The chemical biology of nitric oxide: implications in cellular signaling

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.     

Peroxynitrite- and nitrite-induced oxidation of dopamine: implications for nitric oxide in dopaminergic cell loss

Increased nitric oxide (NO) production has been implicated in many examples of neuronal injury such as the selective neurotoxicity of methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to dopaminergic cells, presumably through the generation of the potent oxidant peroxynitrite (ONOO). Dopamine (DA) is a reactive molecule that, when oxidized to DA quinone, can bind to and inactivate proteins through the sulfhydryl group of the amino acid cysteine. In this study, we sought to determine if ONOO could oxidize DA and participate in this process of protein modification. We measured the oxidation of the catecholamine by following the binding of [3H]DA to the sulfhydryl-rich protein alcohol dehydrogenase. Results showed that ONOO oxidized DA in a concentration- and pH-dependent manner. We confirmed that the resulting DA-protein conjugates were predominantly 5-cysteinyl-DA residues. In addition, it was observed that ONOO decomposition products such as nitrite were also effective at oxidizing DA. These data suggest that the generation of NO and subsequent formation of ONOO or nitrite may contribute to the selective vulnerability of dopaminergic neurons through the oxidation of DA and modification of protein.     

From sparks to spikes: information processing in the electrosensory systems of fish

Recent work on electrosensory systems in fish has combined traditional neuroethological approaches with quantitative methods for characterizing neural coding. These studies have shed light on general issues in sensory processing, including how peripheral sensory receptors encode external stimuli and how these representations are transformed at subsequent stages of processing.     

The role of the reactions of NO with superoxide and oxygen in biological systems: A kinetic approach

In this study we calculate the half-life of ^·NO in its reactions with superoxide and with oxygen under various conditions using the known rate constants for these reactions. The measured half-life of ^·NO in biological systems is 3–5 s, which agrees well with the calculated value for intracellular ^·NO, but not for extracellular ^·NO under normal physiological conditions. The autoxidation of ^·NO to yield NO₂ as a final product cannot be responsible for such a short measured half-life under normal as well as pathologic conditions. Therefore, if there is direct evidence for the occurrence of the reaction of ^·NO with O₂ in the medium, one has to assume that the steady state concentrations of free ^·NO are much lower than those measured. The very low concentrations of free ^·NO in biological systems may result from its reversible strong binding to biological molecules. Simulation of the mechanism of the autoxidation of ^·NO indicates that the binding constants of ^·NO to O₂ or to another ^·NO are too small to account for the very low concentration of free ^·NO in biological systems. Nevertheless, the reaction of ^·NO with oxygen cannot be neglected in biological systems if the intermediate ONOO· reacts rapidly with a biological target. The biological damage caused by ONOO′ is expected to be due to the radical itself and to peroxynitrite, which is most probably formed via the reaction of ONOO· with the biological molecule.     

The analysis of distributed control and information processing in adaptive systems: a biologically motivated approach.

Biological systems have evolved hierarchical, distributed control structures that greatly enhance their adaptability. Two important determinants of biological adaptability considered here are: (i) the pattern of distribution of self-control capabilities; (ii) the degree of programmability of information processing. In this paper we model organizations as goal-oriented, adaptive systems, possessing properties similar to those of biological systems. We use the notion of implicit control (defined as the capability of self-control that is embedded in a system's own dynamics) in the analysis of the impact of specific patterns of distribution of control and information processing on the adaptability of organizations. A principle of design of organizational information systems, that captures important aspects of adaptability-preserving strategies of information processing in biological systems, is stated in terms of the implicit control concept.     

Dioxygen and nitric oxide pathways and affinity to the catalytic site of rubredoxin:oxygen oxidoreductase from Desulfovibrio gigas

Rubredoxin:oxygen oxidoreductase (ROO) is the terminal oxidase of a soluble electron transfer chain found in Desulfovibrio gigas. This protein belongs to the flavodiiron family and was initially described as an oxygen reductase, converting this substrate to water and acting as an oxygen-detoxifying system. However, more recent studies evidenced also the ability for this protein to act as a nitric oxide reductase, suggesting an alternative physiological role. To clarify the apparent bifunctional nature of this protein, we performed molecular dynamics simulations of the protein, in different redox states, together with O₂ and NO molecules in aqueous solution. The two small molecules were parameterized using free-energy calculations of the hydration process. With these simulations we were able to identify specific protein paths that allow the diffusion of both these molecules through the protein towards the catalytic centers. Also, we have tried to characterize the preference of ROO towards the presence of O₂ and/or NO at the active site. By using free-energy simulations, we did not find any significant preference for ROO to accommodate both O₂ and NO. Also, from our molecular dynamics simulations we were able to identify similar diffusion profiles for both O₂ and NO molecules. These two conclusions are in good agreement with previous experimental works stating that ROO is able to catalyze both O₂ and NO. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Self-organization in prebiological systems: Simulations of a model for the origin of genetic information

Summary Computer simulations of a spin glass model for the origin of biological information are discussed. Selection is found to occur among a wide diversity of possible species, and in addition competition, adaptation, and hysteresis are all exhibited.     

Nitrite and nitroglycerin induce rapid release of the vasodilator ATP from erythrocytes: Relevance to the chemical physiology of local vasodilation

Extracellular ATP released from circulating erythrocytes induces vasodilation by stimulating receptor-mediated endothelium NO/EDRF (endothelium-derived relaxing factor) production. We report that pre-stimulation of freshly isolated human erythrocytes with physiological nitrite (100 nM ) or pharmacological nitroglycerin (10 μM) concentrations resulted in >200% spike in ATP release, which was detected on resuspending the cells in fresh medium. The observed response was instantaneous following pre-stimulation but a delay of ∼20 s followed nitroglycerin pre-stimulation, reflecting the time required for prodrug activation within the erythrocyte to its vasoactive metabolites, and NO. The data provided here are consistent with ATP being a conveyor of a NO-induced vasodilatory signal from the erythrocyte to the endothelium. Extended erythrocyte pre-stimulation with the NO donors resulted in a dose-dependent decrease in extracellular ATP, which would attenuate the signal in intact vessels to prevent excessive vasodilation. Importantly, our study constitutes the first report of enhanced vasodilator (ATP) release following human erythrocyte pre-stimulation by an endogenous or pharmacological (nitroglycerin) NO donor. The relevance of our findings to the therapeutic effects of nitroglycerin as well as to nitrate tolerance is discussed.     

Olfactory information processing in the brain: Encoding chemical and temporal features of odors

A fundamental problem in studying the neural mechanisms of odor recognition and discrimination in the olfactory system lies in determining the features or “primitives” of an odor stimulus that are analyzed by glomerular circuits at the first level of processing in the brain. Several recent studies support the idea that it is not simply the molecular features of odors that contain important information, but also the intermittent pattern of their presentation to the olfactory epithelium that helps determine the behavioral response to odor. © 1996 John Wiley & Sons, Inc.     

Environmental decision support systems: A new approach to support the operation and maintenance of horizontal subsurface flow constructed wetlands

This paper describes the development and operation of an Environmental Decision Support System (EDSS) to improve the operation and maintenance of horizontal subsurface-flow constructed wetlands (EDSS-maintenance). Constructed wetlands (CWs) allow wastewater treatment in a sustainable manner since they involve low energy consumption, low construction and functioning costs and low environmental impact. However, operation and maintenance activities are essential to guarantee reliability in CWs performance. The definition of operation and maintenance protocols depends on several quantitative and qualitative aspects such as wastewater treatment plant configuration, CW design, influent characteristics, sensitivity of the receiving media, etc. Bearing this in mind and considering the limited technical knowledge about CWs, the need for a new tool to support CW performance is clear. In this sense, EDSSs offer a new approach because they can tackle problems of complex and uncertain systems. The EDSS-maintenance provides operation and maintenance manuals specifically defined for every CW. To achieve it, the required knowledge was implemented within a rule-based system, which forms the backbone of the EDSS. Several features presented in this paper demonstrate how the EDSS-maintenance provides a proper platform to support the necessary collaborative work in the ecological engineering problem of horizontal subsurface flow CWs operation and maintenance.     

Integrated approach to nitric oxide in animals and plants (mechanism and bioactivity): cell signaling and radicals

Nitric oxide was first the object of extensive investigation in animals. It has been designated as the most widespread signaling molecule. An overview is presented with emphasis on cell signaling, mechanism, and physiological activity. Hence, a basis is provided for comparison of NO in plants with a similar approach. Mechanistically, cell signaling, electron transfer, radicals, and antioxidants are involved. A role is played by NO derivatives, such as peroxynitrite, nitroxyl, nitrite, nitrate, and S-nitroso derivatives. Comparison is made with ethylene. The multifaceted, interdisciplinary approach provides novel insight.     

Nitric Oxide:from a mysterious labile factor to the molecule of the Nobel Prize Recent progress in nitric oxide research

INTR0DUCTI0N:Nitric0xideandN0belPrizeScientificbreakthrough0ftenc0mesfr0munexpected0bserwtions,andimaginativeadventurousreseaxch.Intheearly8O'sattheNewYorkLab0ratoryofSunyHealthCentre,Dr.RobertFurchgottwaspresentedcontradictingresultsbyhistwotechnicians.Onetechnicianalwaysf0undtheacetycholinerelaxedthebloodvessel,whereasthe0therfounditalwayscausedcontraction.Furchg0ttnoticedthatonetechnicianhandledthevesselsroughlyandinadvertentlyrubbedoffthethinlayerofendotheliumfromthesurfaceoftheve…     

Nitric oxide binding to cationic and anionic ferric porphyrins in aqueous solution: reversible formation of ferrous NO species of the cationic porphyrin

Two water-soluble ferric porphyrins, sodium 5α,10β,15α,20β-tetrakis(2-(sulfonatoacetamido)phenyl)porphyrinatoiron(III) (Fe^IIITanP) and 5α,10β,15α,20β-tetrakis(2-(N,N,N-trimethylammoniumacetamido)phenyl)porphyrinatoiron(III) chloride (Fe^IIITcatP), were synthesized. The pK_a values of the coordinated H₂O of Fe^IIITanP and Fe^IIITcatP were evaluated to be 8.0 and 4.1, respectively. Reactions of NO with the ferric porphyrins were examined spectrophotometrically in aqueous solution. Porphyrin Fe^IIITanP binds NO reversibly to give the corresponding ferric NO species at pH 1.3 and pH 3.0, and Fe^IIITcatP reacts similarly with NO at pH 1.3. The thermodynamic data for the NO binding were estimated from van't Hoff plots. At pH 3.0, visible and ESR spectral data indicated that Fe^IIITcatP binds NO reversibly to produce ferrous NO species depending on NO partial pressures. These results were discussed based on through-space intramolecular interactions between the coordinated H₂O or NO and the ionic substituents of the porphyrins.     

Conservation of repeated DNA base sequences in theCrustacea: A molecular approach to decapod phylogeny

Summary Analysis of data obtained from molecular hybridization of³H-labeled repetitious DNA has been utilized to reconstruct the broad outlines of phylogenetic relationships among decapod Crustacea. This molecular reconstruction agrees reasonably well with the paleontological record, and with other schemes obtained by comparative morphological and serological approaches. Preliminary evidence is in line with the hypothesis that continuous addition of new repeated sequence families to the genome over long periods of time may in part account for the correlation observed between percent repetitious DNA hybridized and divergence time. It is tentatively concluded that a core of DNA base sequence homology has been highly conserved throughout the evolution of theCrustacea. Demonstration of inter-species sequence homology has important implications to models which relegate a genetic regulatory function to repeated DNAs.     

Combined in vitro and in silico approach to evaluate the inhibitory potential of an underutilized allium vegetable and its pharmacologically active compounds on multidrug resistant Candida species

Candida infections and related mortality have become a challenge to global health. Nontoxic and natural bioactive compounds from plants are regarded as promising candidates to inhibit these multidrug resistant strains. In the present study, in vitro assays and in silico molecular docking approach was combined to evaluate the inhibitory effect of crude extracts from Allium ampeloprasum and its variety A. porrum on Candida pathogens. Phytochemical screening revealed the presence of phenolic acids and flavonoids in higher quantity. Spectral studies of the extracts support the presence of phenols, flavonoids and organosulfur compounds. Aqueous extract of A. ampeloprasum showed a total antioxidant capacity of 68 ± 1.7 mg AAE/ g and an IC₅₀ value of 0.88 ± 2.1 mg/ml was obtained for DPPH radicals scavenging assay. C. albicans were highly susceptible (19.9 ± 1.1 mm) when treated with aqueous A. ampeloprasum extract. Minimum inhibitory concentrations were within the range of 19–40 μg/ml and the results were significant (p ≤ 0.05). In silico molecular docking studies demonstrated that bioactive phytocompounds of A. ampeloprasum and A. porrum efficiently interacted with the active site of Secreted aspartyl proteinase 2 enzyme that is responsible for the virulence of pathogenic yeasts. Rosmarinic acid and Myricetin exhibited low binding energies and higher number of hydrogen bond interactions with the protein target. Thus the study concludes that A. ampeloprasum and A. porrum that remain as underutilized vegetables in the Allium genus are potential anti-candida agents and their pharmacologically active compounds must be considered as competent candidates for drug discovery.     

Venous endothelium reactivity to Angiotensin II: A study in primary endothelial cultures of rat vena cava and portal vein

The role of the vascular endothelium in modulating the arterial system has been widely investigated, but poorly explored at the venous site. In the present work, primary cultures of venous endothelium from rat Vena Cava (VC) and Portal Vein (PV) were established, characterized and analyzed according to their growth pattern and ability to produce nitric oxide (NO) and prostanoids (PGF_{2 α} and PGI₂), at basal state and after stimulation with Angiotensin II (Ang II, 1 μmol/L). Basal NO was detected in all examined cells in culture. Pre-incubation with Ang II increased NO production in cells from VC (but not in PV cultures), through activation of both AT₁ and AT₂ receptors. Both cultures exhibited detectable levels of PGF_{2 α} at resting conditions, which were similarly enhanced by Ang II. Basal PGI₂ levels were higher in PV, but increased after Ang II treatment in VC, with no further effect on PV cells. We conclude that endothelial cells from VC and PV exhibit important properties and react to Ang II, probably influencing the whole circulatory system. This experimental cell model gives support to further studies concerning intracellular pathways of the venous endothelium, analyzed in separate from the vascular smooth muscle wall.     

Induction of iron regulatory protein 1 RNA-binding activity by nitric oxide is associated with a concomitant increase in the labile iron pool: implications for DNA damage

Iron regulatory protein 1 (IRP1) is a bifunctional [4Fe-4S] protein that controls iron homeostasis. Switching off its function from an aconitase to an apo-IRP1 interacting with iron-responsive element-containing mRNAs depends on the reduced availability of iron in labile iron pool (LIP). Although the modulation of IRP1 by nitric oxide has been characterized, its impact on LIP remains unknown. Here, we show that inhibition of IRP1 aconitase activity and induction of its IRE-binding activity during exposure of L5178Y mouse lymphoma cells to NO are associated with an increase in LIP levels. Removal of NO resulted in a reverse regulation of IRP1 activities accompanied by a decrease of LIP. The increased iron burden in LIP caused by NO exacerbated hydrogen peroxide-induced genotoxicity in L5178Y cells. We demonstrate that the increase in LIP levels in response to chronic but not burst exposure of L5178Y cells to NO is associated with alterations in the expression of proteins involved in iron metabolism.     

Development of NADPH-Diaphorase in the Avian Retina: Regulation by Calcium Ions and Relation to Nitric Oxide Synthase

Abstract: Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit ∼65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 m M calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by N ^G-nitro- l -arginine or l - N ^G-(iminoethyl)ornithine is concentration dependent with IC₅₀ values of ∼1 m M at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC₅₀ values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.     

Estimation of nitric oxide level in vivo by microdialysis with water-soluble iron-N-methyl-d-dithiocarbamate complexes as NO traps: A novel approach to nitric oxide spin trapping in animal tissues

It was found that microdialysis, i.e., passage of aqueous solutions of iron-N-methyl-d-glucamine dithiocarbamate complexes through dialysis fibers implanted into heart, kidney and liver tissues of narcotized rats, was accompanied by effective binding of the complexes to nitric oxide from interstitial fluid. The walls of dialysis fibers used in this study were permeable for compounds with molecular weight not exceeding 5 kDa. The dialyzate samples collected every 20 min and containing diamagnetic nitrosyl Fe³⁺-MGD adducts were reduced to the paramagnetic state with sodium dithionite; their concentration was measured by the EPR method. The basic level of the adducts, which represented mononitrosyl iron complexes with MGD (MNIC–MGD), in the dialyzate samples of all tested organs were similar (1 μМ). Treatment of animals with the water-soluble nitroglycerine analog Isoket or a low-molecular dinitrosyl iron thiosulfate complex as a NO donor increased the concentration of MNIC–MGD with going out into a plateau. The novel approach allows determination of nitric oxide levels in tissue interstitial fluid from concentration of MNIC–MGD formed during microdialysis.