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.     

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.     

一氧化氮合酶FMN结合结构域的电子传递及调控机制研究现状

生物体内NO是在一氧化氮合酶（mitric oxide synthase,NOS）催化下生成的,NOS的结构包括C端还原酶域和N端加氧酶域。还原酶域中的FMN结合结构域既可接受来自NADPH-FAD结构域的电子,又可作为提供电子的供体,在调控催化过程中的电子传递方面发挥着重要作用。主要从FMN结合结构域的构象平衡及其对不同亚型NOS的动力学差异的贡献、FMN结合结构域自身的电荷性质以及NOS中其他结构域对FMN结构域的功能调控三个方面进行了论述,以期揭示NOS独特的电子传递催化机制。     

Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca²⁺, mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca²⁺-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.     

Effects of simulated microgravity on nitric oxide level in cardiac myocytes and its mechanism

The depression of cardiac contractility induced by space microgravity is an important issue of aerospace medicine research, while its precise mechanism is still unknown. In the present study, we explored effects of simulated microgravity on nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) expression and related regulative mechanism using electron spin resonance (ESR) spectroscopy, immunocytochemistry and in situ hybridization. We found a remarkable increase of NO level and up-regulation of iNOS and iNOS mRNA expression in rat cardiac myocytes under simulated microgravity. Staurosporine (a nonselective protein kinase inhibitor), calphostin C (a selective protein kinase C inhibitor), partially inhibited the effect of simulated microgravity. Thus regulative effect of simulated microgravity on iNOS expression is mediated at least partially via activation of protein kinase C. These results indicate that NO system in cardiac myocytes is sensitive to simulated microgravity and may play an important role in the depression of cardiac contractility induced by simulated microgravity.     

Regulation and function of the protein inhibitor of nitric oxide synthase (PIN)/dynein light chain 8 (LC8) in a human mast cell line

The protein inhibitor of nitric oxide synthase (PIN) was independently identified as an inhibitor of nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS), and as a member of the cellular dynein light chain family, dynein light chain 8 (LC8), responsible for intracellular protein trafficking. Mast cells (MC) are involved in several homeostatic and pathological processes and can be regulated by NO. This study describes the expression of PIN/LC8 in the human MC line HMC-1. We also studied if PIN/LC8 binds nNOS, and what role this might have in leukotriene (LT) production. We found that PIN/LC8 mRNA and protein was expressed in HMC-1. Using a GST-PIN construct, we showed PIN binds to nNOS, but not endothelial (e)NOS in HMC-1; in our studies HMC-1 did not express inducible (i)NOS. Intracellular delivery of anti-PIN/LC8 antibody enhanced ionophore (A23187)-induced LT production through an unknown mechanism. Thus we established for the first time expression of PIN/LC8 in human MC, its ability to bind nNOS, and the effect that blocking it has on LT production in a human MC lines.     

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.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.     

Glucocorticoids inhibit the induction of nitric oxide synthase and the related cell damage in adenocarcinoma cells

Lipopolysaccharide (LPS) induced a time-dependent synthesis of nitric oxide (NO) in EMT6 adenocarcinoma cells, assayed by accumulation of NO-derived nitrite in the medium. The induction NO synthesis was inhibited in a concentration-dependent manner by the glucocorticoids dexamethasone (IC₅₀ = 5 nM) and hydrocortisone (IC₅₀ = 20 nM) and this effect was partially antagonized by progesterone and cortexolone. If addition of dexamethasone was delayed 6 h or more, inhibition of nitrite accumulation over 24 h was substantially reduced, indicating a lack of direct effect of glucocorticoids on the NO synthase. Nitrite accumulation was accompanied by cell damage, which was increased by L-arginine and inhibited by $\text{N}{}^{\mathrm{<mtext>G</mtext>}}\text{-}\text{monomethyl-}\text{L}\text{-arginine}$ (L-NMMA) and dexamethasone. These data show that NO is a primary cytotoxic mediator and that suppression of its formation by glucocorticoids explains some of their anti-inflammatory and cytoprotective effects.     

Integrated approach to the mechanisms of thyroid toxins: electron transfer,reactive oxygen species,oxidative stress,cell signaling,receptors, and antioxidants

Although considerable numbers of reviews are available on toxicity of major body organs based on electron transfer (ET), reactive oxygen species (ROS), and oxidative stress (OS), the integrated concept has been less applied to glands. This review represents an interdisciplinary approach to thyroid toxicity, involving ET, ROS, OS, cell signaling, receptors, toxicants, and beneficial effects of antioxidants (AOs). The introductory portion includes general function of the thyroid as well as the mechanism of thyroxine synthesis entailing participation of oxidative events, including the role of iodine. Various ROS, both endogenous and exogenous, are importantly involved in the diverse toxic manifestations. Discussion is centered on hydrogen peroxide and lipid peroxides. There is also treatment of receptor-ligand activity. Cell signaling participates in the various biochemical events taking place in the thyroid, both beneficial and adverse. In addition, the mechanism of cell signaling is discussed based on radicals, ET, relays, conduits, and electrochemistry. In addition to endogenous toxins, various exogenous ones are addressed, falling in diverse classes. Data indicate involvement of ET-ROS-OS in the toxic manifestations. Large numbers of reports reveal the beneficial effects of AOs in countering the toxicity, which is in accord with the mechanistic framework.     

Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10⁻³, 10⁻⁵, 10⁻⁷ and 10⁻⁹ M sodium nitroprusside (SNP). PFs were further cultured with 10⁻⁵ M SNP + 1.0 mM N^ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10⁻³, 10⁻⁵, 10⁻⁷ M) inhibited (P < 0.05) while, lower dose of SNP (10⁻⁹ M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.     

Electromagnetic fields: mechanism,cell signaling,other bioprocesses,toxicity, radicals,antioxidants and beneficial effects

Electromagnetic fields (EMFs) played a role in the initiation of living systems, as well as subsequent evolution. The more recent literature on electrochemistry is documented, as well as magnetism. The large numbers of reports on interaction with living systems and the consequences are presented. An important aspect is involvement with cell signaling and resultant effects in which numerous signaling pathways participate. Much research has been devoted to the influence of man-made EMFs, e.g., from cell phones and electrical lines, on human health. The degree of seriousness is unresolved at present. The relationship of EMFs to reactive oxygen species (ROS) and oxidative stress (OS) is discussed. There is evidence that indicates a relationship involving EMFs, ROS, and OS with toxic effects. Various articles deal with the beneficial aspects of antioxidants (AOs) in countering the harmful influence from ROS-OS associated with EMFs. EMFs are useful in medicine, as indicated by healing bone fractures. Beneficial effects are recorded from electrical treatment of patients with Parkinson’s disease, depression, and cancer.     

JAK／STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila

In multicellular organisms, biological activities are regulated by cell signaling. The various signal transduction pathways regulate cell fate, proliferation, migration, and polarity. Miscoordination of the communicative signals will lead to disasters like cancer and other fatal diseases. The JAK/STAT signal transduction pathway is one of the pathways, which was first identified in vertebrates and is highly conserved throughout evolution. Studying the JAK/STAT signal transduction pathway in Drosophila provides an excellent opportunity to understand the molecular mechanism of the cell regulation during development and tumor formation. In this review, we discuss the general overview of JAK/STAT signaling in Drosophila with respect to its functions in the eye development and stem cell fate determination.     

Nitric oxide and cell signaling; modulation of redox tone and protein modification

Summary. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have an impact on many cellular processes, often serving as signal transducers in both physiological and pathological situations. These small molecules can act as ligands for receptors as is the case for nitric oxide and guanylate cyclase. However, they can also modify proteins, changing their function and establishing a baseline for other signals in a process that we have termed redox tone. In this review, we discuss the different mechanisms of redox cell signaling, and give specific examples of RNS participation in cell signaling via classical and redox tone pathways.     

Growth hormone induces eNOS expression and nitric oxide release in a cultured human endothelial cell line

Growth hormone deficiency is linked to cardiovascular disease and particularly increased peripheral vascular resistance. Surprisingly, its role in endothelial nitric oxide (NO) synthetase (eNOS) regulation and NO release is basically unknown. We therefore studied the effects of different doses of somatotropin in cultures of a human endothelial cell line (EAhy926). We investigated expression and activity of eNOS, as well as other target genes known to be deregulated in cardiovascular disease including E-selectin and the lectin-like oxidized low density lipoprotein receptor. Treatment of cultured human endothelial cells with somatotropin resulted in significant (P<0.05) increases of eNOS gene and protein expression, as well as NO release, whereas production of intracellular reactive oxygen species was significantly reduced, at the highest somatotropin dose level. The enhanced eNOS gene/protein expression and enzyme activity correlate well. Our findings are suggestive for a novel role of growth hormone in endothelial biology, and particularly NO production.     

Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: Influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H₂O₂ production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.     

The olfactory responses of the antenna and maxillary palp of the fleshfly,Neobellieria bullata (Diptera: Sarcophagidae), and their sensitivity to blockage of nitric oxide synthase

The relative sensitivities of the olfactory receptors in the antenna and maxillary palp of the fleshfly, Neobellieria bullata, were assessed using simultaneous electroantennograms (EAGs) and electropalpograms (EPGs). In general, the antennae and maxillary palps were more sensitive to odors related to animals (blood extract and saturated carboxylic acid) than to odors that were plant-derived (citral, hexenol, hexenal). In addition, the maxillary palps were relatively less sensitive to plant-derived odorants than the antennae, perhaps related to their anatomical position. Scanning electron microscopy was also used to assess the types of sensilla found on the two organs. In addition, NADPH-diaphorase histochemistry was used in an attempt to localize the enzyme nitric oxide synthase (NOS) in the antenna and the maxillary palps. We found evidence of NADPH-diaphorase staining in both organs, with localized staining in the antennal cells and more general staining in the maxillary palps. When NOS was selectively blocked using the antagonist L-NAME, the amplitude of the EAGs and EPGs to odorants fell by 30-50%. In contrast, application of the inactive enantiomer, D-NAME, did not change the amplitude of the EAGs or the EPGs. Our results indicate that NOS is involved in the function of olfactory receptor cells in the fleshfly.     

Unifying mechanism for metals in toxicity,carcinogenicity and therapeutic action: integrated approach involving electron transfer,oxidative stress,antioxidants, cell signaling and receptors

This comprehensive review of biometals involves the unifying theme of electron transfer, reactive oxygen species, and oxidative stress (OS) applied to toxicity, carcinogenicity, and therapeutic action. The beneficial effect of antioxidants supports the participation of OS. The metals involved are mainly in the heavier category. An important aspect is the favorable reduction potential exhibited by the bioactive materials that permits redox cycling in vivo with the generation of oxy radicals. The basic mechanistic theme is applicable to other electron transfer (ET) functionalities. Appreciable evidence indicates the participation of cell signaling in various ways. Also, a simplifying framework is provided based on radical species and electrochemistry (ET and molecular electrostatic potential). This review also discusses receptor participation with focus on binding to proteins. Resultant physiological effects are summarized. The overview provides an integrated approach to metal bioactivation.     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.