Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

Reactive oxygen species and anti-oxidant defenses in tail of tadpoles, Xenopus laevis

Tail regression in tadpoles is one of the most spectacular events in anuran metamorphosis. Reactive oxygen species and oxidative stress play an important role during this process. Presently, the cell- and tissue-specific localization of antioxidant enzymes such as superoxide dismutase (SOD) and catalase as well as neuronal and inducible nitric oxide synthase isoforms (nNOS and iNOS) responsible for production of nitric oxide (NO) were carried out during different stages of metamorphosis in tail of tadpole Xenopus laevis. NO also has profound effect on the mitochondrial function having its own nitric oxide NOS enzyme. Hence, in situ staining for NO and mitochondria also was investigated. The distribution of nNOS and iNOS was found to be stage specific, and the gene expression of nNOS was up-regulated by thyroxin treatment. In situ staining for NO and mitochondria shows co-localization, suggesting mitochondria being one of the sources of NO. SOD and catalase showed significant co-localization during earlier stages of metamorphosis, but before the tail regression begins, there was a significant decrease in activity as well as co-localization suggesting increased ROS accumulation. These findings are discussed in terms of putative functional importance of ROS and cytoplasmic as well as mitochondrial derived NO in programmed cell death in tail tissue.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

Modulation of nitric oxide (NO) biosynthesis in lactobacilli

We characterized effects of nitric oxide synthase (NOS) substrate L-arginine and classical inhibitors of mammalian NOS on nitric oxide (NO) biosynthesis in probiotic bacteria Lactobacillus plantarum 8P-A3. NO-synthase origin of nitric oxide detected by fluorescent NO indicator 1,2-diaminoanthraquinone (DAA) was confirmed by induction of NO production by exogenous L-arginine. None of the used inhibitors of three isoforms of mammalian NOSs (L-NAME, L-NIL, nNOS inhibitor I) showed significant inhibitory effect of lactobacillar NO-synthase activity.     

Extremely low-frequency magnetic fields modulate nitric oxide signaling in rat brain

Our previous study has shown that an extremely low‐frequency magnetic field (ELF‐MF) induces nitric oxide (NO) synthesis by Ca²⁺‐dependent NO synthase (NOS) in rat brain. The present study was designed to confirm that ELF‐MF affects neuronal NOS (nNOS) in several brain regions and to investigate the correlation between NO and nNOS activation. The exposure of rats to a 2 mT, 60 Hz ELF‐MF for 5 days resulted in increases of NO levels in parallel with cGMP elevations in the cerebral cortex, striatum, and hippocampus. Cresyl violet staining and electron microscopic evaluation revealed that there were no significant differences in the morphology and number of neurons in the cerebral cortex, striatum, and hippocampus. Differently, the numbers of nNOS‐immunoreactive (IR) neurons were significantly increased in those cerebral areas in ELF‐MF‐exposed rats. These data suggest that the increase in NO could be due to the increased expression and activation of nNOS in cells. Based on NO signaling in physiological and pathological states, ELF‐MF created by electric power systems may induce various physiological changes in modern life. Bioelectromagnetics 33:568–574, 2012. © 2012 Wiley Periodicals, Inc.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K_m for arginine 2.8, 2.0 and 2.3 μM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Nitric oxide and neuromodulation in the caudal neurosecretory system of teleosts

Although evidence exists that nitric oxide (NO) mediates neuroendocrine secretion in mammals, the involvement of NO in the neuroendocrine regulation of non-mammalian vertebrates has yet to be investigated in detail. The present review conveys several recent data, suggesting that NO plays a modulatory role in the caudal neurosecretory system (CNSS) of teleosts. The presence and distribution of neuronal NO synthase (nNOS) was demonstrated in the CNSS of the Nile tilapia Oreochromis niloticus by means of NADPHd histochemistry, NOS immunohistochemistry, NOS immunogold electron microscopy, the citrulline assay for NOS activity and Western blot analysis. NO production by the caudal spinal cord homogenates was also evaluated by the oxyhemoglobin assay. On the whole, these findings indicate that caudal neurosecretory cells express NOS enzymes and presumably produce NO as a cotransmitter. Moreover, the comparison of the nNOS distribution with that of urotensins I and II (UI and UII) suggests that neurosecretory Dahlgren cells belong to two different functional subpopulations: a population of UI/UII secreting nitrergic neurons and a population of non-nitrergic neurons, which principally secrete UII. These results implicate NO as a putative modulator of the release of urotensins from the neurosecretory axon terminals. Therefore, like in mammals, NO appears to influence neuroendocrine secretion in teleosts.     

Relationship between structure and inhibitory effect of arginine analogues on neuronal nitric oxide synthase activity

Since nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) froml-arginine (Arg) which has an amidino group in its molecule, we, examined the effect of 29 kinds of Arg analogues on neuronal NOS (nNOS) activity in the rat brain. None of the Arg analogues acted as a substrate for nNOS. Diamidinocystamine, hirudonine, and guanethidine inhibited nNOS activity to 67.3%, 64.2% and 74.1%, respectively, but their inhibitory efficiency was lower than N^G-monomethyl-l-arginine (to 36.5%) which is a well known NOS inhibitor. Dimethylguanidine and N-benzoylguanidine also significantly inhibited nNOS activity to 88.0% and 90.7%, respectively. Whereas almost all of the NOS inhibitors previously reported were synthesizdd by substituting the amidino nitrogen of Arg, none of these new inhibitors were substituted at this position. Furthermore, hirudonine, which is a naturally occurring compound, was thought to act as an agonist at polyamine binding site of the N-methyl-d-aspartate type of glutamate receptor complex. It is also interesting that guanethidine, an antihypertensive agent, inhibit nNOS activity. These new drugs are useful for the investigation not only of the chemical nature of nNOS but also of the physiologic function of NO.     

Structural studies of constitutive nitric oxide synthases with diatomic ligands bound

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)–arginine–CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with l-arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY} ⁿ formalism developed from the inorganic diatomic–metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O₂) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.     

Role of Nitric Oxide in Pathogenesis Underlying Ischemic Cerebral Damage

1. Based upon the intriguing report that nitric oxide synthase (NOS) inhibitor dose-dependently reverses N-methyl-D-aspartate (NMDA)-induced neurotoxicity observed in primary cortical cell cultures, many laboratories have investigated whether NOS inhibition is beneficial as a treatment for cerebral ischemia.2. Although the results are variable, it is likely thought that nitric oxide plays a key role in pathomechanism underlying ischemic brain damage.3. We review the experimental studies on effects of NOS inhibition on cerebral ischemia and measuring nitric oxide produced in the brain subjected to cerebral ischemia.4. Finally, the possibility of NOS inhibitors as a therapeutical tool is discussed.     

Bcl-2-linked apoptosis due to increase in NO synthase in brain of SAMP10

We examined the linkage of nitric oxide (NO)-induced apoptosis to acceleration of brain aging of senescence-accelerated mouse prone 10 (SAMP10). The expression of neuronal nitric oxide synthase (nNOS) increased in the cerebral cortex of the brain of SAMP10 in an age-dependent manner and significantly higher levels of neuronal nitric oxide synthase (nNOS) were observed in both young and old SAMP10 as compared to age-matched controls. Moreover, a lower level of anti-apoptotic protein Bcl-2 and a higher level of pro-apoptotic protein cytochrome c in cytosol were observed in SAMP10 compared to the control. However, there was no significant difference in the expression of pro-apoptotic protein p53 between SAMP10 and the control. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive apoptotic cells were more abundant in the cerebral cortex of aged SAMP10 than in the control. The present results suggest that an age-dependent increase of NO by up-regulation of nNOS promotes the Bcl-2-linked apoptosis in the cerebral cortex of SAMP10 and this may cause the acceleration of brain aging of SAMP10.     

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action

N^α-vanillyl-N^ω-nitroarginine (N ? 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N ? 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide ‘NO’ production induced by calcium ionophore in NG 108-15 cells. N ? 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy ( ? 10.2 kcal/mol) obtained from docking N ? 1 to nNOS supported the additional mode of action of N ? 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N ? 1 at 75 μmol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Deregulation of Hepatic Insulin Sensitivity Induced by Central Lipid Infusion in Rats Is Mediated by Nitric Oxide

Background

Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis.

Methodology

We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats).

Principal Findings

Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid).

Conclusions/Significance

We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production.     

Roles of nitric oxide in brain hypoxia-ischemia.

A large body of evidence has appeared over the last 6 years suggesting that nitric oxide biosynthesis is a key factor in the pathophysiological response of the brain to hypoxia-ischemia. Whilst studies on the influence of nitric oxide in this phenomenon initially offered conflicting conclusions, the use of better biochemical tools, such as selective inhibition of nitric oxide synthase (NOS) isoforms or transgenic animals, is progressively clarifying the precise role of nitric oxide in brain ischemia. Brain ischemia triggers a cascade of events, possibly mediated by excitatory amino acids, yielding the activation of the Ca2+-dependent NOS isoforms, i.e. neuronal NOS (nNOS) and endothelial NOS (eNOS). However, whereas the selective inhibition of nNOS is neuroprotective, selective inhibition of eNOS is neurotoxic. Furthermore, mainly in glial cells, delayed ischemia or reperfusion after an ischemic episode induces the expression of Ca2+-independent inducible NOS (iNOS), and its selective inhibition is neuroprotective. In conclusion, it appears that activation of nNOS or induction of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through eNOS activation within the endothelium of blood vessels, which mediates vasodilation and hence increases blood flow to the damaged brain area.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

Alterations of NO synthase isoforms in brain and kidney of rats with genetic and salt hypertension

Both brain and peripheral nitric oxide (NO) play a role in the control of blood pressure and circulatory homeostasis. Central NO production seems to counteract angiotensin II-induced enhancement of sympathetic tone. The aim of our study was to evaluate NO synthase (NOS) activity and protein expression of its three isoforms--neuronal (nNOS), endothelial NOS (eNOS) and inducible (iNOS)--in two brain regions involved in blood pressure control (diencephalon and brainstem) as well as in the kidney of young adult rats with either genetic (12-week-old SHR) or salt-induced hypertension (8-week-old Dahl rats). We have demonstrated reduced nNOS and iNOS expression in brainstem of both hypertensive models. In SHR this abnormality was accompanied by attenuated NOS activity and was corrected by chronic captopril treatment which prevented the development of genetic hypertension. In salt hypertensive Dahl rats nNOS and iNOS expression was also decreased in the diencephalon where neural structures important for salt hypertension development are located. As far as peripheral NOS activity and expression is concerned, renal eNOS expression was considerably reduced in both genetic and salt-induced hypertension. In conclusions, we disclosed similar changes of NO system in the brainstem (but not in the diencephalon) of rats with genetic and salt-induced hypertension. Decreased nNOS expression was associated with increased blood pressure due to enhanced sympathetic tone.     

Augmentation of Nitric Oxide,Superoxide, and Peroxynitrite Production During Cerebral Ischemia and Reperfusion in the Rat

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, N-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.