conjugation may be a new delivery system for SOD1 into central neurons and therapeutically beneficial for AngII-related cardiovascular diseases., (Published by Elsevier Inc.)

Reactive oxygen species (ROS), particularly superoxide (O(2)(.-)), have been identified as key signaling intermediates in ANG II-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O(2)(.-) in ANG II-stimulated neurons; however, additional sources of O(2)(.-), including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O(2)(.-) in neurons and that increased scavenging of mitochondria-produced O(2)(.-) attenuates ANG II-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG II (100 nM) increased mitochondria-localized O(2)(.-) levels, as measured by MitoSOX Red fluorescence. This response was significantly attenuated in neurons overexpressing the mitochondria-targeted O(2)(.-)-scavenging enzyme Mn-SOD. To examine the biological significance of the ANG II-mediated increase in mitochondria-produced O(2)(.-), we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II-induced inhibition of voltage-gated K(+) current (I(Kv)) in neurons. Adenovirus-mediated Mn-SOD overexpression or pretreatment with the cell-permeable antioxidant tempol (1 mM) significantly attenuated ANG II-induced inhibition of I(Kv). In contrast, pretreatment with extracellular SOD protein (400 U/ml) had no effect. Mn-SOD overexpression also inhibited ANG II-induced activation of Ca(2+)/calmodulin kinase II, a redox-sensitive protein known to modulate I(Kv). These data indicate that ANG II increases mitochondrial O(2)(.-), which mediates, at least in part, ANG II-induced activation of Ca(2+)/calmodulin kinase II and inhibition of I(Kv) in neurons.

Peripheral chemoreflex sensitivity is enhanced in both clinical and experimental chronic heart failure (CHF). Here we investigated the role of manganese superoxide dismutase (MnSOD), the SOD isoform specially targeted to mitochondria, and mitochondrial superoxide levels in the enhanced chemoreceptor activity and function of the carotid body (CB) in CHF rabbits. CHF suppressed MnSOD protein expression and elevated mitochondrial superoxide levels in CB compared with that in sham CB. Adenovirus (Ad) MnSOD (1 x 10(8) plaque-forming units/ml) gene transfer selectively to the CBs normalized mitochondrial superoxide levels in glomus cells from CHF CB. In addition, Ad MnSOD reduced the elevation of superoxide level in CB tissue from CHF rabbits. Ad MnSOD significantly increased MnSOD expression in CHF CBs and normalized the baseline renal sympathetic nerve activity and the response of renal sympathetic nerve activity to hypoxia in CHF rabbits. Ad MnSOD decreased baseline single-fiber discharge from CB chemoreceptors compared with Ad Empty (6.3 + or - 1.5 vs. 12.7 + or - 1.4 imp/s at approximately 100-Torr Po(2), P < 0.05) and in response to hypoxia (20.5 + or - 1.8 vs. 32.6 + or - 1.4 imp/s at approximately 40-Torr Po(2), P < 0.05) in CHF rabbits. Compared with Ad Empty, Ad MnSOD reversed the blunted K(+) currents in CB glomus cells from CHF rabbits (385 + or - 11 vs. 551 + or - 20 pA/pF at +70 mV, P < 0.05). The results suggest that decreased MnSOD in the CB and elevated mitochondrial superoxide levels contribute to the enhanced CB chemoreceptor activity and peripheral chemoreflex function in CHF rabbits.

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.

We recently demonstrated that superoxide (O2*-) is a key signaling intermediate in central angiotensin II (Ang II)-elicited blood pressure and drinking responses, and that hypertension caused by systemic Ang II infusion involves oxidative stress in cardiovascular nuclei of the brain. Intracellular Ca2+ is known to play an important role in Ang II signaling in neurons, and it is also linked to reactive oxygen species mechanisms in neurons and other cell types. However, the potential cross-talk between Ang II, O2*-, and Ca2+ in neural cells remains unknown. Using mouse neuroblastoma Neuro-2A cells, we tested the hypothesis that O2*- radicals are involved in the Ang II-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in neurons. Ang II caused a rapid time-dependent increase in [Ca2+]i that was abolished in cells bathed in Ca2+-free medium or by pretreatment with the nonspecific voltage-gated Ca2+ channel blocker CdCl2, suggesting that voltage-sensitive Ca2+ channels are the primary source of Ang II-induced increases in [Ca2+]i in this cell type. Overexpression of cytoplasm-targeted O2*- dismutase via an adenoviral vector (AdCuZnSOD) efficiently scavenged Ang II-induced increases in intracellular O2*- and markedly attenuated the increase in [Ca2+]i caused by this peptide. Furthermore, adenoviral-mediated expression of a dominant-negative isoform of Rac1 (AdN17Rac1), a critical component for NADPH oxidase activation and O2*- production, significantly inhibited the increase in [Ca2+]i after Ang II stimulation. These data provide the first evidence that O2*- is involved in the Ang II-stimulated influx of extracellular Ca2+ in neural cells and suggest a potential intracellular signaling mechanism involved in Ang II-mediated oxidant regulation of central neural control of blood pressure.

Hypertension caused by angiotensin II (Ang II) infusion is associated with oxidative stress in the peripheral vasculature and kidney. The role of redox mechanisms in the central nervous system (CNS), a tissue known to be pivotal in Ang II-dependent hypertension, has not been investigated. We recently identified superoxide (O2*-) in the brain as a key signaling intermediate in the transient pressor response elicited by acute injection of Ang II directly into the CNS. Here we tested the hypothesis that hypertension caused by chronic systemic infusion of Ang II is mediated by a central neurogenic mechanism involving O2*-. Infusion of Ang II (600 ng x kg(-1) x min(-1)) over a 2-week period in mice caused a gradually developing hypertension that was correlated with marked elevations in O2*- production specifically in the subfornical organ (SFO), a brain region lying outside the blood-brain barrier and known to be a primary sensor for blood-borne Ang II. Adenoviral-mediated delivery of cytoplasmically targeted superoxide dismutase (SOD) selectively to this site prevented the hypertension and the increased O2*- production, whereas gene transfer of SOD targeted to the extracellular matrix had no effect. These data suggest that increased intracellular O2*- production in the SFO is critical in the development of Ang II-induced hypertension.

Angiotensin II (Ang II) has profound effects in the central nervous system (CNS), including promotion of thirst, regulation of vasopressin secretion, and modulation of sympathetic outflow. Despite its importance in cardiovascular and volume homeostasis, angiotensinergic mechanisms are incompletely understood in the CNS. Recently, a novel signaling mechanism for Ang II involving reactive oxygen species (ROS) has been identified in a variety of peripheral tissues, but the involvement of ROS as second messengers in Ang II-mediated signaling in the CNS has not been reported. The hypothesis that superoxide is a key mediator of the actions of Ang II in the CNS was tested in mice using adenoviral vector-mediated expression of superoxide dismutase (AdSOD). Changes in blood pressure, heart rate, and drinking elicited by injection of Ang II in the CNS were abolished by prior treatment with AdSOD in the brain, whereas the cardiovascular responses to carbachol, another central vasopressor agent, were unaffected. In addition, Ang II stimulated superoxide generation in primary CNS cell cultures, and this was prevented by the Ang II receptor (Ang II type 1 subtype) antagonist losartan or AdSOD. These results identify a novel signaling mechanism mediating the actions of Ang II in the CNS. Dysregulation of this signaling cascade may be important in hypertension and heart failure triggered by Ang II acting in the CNS.

New Findings What is the central question of this study? This study evaluated the following central question: does N-acetylcysteine (N-AC), an antioxidant that readily penetrates the blood-brain barrier, have the capability to reduce the increase in sympathetic nerve activity observed during hyperacute intermittent hypoxia?, What is the main finding and its importance? We demonstrate that N-AC decreases muscle sympathetic nerve activity in response to hyperacute intermittent hypoxia versus placebo control. This finding suggests that antioxidants, such as N-AC, have therapeutic potential in obstructive sleep apnoea., This investigation tested the following hypotheses: that (i) N-acetylcysteine (N-AC) attenuates hyperacute intermittent hypoxia-induced sympathoexcitation, (ii) without elevating superoxide measured in peripheral venous blood. Twenty-eight healthy human subjects were recruited to the study. One hour before experimentation, each subject randomly ingested either 70 mg kg−1 of N-AC ( n = 16) or vehicle placebo ( n = 12). Three-lead ECG and arterial blood pressure, muscle sympathetic nerve activity ( n = 17) and whole-blood superoxide concentration (using electron paramagnetic resonance spectroscopy; n = 12) were measured. Subjects underwent a 20 min hyperacute intermittent hypoxia training (hAIHT) protocol that consisted of cyclical end-expiratory apnoeas with 100% nitrogen. N-AC decreased muscle sympathetic nerve activity after hAIHT compared with placebo ( P < 0.02). However, N-AC did not alter superoxide concentrations in venous blood compared with placebo ( P > 0.05). Moreover, hAIHT did not increase superoxide concentrations in the peripheral circulation as measured by electron paramagnetic resonance ( P > 0.05). Based on these findings, we contend that (i) hAIHT and (ii) the actions of N-AC in hAIHT are primarily mediated centrally rather than peripherally, although central measurements of reactive oxygen species are difficult to obtain in human subjects, thus making this assertion difficult to verify. This investigation suggests the possibility of developing a pharmaceutical therapy to inhibit the sympathoexcitation associated with obstructive sleep apnoea. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS), particularly superoxide (O2-), have been identified as key signaling intermediates in ANG Il-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O2- in ANG 11-stimulated neurons; however, additional sources of O2-, including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O2- in neurons and that increased scavenging of mitochondria-produced O2-' attenuates ANG Il-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG H (100 nM) increased mitochondria-localized O2- levels, as measured by MitoSOX Red flu- orescence. This response was significantly attenuated in neurons overex- pressing the mitochondria-targeted O2- -scavenging enzyme Mn-SOD. To examine the biological significance of the ANG 11-mediated increase in mitochondria-produced O2-, we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II- induced inhibition of voltage-gated K+ current (IKv) in neurons. Adeno- virus-mediated Mn-SOD overexpression or pretreatment with the cell- permeable antioxidant tempol (1 nM) significantly attenuated ANG Il-induced inhibition of IKv. In contrast, pretreatment with extracellular SOD protein (400 U/mI) had no effect. Mn-SOD overexpression also inhibited ANG 11-induced activation of Ca2+/calmodulin kinase H, a redox-sensitive protein known to modulate 1Kv. These data indicate that ANG 11 increases mitochondrial O2-, which mediates, at least in part, ANG Il-induced activation of Ca2+/calmodulin kinase II and inhibition of IKv in neurons. [ABSTRACT FROM AUTHOR]

We investigated the association of superoxide in patients undergoing right heart catheterization for the evaluation of pulmonary hypertension. B METHODS: b Twenty two patients over age 19 undergoing right heart catheterization had blood samples taken from a peripheral vein and from their pulmonary artery. [Extracted from the article]