Gamma-irradiation potentiates L-arginine-dependent nitric oxide formation in mice.

Gamma-irradiation of mongrel mice at a sublethal dose (700 Roentgen) enhanced the formation of nitric oxide (NO) in the liver, intestine, lung, kidney, brain, spleen or heart of the animals. NO formation was determined by the increase in intensity of the EPR signal due to trapping of NO into mononitrosyl iron complexes (MNIC) with exogenous diethyldithiocarbamate (DETC) injected intraperitoneally. The EPR signal of these MNIC-DETC complexes was characterized by g-factor values at g perpendicular values at g perpendicular = 2.035 and g parallel = 2.02 and a triplet hyperfine structure at g perpendicular. The NO synthase inhibitor, NG-nitro-L-arginine, prevented MNIC-DETC complex formation both in liver and intestine, demonstrating the involvement of endogenous NO formed. Thus, gamma-irradiation may enhance endogenous NO biosynthesis in these tissues, presumably by facilitating the entry of Ca2+ ions into the membrane as well as the cytosol of NO-producing cells through irradiation-induced membrane lesions.     

Restrained whole body plethysmography for measure of strain-specific and allergen-induced airway responsiveness in conscious mice.

The mouse is the most extensively studied animal species in respiratory research, yet the technologies available to assess airway function in conscious mice are not universally accepted. We hypothesized that whole body plethysmography employing noninvasive restraint (RWBP) could be used to quantify specific airway resistance (sRaw-RWBP) and airway responsiveness in conscious mice. Methacholine responses were compared using sRaw-RWBP vs. airway resistance by the forced oscillation technique (Raw-FOT) in groups of C57, A/J, and BALB/c mice. sRaw-RWBP was also compared with sRaw derived from double chamber plethysmography (sRaw-DCP) in BALB/c. Finally, airway responsiveness following allergen challenge in BALB/c was measured using RWBP. sRaw-RWBP in C57, A/J, and BALB/c mice was 0.51 +/- 0.03, 0.68 +/- 0.03, and 0.63 +/- 0.05 cm/s, respectively. sRaw derived from Raw-FOT and functional residual capacity (Raw*functional residual capacity) was 0.095 cm/s, approximately one-fifth of sRaw-RWBP in C57 mice. The intra- and interanimal coefficients of variations were similar between sRaw-RWBP (6.8 and 20.1%) and Raw-FOT (3.4 and 20.1%, respectively). The order of airway responsiveness employing sRaw-RWBP was AJ > BALBc > C57 and for Raw-FOT was AJ > BALB/c = C57. There was no difference between the airway responsiveness assessed by RWBP vs. DCP; however, baseline sRaw-RWBP was significantly lower than sRaw-DCP. Allergen challenge caused a progressive decrease in the provocative concentration of methacholine that increased sRaw to 175% postsaline values based on sRaw-RWBP. In conclusion, the technique of RWBP was rapid, reproducible, and easy to perform. Airway responsiveness measured using RWBP, DCP, and FOT was equivalent. Allergen responses could be followed longitudinally, which may provide greater insight into the pathogenesis of chronic airway disease.     

Iron as an inducer of nitric oxide formation in the animal body

Citrate iron complex injections to mice or rats resulted in the nitric oxide formation detected by nitric oxide binding to iron-diethyldithiocarbomate complexes. The mononitrosyl iron complexes formed were paramagnetic and EPR active. The maximal nitric oxide concentrations in rat livers were 15-20 nm per gram of tissue. Phenosan-K (an antioxidant) inhibited partly the iron capacity to nitric oxide formation in animal organisms. The nitric oxide formation was proposed to be due to some endogenic amino groups oxidation by active oxygen agents or products of lipid or non-saturated fatty acid production under the prooxidant action of the iron.     

Role of nitric oxide in tolerance to lipopolysaccharide in mice.

The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the febrile response, which is called endotoxin tolerance. We tested the hypothesis that nitric oxide (NO) arising from inducible NO synthase (iNOS) plays a role in endotoxin tolerance, using not only pharmacological trials but also genetically engineered mice. Body core temperature was measured by biotelemetry in mice treated with NG-monomethyl-L-arginine (L-NMMA, 40 mg/kg; a nonselective NO synthase inhibitor) or aminoguanidine (AG, 10 mg/kg; a selective iNOS inhibitor) and in mice deficient in the iNOS gene (iNOS KO) mice. Tolerance to LPS was induced by means of three consecutive LPS (100 microg/kg) intraperitoneal injections at 24-h intervals. In wild-type mice, we observed a significant reduction of the febrile response to repeated administration of LPS. Injection of L-NMMA and AG markedly enhanced the febrile response to LPS in tolerant animals. Conversely, iNOS-KO mice repeatedly injected with LPS did not become tolerant to the pyrogenic effect of LPS. These data are consistent with the notion that NO modulates LPS tolerance in mice and that iNOS isoform is involved in NO synthesis during LPS tolerance.     

Peroxynitrite formation from macrophage-derived nitric oxide.

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.     

The use of body mass loss to estimate metabolic rate in birds

During starvation, energy production occurs at the expense of body reserve utilisation which results in body mass loss. Knowing the role of the fuels involved in this body mass loss, along with their energy density, can allow an energy equivalent of mass loss to be calculated. Therefore, it is possible to determine daily energy expenditure (DEE) if two body mass loss measurements at an interval of a few days are obtained. The technique can be cheap, minimally stressful for the animals involved, and the data relatively simple to gather. Here we review the use of body mass loss to estimate DEE in birds through critiquing the strengths and weaknesses of the technique, and detail the methodology and considerations that must be adhered to for accurate measures of DEE to be obtained. Owing to the biology of the species, the use of the technique has been used predominantly in Antarctic seabirds, particularly penguins and albatrosses. We demonstrate how reliable the technique can be in predicting DEE in a non-Antarctic species, common eiders (Somateria mollissima), the female of which undergoes a fasting period during incubation. We conclude that using daily body mass loss to estimate DEE can be a useful and effective approach provided that (1) the substrate being consumed during mass loss is known, (2) the kinetics of body mass loss are understood for the species in question and (3) only species that enter a full phase II of a fast (where substrate catabolism reaches a steady state) and are not feeding for a period of time are appropriate for this method.     

Platelet activation and modulation of the induction of nitric oxide synthase in the conscious rat.

Injection of lipopolysaccharide (LPS) (Salmonella W. Typhosa i.v. bolus) into conscious rats, induced a rapid drop of circulating platelets analogous to that induced by ADP. The animals showed a small fall in mean arterial blood pressure (MABP), an increase in heart rate and a significant increase in plasma nitrite and nitrate level. This result is consistent with the stimulation of an inducible NO synthase (i-NOS). The administration of the stable prostacyclin analogue, iloprost plus ADP or LPS, significantly protected against the decrease in free platelet number induced by ADP or LPS. The plasma nitrite and nitrate level stimulated by LPS was significantly reduced by iloprost and also by prostacyclin. These results are consistent with an inhibition of i-NOS by agents that increase the intracellular level of cAMP. The administration of the NO donor S-Nitroso-N-acyl-D-penicillamine (SNAP) plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP and by LPS. SNAP did not decrease the plasma nitrite and nitrate level stimulated by LPS; furthermore it induced a significant increase of heart rate, without affecting MABP, suggesting a direct accelerating effect of NO on the sino-atrial node. The administration of S-nitroso-glutathione (GSNO), a stable nitrosothiol, plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP but not by LPS. GSNO significantly reduced the plasma nitrite and nitrate level stimulated by LPS. These data demonstrate that the L-Arginine: NO pathway in vivo may be modulated by prostanoids and that compounds which increase cAMP, such as iloprost, are able to protect against LPS-induced early thrombocytopenia.     

A neural mechanism of synergy formation for whole body reaching

The present study proposes a computational model for the formation of whole body reaching synergy, i.e., coordinated movements of lower and upper limbs, characterized by a focal component (the hand must reach a target) and a postural component (the center of mass must remain inside the support base). The model is based on an extension of the equilibrium point hypothesis that has been called Passive Motion Paradigm (PMP), modified in order to achieve terminal attractor features and allow the integration of multiple constraints. The model is a network with terminal attractor dynamics. By simulating it in various conditions it was possible to show that it exhibits many of the spatio-temporal features found in experimental data. In particular, the motion of the center of mass appears to be synchronized with the motion of the hand and with proportional amplitude. Moreover, the joint rotation patterns can be accounted for by a single functional degree of freedom, as shown by principal component analysis. It is also suggested that recent findings in motor imagery support the idea that the PMP network may represent the motor cognitive part of synergy formation, uncontaminated by the effect of execution.     

Modulation of nitric oxide formation by endothelial nitric oxide synthase gene haplotypes

Nitric oxide (NO) is a major regulator of the cardiovascular system. However, the effects of endothelial nitric oxide synthase (eNOS) gene polymorphisms or haplotypes on the circulating concentrations of nitrite (a sensitive marker of NO formation) and cGMP are unknown. Here we examined the effects of eNOS polymorphisms in the promoter region (T-786C), in exon 7 (Glu298Asp), and in intron 4 (4b/4a) and eNOS haplotypes on the plasma levels of nitrite and cGMP. We hypothesized that eNOS haplotypes could have a major impact on NO formation. We genotyped 142 healthy subjects by PCR-RFLP. To assess NO formation, the plasma concentrations of nitrite and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Haplotypes were inferred using the PHASE 2.1 program. No significant differences were found in age, body mass index, systolic and diastolic arterial blood pressure, heart rate, total cholesterol, triglycerides, cGMP, or nitrite among the genotype groups for the three polymorphisms studied here (all p>0.05). Interestingly, the C-4b-Glu haplotype was associated with lower plasma nitrite concentrations than those found in the other haplotype groups (p<0.05), but not with different cGMP levels (p>0.05). These findings suggest that eNOS gene variants combined within a specific haplotype modulate NO formation, although individual eNOS polymorphisms probably do not have major effects.     

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

BACKGROUND: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. METHODS: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. RESULTS: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. CONCLUSION: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.     

Dose- and time-dependence of radiation-induced nitric oxide formation in mice as quantified with electron paramagnetic resonance.

In vivo nitric oxide (NO) formation was quantified in mice after exposure to high-dose whole-body X-ray irradiation. NO produced and accumulated in the livers of irradiated mice was determined using NO trapping method with iron-dithiocarbamate complex combined with electron paramagnetic resonance (EPR) spectroscopy. When mice were irradiated with 50 Gy X-ray, NO formation peaked in approximately 3 h after the irradiation was terminated. Dose-dependence study indicated that NO formation measured 5 h after irradiation was leveled off at the dose higher than 50 Gy. Administration of NO synthase inhibitor, N(G)-monomethyl L-arginine (L-NMMA) shortly after irradiation completely abolished the NO signal, indicating that radiation-induced NO is produced through L-arginine-dependent NO synthase pathways. These results suggest that irradiation of X-ray initiates inflammation processes, resulting in delayed NO synthase expression and NO formation.     

The neurokinin-1 receptor modulates the methamphetamine-induced striatal apoptosis and nitric oxide formation in mice

In a previous study we showed that pharmacological blockade of the neurokinin-1 receptors attenuated the methamphetamine (METH)-induced toxicity of the striatal dopamine terminals. In the present study we examined the role of the neurokinin-1 receptors on the METH-induced apoptosis of some striatal neurons. To that end, we administered a single injection of METH (30 mg/kg, i.p.) to male mice. METH induced the apoptosis (terminal deoxyncleotidyl transferase-mediated dUTP nick end labeling) of approximately 20% of striatal neurons. This percentage of METH-induced apoptosis was significantly attenuated by either a single injection of the neurokinin-1 receptor antagonist, 17-β-hydroxy-17-a-ethynyl-5-a-androstano[3,2-β]pyrimido[1,2-a]benzimidazole (WIN-51,708) (5 mg/kg, i.p.), or the ablation of the striatal interneurons expressing the neurokinin-1 receptors (cholinergic and somatostatin) with the selective neurotoxin [Sar⁹,Met(O₂)¹¹] substance P–saporin. Next we assessed the levels of striatal 3-nitrotyrosine (3-NT) by HPLC and immunohistochemistry. METH increased the levels of striatal 3-NT and this increase was attenuated by pre-treatment with WIN-51,708. Our data support the hypothesis that METH-induced striatal apoptosis occurs via a mechanism involving the neurokinin-1 receptors and the activation of nitric oxide synthesis. Our findings are relevant for the treatment of METH abuse and may be relevant to certain neurological disorders involving the dopaminergic circuitry of the basal ganglia.     

Transport of L-arginine and nitric oxide formation in human platelets.

The results of the present study show that human platelets take up l-arginine by two transport systems which are compatible with the systems y+ and y+L. These Na+independent transporters have been distinguished by treating platelets with N-ethylmaleimide that blocks selectively system y+. System y+, that accounts for 30-40% of the total transport, is characterized by low affinity for l-arginine, is unaffected by l-leucine, is sensitive to changes of membrane potential and to trans-stimulation. The other component of l-arginine transport identified with the system y+L (approximately 60-70% of the total flux) shows high affinity for l-arginine, is insensitive to N-ethylmaleimide treatment, unaffected by changes in membrane potential, sensitive to trans-stimulation and inhibited by l-leucine in the presence of Na+. Moreover a strict correlation between l-arginine transport and nitric oxide (NO) production in whole cells was found. N-ethylmaleimide and l-leucine decreased NO production as well as cGMP elevation, and the effect on NO and cGMP were closely related. It is likely that the l-arginine transport systems y+ and y+L are both involved in supplying substrate for NO production and regulation in human platelets.     

A new and more accurate technique to characterize airway nitric oxide using different breath-hold times.

Exhaled nitric oxide (NO) arises from both airway and alveolar regions of the lungs, which provides an opportunity to characterize region-specific inflammation. Current methodologies rely on vital capacity breathing maneuvers and controlled exhalation flow rates, which can be difficult to perform, especially for young children and individuals with compromised lung function. In addition, recent theoretical and experimental studies demonstrate that gas-phase axial diffusion of NO has a significant impact on the exhaled NO signal. We have developed a new technique to characterize airway NO, which requires a series of progressively increasing breath-hold times followed by exhalation of only the airway compartment. Using our new technique, we determined values (means +/- SE) in healthy adults (20-38 yr, n = 8) for the airway diffusing capacity [4.5 +/- 1.6 pl.s(-1).parts per billion (ppb)(-1)], the airway wall concentration (1,340 +/- 213 ppb), and the maximum airway wall flux (4,350 +/- 811 pl/s). The new technique is simple to perform, and application of this data to simpler models with cylindrical airways and no axial diffusion yields parameters consistent with previous methods. Inclusion of axial diffusion as well as an anatomically correct trumpet-shaped airway geometry results in significant loss of NO from the airways to the alveolar region, profoundly impacting airway NO characterization. In particular, the airway wall concentration is more than an order of magnitude larger than previous estimates in healthy adults and may approach concentrations (approximately 5 nM) that can influence physiological processes such as smooth muscle tone in disease states such as asthma.     

Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase

H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.     

The rational design of inhibitors of nitric oxide formation by inducible nitric oxide synthase

A series of compounds was rationally designed as inhibitors of dimer formation of the inducible isoform of nitric oxide synthase, and subsequent nitric oxide production. The conformation of two fragments obtained from a crystal structure was utilized to design a tether connecting those same two fragments. The resulting compounds were potent dimerization inhibitors that bound to the enzyme in a similar conformation as the fragments.     

A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.