Role Of Endogenous Nitric Oxide Research Articles

Synergy of Plant Growth Promoting Rhizobacteria and Silicon in regulation of AgNPs induced stress of rice seedlings.

Silver Nanoparticles (AgNPs), as an emerging pollutant, have been receiving significant attention as they deepen the concern regarding the issue of food security. Silicon (Si) and plant growth-promoting rhizobacteria (PGPR) are likely to serve as a sustainable approach to ameliorating abiotic stress and improving plant growth through various mechanisms. The present study aims to evaluate the synergistic effect of Si and PGPRs on growth, physiological, and molecular response in rice seedlings (Oryza sativa) under AgNPs stress. Data suggested that under AgNPs exposure, the root and shoot growth, photosynthetic pigments, antioxidant enzymes (CAT and APX), expression of antioxidant genes (OsAPX and OsGR), silicon transporter (OsLsi2), and auxin hormone-related genes (OsPIN10 and OsYUCCA1) were significantly decreased which accompanied with the overproduction of reactive oxygen species (ROS), nitric oxide (NO) and might be due to higher accumulation of Ag in plant cells. Interestingly, the addition of Si along with the AgNPs enhances the level of ROS generation, thus oxidative stress, which causes severe damage in all the above-tested parameters. On the other hand, application of PGPR alone and along with Si reduced the toxic effect of AgNPs through the improvement of growth, biochemical, and gene regulation (OsAPX and OsGR, OsPIN10 and OsYUCCA1). However, the addition of L-NAME along with PGPRs and silicon drastically lowered the AgNPs induced toxicity through lowering the oxidative stress and maintained the overall growth of rice seedlings, which suggests the role of endogenous NO in Si and PGPRs mediated management of AgNPs toxicity in rice seedlings.

Loss of S-Nitrosoglutathione reductase disturbs phytohormone homeostasis and regulates shoot side branching and fruit growth in tomato.

S-nitrosoglutathione (GSNO) plays a central role in nitric oxide (NO) homeostasis, and GSNO reductase (GSNOR) regulates the cellular levels of GSNO across kingdoms. Here, we investigated the role of endogenous NO in shaping shoot architecture and controlling fruit set and growth in tomato (Solanum lycopersicum). SlGSNOR silencing promoted shoot side branching and led to reduced fruit size, negatively impacting fruit yield. Greatly intensified in slgsnor knockout plants, these phenotypical changes were virtually unaffected by SlGSNOR overexpression. SlGSNOR silencing or knockout intensified protein tyrosine nitration and S-nitrosation and led to aberrant auxin production and signaling in leaf primordia and fruit-setting ovaries, besides restricting the shoot basipetal polar auxin transport stream. SlGSNOR deficiency triggered extensive transcriptional reprogramming at early fruit development, reducing pericarp cell proliferation due to restrictions on auxin, gibberellin and cytokinin production and signaling. Abnormal chloroplast development and carbon metabolism were also detected in early-developing NO-overaccumulating fruits, possibly limiting energy supply and building blocks for fruit growth. These findings provide new insights into the mechanisms by which endogenous NO fine-tunes the delicate hormonal network controlling shoot architecture, fruit set and post-anthesis fruit development, emphasizing the relevance of NO-auxin interaction for plant development and productivity.

Spontaneous pulmonary emphysema in mice lacking all three nitric oxide synthase isoforms

The roles of endogenous nitric oxide (NO) derived from the entire NO synthases (NOSs) system have yet to be fully elucidated. We addressed this issue in mice in which all three NOS isoforms were deleted. Under basal conditions, the triple n/i/eNOSs−/− mice displayed significantly longer mean alveolar linear intercept length, increased alveolar destructive index, reduced lung elastic fiber content, lower lung field computed tomographic value, and greater end-expiratory lung volume as compared with wild-type (WT) mice. None of single NOS−/− or double NOSs−/− genotypes showed such features. These findings were observed in the triple n/i/eNOSs−/− mice as early as 4 weeks after birth. Cyclopaedic and quantitative comparisons of mRNA expression levels between the lungs of WT and triple n/i/eNOSs−/− mice by cap analysis of gene expression (CAGE) revealed that mRNA expression levels of three Wnt ligands and ten Wnt/β-catenin signaling components were significantly reduced in the lungs of triple n/i/eNOSs−/− mice. These results provide the first direct evidence that complete disruption of all three NOS genes results in spontaneous pulmonary emphysema in juvenile mice in vivo possibly through down-regulation of the Wnt/β-catenin signaling pathway, demonstrating a novel preventive role of the endogenous NO/NOS system in the occurrence of pulmonary emphysema.

Nitric oxide: a new role in intensive care

Inhaled nitric oxide has been used for 30 years to improve oxygenation and decrease pulmonary vascular resistance. In the past 15 years, there has been increased understanding of the role of endogenous nitric oxide on cell surface receptors, mitochondria, and intracellular processes involving calcium and superoxide radicals. This has led to several animal and human experiments revealing a potential role for administered nitric oxide or nitric oxide donors in patients with systemic inflammatory response syndrome or ischaemia-reperfusion injury, and in patients for whom exposure of blood to artificial surfaces has occurred.

The role of endogenous nitric oxide in salicylic acid-induced up-regulation of ascorbate-glutathione cycle involved in salinity tolerance of pepper (Capsicum annuum L.) plants

An experimentation was carried out to appraise whether or not nitric oxide (NO) contributes to salicylic acid (SA)-induced salinity tolerance particularly by regulating ascorbate-glutathione (AsA-GSH) cycle. Before starting salinity stress (SS), SA (0.5 mM) was sprayed to the foliage of plants once every other day for a week and then seedlings were grown under control or SS (100 mM NaCl), for five weeks. Salinity stress enhanced the AsA-GSH cycle-related enzymes, glutathione reductase (GR), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR). Furthermore, SS caused substantial decreases in plant physiological-related traits such as leaf potassium (K) contents, K+/Na+ ratio, the ratios of reduced ascorbate/dehydroascorbic acid (AsA/DHA) and reduced glutathione/oxidized glutathione (GSH/GSSG), but in contrast, significant increases occurred in leaf hydrogen peroxide, malondialdehyde, electron leakage, proline, the premier antioxidant enzymes’ activities, Na+ and NO. SA reduced leaf Na+ content and oxidative stress-related traits, but improved all earlier-mentioned traits compared with those in plants treated with SS alone. All positive effects of SA were eliminated by NO scavenger, 0.1 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (c-PTIO) by reducing NO, suggesting that NO produced by SA up-regulated the activities of AsA-GSH cycle and antioxidant enzymes, so it could play a central function as a signal molecule in salt tolerance of pepper plants.

The putative role of endogenous nitric oxide in brassinosteroid-induced antioxidant defence system in pepper (Capsicum annuum L.) plants under water stress

Brassinosteroids (BRs) have been rarely tested for their effective roles in mitigation of deleterious effects of water stress (WS) on plants. In addition, the contribution of nitric oxide (NO) in BR-improved plant tolerance to water stress needs to be elucidated. So, a trial was carried out to uncover the contribution of NO in BR-induced tolerance of pepper plants to WS. For well-watered and water-stressed plants, soil water availability was sustained at 80% and 40% of the full water storage capacity, respectively. BR (24-epibrassinolide, EB; 1.0 μM) was sprayed to the leaves of both well-watered and water stressed-pepper plants every two days for 10 days prior to the initiation of stress treatment. After starting WS treatment, cPTIO was sprayed to plant leaves twice a week for four weeks. Water stress caused a reduced plant growth and oxidative stress, but the application of EB increased plant growth and reversed the oxidative stress. The EB treatment increased endogenous NO and reinforced antioxidant defence systems, but the cPTIO application reversed the NO levels, downregulated the antioxidant defence systems, and aggravated oxidative damages caused by WS. These results show that EB-induced NO generation and NO-mediated antioxidant defence systems might be crucial mechanisms for EB-improved tolerance of pepper plants to WS. So, both EB and NO jointly are responsible for achieving improved tolerance of pepper plants to water stress.

The role of endogenous nitric oxide in melatonin-improved tolerance to lead toxicity in maize plants.

Melatonin (MT) and nitric oxide (NO) are known as scavengers of free radicals and an antioxidant against biotic and abiotic stresses in plant defense systems. However, whether NO interplays role in MT-induced antioxidant defense remains to be determined in the plants exposed to lead (Pb) toxicity. So, two experiments were designed to evaluate the role of NO in MT-mediated tolerance of maize plants to Pb stress. In the initial experiment, prior to starting different treatments, a solution of 0.05- or 0.10-mM MT was sprayed every other day for a period of 10days to the leaves of maize plants exposed to Pb stress (0.1-mM PbCl2). Pb toxicity significantly caused reduction in plant biomass (both fresh and dry), PSII maximum efficiency (Fv/Fm), total chlorophyll, leaf potassium (K), calcium (Ca), and leaf water potential, but it resulted in increased levels of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), electron leakage (EL), leaf Pb, and endogenous NO. An addition experiment was set up to further understand whether NO played role in mitigation of Pb toxicity in maize plants by MT using scavengers of NO and cPTIO combined with the MT treatments. MT-induced tolerance to Pb toxicity was totally eliminated by cPTIO by reversing endogenous NO. The present results clearly indicated that MT mediated the endogenous NO to improve tolerance of maize plants to Pb toxicity. This evidence was also supported by the increases of H2O2 and MDA and reduces some antioxidant enzyme activities tested as well as the plant growth inhibition and increased leaf Pb content by application of MT combined with cPTIO.

Protein disulfide isomerase regulation by nitric oxide maintains vascular quiescence and controls thrombus formation

Essentials Nitric oxide synthesis controls protein disulfide isomerase (PDI) function. Nitric oxide (NO) modulation of PDI controls endothelial thrombogenicity. S-nitrosylated PDI inhibits platelet function and thrombosis. Nitric oxide maintains vascular quiescence in part through inhibition of PDI. SUMMARY: Background Protein disulfide isomerase (PDI) plays an essential role in thrombus formation, and PDI inhibition is being evaluated clinically as a novel anticoagulant strategy. However, little is known about the regulation of PDI in the vasculature. Thiols within the catalytic motif of PDI are essential for its role in thrombosis. These same thiols bind nitric oxide (NO), which is a potent regulator of vessel function. To determine whether regulation of PDI represents a mechanism by which NO controls vascular quiescence, we evaluated the effect of NO on PDI function in endothelial cells and platelets, and thrombus formation in vivo. Aim To assess the effect of S-nitrosylation on the regulation of PDI and other thiol isomerases in the vasculature. Methods and results The role of endogenous NO in PDI activity was evaluated by incubating endothelium with an NO scavenger, which resulted in exposure of free thiols, increased thiol isomerase activity, and enhanced thrombin generation on the cell membrane. Conversely, exposure of endothelium to NO+ carriers or elevation of endogenous NO levels by induction of NO synthesis resulted in S-nitrosylation of PDI and decreased surface thiol reductase activity. S-nitrosylation of platelet PDI inhibited its reductase activity, and S-nitrosylated PDI interfered with platelet aggregation, α-granule release, and thrombin generation on platelets. S-nitrosylated PDI also blocked laser-induced thrombus formation when infused into mice. S-nitrosylated ERp5 and ERp57 were found to have similar inhibitory activity. Conclusions These studies identify NO as a critical regulator of vascular PDI, and show that regulation of PDI function is an important mechanism by which NO maintains vascular quiescence.

Nitric oxide contributes to methane-induced osmotic stress tolerance in mung bean

BackgroundOsmotic stress is a major abiotic stress limiting crop production by affecting plant growth and development. Although previous reports discovered that methane (CH4) has a beneficial effect on osmotic stress, the corresponding downstream signal(s) is still elusive.ResultsPolyethylene glycol (PEG) treatment progressively stimulated the production of CH4 in germinating mung bean seeds. Exogenous CH4 and sodium nitroprusside (SNP) not only triggered nitric oxide (NO) production in PEG-stressed plants, but also alleviated the inhibition of seed germination. Meanwhile, amylase activity was activated, thus accelerating the formation of reducing sugar and total soluble sugar. Above responses could be impaired by NO scavenger(s), suggesting that CH4-induced stress tolerance was dependent on NO. Subsequent tests showed that CH4 could reestablish redox balance in a NO-dependent fashion. The addition of inhibitors of the nitrate reductase (NR) and NO synthase in mammalian (NOS), suggested that NR and NOS-like protein might be partially involved in CH4-alleviated seed germination inhibition. In vitro and scavenger tests showed that NO-mediated S-nitrosylation might be associated with above CH4 responses.ConclusionsTogether, these results indicated an important role of endogenous NO in CH4-enhanced plant tolerance against osmotic stress, and NO-regulated redox homeostasis and S-nitrosylation might be involved in above CH4 action.

Achieving Long-Term Biocompatible Silicone via Covalently Immobilized S-Nitroso- N-acetylpenicillamine (SNAP) That Exhibits 4 Months of Sustained Nitric Oxide Release.

Ever since the role of endogenous nitric oxide (NO) in controlling a wide variety of biological functions was discovered approximately three decades back, multiple NO-releasing polymeric materials have been developed. However, most of these materials are typically short lived due to the inefficient incorporation of the NO donor molecules within the polymer matrix. In the present study, S-nitroso- N-acetyl penicillamine (SNAP) is covalently attached to poly(dimethylsiloxane) (PDMS) to create a highly stable nitric oxide (NO) releasing material for biomedical applications. By tethering SNAP to the cross-linker of PDMS, the NO donor is unable to leach into the surrounding environment. This is the first time that a sustainable NO release and bacterial inhibition for over 125 days has been achieved by any NO-releasing polymer with supporting evidence of potential long-term hemocompatibility and biocompatibility. The material proves to have very high antibacterial efficacy against Staphylococcus aureus by demonstrating a 99.99% reduction in the first 3 days in a continuous flow CDC bioreactor, whereas a similar inhibitory potential of 99.50% was maintained by the end of 1 month. Hemocompatibility of SNAP-PDMS was tested using a rabbit extracorporeal circuit (ECC) model over a 4 h period. Thrombus formation was greatly reduced within the SNAP-PDMS-coated ECCs compared to the control circuits, observing a 78% reduction in overall thrombus mass accumulation. These results demonstrate the potential of utilizing this material for blood and tissue contacting biomedical devices in long-term clinical applications where infection and unwanted clotting are major issues.

Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells

Main conclusionA simple and rapid method involving flow cytometry and NO-specific probe (DAF-FM DA) proved useful for detection and determination of intracellular NO production in Medicago truncatula suspension cells and leaves as well as in cells of Avena fatua, Amaranthus retroflexus embryos and leaves.The measurement of nitric oxide (NO) in plant material is important for examining the regulatory roles of endogenous NO in various physiological processes. The possibility of detecting and determining intracellular NO production by flow cytometry (FCM) with 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM DA), an NO-specific probe in Medicago truncatula cells in suspension and leaves as well as in cells of embryos and leaves of Avena fatua L. or Amaranthus retroflexus L. was explored. To detect and measure NO production by cell suspension or embryos and leaves, the recommended DAF-FM DA concentration is 5 or 10 µM, respectively, applied for 30 min. Exogenous NO increased the intensity of the fluorescent signal in embryos and leaves of both plants, while carboxy-PTIO (cPTIO), an NO scavenger, decreased it. Thus, these results demonstrate that NO can be detected and an increase and a decrease of its intracellular level can be estimated. Wounding was observed to increase the fluorescence signal, indicating an increase in the intracellular NO level. In addition, the levels of exogenous and endogenous ascorbic acid were demonstrated to have no effect on the NO-related fluorescence signal, indicating the signal’s specificity only in relation with NO. The applicability of the proposed method for detection and determination of NO was confirmed (1) by in situ NO imaging in cell suspensions and (2) by determining the NO concentration in embryos and leaves using the Griess reagent. In view of the data obtained, FCM is recommended as a rapid and simple method with which to detect and determine intracellular NO production in plant cells.

Hypothalamic paraventricular nucleus neuronal nitric oxide synthase activity is a major determinant of renal sympathetic discharge in conscious Wistar rats.

What is the central question of this study? Does chronic reduction of neuronally generated nitric oxide in the hypothalamic paraventricular nucleus affect the set-point regulation of blood pressure and sympathetic activity destined to the kidneys? What is the main finding and its importance? Within the hypothalamic paraventricular nucleus, nitric oxide generated by neuronal nitric oxide synthase plays a major constitutive role in suppressing long term the levels of both ongoing renal sympathetic activity and arterial pressure in conscious Wistar rats. This finding unequivocally demonstrates a mechanism by which the diencephalon exerts a tonic influence on sympathetic discharge to the kidney and may provide the basis for both blood volume and osmolality homeostasis. The paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in cardiovascular and neuroendocrine regulation. Application of nitric oxide donors to the PVN stimulates GABAergic transmission, and may suppress sympathetic nerve activity (SNA) to lower arterial pressure. However, the role of endogenous nitric oxide within the PVN in regulating renal SNA chronically remains to be established in conscious animals. To address this, we used our previously established lentiviral vectors to knock down neuronal nitric oxide synthase (nNOS) selectively in the PVN of conscious Wistar rats. Blood pressure and renal SNA were monitored simultaneously and continuously for 21days (n=14) using radio-telemetry. Renal SNA was normalized to maximal evoked discharge and expressed as a percentage change from baseline. The PVN was microinjected bilaterally with a neurone-specific tetracycline-controllable lentiviral vector, expressing a short hairpin miRNA30 interference system targeting nNOS (n=7) or expressing a mis-sense as control (n=7). Recordings continued for a further 18days. The vectors also expressed green fluorescent protein, and successful expression in the PVN and nNOS knockdown were confirmed histologically post hoc. Knockdown of nNOS expression in the PVN resulted in a sustained increase in blood pressure (from 95±2 to 104±3mmHg, P<0.05), with robust concurrent sustained activation of renal SNA (>70%, P<0.05). The study reveals a major role for nNOS-derived nitric oxide within the PVN in chronic set-point regulation of cardiovascular autonomic activity in the conscious, normotensive rat.

Pancreatic-β-cell survival and proliferation are promoted by protein kinase G type Iα and downstream regulation of AKT/FOXO1.

Early studies showed nitric oxide as a pro-inflammatory-cytokine-induced toxin involved in pancreatic β-cell destruction during pathogenesis of type-1 diabetes. However, nitric oxide has both cytotoxic and cytoprotective effects on mammalian cells, depending on concentration and micro-environmental surroundings. Our studies have shown that low/physiological-level nitric oxide selectively activates protein kinase G type Iα isoform, promoting cytoprotective/pro-cell-survival effects in many cell types. In bone marrow-derived stromal/mesenchymal stem cells, protein kinase G type Iα mediates autocrine effects of nitric oxide and atrial natriuretic peptide, promoting DNA-synthesis/proliferation and cell survival. In this study, endothelial nitric oxide synthase/neuronal nitric oxide synthase inhibitor L-NIO (L-N(5)-(1-iminoethyl)ornithine), soluble guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one), atrial natriuretic peptide-receptor inhibitor A71915 and protein kinase G type Iα kinase activity inhibitor DT-2 all increased apoptosis and decreased insulin secretion in RINm5F pancreatic β-cells, suggesting autocrine regulatory role for endogenous nitric oxide- and atrial natriuretic peptide-induced activation of protein kinase G type Iα. In four pancreatic β-cell lines, Beta-TC-6, RINm5F, INS-1 and 1.1B4, protein kinase G type Iα small-interfering RNA decreased phospho-serine-239-VASP (indicator of endogenous protein kinase G type Iα kinase activity), increased apoptosis and decreased proliferation. In protein kinase G type Iα-knockdown β-cell lines, expressions of phospho-protein kinase B (PKB/AKT) (AKT), phospho-Forkhead box protein O1 (FOXO1) (transcriptional repressor of pancreas duodenum homobox-1) and pancreas duodenum homobox-1 were decreased, suppressing proliferation and survival in pancreatic β-cells. The data suggest autocrine nitric oxide/atrial natriuretic peptide-induced activation of protein kinase G type Iα/p-AKT/p-FOXO1 promotes survival and proliferation in pancreatic β-cells, providing therapeutic implications for development of new therapeutic agents for diabetes.

Characterization of the role of endogenous nitric oxide in myogenic vascular oscillations during cooling-evoked hemodynamic perturbations of rats.

Rapid immersion of a rat's limbs into 4 °C water, a model of cold stress, can elicit hemodynamic perturbations (CEHP). We previously reported that CEHP is highly relevant to sympathetic activation and nitric oxide production. This study identifies the role of nitric oxide in CEHP. Conscious rats were pretreated with the nitric oxide synthase inhibitor L-NAME (NG-nitro-l-arginine methyl ester) alone or following the removal of sympathetic influences using hexamethonium or guanethidine. Rats were then subjected to a 10 min cold-stress trial. Hemodynamic indices were telemetrically monitored throughout the experiment. The analyses included measurements of systolic blood pressure; heart rate; dicrotic notch; short-term cardiovascular oscillations and coherence between blood pressure variability and heart rate variability in regions of very low frequency (0.02-0.2 Hz), low frequency (0.2-0.6 Hz), and high frequency (0.6-3.0 Hz). We observed different profiles of hemodynamic reaction between hexamethonium and guanethidine superimposed on L-NAME, suggesting an essential role for a functional adrenal medulla release of epinephrine under cold stress. These results indicate that endogenous nitric oxide plays an important role in the inhibition of sympathetic activation and cardiovascular oscillations in CEHP.

Endogenous nitric oxide mediates He-Ne laser-induced adaptive responses in salt stressed-tall fescue leaves.

The aim of this study was to investigate the role of endogenous nitric oxide in protective effects of He-Ne laser on salt stressed-tall fescue leaves. Salt stress resulted in significant increases of membrane injury, reactive oxygen species (ROS) production, polyamine accumulation, and activities of SOD, POD, and APX, while pronounced decreases of antioxidant contents, CAT activity and intracellular Ca(2+) concentration in seedlings leaves. He-Ne laser illumination caused a distinct alleviation of cellular injury that was reflected by the lower MDA amounts, polyamine accumulation and ROS levels at the stress period. In contrast, the laser treatment displayed a higher Ca(2+) concentration, antioxidant amounts, NO release, antioxidant enzyme, and NOS activities. These responses could be blocked due to the inhibition of NO biosynthesis by PTIO (NO scavenger) or LNNA (NOS inhibitor). The presented results demonstrated that endogenous NO might be involved in the progress of He-Ne laser-induced plant antioxidant system activation and ROS degradation in order to enhance adaptive responses of tall fescue to prolonged saline conditions.

Cellular inactivation of nitric oxide induces p53-dependent apoptosis in human melanoma cells

Purpose: To examine the role of endogenous nitric oxide (NO • ) and influence of p53 status during apoptosis induced by a selective iNOS inhibitor, N-[(3-aminomethyl) benzyl] acetamidine (1400W), and/or an NO • scavenger carboxy-PTIO (c-PTIO) in two isogenic human melanoma cell lines, wild-type p53 (A375) and p53 mutant (SK mel-28) cells. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Annexin V/propidium iodide assay were used to test for antiproliferation and apoptosis, respectively. Griess and reverse transcription-polymerase chain reaction (RT-PCR) reactions were carried out to assay NO • production and the mRNA levels of inhibitors of apoptosis (IAP). Results: c-PTIO and 1400W, alone or in combination, inhibited cell growth and promoted apoptosis via sub-G1 cell cycle arrest mediated by decrease in NO • . Apoptosis was delayed and greatly reduced in magnitude in SK mel-28 cells, underscoring the importance of p53 modulation of the response. In both cell types, apoptosis induced by iNOS inhibition and/or NO • depletion was blocked by an exogenous NO • donor, sodium nitroprusside. It was also found that inhibitors of apoptosis family (survivin, XIAP and cIAP1) were significantly depressed, which appear to play an important role in the regulation of p53- mediated apoptotic response under these conditions. Conclusion: The data obtained provide insight into the mechanism of cell proliferation action of endogenous NO • , based on p53 status, and indicate manipulation of iNOS may offer exciting opportunities to improve the effectiveness of melanoma treatment. Keywords: Apoptosis, Human melanoma cells, Inducible nitric oxide synthase, p53

Role of Endogenous Nitric Oxide in Hyperaggressiveness of Tumor Cells that Survive a Photodynamic Therapy Challenge.

Many malignant tumors exploit nitric oxide (NO) for a survival, growth, and migration/invasion advantage, and also to withstand the cytotoxic effects of chemo- and radiotherapies. Endogenous NO has also been shown to antagonize photodynamic therapy (PDT), a unique minimally invasive modality involving a photosensitizing (PS) agent, PS-exciting light in the visible- to near-infrared range, and molecular oxygen. The anti-PDT effects of NO were discovered about 20 years ago, but the underlying mechanisms are still not fully understood. More recent studies in the author's laboratory using breast, prostate, and brain cancer cell lines have shown that inducible NO synthase (iNOS/NOS2) is dramatically upregulated after a PDT challenge using 5-aminolevulinic acid (ALA-) -induced protoporphyrin IX as the PS. The parallel increase in NO resulted not only in a greater resistance to cell killing but also in a striking increase in the growth and migration/invasion rate of surviving cells. These in vitro findings and their recent recapitulation at the in vivo level are discussed in this article, along with how iNOS/NO's negative effects on PDT can be attenuated by the use of select iNOS inhibitors as PDT adjuvants.

Abstract 15229: Critical Protective Role of Endogenous Nitric Oxide in Established Severe Pulmonary Arterial Hypertension in Rats

Background: It is widely believed that impared bioavailability of endothelium-derived nitric oxide (NO) contributes to the development of pulmonary arterial hypertension (PAH). However, there are reports that show high pulmonary endothelial NO synthase (eNOS) expression in PAH patients, and the functional roles of endogenous NO in established PAH remain unclear. Hypothesis: We tested the hypothesis that pulmonary eNOS expression is increased, and that endogenous NO plays an important functional role in a rat model of PAH. Method and Results: Adult male Sprague Dawley rats (BW: 150 - 200g) were injected subcutaneously with Sugen5416 (20 mg/kg; VEGF receptor blocker), and exposed to hypoxia (10% O2) for 3 weeks followed by a return to normoxia for 5 weeks. Hemodynamic examination was performed at the 1-, 3-, 5- and 8-week (wk) time points after Sugen5416 injection. RV systolic pressure increased time-dependently (RVSP: 47 ± 6, 83 ± 14, 101 ± 22, and 122 ± 16 mmHg at 1-, 3-, 5- and 8-wk time points, n=4-6) compared with normal rats (31 ± 2 mmHg, n=4, p&lt;0.01, respectively). Blockade of NOS with Nω-nitro-L- arginine methyl ester (L-NAME, 30 mg/kg, iv) further increased RVSP acutely (ΔRVSP from each baseline: 7 ± 11, 25 ± 11, 52± 21, and 43 ± 13 at 1-, 3-, 5- and 8-wk time points) compared with normal rats (0.8 ± 1.1 mmHg). At the 8-wk time point, inhibition of NOS caused a marked RVSP elevation (165 ± 11 mmHg), leading to RV failure and death in all rats tested. In vivo pulmonary angiogram using the synchrotron radiation microangiogram system showed severe small pulmonary arterial narrowing in response to L-NAME. Western blot analysis showed increased eNOS protein levels in PAH rat lungs (n=4 each time point; p&lt;0.01 vs. normal) and immunohistochemisty revealed high expression of eNOS in remodeled pulmonary arteries at the 5- and 8-time points. Conclusion: Based on our findings that L-NAME caused marked pulmonary vasoconstriction and further elevation in RVSP and that there was high lung eNOS protein levels in advanced PAH rats, we conclude, contrary to the general assumption, that endogenous NO presumably via eNOS may play a critical protective role by counteracting severe pulmonary vasoconstriction in established PAH.

High nitric oxide production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the tumour‐initiating properties of colon cancer stem cells

Chronic inflammation is a leading cause of neoplastic transformation in many human cancers and especially in colon cancer (CC), in part due to tumour promotion by nitric oxide (NO) generated at inflammatory sites. It has also been suggested that high NO synthesis, secondary to inducible NO synthase (iNOS) expression, is a distinctive feature of cancer stem cells (CSCs), a small subset of tumour cells with self-renewal capacity. In this study we explored the contribution of NO to the development of colon CSC features and evaluated potential strategies to treat CC by modulating NO production. Our data show an integral role for endogenous NO and iNOS activity in the biology of colon CSCs. Indeed, colon CSCs with high endogenous NO production (NO(high)) displayed higher tumourigenic abilities than NO(low) fractions. The blockade of endogenous NO availability, using either a specific iNOS inhibitor or a genetic knock-down of iNOS, resulted in a significant reduction of colon CSC tumourigenic capacities in vitro and in vivo. Interestingly, analysis of genes altered by iNOS-directed shRNA showed that the knockdown of iNOS expression was associated with a significant down-regulation of signalling pathways involved in stemness and tumour progression in colon CSCs. These findings confirm that endogenous NO plays an important role in defining the stemness properties of colon CSCs through cross-regulation of several cellular signalling pathways. This discovery could shed light on the mechanisms by which NO induces the growth and invasiveness of CC, providing new insights into the link between inflammation and colon tumourigenesis.

一氧化氮对植物重金属胁迫抗性影响的研究进展

Heavy metal pollution has become an increasingly serious environmental problem because heavy metal can be easily taken up by plants,leading to inhibition of plant growth and development. Hence,it is necessary to investigate resistances of plants to heavy metal stress. As an important signaling molecule,nitric oxide( NO) is involved in the regulation of multiple plant responses to a variety of abiotic and biotic stresses. Recently,an increasing number of articles have reported the effects of NO on resistances of plants to heavy metal stress. However,studies which systematically summarize the molecular mechanisms of NO on resistances of plants to heavy metal stress are quite limited. This research mainly reviews the pathways of NO production,changes of endogenous NO contents under heavy metal stress and influences of exogenous and endogenous NO on resistances to heavy metal stress. The sources of NO production in plants involve not only enzymatic reaction pathways which include nitric oxide synthase( NOS),nitrate reductase( NR),nitrite reductase( Ni-NOR) pathways and etc. but also non- enzymatic reaction pathways. Many authors have noted discrepant reports on the effects of heavy metal stress on endogenous NO content in plants and the observed differences in endogenous NO accumulation are frequently ascribed to the use of different duration of treatment,heavy metal concentrations,species and genotypes of plants and varieties of plant tissues. Interestingly,all plant cell suspensions show a visible increase in endogenous NO accumulation under heavy metal stress,which is attributed to the fact that they are lacking of network regulation between cells and cells. Meanwhile,it has been demonstrated that exogenous NO could enhance antioxidant defence system of plant cells,act as an antioxidant promoting direct scavenging of reactive oxygen species,induce metallothioneins( MTs) gene expression by displacing heavy metal bound to MTs,and affect the uptake of heavy metal intoplants and the distribution of heavy metal in plant cells though regulating the metabolism of cell wall composition,consequently relieve heavy metal toxicity and enhance resistances of plants to heavy metal stress. However,the functional roles of endogenous NO in regulating resistances of plants to heavy metal stress are controversial. Some research show that endogenous NO is helpful for alleviating heavy metal-induced toxicity. On the contrary,some evidences indicate that endogenous NO is harmful,and participates in heavy metal-induced cell toxicity and programmed cell death through accelerating the absorption of heavy metal and S-nitrosylation of phytochelatins. These conflicting data suggest that NO may have a dual effect on resistances to heavy metal stress,but great efforts are required in order to clarify this speculation.Finally,interrelated perspectives are also discussed to further study the relationship between NO and resistances to heavy metal stress.