Effect of elevated temperature and of hypoxia on activity of NO-ergic system of the central nervous system of bivalve molluscs

By light and electron microscopy methods the effect of changes of environmental conditions on the state of the nitroxidergic system has been studied in molluscs on the background of action of elevated temperature and hypoxia. Analysis is performed of biological effect of isolated and combined effects of the studied factors on dynamics of NO synthesis. A higher resistance of CNS neurons to the combined action of hyperthermia and hypoxia is revealed in molluscs with the initially high level of nitrogen oxide production. In molluscs with the initially low level of development of the nitroxidergic system, induction of NO formation in stress has been found to be accompanied by a change of morphology of nervous structures. It is suggested that nitrogen oxide participates in evolutionary established mechanisms of protection of mollusc nerve cells from hypoxia, while the initial high level of NO production reflects larger adaptational possibilities characteristic of these organisms.     

Production and storage of nitric oxide in adaptation to hypoxia.

Adaptation to hypobaric hypoxia is known to exert multiple protective effects related with nitric oxide (NO). However the effect of adaptation to hypoxia on NO metabolism has remained unclear in many respects. In the present work we studied the interrelation between NO production and storage in the process of adaptation to hypoxia. The NO production was determined by the total nitrite/nitrate concentration in rats plasma. The volume of NO store was evaluated in vitro by the magnitude of isolated aorta relaxation to diethyldithiocarbamate. It was shown that both the nitrite/nitrate level and the NO store increased as adaptation to hypoxia developed. Furthermore, the NO store volume significantly correlated with plasma nitrite/nitrate. Therefore, adaptation to hypoxia stimulates NO production and storage and these effects can potentially underlie NO-dependent beneficial effects of adaptation.     

NO-dependent effects during adaptation of rats to intermittent hypoxia

In experiments on Wistar rats processes nitric oxide production on concentration of anions (NO2-, NO3-), carbamide and polyamines contents were investigated in processes of rats adaptation to acute hypoxia (7% O2 in N2, 30 min) and intermittent hypoxia training (10% O2 in N2, 15 min, 5 cycles daily) during 14 days. NO production by oxygen-dependent and oxygen-independent metabolites paths has been investigated. It is concluded that the disturbances in nitric oxide system induced by acute hypoxia by L-arginine injections may result in acute hypoxia.     

Effect of copper ions on spatial density of NO synthase-positive cells in intestine of the mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (mollusca: bivalvia: mytilidae). A histochemical study

By the histochemical method of detection of NADPH-diaphorase (NADPH-d) (EC1.6.99.1) [1] the state of nitroxidergic enteric nervous system of the mussel Crenomytilus grayanus was studied under conditions of an increased copper concentration in water. Under the action of copper ions the density of distribution of NADPH-d-positive cells has been established to be changed as compared with control throughout 28 days. A sharp rise of proportion of the labeled cells and their enzyme activity was noted after one day of the experiment. The labeled bipolar cells were of dark blue color and were located within the epithelium. There were revealed numerous nerve fibers penetrating the intestinal epithelium throughout its entire length as well as bipolar nerve cells in epithelium of the minor typhlosole and of crystalline style sac; in control molluscs the NADPH-d-positive cells in these parts were absent. After 7 days the difference between control and experimental decreased and remained at this level after 14 days, while after 21 days of exposition the proportion of labeled cells in the experimental mussels was lower than in control, but increased again after 28 days. It is suggested that nitric oxide is an important protective factor of the intestinal epithelium of the mussel C. grayanus and participates in adaptation of this mollusc to action of the elevated concentration of copper ions in water.     

Modulation of mitochondria and NADPH oxidase function by the nitrate-nitrite-NO pathway in metabolic disease with focus on type 2 diabetes

Mitochondria play fundamental role in maintaining cellular metabolic homeostasis, and metabolic disorders including type 2 diabetes (T2D) have been associated with mitochondrial dysfunction. Pathophysiological mechanisms are coupled to increased production of reactive oxygen species and oxidative stress, together with reduced bioactivity/signaling of nitric oxide (NO). Novel strategies restoring these abnormalities may have therapeutic potential in order to prevent or even treat T2D and associated cardiovascular and renal co-morbidities. A diet rich in green leafy vegetables, which contains high concentrations of inorganic nitrate, has been shown to reduce the risk of T2D. To this regard research has shown that in addition to the classical NO synthase (NOS) dependent pathway, nitrate from our diet can work as an alternative precursor for NO and other bioactive nitrogen oxide species via serial reductions of nitrate (i.e. nitrate-nitrite-NO pathway). This non-conventional pathway may act as an efficient back-up system during various pathological conditions when the endogenous NOS system is compromised (e.g. acidemia, hypoxia, ischemia, aging, oxidative stress). A number of experimental studies have demonstrated protective effects of nitrate supplementation in models of obesity, metabolic syndrome and T2D. Recently, attention has been directed towards the effects of nitrate/nitrite on mitochondrial functions including beiging/browning of white adipose tissue, PGC-1α and SIRT3 dependent AMPK activation, GLUT4 translocation and mitochondrial fusion-dependent improvements in glucose homeostasis, as well as dampening of NADPH oxidase activity. In this review, we examine recent research related to the effects of bioactive nitrogen oxide species on mitochondrial function with emphasis on T2D.     

Nitric oxide production in the hypoxic lung

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2 to be in the physiological range, it has been suggested that O2 substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.     

Nitric oxide metabolites level in human serum in acute normobaric hypoxia

The influence of acute normobaric hypoxia on NO metabolites level of the blood serum in volunteers at respiration of hypoxic gas mixture containing 8 % of O2 during 25 min was investigated. Health status of participants and the hypoxia intensity were monitored with a complex of indexes: EEC, ECG, blood pressure, oxygen saturation of haemoglobin, cardiac output, gas composition of exhaled air. Cluster analysis (k-means clustering) conducted among volunteers that have successfully passed the test has shown presence of two groups differing in NO metabolites level during experiment. Statistically significant differences on NO metabolites level between groups were observed before hypoxia exposure, on 10th minute of acute hypoxia (maximum difference) and on 5th minute of recovery. Differences on NO metabolites level between groups have been caused by changes in nitrates concentration whereas nitrites level did not differ. The least NO and nitrates levels have been revealed in volunteers that have been in volunteers that had interrupted performance of the test after 10 minutes of respiration of hypoxic gas mixture. Thus the moderate increase of NO metabolites level due to accumulation of nitrates at acute hypoxia testifies to good adaptive reserves of system of nitric oxide generation in organism.     

NADPH-diaphorase activity in the digestive system of gastropod molluscs <Emphasis Type="Italic">Achatina fulica</Emphasis> and <Emphasis Type="Italic">Littorina littorea</Emphasis>

Distribution and morphological peculiarities of nitroxidergic elements throughout the entire length of digestive tract was studied for the first time in gastropod molluscs Littorina littorea (Prosobranchia) and Achatina fulica (Pulmonata) using histochemical detection of NADPH-diaphorase (NADPHd). NO-ergic cells and fibers were revealed in all parts of the mollusc digestive system beginning from esophagus. Intensive NADPHd activity is found in a great number of intraepithelial cells of the open type and their processes in the intraand subepithelial nerve plexuses, subepithelial neurons, granular connective tissue cells, and multiple nervous fibers distributed among muscular elements of digestive tract as well as those in nerves innervating the tract. NADPHd was also revealed in receptor cells in the oral area and in the A. fulica CNS ganglia innervating the digestive tract. A. fulica has a more complicated organization of A. fulica nitroxidergic system of the digestive tract. A system of glomerular structures formed by thin NADPHd-positive neural fibers coming from epithelium is found directly beneath the epithelium in esophagus, stomach, and midgut of the mollusc. More superficially under the main groups of muscular elements there are revealed small clusters of NADPHd-positive neurons that can be classified as primitive, non-structured microganglia. The distribution pattern and a possible functional role of nitroxidergic elements in digestive tract of molluscs as compared with other invertebrate and vertebrate animals are discussed.     

Nitric oxide production by haemocytes from Mytilus galloprovincialis shows seasonal variations

Nitric oxide (NO) has been identified as an important physiological modulator, with evidence of its role as a signalling molecule throughout the whole phylogenetic scale. In marine molluscs, it intervenes in processes related to the immune function of haemocytes. The presented results indicate that basal NO production by haemocytes of Mytilus galloprovincialis shows seasonal variations, with summer values statistically higher than those of winter. The presence of IL-2 increased NO production in winter. In summer, incubating the haemocytes with TNF-alpha for 6h slightly increased NO production. LPS, TGF-beta1 or PDGF did not induce significant effects on NO production by the haemocytes. Immunoblotting experiments detected two proteins that bind to vertebrate iNOS and eNOS antibodies, with different seasonal expression: the protein that binds to anti-iNOS antibody was expressed throughout the year, whereas the anti-eNOS antibody bound with a protein that was only detected in winter. IL-2 is suggested to start a signalling system dependent on the seasonal presence of winter protein. Such a system would activate the enzyme, thus favouring the higher NO production detected in winter.     

Regulatory interconnections of cyclooxigenase and inducible no-synthase in urinary bladder epithelial cells of the frog Rana temporaria under action of bacterial stimuli

Earlier we have shown that in epithelial cells of the frog urinary bladder under action of bacterial lipopolysaccharides (LPS) there is activated expression of inducible NO-synthase (iNOS) and there is increased the NO production, which can play an important role in providing protective cell reactions from pathogens. The goal of the present work consisted in study of cyclooxigenase (cOG) products and mechanisms of their regulatory effect on expression of iNOS under action of LPS. In experiments on urinary bladder epithelial cells on the frog Rana temporaria it has been shown that incubation of the cells for 21 h with LPS leads to a rise in production of PGE2 and nitrites, stable NO metabolites. Inhibitor of iNOS 1400W decreased sharply production of nitrites, but did not affect the PGE2 level. Both the basal and the LPS-stimulated level of PGE2 and nitrites were inhibited in the presence of selective cOG inhibitors--SC-560 (cOG-1) and NS-398 (cOG-2). The IC50 value amounted to 90, 220, and 470 microM for NS-398, SC-560, and diclofenac (unspecific inhibitor of both isoforms), respectively. PGE2 and butaprost, the EP2-receptor agonist, but not agonists of EP1/EP3 or EP1 receptors, partially eliminated the inhibitory action of diclofenac on production of nitrites. Action of PGE2 was accompanied by an increase in the intracellular cAMP. Analysis of expression of iNOS mRNA in the epithelial cells incubated with LPS or LPS + inhibitor of cOG has shown the LPS-stimulated rise in expression of iNOS mRNA to decrease sharply in the presence of SC-560 or NS-398. Thus, the epithelial cells of the frog urinary bladder have the effectively functioning system of the congenital immune protection against bacterial pathogens, the most important component of this system being PGE2 and NO. Analysis of mechanisms of regulatory interactions of cOG and iNOS indicates that in this cell type the main regulators of iNOS expression and of the nitrogen oxide level are products of the cOG catalytic activity.     

Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms

We tested the hypothesis that nitric oxide (NO) produced within the carotid body is a tonic inhibitor of chemoreception and determined the contribution of neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the inhibitory NO effect. Accordingly, we studied the effect of NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the selective nNOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to hypoxia (Po(2) approximately 30 Torr). CBs excised from pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at 38 degrees C and pH 7.40, and chemosensory discharges were recorded from the carotid sinus nerve. SNAP (100 microM) reduced the responses to nicotine and NaCN. l-NAME (1 mM) enhanced the responses to nicotine and NaCN by increasing their duration, but TRIM (100 microM) only enhanced the responses to high doses of NaCN. The amplitude of the response to hypoxia was enhanced by l-NAME but not by TRIM. Our results suggest that both isoforms contribute to the NO action, but eNOS being the main source for NO in the cat CB and exerting a tonic effect upon chemoreceptor activity.     

PDT诱导的凋亡细胞对巨噬细胞NO合成的影响

NO作为细胞间信息传递的重要调节因子,在肿瘤的发生、发展以及转移过程中被广泛研究。一氧化氮合酶是合成NO的关键酶,诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)通常在应激、荷瘤等病理状态下被激活,产生大量NO。NO具有细胞毒性,与机体免疫反应及细胞凋亡有关,在许多致癌和抑癌机制中扮演着重要角色。实验探讨了光动力学疗法(photodynamic therapy,PDT)处理产生的小鼠乳腺癌凋亡细胞对巨噬细胞产生NO的影响,从而确定活化的巨噬细胞在肿瘤生长中的作用。     

Nitric oxide and carotid body chemoreception.

Nitric oxide (NO) has been proposed as an inhibitory modulator of carotid body chemosensory responses to hypoxia. It is believed that NO modulates carotid chemoreception by several mechanisms, which include the control of carotid body vascular tone and oxygen delivery and reduction of the excitability of chemoreceptor cells and petrosal sensory neurons. In addition to the well-known inhibitory effect, we found that NO has a dual (dose-dependent) effect on carotid chemoreception depending on the oxygen pressure level. During hypoxia, NO is primarily an inhibitory modulator of carotid chemoreception, while in normoxia NO increased the chemosensory activity. This excitatory effect produced by NO is likely mediated by an impairment of mitochondrial electron transport and oxidative phosphorylation, which increases the chemosensory activity. The recent findings that mitochondria contain an isoform of NO synthase, which produces significant amounts of NO for regulating their own respiration, suggest that NO may be important for the regulation of mitochondrial energy metabolism and oxygen sensing in the CB.     

Why is the Pathology of Falciparum Worse than that of Vivax Malaria?

Here, Ian Clark and Bill Cowden summarize new evidence suggesting that nitric oxide (NO) generated by inducible NO synthase (iNOS) provides a functional link between the previously competing approaches to malarial disease pathogenesis: ischaemic hypoxia and NO. When combined with the newly recognized roles of iNOS in renal and pulmonary function and glucose metabolism, synergy between inflammatory cytokines and hypoxia in iNOS induction provides a framework to help explain, at a molecular level, the differences in the pathology seen in falciparum and vivax malaria. Thus sequestration, through localized hypoxia, might contribute to pathology by enhancing cytokine-induced iNOS. Generalized hypoxia might have the same effect.     

Nitric oxide is involved in the protective effects of short-term adaptaion to hypoxia in the course of stress-induced disorders in Krushinsky-Molodkina rats

A possible involvement of nitric oxide in the protective effect of short-term adaptation of Krushinsky-Molodkina rats to mild hypoxia simulating 5000 m above sea level was studied. Nitric oxide proved to have a considerable protective effect on stress-induced disorders in Krushinsky-Molodkina rats as demonstrated using NO-synthase inhibitors and NO monitoring by electron spin resonance under different experimental conditions.     

Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-gamma induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 +/- 14 micro M, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 +/- 1.7 micro M, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and alpha-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.     

Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells

BACKGROUND: Empirical observations have shown that ozonated autohemotherapy markedly improves the symptoms of chronic limb ischemia (muscular pain at rest, intermittent claudication, etc) in atherosclerotic patients, but mechanisms of action remain unclear. AIMS: Human endothelial cells (HUVECs) are known to release nitrogen monoxide (NO) and we investigated the biological effects of human ozonated serum on HUVECs in culture. METHODS: We assessed the relevance of peroxidation, the release of NO as nitrite and of three classical cytokines. RESULTS: The treatment of HUVECs with ozonated serum yields a dose dependent increase of thiobarbituric acid reactive substances (TBARS) and of hydrogen peroxide (H2O2) and a decrease of protein thiol groups (PTG). Concomitantly, in comparison to either the control or the oxygenated sample, there is a significant and steady increase of nitric oxide (NO) production; this is markedly enhanced by the addition of L-arginine (20 microM) and inhibited in the presence of the NO inhibitor, L-NAME (20 mM). The main mediator of ozone action is H2O2 as it has been shown either after its direct measurement or by the addition of 20, 40 and 100 microM. Moreover, during 24 hours incubation we have investigated the production of endothelin 1 (ET-1), E-selectin and Interleukin 8 (IL-8) and it appears that ozonation enhances IL-8, inhibits E-selectin and hardly modifies ET-1 production. CONCLUSIONS: It appears that reinfusion of ozonated blood, by enhancing release of NO, may induce vasodilation in ischemic areas and reduce hypoxia.