Combined Effects Of Nitric Oxide Research Articles

Root NRT, NiR, AMT, GS, GOGAT and GDH expression levels reveal NO and ABA mediated drought tolerance in Brassica juncea L.

Little is known about the interactive effects of exogenous nitric oxide (NO) and abscisic acid (ABA) on nitrogen (N) metabolism and related changes at molecular and biochemical levels under drought stress. The present study highlights the independent and combined effect of NO and ABA (grouped as “nitrate agonists”) on expression profiles of representative key genes known to be involved in N-uptake and assimilation, together with proline metabolism, N–NO metabolism enzyme’s activity and nutrient content in polyethylene glycol (PEG) treated roots of Indian mustard (B. juncea cv. Varuna). Here we report that PEG mediated drought stress negatively inhibited growth performance, as manifested by reduced biomass (fresh and dry weight) production. Total N content and other nitrogenous compounds (NO3−, NO2−) were decreased; however, NH4+, NH4+/ NO3− ratio and total free amino acids content were increased. These results were positively correlated with the PEG induced changes in expression of genes and enzymes involved in N-uptake and assimilation. Also, PEG supply lowered the content of macro- and micro-nutrients but proline level and the activity of ∆1-pyrroline-5-carboxylate synthetase increased indicating increased oxidative stress. However, all these responses were reversed upon the exogenous application of nitrate agonists (PEG + NO, PEG + NO + ABA, and PEG + ABA) where NO containing nitrate agonist treatment i.e. PEG + NO was significantly more effective than PEG + ABA in alleviating drought stress. Further, increases in activities of L-arginine dependent NOS-like enzyme and S-nitrosoglutathione reductase were observed under nitrate agonist treatments. This indicates that the balanced endogenous change in NO and ABA levels together during synthesis and degradation of NO mitigated the oxidative stress in Indian mustard seedlings. Overall, our results reveal that NO independently or together with ABA may contribute to improved crop growth and productivity under drought stress.

Photoactive NO hybrids with pseudo-zero-order release kinetics for antimicrobial applications.

Bacterial infection is a major threat to the health and life of humans due to the development of drug resistance, which is related to biofilm formation. Nitric oxide (NO) has emerged as an important factor in regulating biofilm formation. In order to harness the potential benefits of NO and develop effective antibacterial agents, we designed and synthesized a new class of NO hybrids in which the active scaffold benzothienoazepine was tagged with a nitroso group and further conjugated with quaternary ammoniums or phosphoniums. The temporal release of NO from these hybrids can be achieved by photoactivation. Interestingly, the NO release follows a pseudo-zero-order kinetics, which is easily determined by measuring the fluorescent benzothienoazepine or NO. Compared to the positive control ciprofloxacin, the NO hybrid with triphenyl phosphonium (TPP) exhibited more effective activity against S. aureus biofilm in darkness. Irradiation of the NO hybrid led to higher inhibition against S. aureus biofilm compared to the parental NO hybrid in darkness or the corresponding NO-released product, indicating the combined effect of NO and the NO-released product. Therefore, this new class of NO hybrids includes very promising antimicrobial agents and this work provides a new way for the design of highly effective antimicrobial agents.

Nitric oxide and ROS mediate autophagy and regulate Alternaria alternata toxin-induced cell death in tobacco BY-2 cells

Synergistic interaction of nitric oxide (NO) and reactive oxygen species (ROS) is essential to initiate cell death mechanisms in plants. Though autophagy is salient in either restricting or promoting hypersensitivity response (HR)-related cell death, the crosstalk between the reactive intermediates and autophagy during hypersensitivity response is paradoxical. In this investigation, the consequences of Alternaria alternata toxin (AaT) in tobacco BY-2 cells were examined. At 3 h, AaT perturbed intracellular ROS homeostasis, altered antioxidant enzyme activities, triggered mitochondrial depolarization and induced autophagy. Suppression of autophagy by 3-Methyladenine caused a decline in cell viability in AaT treated cells, which indicated the vital role of autophagy in cell survival. After 24 h, AaT facilitated Ca2+ influx with an accumulation of reactive oxidant intermediates and NO, to manifest necrotic cell death. Inhibition of NO accumulation by 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) decreased the level of necrotic cell death, and induced autophagy, which suggests NO accumulation represses autophagy and facilitates necrotic cell death at 24 h. Application of N-acetyl-L-cysteine at 3 h, confirmed ROS to be the key initiator of autophagy, and together with cPTIO for 24 h, revealed the combined effects of NO and ROS is required for necrotic HR cell death.

Abstract 5111: Nitric oxide and its role in photodynamic therapy

Abstract Porphyrinic pigments are used as photosensitizers (PS) in photodynamic detection (PDD) and therapy (PDT) that is a minimally invasive modality in the fight against cancer. When the PS is activated by visible light at a given wavelength, reactive oxygen species (ROS) are generated, which cause cancer cells to undergo cell death. Despite significant advances, drawbacks of the PSs in clinical use include their non-selectivity in cellular-targeting causing cell death by necrosis leading to tissue inflammation. Nitric oxide (NO) has been shown to play a key role in modulating apoptotic cell death pathways and to react with reactive oxygen species to form additional lethal reactive nitrogen species (RNS). In our efforts to enhance the effectiveness of PDT, we set out to investigate the role of NO in PDT. We hypothesized that NO delivered to cancer cells at the time that the PS was administered would enhance the efficacy of PDT by promoting mitochondria-mediated apoptosis. To this end, we incubated androgen-sensitive human prostate adenocarcinoma (LNCaP) cells with both a PS and an NO releasing agent that was most effective in NO release as was spectrophotometrically determined by its oxidation of hemoglobin. Phototoxicity experiments were carried out at 37 OC with a noncoherent light source. Cell viability, damage and death were assessed in both illuminated and non-illuminated cells and were quantified by MTT staining as well as trypan blue and propidium iodide exclusion. To corroborate the cell viability results, we assayed clonogenic recovery in response to PDT in pigmented cells both in the presence and absence of NO. Our results indicate that the effectiveness of PDT in causing cell death depends on the NO concentration. PDT with NO alone was toxic to the cancer cells at high concentration of NO, whereas at low NO concentration no significant cell damage was observed after light illumination. PDT with the PS alone was not as effective in promoting cell death as PDT in the presence of both NO and the PS. Depending on the concentrations of the PS and NO, we observed that either necrosis or apoptosis were the prevailing modes of cell death after PDT. Our results indicate that the phototoxicity of the compounds is mainly determined by their intracellular concentration. Thus, understanding the combined effects of NO and the PSs in enhancing cell phototoxicity will aid in determining the roles that NO plays in improving the efficacy of PDT and may provide an alternate regimen to enhance PDT efficacy. Citation Format: Marco Monroy, Pooncharas Tipgunlakant, Ursula Simonis, Raymond Esquerra. Nitric oxide and its role in photodynamic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2014-5111

Lisinopril augments the combined effect of nitric oxide and potassium channels on endothelium‐dependent relaxation in spontaneously hypertensive rats

Endothelium‐dependent relaxation is reported to be impaired in hypertension. However, there is equivocal evidence on the exact nature of the underlying dysfunction and whether it could be affected by antihypertensive drugs specifically angiotensin‐converting enzyme inhibitors. This study was conducted to evaluate the contribution of NO and potassium channels to endothelial function after lisinopril treatment in spontaneously hypertensive rats. Male animals (10–12 weeks old) were treated with lisinopril (20mg/Kg/day) for 10 weeks. ACh (0.1 nM–10μM)‐induced relaxation of third order mesenteric arteries was measured on a wire myograph with isometric recording in the presence and absence of different blockers. In the presence of inhibitor of nitric oxide synthase (LNMMA 100uM), the maximum relaxation of arteries from untreated (UT) and treated (T) rats to ACh was reduced by 19±3.5% and 5.0±01.6% respectively, while in the presence of SK and IK channel blockers (apamin 100nM and TRAM‐34 10μM) it was reduced by 35±3.5% and 34%±6.25%; respectively (n=10–22). With combined blockade, ACh‐induced relaxation was inhibited by 29±6.2% UT and 65±3.2 % T. Our results suggest that chronic treatment with lisinopril, although it reduced the relative contribution of nitric oxide alone it enhanced the combined effect of nitric oxide and potassium channels towards more endothelium‐dependent relaxation.

Opposing effects of nitric oxide and prostaglandin inhibition on muscle mitochondrial V̇o2 during exercise

Nitric oxide (NO) and prostaglandins (PG) together play a role in regulating blood flow during exercise. NO also regulates mitochondrial oxygen consumption through competitive binding to cytochrome-c oxidase. Indomethacin uncouples and inhibits the electron transport chain in a concentration-dependent manner, and thus, inhibition of NO and PG synthesis may regulate both muscle oxygen delivery and utilization. The purpose of this study was to examine the independent and combined effects of NO and PG synthesis blockade (L-NMMA and indomethacin, respectively) on mitochondrial respiration in human muscle following knee extension exercise (KEE). Specifically, this study examined the physiological effect of NO, and the pharmacological effect of indomethacin, on muscle mitochondrial function. Consistent with their mechanism of action, we hypothesized that inhibition of nitric oxide synthase (NOS) and PG synthesis would have opposite effects on muscle mitochondrial respiration. Mitochondrial respiration was measured ex vivo by high-resolution respirometry in saponin-permeabilized fibers following 6 min KEE in control (CON; n = 8), arterial infusion of N(G)-monomethyl-L-arginine (L-NMMA; n = 4) and Indo (n = 4) followed by combined inhibition of NOS and PG synthesis (L-NMMA + Indo, n = 8). ADP-stimulated state 3 respiration (OXPHOS) with substrates for complex I (glutamate, malate) was reduced 50% by Indo. State 3 O(2) flux with complex I and II substrates was reduced less with both Indo (20%) and L-NMMA + Indo (15%) compared with CON. The results indicate that indomethacin reduces state 3 mitochondrial respiration primarily at complex I of the respiratory chain, while blockade of NOS by L-NMMA counteracts the inhibition by Indo. This effect on muscle mitochondria, in concert with a reduction of blood flow accounts for in vivo changes in muscle O(2) consumption during combined blockade of NOS and PG synthesis.

The Comparative and Combined Effects of Nitric Oxide and Higher Alkanes in Sensitizing Methane Oxidation

The comparative and combined effects of nitric oxide (NO) and higher alkanes on methane oxidation were examined by experimentation and kinetic modeling. Experiments were conducted using fuel-lean, lower-alkanes mixtures with NO (0–400 ppm v/v) in an atmospheric flow reactor at residence time of 2 s over the temperature range of 820–950 K. NO was found to greatly promote methane conversion, and its sensitizing effect strengthened with the increasing concentration that was added to the system. The promoting effect of higher alkanes on methane conversion was also evident, particularly at zero or low NO concentrations. A strong dependency of the sensitizing effect on the concentration of higher alkanes present was also observed. The kinetic mechanism from Le Cong et al., performed reasonably well in reproducing the experimental trends. However, the sensitizing propensity of higher alkanes in the presence of NO could not be unambiguously ranked under all conditions. Modification to the kinetic mechanism of Le Cong et al., was attempted. Specifically, the submechanism of C3 peroxy radicals was added, but the modeling results indicated a lower impact on methane conversion than was initially expected. The most sensitive reactions were revealed, and the generalized reaction pathways for methane oxidation sensitized by higher alkanes, with or without the presence of NO, were also proposed, following detailed sensitivity analysis.

Erratum to: Improvement in oxygenation by prone position and nitric oxide in patients with acute respiratory distress syndrome

Inhaled nitric oxide (NO) and prone position improve arterial oxygenation in patients with the acute respiratory distress syndrome. This study was undertaken to assess the combined effects of NO and prone position in these patients.Prospective clinical study.General intensive care service in a community teaching hospital.14 mechanically ventilated adult patients with the acute respiratory distress syndrome (mean lung injury score 3.23+/-0.27).We measured hemodynamic and oxygenation parameters in the supine position and 2 h later in the prone position, before and during inhalation of 10 ppm NO. A positive response in oxygenation was defined as a > or =20% increment in the arterial oxygen tension/fractional inspired oxygen ratio (PaO2/FIO2). In the prone position PaO2/FIO2 increased significantly (from 110+/-55 to 161+/-89 mm Hg, p<0.01) and venous admixture decreased (from 38+/-12 to 30+/-7%, p<0.01) compared to the supine position. Ten of the 14 patients were responders in the prone position. In the supine position, inhalation of NO improved oxygenation to a lesser extent, increasing PaO2/FIO2 to 134+/-64 mm Hg (p<0.01) and decreasing venous admixture to 35+/-12%, (p<0.01). Five of the 14 patients responded to NO inhalation supine and 8 of 14 responded prone (p = 0.22). The combination of NO therapy and prone positioning was additive in increasing PaO2/FIO2 (197+/-92 mm Hg) and decreasing venous admixture (27+/-8%) (p<0.01). This combination also showed a positive oxygenation response on compared to the supine value without NO in 13 of the 14 patients (93 %). NO-induced changes in PaO2/FIO2 were correlated to changes in pulmonary vascular resistance only in the prone position.In patients with the acute respiratory distress syndrome, the combination of NO and prone position is a valuable adjunct to mechanical ventilation.

Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes

Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS−/− mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS−/− mice suggesting that increased mitochondrial sensitivity to NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.

Blockade Of Prostaglandins And Nitric Oxide In-vivo Reduces State 3 Mitochondrial Respiration In Human Skeletal Muscle

Combined inhibition of nitric oxide (NO) and prostaglandins (PG) reduces skeletal muscle blood flow and oxygen delivery at rest and during exercise. It has recently been found that combined inhibition of NO and PG blockade reduces muscle oxygen uptake during exercise (Mortensen et al. 2007) suggesting a partial inhibition of aerobic metabolism or improved muscle efficiency of oxidative phosphorylation or actin-myosin cycling. PURPOSE: The purpose of this study was to examine in humans the independent and combined effect of NO and PG blockade with L-NMMA and Indomethacin (Indo) on mitochondrial respiration in muscle from biopsies of the vastus lateralis (VL) following knee extension (KE) exercise. METHODS: Mitochondrial respiration was measured ex-vivo by high resolution respirometry in saponin-permeabilized fibers following 6 min KE in control (CON, n=8), arterial infusion of LNMMA (n=4) and Indo (n=4) randomized in order, followed by combined inhibition of NO and PG (L-NMMA + Indo, n=8). RESULTS. ADP-stimulated state 3 respiration with substrates for complex I (glutamate, malate) was reduced 50% by Indo compared to CON. State 3 O2 flux with convergent electron input through both complex I and II with addition of succinate was equally reduced with Indo (20%) and L-NMMA + Indo (15%) compared to CON and LNMMA alone. Uncoupled O2 flux determined by titration of the ionophore FCCP was not increased above state 3 respiration with Indo suggesting tight coupling to oxidative phosphorylation. Graded titrations of L-NMMA into the respirometer in CON muscle reduced state 3 respiration by 12% only at a supra-physiological dose, while titration of Indo caused a linear reduction at all doses within and above the physiological range. CONCLUSION: The results indicate that inhibition of PG by indomethacin inhibits state 3 mitochondrial respiration primarily at complex I of the respiratory chain. This effect may in part explain the in-vivo reduction in muscle O2 uptake during exercise with double blockade of NO and PG, since blockade of NO by L-NMMA counteracts the inhibitory effect of Indo.

Modeling Nitric Oxide Regulation Of Ec Coupling In Cardiac Myocytes

Nitric oxide (NO) signaling is a potent modulator of cardiac contractility in conditions of increased heart rate or ß-adrenergic signaling. Changes in nitric oxide synthase (NOS), the enzyme responsible for NO production, play a significant role in EC coupling observed in heart failure following myocardial infarction. NO signaling is thought to modulate cardiac function by targeting a range of EC coupling proteins including ryanodine receptor, phospholamban, L-type Ca2+ channel and myosin. However, the mechanisms underlying NO signaling and the relative importance of NO targets are unclear. Previous computational models of ß-adrenergic signaling and EC coupling have not accounted for NO regulation. We propose a new model that incorporates NO metabolism and effects of eNOS and nNOS activity on EC coupling. This integrated model provides a consistent framework to quantitatively predict the combined effects of NO on EC coupling and explain discrepancies in prior experimental results.

A Genetic Analysis of Nitrosative Stress

Nitrosative stress is induced by pathophysiological levels of nitric oxide (NO) and S-nitrosothiols (e.g., S-nitrosoglutathione, GSNO) and arises, at least in significant part, from the nitrosylation of critical protein Cys thiols (S-nitrosylation) and metallocofactors. However, the mechanisms by which NO and GSNO mediate nitrosative stress are not well understood. Using yeast Saccharomyces cerevisiae strains lacking NO- and/or GSNO-consuming enzymes (flavohemoglobin and GSNO reductase, respectively), we measured the individual and combined effects of NO and GSNO on both cell growth and the formation of protein-bound NO species. Our results suggest an intracellular equilibrium between NO and GSNO, dependent in part on cell-catalyzed release of NO from GSNO (i.e., "SNO-lyase" activity). However, whereas NO induces multiple types of protein-based modifications, levels of which correlate with inhibition of cell growth, GSNO mainly affects protein S-nitrosylation, and the relationship between S-nitrosylation and nitrosative stress is more complex. These data support the idea of multiple classes of protein-SNO, likely reflected in divergent routes of synthesis and degradation. Indeed, a significant fraction of protein S-nitrosylation by NO occurs in the absence of O(2), which is commonly assumed to drive this reaction but instead is apparently dependent in substantial part upon protein-bound transition metals. Additionally, our findings suggest that nitrosative stress is mediated principally via the S-nitrosylation of a subset of protein targets, which include protein SNOs that are stable to cellular glutathione (and thus are not metabolized by GSNO reductase). Collectively, these results provide new evidence for the mechanisms through which NO and GSNO mediate nitrosative stress as well as the cellular pathways of protein S-nitrosylation and denitrosylation involving metalloproteins, SNO lyase(s) and GSNO reductase.

3,4-Dihydroxyphenylacetic acid (DOPAC) modulates the toxicity induced by nitric oxide in PC-12 cells via mitochondrial dysfunctioning

It has been postulated that dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, and nitric oxide ( NO) induce mitochondrial dysfunction in a synergistic manner. We examined the combined effects of NO and DOPAC on PC-12 cells in terms of cell viability, nuclear morphology, mitochondrial parameters and cell death mechanisms. The apoptotic cell death induced by the NO-donor, S-nitroso- N-acetylpenicillamine (SNAP), was differently modulated by DOPAC as a function of DOPAC/cell ratios. Whereas below 200 nmol/10 6 cells, DOPAC inhibited a typical apoptotic pathway induced by exposure the cells to the NO donor, above 200 nmol DOPAC/10 6 cells, the cell death was not only enhanced but encompassed a distinct mechanism. Loading the cells with dopamine mimicked the effects of DOPAC. Specifically, the combination of DOPAC and NO induced an early mitochondrial membrane potential dissipation and ATP depletion followed by loss of cellular membrane integrity. Mitochondrial dysfunction was accompanied by the release of cytochrome c in both cases, NO individually and in combination with DOPAC, but caspase-3 and caspase-9 activation were only observed in the absence of DOPAC. DOPAC alone was ineffective. Thus, our results suggest a role for DOPAC as a modulator of cell fate and point to a pathway of cell death involving DOPAC and NO, via mechanisms that include mitochondrial dysfunction but do not involve the activation of the typical apoptotic caspase cascade. The significance of these results is discussed in connection with the mechanisms of cell death underlying Parkinson's disease.

Synergistic inhibition of respiration in brain mitochondria by nitric oxide and dihydroxyphenylacetic acid (DOPAC): Implications for Parkinson's disease

The inhibition of mitochondrial respiration by nitric oxide ( NO) at cytochrome c oxidase level has been established as a physiological regulatory mechanism of mitochondrial function. Given, on the one hand, the potential involvement of NO and dopamine metabolism in mitochondrial dysfunction associated with neurodegeneration and, on the other hand, the reported interaction of NO with dihydroxyphenylacetic acid (DOPAC), a major mitochondrial-associated dopamine metabolite, we examined the combined effects of NO and DOPAC on the respiratory chain of isolated rat brain mitochondria. Whereas dopamine or DOPAC induced no measurable effects on the mitochondrial respiration rate, a mixture of NO with DOPAC inhibited the rate in a way stronger than that exerted by NO. This effect was noticed with actively respiring (state 3) and resting (state 4) mitochondria. At variance with DOPAC, dopamine failed to potentiate NO inhibitory effects. The inhibition was dependent on the concentration of both compounds, NO and DOPAC, and exhibited characteristics similar to those exerted by NO, namely: it was reversible and dependent on the concentration of oxygen. Analysis of respiratory enzymatic activities demonstrated a selective inhibition at the level of cytochrome c oxidase (complex IV). Insights into the chemical mechanisms underlying the inhibitory effect were inferred from experiments using metmyoglobin (a ligand for NO and derived species, such as nitroxyl anion) and ferrocyanide (a reductant of NO, producing nitroxyl anion). Whereas metmyoglobin decreased the inhibition, ferrocyanide potentiated the inhibition. Moreover, a mixture of ferrocyanide with NO reproduced the effects exerted by the mixture of NO with DOPAC. The results are consistent with the notion of a reaction of NO with DOPAC producing a nitric oxide-derived compound(s), which inhibit O 2 uptake at the cytochrome oxidase level. Although the mechanism in question remains to be clearly elucidated it is suggested that the NO/DOPAC-dependent inhibition of cytochrome oxidase may involve nitroxyl anion. The significance of these observations for mitochondrial dysfunction inherent in Parkinson's disease is discussed.

Effects of Nitric Oxide (NO) and Soluble Nucleoside Triphosphate Diphosphohydrolase (NTPDase) on Inhibition of Platelet Deposition In Vitro

Vascular thrombosis is regulated via the release of several constituents from the vascular endothelium, including nucleoside triphosphate diphosphohydrolases (NTPDases or ectonucleotidases), nitric oxide (NO), and eicosanoids. Currently, it is unknown how these constituents interact in the inhibition of platelet aggregation and adhesion. To investigate the combined effects of NO and NTPDase on platelet deposition sequestration, an in vitro study was performed to compare inhibition of platelet deposition to a biomaterial by NO in the absence or presence of soluble NTPDase. Results of the platelet inhibition studies with NO and NTPDase conclusively show that the inhibitory effects of NTPDase and NO are additive. The platelet inhibitory potency in the presence of NO was enhanced by NTPDase in a dose-dependent manner, for a given NO exposure. This augmentation is independent of aspirin; the ability of NTPDase or NO alone to inhibit platelet deposition is also independent of aspirin. Clearly, NO and NTPDase independently contribute to platelet inhibition via different mechanisms. The inaction of NO on the activity of NTPDase confirmed that NO or reaction products in the presence of O 2 do not interact with NTPDase directly.

Glomerular Basement Membrane Anionic Sites in Adriamycin Nephropathy: Effect of Saline Loading and Nitric Oxide Modulation

Background: In previous studies we found that experimental Adriamycin (ADR) nephropathy is associated with the loss of glomerular basement membrane (GBM) anionic sites provided by heparan sulfate proteoglycans. Chronic saline loading in normal rats resulted in a similar effect on the GBM anionic sites. The L-arginine-nitric oxide synthase-nitric oxide system is involved in the pathogenesis of experimental chronic renal failure. The present study was performed to determine the combined effect of nitric oxide (NO) modulation and chronic saline loading in ADR nephropathy. The modulation of NO was done by chronic administration of L-arginine (NO donor) or N^w-nitro-L-arginine, a known nitric oxide synthase inhibitor. Methods: Systolic blood pressure was measured in awake rats by a tail-cuff method. Renal function was assessed by creatinine clearance, FeNa%, and daily protein excretion. The change of mean GBM widths and anionic sites distribution were assessed by electron microscopy. The localization of anionic sites was carried out by cationic colloidal gold. Plasma and urinary nitrates (NO_x) were measured by nitrite (NO₂) + nitrate (NO₃), stable metabolites of NO. Results: Two weeks after the ADR administration (3.5 mg/kg BW iv) the rats had severe renal failure (creatinine clearance 134 ± 31 µl/min/100 g BW vs. initial values 670 ± 29 µl/min/ 100 g BW, p < 0.001), high FeNa%, severe proteinuria, increased GBM width, significant reduction of GBM anionic sites and low urinary NO_x excretion. The saline loading resulted in further reduction of GBM anionic sites count and blood pressure elevation. The inhibition of NO did not change the course of ADR nephropathy. The main finding of the present study is that chronic administration of L-arginine significantly alleviates the renal failure in the ADR (+/– saline loading) nephropathy. The L-arginine-treated rat had higher creatinine clearance, lower FeNa% and protein excretion and complete normalization of GBM anionic sites distribution. Conclusions: Sodium loading has a deleterious effect on GBM permselectivity. L-Arginine prevents the reduction of GBM anionic sites, decreases proteinuria and alleviates the renal insufficiency in ADR nephropathy.

Nitric Oxide Inhibits Neonatal Hepatocyte Oxidative Metabolism

Liver function is frequently impaired in neonates with sepsis. Nitric oxide (NO) is thought to be a mediator of organ dysfunction and liver oxidative metabolism during sepsis. The authors developed an in vitro model to investigate the effect of NO and the combined effect of NO plus H2O2 on neonatal hepatocyte oxidative metabolism. Hepatocytes were isolated from neonatal rats. Oxygen consumption was measured polarographically. In Study A, cells were exposed to S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, at various concentrations. In study B, myxothiazol and oligomycin, inhibitors of mitochondrial respiration, were added to investigate the site of action of NO. In study C, hepatocytes were incubated in the presence of both SNAP (300 micromol/L) and H2O2 (1.5 mmol/L). In study D, morphological alterations induced by NO and NO plus H2O2 were investigated by hepatocyte electron microscopy. In study A, SNAP caused a dose-dependent decrease in oxygen consumption. A significant inhibition was reached at 300 micromol/L SNAP. In study B, the lack of further inhibition when SNAP was given together with myxothiazol indicates that NO acts intramitochondrially. Similarly, no further inhibition occurred when the NO donor was given together with oligomycin, suggesting that the effect of NO is mainly at the level of ATP synthase. In study C, concomitant addition of 300 micromol/L SNAP and 1.5 mmol/L H2O2 to hepatocytes caused further inhibition of oxygen consumption compared with either SNAP or H2O2 alone. In study D, mild alterations in hepatocyte morphology were noted in the presence of SNAP or SNAP plus H2O2. In neonatal hepatocytes, NO significantly inhibits mitochondrial oxygen consumption, possibly at the level of ATP synthase. The effect of NO is additive to that of H2O2. Morphological findings were consistent with these biochemical effects and suggest that NO and H2O2 are important mediators of liver damage during sepsis.

High-dose nitric oxide inhalation increases lung injury after gastric aspiration.

Inhaled nitric oxide is often used in patients with adult respiratory distress syndrome. However, nitric oxide also may be significantly toxic, especially if administered concurrently with hyperoxia. The authors evaluated the isolated effect of nitric oxide and the combined effects of nitric oxide and hyperoxia on lung injury in rats after acid aspiration. Animals were injured by instillation of 1.2 ml/kg hydrogen chloride in low-pH saline (the acid group) or acidified gastric particles (the casp group) into the lungs under halothane anesthesia via a tracheal catheter. Controls received no injury vehicle but rather underwent the surgical process. After recovery from anesthesia, the animals were exposed to 20% or 90% oxygen with or without 20, 40, or 80 ppm nitric oxide for 5 h. The permeability index, alveolar-arterial oxygen difference, the ratio of oxygen pressure to the inspired fraction of oxygen, and the ratio of wet to dry weight were assessed 5 h after injury as indices of lung injury. Data were assessed using analysis of variance. Each group included 6-10 rats. Exposure to nitric oxide (80 ppm) in air increased protein permeability in the lungs to a permeability index of 1.42+/-0.12 after acid aspiration. The combination of nitric oxide (80 ppm) and hyperoxia further increased protein leakage to a permeability index of 2.1+/-0.25. Exposure to lower concentrations of nitric oxide (e.g., 20 and 40 ppm) increased the permeability index of the lungs (1.44+/-0.21, 1.75+/-0.29, respectively) in the presence of hyperoxia, although it did not affect the permeability index of the lungs during exposure to air. Pretreatment of animals with deferoxamine and methylene blue partially inhibited the adverse effect of hyperoxia and nitric oxide, which suggested a complex underlying mechanism involving both reactive-species generation and pulmonary vasomotor changes. These results show that inhaled nitric oxide at 80 ppm for a short duration (5 h) increases the severity of the inflammatory microvascular lung injury after acid aspiration. The pulmonary damage is exacerbated further in the presence of high oxygen concentrations. Although lower concentrations of nitric oxide did not increase the extent of lung injury, longer exposure times need to be assessed.

Nitric oxide-induced potentiation of the killing of Burkholderia cepacia by reactive oxygen species: implications for cystic fibrosis.

Burkholderia (formerly Pseudomonas) cepacia has emerged as an important pulmonary pathogen in cystic fibrosis, and survives within the lung despite a vigorous neutrophil-dominated immune response. Nitric oxide (NO) contributes to the antimicrobial activity of reactive oxygen species in the normal lung, but recent evidence suggests that inducible NO synthase is not expressed in the airway epithelial cells of cystic fibrosis (CF) patients. This may explain the failure of the neutrophil response to eliminate B. cepacia. To test this hypothesis, the present study examined the combined effect of NO, superoxide and H2O2 against B. cepacia. There was no killing of a highly transmissible strain by either superoxide or NO alone, but their combination reduced the bacterial count by >1000-fold over 75 min. This bactericidal activity was not sensitive to addition of superoxide dismutase, but was abrogated completely by catalase, suggesting that NO and hydrogen peroxide were the bactericidal mediators. Increased killing by NO in combination with H2O2 was seen for seven of a further 11 strains examined. The lack of NO in the lungs of CF patients may contribute to the survival of B. cepacia.

Combined effects of nitric oxide and oxygen during acute pulmonary vasodilator testing

OBJECTIVESWe compared the ability of inhaled nitric oxide (NO), oxygen (O2) and nitric oxide in oxygen (NO+O2) to identify reactive pulmonary vasculature in pulmonary hypertensive patients during acute vasodilator testing at cardiac catheterization.BACKGROUNDIn patients with pulmonary hypertension, decisions regarding suitability for corrective surgery, transplantation and assessment of long-term prognosis are based on results obtained during acute pulmonary vasodilator testing.METHODSIn group 1, 46 patients had hemodynamic measurements in room air (RA), 100% O2, return to RA and NO (80 parts per million [ppm] in RA). In group 2, 25 additional patients were studied in RA, 100% O2and 80 ppm NO in oxygen (NO+O2).RESULTSIn group 1, O2decreased pulmonary vascular resistance (PVR) (mean ± SEM) from 17.2 ± 2.1 U·m2to 11.1 ± 1.5 U·m2(p < 0.05). Nitric oxide caused a comparable decrease from 17.8 ± 2.2 U·m2to 11.7 ± 1.7 U·m2(p < 0.05). In group 2, PVR decreased from 20.1 ± 2.6 U·m2to 14.3 ± 1.9 U·m2in O2(p < 0.05) and further to 10.5 ± 1.7 U·m2in NO+O2(p < 0.05). A response of 20% or more reduction in PVR was seen in 22/25 patients with NO+O2compared with 16/25 in O2alone (p = 0.01).CONCLUSIONSInhaled NO and O2produced a similar degree of selective pulmonary vasodilation. Our data suggest that combination testing with NO+O2provides additional pulmonary vasodilation in patients with a reactive pulmonary vascular bed in a selective, safe and expeditious fashion during cardiac catheterization. The combination of NO+O2identifies patients with significant pulmonary vasoreactivity who might not be recognized if O2or NO were used separately.