Nitric oxide, a biological double-faced janus--is this good or bad?

Nitric oxide (NO) is a biological messenger molecule produced by one of the essential amino acids L-arginine by the catalytic action of the enzyme NO synthase (NOS). The dual role of NO as a protective or toxic molecule is due to several factors, such as; the isoform of NOS involved, concentration of NO and the type of cells in which it is synthesised, the availability of the substrate L-arginine, generation of guanosine 3,5'-cyclic monophosphate (cGMP) from soluble guanylate cyclase and the overall extra and intracellular environment in which NO is produced. NOS activation as a result of trauma (calcium influx) or infection leads to NO production, which activates its downstream receptor sGC to synthesise cGMP and/or leads to protein nitrosylation. This may lead to one or more systemic effects including altered neurotransmission which can be protective or toxic, vaso/bronchodilatation in the cardiovascular and respiratory systems and enhanced immune activity against invading pathogens. In addition to these major functions, NO plays important role in thermoregulation, renal function, gastrointestinal motility, endocrine function, and various functions of the urogenital system ranging from renin secretion to micturation; spermatogenesis to penile erection; and ovulation to implantation and parturition. A schematic summary of the functions of NO and the various isoforms of NOS expressed in body systems is shown in figure 1. In this review, the historical background, biochemistry and biosynthesis of NO and its enzymes together with the mechanism of NO actions in physiology and pathophysiology are discussed.     

The inhibitory effect of the fungal toxin, destruxin A, on behavioural fever in the desert locust

During an infection locusts behaviourally fever by seeking out higher environmental temperatures. This behaviour places the pathogen at sub-optimal growth temperatures while improving the efficiency of the immune system, thereby prolonging the lifespan of the host. It is therefore in the interest of the pathogen to either adapt to fever-like temperatures or to evolve mechanisms to interfere with, or inhibit fever. We investigated the behavioural fever response of desert locusts to two fungal pathogens. A prolonged fever was observed in locusts infected with Metarhizium acridum. However, fever was comparatively short-lived during infection with Metarhizium robertsii. In both cases restriction of thermoregulation reduced lifespan. Destruxin A (dtx A) produced by M. robertsii, but not M. acridum has previously been associated with the inhibition of the insect immune system. Injection of dtx A during infection with the fever-causing M. acridum inhibited fever and was particularly effective when administered early on in infection. Furthermore, locusts injected with dtx A were more susceptible to M. acridum infection. Therefore engineering M. acridum isolates currently used for locust biocontrol, to express dtx A may improve efficiency of control by interfering with fever.     

Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1β

Interleukin-1β (IL-1β) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca²⁺ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1β and other pyrogens. Data from different labs indicate that Ca²⁺ and Na⁺ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca²⁺ and PGE₂ concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1β-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204–212) peptide counteract IL-1β-induced fever and abolish changes in Ca²⁺ and PGE₂ concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca²⁺ mobilization in response to IL-1β and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP₃)-sensitive pools are the main source of the mobilized Ca²⁺. It is concluded that the NO/cGMP/Ca²⁺ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1β.     

Nitrite modulates contractility of teleost (Anguilla anguilla and Chionodraco hamatus, i.e. the Antarctic hemoglobinless icefish) and frog (Rana esculenta) hearts

Being the largest form of intravascular and tissue storage of nitric oxide (NO) and a signalling molecule itself, the nitrite anion (NO₂⁻) has emerged as a key player in many biological processes. Since the heart is under an important NO-mediated autocrine-paracrine control, in mammals the cardiac effects of nitrite are under intensive investigation. In contrast, nothing is known in non-mammalian vertebrates. We evaluated nitrite influence on cardiac performance in the perfused beating heart of three different cold-blooded vertebrates, i.e. two teleost fishes, the temperate red-blooded Anguilla anguilla, the Antarctic stenotherm, hemoglobinless Chionodraco hamatus (icefish), and the frog Rana esculenta. We showed that, under basal conditions, in all animals nitrite influences cardiac mechanical performance, inducing negative inotropism in eel and frog, while being a positive inotrope in C. hamatus. In all species, these responses parallel the inotropic effects of authentic NO. We also demonstrated that the nitrite-dependent inotropic effects are i) dependent from NO synthase (NOS) activity in fish; ii) sensitive to NO scavenging in frog; iii) cGMP/PKG-dependent in both eel and frog. Results suggest that nitrite is an integral physiological source of NO and acts as a signalling molecule in lower vertebrate hearts, exerting relevant inotropic actions through different species-specific mechanisms.     

Modulation of nitrate reductase activity by photosynthetic electron transport chain and nitric oxide balance in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta)

Nitrate reductase (NR), a key enzyme in nitrogen metabolism, has been implicated in the production of nitric oxide (NO) in plants. The effect of photosynthetic electron transport chain inhibitors and NO scavengers or donors on NR activity of Gracilaria chilensis was studied under experimental laboratory conditions. Effective quantum yield (Φ _PSII) and NR activity were significantly diminished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, two photosynthetic electron flux inhibitors of photosystem (PS) II and PSI, respectively, but not by diphenyleneiodonium, a NADPH oxidase inhibitor, indicating a direct dependence of NR activity on the PSII and PSI electron flux. Nitrate reductase activity was sensitive to a decrease or increase of NO levels when NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO donor (sodium nitroprusside) were added. Moreover, the addition of 8Br-cGMP, a secondary signal molecule, stimulated NR activity. These results evidence a modulation of the photosynthetic electron transport chain and NO balance on G. chilensis NR activity. This association could be linked to the crucial tight modulation of nitrogen assimilation and carbon metabolism to guarantee nitrite incorporation into organic compounds and to avoid toxicity by nitrite, reactive oxygen species, or nitric oxide in the cells. Nitric oxide showed to be an important signaling molecule regulating NR activity and cGMP could participate as secondary messenger on this regulation by phosphorylation and desphosphorylation processes.     

Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling

Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function.     

Exogenous Nitric Oxide (NO) Interferes with Lead (Pb)-Induced Toxicity by Detoxifying Reactive Oxygen Species in Hydroponically Grown Wheat (Triticum aestivum) Roots

Nitric Oxide (NO) is a bioactive signaling molecule that mediates a variety of biotic and abiotic stresses. The present study investigated the role of NO (as SNP [sodium nitroprusside]) in ameliorating lead (Pb)-toxicity in Triticum aestivum (wheat) roots. Pb (50 and 250 μM) alone and in combination with SNP (100 μM) was given to hydroponically grown wheat roots for a period of 0–8 h. NO supplementation reduced the accumulation of oxidative stress markers (malondialdehyde, conjugated dienes, hydroxyl ions and superoxide anion) and decreased the antioxidant enzyme activity in wheat roots particularly up to 6 h, thereby suggesting its role as an antioxidant. NO ameliorated Pb-induced membrane damage in wheat roots as evidenced by decreased ion-leakage and in situ histochemical localization. Pb-exposure significantly decreased in vivo NO level. The study concludes that exogenous NO partially ameliorates Pb-toxicity, but could not restore the plant growth on prolonged Pb-exposure.     

Developmental stage- and concentration-specific sodium nitroprusside application results in nitrate reductase regulation and the modification of nitrate metabolism in leaves of Medicago truncatula plants

Nitric oxide (NO) is a bioactive molecule involved in numerous biological events that has been reported to display both pro-oxidant and antioxidant properties in plants. Several reports exist which demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the expression regulation of many antioxidant enzymes. This study attempts to provide a novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on the nitrosative status and nitrate metabolism of mature (40 d) and senescing (65 d) Medicago truncatula plants. Higher concentrations of SNP resulted in increased NO content, cellular damage levels and reactive oxygen species (ROS) concentration, further induced in older tissues. Senescing M. truncatula plants demonstrated greater sensitivity to SNP-induced oxidative and nitrosative damage, suggesting a developmental stage-dependent suppression in the plant’s capacity to cope with free oxygen and nitrogen radicals. In addition, measurements of the activity of nitrate reductase (NR), a key enzyme involved in the generation of NO in plants, indicated a differential regulation in a dose and time-dependent manner. Furthermore, expression levels of NO-responsive genes (NR, nitrate/nitrite transporters) involved in nitrogen assimilation and NO production revealed significant induction of NR and nitrate transporter during long-term 2.5 mM SNP application in mature plants and overall gene suppression in senescing plants, supporting the differential nitrosative response of M. truncatula plants treated with different concentrations of SNP.     

Neurophysiology of fever

Fever is a primary disorder of thermoregulation and a common clinical sign in many diseases. It is characterized by an upward displacement in the level at which body temperature is regulated. Early attempts to study hypothalamic neuronal activity in relation to fever described the behavior of isolated single units after intravenous injections of endotoxin pyrogen. It was concluded that the thermosensitivity of many warm-sensitive units was depressed after pyrogen injections, but due to the indirect technique employed, it is not possible to distinguish whether this observation is the cause or result of fever. A decrease in hypothalamic thermosensitivity is contrary to observations made during fever in conscious animals. More specific applications of pyrogenic stimuli such as prostaglandin E1 onto individual hypothalamic neurons using the technique of microelectrophoresis have not borne out these earlier observations. A major obstacle to studying the neurophysiology of thermoregulation and fever is the absence of any obvious correlation between neuroanatomy and function in the hypothalamus. Present methods of identifying and classifying hypothalamic cells as participants in thermoregulation are patently inadequate. Until a more specific correlation between anatomy and function is established, the neurophysiological mechanisms of fever will remain obscure.     

Azide-dependent nitric oxide emission from the water fern Azolla pinnata

Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (N O ₂ ^? ) the amino acid L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN₃). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.     

Effects of nitric oxide on sunflower seedlings: A balance between defense and development

Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen interactions and plant development. Previous work showed the participation of NO in the development and lignin composition of sunflower roots. Thereby, we have hypothesized that NO applications could control the attack of the fungal pathogen Verticillium dahliae in sunflowers. Seedlings growing hydroponically were pretreated with NO donors and further inoculated with the fungus. Evaluation of disease symptoms showed that NO pretreatments could not reduce Verticillium wilt. Strikingly, NO donors appear to promote the fungal infection. These results indicate that NO applications were unable to protect sunflowers from Verticillium attack and highlight the role played by the fine tuning regulation of NO levels required to balance plant responses between development and defense.     

Control of NO level in rhizobium-legume root nodules: Not only a plant globin story

Nitric oxide (NO) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence. Here we discuss the importance of the bacterial Sinorhizobium meliloti flavohemoglobin to finely tune the NO level inside nodules and further, we demonstrate that S. meliloti possesses at least two non redundant ways to control NO and that both systems are necessary to maintain efficient nitrogen fixing activity.     

Unbalanced thermoregulation in experimental autoimmune encephalitis induced in Lewis rats

Thermoregulation in patients suffering from multiple sclerosis (MS) is impaired and may result in either increases or decreases in body temperature. We have found that rat experimental autoimmune encephalitis (EAE), being a model of MS, is associated with body temperature disturbances as well.The purpose of the current study was to examine whether the altered body temperature in EAE-induced rats is due to either a deficit in thermoregulation or a controlled change in its set point.Subcutaneous injection of encephalitogenic emulsion into both pads of hind feet of the Lewis rats provoked EAE symptoms. Body temperature (Tb) of 6 rats was measured using biotelemetry system, and ambient temperature (Ta) preferred by 6 rats of another group was analyzed using thermal gradient system.Symptoms of EAE started 11 days postinjection and progressed quickly, culminating in a complete paralysis in rats placed in the gradient, which was associated with behavioural fever (accordingly, selected Ta raised to as much as 32.8 ± 0.5 °C vs 27.2 ± 0.6 °C in control rats). On the other hand, EAE rats, placed at a constant Ta of 24 °C, were able to generate fever (Tb of 37.8 ± 0.1 °C) at the start of the illness and then paralysis compromised fever (most likely due to an impairment of thermogenesis), which, surprisingly, resulted in recovery.We conclude that EAE onset in rats is associated with fever and its behavioural supporting leads to aggravation of the autoimmune neurotoxicity.     

Host-pathogen interactions in a varying environment: temperature,behavioural fever and fitness

We demonstrate how variable temperatures, mediated by host thermoregulation and behavioural fever, critically affect the interaction between a host (the desert locust, Schistocerca gregaria) and a pathogen (the fungus Metarhizium anisopliae var. acridum). By means of behavioural thermoregulation, infected locusts can raise their body temperatures to fever levels. The adaptive value of this behaviour was examined using three thermal regimes wherein maximum body temperatures achievable were: (i) below, or (ii) at normally preferred temperatures, or were (iii) unrestricted, allowing heightened fever temperatures. All infected locusts ultimately succumbed to disease, with median survival times of 8, 15 and 21 days post-infection, respectively. Crucially, only those locusts able to fever produced viable offspring. This represents, to our knowledge, the first demonstration of the adaptive value of behavioural fever following infection with a naturally occurring pathogen. By contrast, although normal host thermoregulation moderately reduced pathogen reproduction (by 35%), there was no additional negative effect of fever, resulting in an asymmetry in the fitness consequences of fever for the host and the pathogen. The dependency of the host-pathogen interaction upon external abiotic conditions has implications for how virulence and resistance are treated both theoretically and in the management of pests and diseases.     

Nitric oxide and heat shock protein 90 co-regulate temperature-induced bleaching in the soft coral Eunicea fusca

Coral bleaching represents a complex physiological process that is affected not only by environmental conditions but by the dynamic internal cellular biology of symbiotic dinoflagellates (Symbiodinium spp.) and their cnidarian hosts. Recently, nitric oxide (NO) has emerged as a key molecule involved with the expulsion of Symbiodinium from host cnidarian cells. However, the site of production remains under debate, and the corresponding signaling pathways within and between host and endosymbiont remain elusive. In this study, using freshly isolated Symbiodinium from the soft coral Eunicea fusca, I demonstrate that thermally induced stress causes an upregulation in Symbiodinium heat shock protein 90 (Hsp90). In turn, Hsp90 shows a concomitant ability to enhance the activity of a constitutively expressed isoform of NO synthase. The resulting production of NO constitutes a signaling molecule capable of inducing Symbiodinium expulsion. Using nitric oxide synthase (NOS) and Hsp90 polyclonal antibodies, thermal stress-induced Hsp90 was shown to co-immunoprecipitate with a constitutive isoform of NOS. The specific blocking of Hsp90 activity, with the Hsp90 inhibitor geldanamycin, was capable of inhibiting NO production implicating the involvement of a coordinated regulatory system. These results have strong evolutionary implications for Hsp90–NOS chaperone complexes among biological kingdoms and provide evidence for a new functional role in symbiotic associations.     

Living in sympatry: The effect of habitat partitioning on the thermoregulation of three Mediterranean lizards

The ability for effective, accurate and precise thermoregulation is of paramount importance for ectotherms. Sympatric lizards often partition their niche and select different microhabitats. These microhabitats, however, usually differ in their thermal conditions and lizards have to adapt their thermoregulation behavior accordingly. Here, we evaluated the impact of habitat partitioning on the thermal biology of three syntopic, congeneric lacertids (Podarcis peloponnesiacus, P. tauricus and P. muralis) from central Peloponnese, Greece. We assessed thermoregulation effectiveness (E) using the three standard thermal parameters: body (T_b), operative (T_e) and preferred (T_pref) temperatures. We hypothesized that the microhabitats used by each species would differ in thermal quality. We also predicted that all species would effectively thermoregulate, as they inhabit a thermally challenging mountain habitat. As expected, the partition of the habitat had an effect on the thermoregulation of lizards since microhabitats had different thermal qualities. All three species were effective and accurate thermoregulators but one of them achieved smaller E values as a result of the lower T_b in the field. This discrepancy could be attributed to the cooler (but more benign) thermal microhabitats that this species occupies.     

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.