Non-enzymatic Nitric Oxide Research Articles

5-Aminolevulinic Acid Induces Chromium [Cr(VI)] Tolerance in Tomatoes by Alleviating Oxidative Damage and Protecting Photosystem II: A Mechanistic Approach.

Chromium [Cr(VI)] pollution is a major environmental risk, reducing crop yields. 5-Aminolevunic acid (5-ALA) considerably improves plant abiotic stress tolerance by inducing hydrogen peroxide (H2O2) and nitric oxide (NO) signalling. Our investigation aimed to uncover the mechanism of tomato tolerance to Cr(VI) toxicity through the foliar application of 5-ALA for three days, fifteen days before Cr treatment. Chromium alone decreased plant biomass and photosynthetic pigments, but increased oxidative stress markers, i.e., H2O2 and lipid peroxidation (as MDA equivalent). Electrolyte leakage (EL), NO, nitrate reductase (NR), phytochelatins (PCs), glutathione (GSH), and enzymatic and non-enzymatic antioxidants were also increased. Foliar application of 5-ALA before Cr treatment improved plant growth and photosynthetic pigments, diminished H2O2, MDA content, and EL, and resulted in additional enhancements of enzymatic and non-enzymatic antioxidants, NR activity, and NO synthesis. In Cr-treated tomato seedlings, 5-ALA enhanced GSH and PCs, which modulated Cr sequestration to make it nontoxic. 5-ALA-induced Cr tolerance was further enhanced by sodium nitroprusside (SNP), a NO donor. When sodium tungstate (ST), a NR inhibitor, was supplied together with 5-ALA to Cr-treated plants, it eliminated the beneficial effects of 5-ALA by decreasing NR activity and NO synthesis, while the addition of SNP inverted the adverse effects of ST. We conclude that the mechanism by which 5-ALA induced Cr tolerance in tomato seedlings is mediated by NR-generated NO. Thus, NR and NO are twin players, reducing Cr toxicity in tomato plants via antioxidant signalling cascades.

Skeletal Muscle Nitrate as a Regulator of Systemic Nitric Oxide Homeostasis.

Nonenzymatic nitric oxide (NO) generation via the reduction of nitrate and nitrite ions, along with remarkably high levels of nitrate ions in skeletal muscle, have been described recently. Skeletal muscle nitrate storage may be critical for maintenance of NO homeostasis in healthy aging, and nitrate supplementation may be useful for the treatment of specific pathophysiologies and for enhancing normal functions.

Does dietary nitrate say NO to cardiovascular ageing? Current evidence and implications for research.

CVD are characterised by a multi-factorial pathogenesis. Key pathogenetic steps in the development of CVD are the occurrence of endothelial dysfunction and formation of atherosclerotic lesions. Reduced nitric oxide (NO) bioavailability is a primary event in the initiation of the atherosclerotic cascade. NO is a free radical with multiple physiological functions including the regulation of vascular resistance, coagulation, immunity and oxidative metabolism. The synthesis of NO proceeds via two distinct pathways identified as enzymatic and non-enzymatic. The former involves the conversion of arginine into NO by the NO synthases, whilst the latter comprises a two-step reducing process converting inorganic nitrate into nitrite and subsequently NO.Inorganic is present in water and food, particularly beetroot and green leafy vegetables. Several investigations have therefore used the non-enzymatic NO pathway as a target for nutritional supplementation ( salts) or dietary interventions (high- foods) to increase NO bioavailability and impact on cardiovascular outcomes. Some studies have reported positive effects of dietary on systolic blood pressure and endothelial function in patients with hypertension and chronic heart failure. Nevertheless, results have been inconsistent and the size of the effect appears to be declining in older individuals. Additionally, there is a paucity of studies for disorders such as diabetes, CHD and chronic kidney failure. Thus, whilst dietary supplementation could represent an effective and viable strategy for the primary and secondary prevention of age-related cardiovascular and metabolic diseases, more large-scale, robust studies are awaited to confirm or refute this notion.

Suppression and inhibition of Acetylcholinesterase (AChE) Gene Expression and Adenosine Deaminase (ADA) respectively in Cadmium Treated Rats by curcumin administration

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes have been reported to exert anticholinesterase and cognitive enhancing potentials with limited information on their scientific basis and how they regulate gene expression.This study sought to evaluate the in vitro effect of curcumin on cerebral cortex acetylcholinesterase (AChE) activity, their mRNA gene expression level and adenosine deaminase (ADA) in cadmium (Cd)‐treated rats. Animals were divided into six groups (n=6): group 1 [(control) received saline/vehicle], group 2 [saline plus curcumin (25 mg/kg)], group 3 [saline plus curcumin (50 mg/kg)], group 4 [Cd (2.5 mg/kg) plus vehicle], group 5 [Cd (2.5 mg/kg) plus curcumin (25 mg/kg)] and group 6 [Cd (2.5 mg/kg) plus curcumin (50 mg/kg)] by oral gavage for 7 days. Acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities were determined and AChE expression was analyzed out using real time polymerase chain reaction (RT‐qPCR).The results of this study revealed that ADA activity was increased in Cd‐poisoned rats, and curcumin co‐treatment reversed this effect comparable to the control. Also, it was observed that acute administration of Cd increased AChE activity and caused a significant (P<0.05) increase in AChE mRNA levels in whole cerebral cortex when compared to control group. However, co‐treatment with curcumin inhibited AChE activity and alters AChE mRNA levels when compared to Cd‐treated rats. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfhydryl (−SH) group and nitric oxide (NO). However, the co‐treatment with curcumin at 25 and 50 mg/kg, respectively increased the non‐enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level.In conclusion, inhibition of AChE and ADA activities, suppression of AChE mRNA gene expression level in Cd exposed rats, as well as increased antioxidant status by curcumin in Cd‐induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties, thus providing some biochemical and molecular evidence on the therapeutic effect of this turmeric‐derived compound in treating neurological disorders including Alzheimer's disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non-enzymatic nitric oxide release from biodegradable S-nitrosothiol bound polymer: synthesis, characterization, and antibacterial effect

Nitric oxide (NO) delivery is an effective method for the development of NO-mediated therapy under the physiological conditions for biological applications. S-nitrosothiol is the most widely used NO donor for therapeutics which is capable of spontaneously releasing NO under physiological conditions and acts as a potential stable source of NO and as an S-nitrosating agent. In this present work, the preparation of a biodegradable and biocompatible polymer poly(acrylic acid) (PAA–CANO) cross linked with the S-nitrosothiol,cysteamine (CANO) is reported. Thus, prepared polymer showed a controlled maximum release of NO up to 6 days and it provided a potential defense against pathogenic bacteria. The prepared PAA–CANO was characterized by various techniques such as Fourier transform infrared spectroscopy, ultraviolet–visible spectrophotometry, nuclear magnetic resonance spectroscopy, and thermogravimetric analysis. In addition, loading of PAA–CANO into spongy scaffolds consisting of chitosan and alginate in 1:2 ratio resulting in a new antibacterial active compound suitable for providing infection-free environment on surgical covers, devices, and apparel is also reported. This polymeric scaffold showed a controlled NO release of 2.43 × 10−10 M/s/cm2. The morphology of the scaffolds is a micro-fibrous structure with a porosity of 57% and it was characterized by scanning electron microscopy. Covalent tethering of nitric-oxide-releasing compound S-nitrosocysteamine to poly(acrylic acid) improves its stability and leads to controlled release of nitric oxide useful for the antibacterial activity.

Curcumin improves episodic memory in cadmium induced memory impairment through inhibition of acetylcholinesterase and adenosine deaminase activities in a rat model.

Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes has been reported to exert cognitive enhancing potential with limited scientific basis. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in cadmium (Cd)-induced memory impairment in rats. Animals were divided into six groups (n=6): saline/vehicle, saline/curcumin 12.5mg/kg, saline/curcumin 25mg/kg, Cd/vehicle, Cd/curcumin 12.5mg/kg, and Cd/curcumin 25mg/kg. Rats received Cd (2.5mg/kg) and curcumin (12.5 and 25mg/kg, respectively) by gavage for 7days. The results of this study revealed that cerebral cortex AChE and ADA activities were increased in Cd-poisoned rats, and curcumin co-treatment reversed these activities to the control levels. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfuhydryl (-SH) group and nitric oxide (NO), a potent neurotransmitter and neuromodulatory agent. However, the co-treatment with curcumin at 12.5 and 25mg/kg, respectively increased the non-enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level. Therefore, inhibition of AChE and ADA activities as well as increased antioxidant status by curcumin in Cd-induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties.

Curcumin inhibits adenosine deaminase and arginase activities in cadmium-induced renal toxicity in rat kidney

In this study, the effect of enzymes involved in degradation of renal adenosine and l-arginine was investigated in rats exposed to cadmium (Cd) and treated with curcumin, the principal active phytochemical in turmeric rhizome. Animals were divided into six groups (n = 6): saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. The results of this study revealed that the activities of renal adenosine deaminase and arginase were significantly increased in Cd-treated rats when compared with the control (p < 0.05). However, co-treatment with curcumin inhibits the activities of these enzymes compared with Cd-treated rats. Furthermore, Cd intoxication increased the levels of some renal biomarkers (serum urea, creatinine, and electrolytes) and malondialdehyde level with a concomitant decrease in functional sulfhydryl group and nitric oxide (NO). However, co-treatment with curcumin at 12.5 mg/kg and 25 mg/kg, respectively, increases the nonenzymatic antioxidant status and NO in the kidney, with a concomitant decrease in the levels of malondialdehyde and renal biomarkers. Therefore, our results reinforce the importance of adenosine deaminase and arginase activities in Cd poisoning conditions and suggest some possible mechanisms of action by which curcumin prevent Cd-induced renal toxicity in rats.

Redox-mediated mechanisms and biological responses of copper-catalyzed reduction of the nitrite ion in vitro

During ischemia nitrite may be converted into nitric oxide (NO) by reaction with heme-carrying proteins or thiol-containing enzymes. NO acts as a regulator of vasodilation and protector against oxidative stress-induced tissue injuries. As a result of ischemia-induced oxidative stress, hypoxia and/or acidosis bivalent copper ions (Cu2+) can dissociate from their physiological carrier proteins. Reduced by the body’s own antioxidants, the resultant Cu1+ might represent an effective reductant of nitrite. Here we have evaluated in vitro copper-dissociation from copper/BSA (bovine serum albumin) complexes under ischemic conditions. Furthermore, using physiological concentrations, we have characterized the capacity of antioxidants and bivalent copper ions to serve as Cu1+-agitated catalytic sites for nitrite reduction and also the biological responses of this mechanism in vitro. We found that as a consequence of an acidic milieu and/or oxidative stress the copper-binding capacity of serum albumin strongly declined, leading to significant dissociation of copper ions into the ambient solution. At physiologically relevant pH-values Cu2+ ions in combination with physiologically available copper reductants (i.e., ascorbate, glutathione, Fe2+) significantly enhanced nitrite reduction and subsequent non-enzymatic NO generation under hypoxic but also normoxic conditions. Our data demonstrate for the first time that upon ischemic conditions carrier protein-dissociated copper ions combined with appropriate reductants may serve as Cu1+-driven catalytic sites for nitrite reduction, leading to the formation of biologically relevant NO formation. Thus, in addition to the action of heme proteins, copper-catalyzed non-enzymatic NO formation from nitrite might represent a further physiologically relevant vasodilating and NO-dependent protective principle to ischemic stress.

Mechanism and biological relevance of blue-light (420–453 nm)-induced nonenzymatic nitric oxide generation from photolabile nitric oxide derivates in human skin in vitro and in vivo

Human skin contains photolabile nitric oxide (NO) derivates such as nitrite and S-nitrosothiols, which upon UVA radiation decompose under high-output NO formation and exert NO-specific biological responses such as increased local blood flow or reduced blood pressure. To avoid the injurious effects of UVA radiation, we here investigated the mechanism and biological relevance of blue-light (420–453nm)-induced nonenzymatic NO generation from photolabile nitric oxide derivates in human skin in vitro and in vivo. As quantified by chemiluminescence detection (CLD), at physiological pH blue light at 420 or 453nm induced a significant NO formation from S-nitrosoalbumin and also from aqueous nitrite solutions by a to-date not entirely identified Cu1+-dependent mechanism. As detected by electron paramagnetic resonance spectrometry in vitro with human skin specimens, blue light irradiation significantly increased the intradermal levels of free NO. As detected by CLD in vivo in healthy volunteers, irradiation of human skin with blue light induced a significant emanation of NO from the irradiated skin area as well as a significant translocation of NO from the skin surface into the underlying tissue. In parallel, blue light irradiation caused a rapid and significant rise in local cutaneous blood flow as detected noninvasively by using micro-light-guide spectrophotometry. Irradiation of human skin with moderate doses of blue light caused a significant increase in enzyme-independent cutaneous NO formation as well as NO-dependent local biological responses, i.e., increased blood flow. The effects were attributed to blue-light-induced release of NO from cutaneous photolabile NO derivates. Thus, in contrast to UVA, blue-light-induced NO generation might be therapeutically used in the treatment of systemic and local hemodynamic disorders that are based on impaired physiological NO production or bioavailability.

Cardiac Shock Wave Therapy for Chronic Refractory Angina Pectoris. A Prospective Placebo‐Controlled Randomized Trial

Cardiac shock wave therapy (CSWT) delivered to the myocardium increases capillary density and regional myocardial blood flow in animal experiments. In addition, nonenzymatic nitric oxide production and the upregulation of vascular growth factor's mRNA by CSWT have been described. The aim of the study was therefore to test its potential to relieve symptoms in patients with chronic stable angina pectoris. Twenty-one patients (mean age 68.2 ± 8.3 years, 19 males) with chronic refractory angina pectoris and evidence of inducible myocardial ischemia during MIBI-SPECT imaging, were randomized into a treatment (n = 11) and a placebo arm (n = 10). The region of exercise-induced ischemia was treated with echocardiographic guidance during nine sessions over a period of 3 months. One session of CSWT consisted of 200 shots/spot (9--12 spots/session) with an energy intensity of 0.09 mJ/mm(2) . In the control group acoustic simulation was performed without energy application. Medication was kept unchanged during the whole treatment period. In the treatment group, symptoms improved in 9/11 patients, and the ischemic threshold, determined by cardiopulmonary exercise stress testing, increased from 80 ± 28 to 95 ± 28 W (P= 0.036). In the placebo arm, only 2/10 patients reported an improvement and the ischemic threshold remained unchanged (98 ± 23 to 107 ± 23 W; P= 0.141). The items "physical functioning" (P= 0.043), "general health perception" (P= 0.046), and "vitality" (P= 0.035) of the SF-36 questionnaire significantly improved in the treatment arm, whereas in the placebo arm, no significant change was noted. Neither arrhythmias, troponin rise nor complications were observed during treatment. This placebo controlled trial shows a significant improvement in symptoms, quality of life parameters and ischemic threshold during exercise in patients with chronic refractory angina pectoris treated with CSWT. Thus, CSWT represents a new option for the treatment of patients with refractory AP.

Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells

Red blood cells (RBC) play an important role in the balance between generation and scavenging of nitric oxide (NO) and hence its local bioavailability and influence on vasomotor control. Previous studies have reported increased NO levels in RBC suspensions subsequent to exposure to shear forces; the present study was designed to further investigate changes in intracellular NO concentration and possible mechanisms involved for RBC exposed to well-controlled shear forces. Attached human RBC were subjected to shear stresses up to 0.1Pa in a parallel-plate flow channel; fluorescent methods were used to monitor changes in intracellular NO and calcium concentrations. Intracellular NO concentration, estimated by the fluorescence level of 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM), increased sharply within 30s following the application of shear stress between 0.013 and 0.1Pa. This increase was only partially prevented by the absence of l-arginine and by the presence of l-N-acetyl-methyl-arginine (L-NAME), strongly suggesting that this response was in part related to the activation of NO-synthase (NOS) enzyme. The increase in intracellular NO concentration under shear stress was also inhibited by calcium chelation in the suspending medium, indicating the role of calcium entry for NOS activation. Increases of intracellular calcium concentrations under the same shearing conditions were demonstrated by monitoring Fluo-3/AM fluorescence in RBC exposed to shear stress. Serine 1177 phosphorylated NOS protein, the activated form of the enzyme determined by immunohistochemistry, was found to be significantly increased following the exposure of RBC to 0.1Pa shear stress for 1min. These data confirm that RBC possess a NOS enzyme that is actively synthesizing NO and activated by effective shear forces. The data also suggest that there may be additional (e.g., non-enzymatic) NO generating mechanisms in RBC that are also enhanced under shear stress.

Nitric oxide and nitrosative stress tolerance in yeast.

The opportunistic human fungal pathogen Candida albicans encounters diverse environmental stresses when it is in contact with its host. When colonizing and invading human tissues, C. albicans is exposed to ROS (reactive oxygen species) and RNIs (reactive nitrogen intermediates). ROS and RNIs are generated in the first line of host defence by phagocytic cells such as macrophages and neutrophils. In order to escape these host-induced oxidative and nitrosative stresses, C. albicans has developed various detoxification mechanisms. One such mechanism is the detoxification of NO (nitric oxide) to nitrate by the flavohaemoglobin enzyme CaYhb1. Members of the haemoglobin superfamily are highly conserved and are found in archaea, eukaryotes and bacteria. Flavohaemoglobins have a dioxygenase activity [NOD (NO dioxygenase domain)] and contain three domains: a globin domain, an FAD-binding domain and an NAD(P)-binding domain. In the present paper, we examine the nitrosative stress response in three fungal models: the pathogenic yeast C. albicans, the benign budding yeast Saccharomyces cerevisiae and the benign fission yeast Schizosaccharomyces pombe. We compare their enzymatic and non-enzymatic NO and RNI detoxification mechanisms and summarize fungal responses to nitrosative stress.

Photolytically generated nitric oxide inhibits caspase activity and results in AIF-mediated cell death

In human skin tissue nitrite is found at relatively high concentrations and represents the main source for cutaneous non-enzymatic nitric oxide (NO) formation during UVA exposure due to photolytical decomposition. Since NO has been repeatedly shown to act pro- as well as anti-apoptotic we here studied the effects of UVA irradiation on human keratinocytes in the presence of nitrite. We show that UVA-induced nitrite photodecomposition effectively inactivated caspase activity. In parallel, we observed in human skin keratinocytes, UVA-irradiated in the presence of nitrite, a proteolytic processing of apoptosis-inducing factor (AIF) followed by translocation from the mitochondrion into the nucleus. This translocation resulted in a characteristic apoptotic nuclear phenotype, which differs from the known nuclear phenotype of caspase-mediated chromatin condensation and apoptotic body formation. Interestingly both, AIF translocation and AIF-induced nuclear phenotype changes can be inhibited by NO scavengers, demonstrating the distinct role of nitrite-derived NO in the observed processes. This mode of UVA-induced apoptosis is AIF-dependent and NO-mediated and strongly depends on the presence of nitrite, abundantly present in skin tissue. Thus, photolysis of nitrite in the skin appears to represent an important backup mechanism, which ensures removal of UVA-damaged cells even in the absence of caspase activation.

NO detection in biological samples: Differentiation of 14NO and 15NO using infrared laser spectroscopy

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between 14NO and 15NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples. With the cavity leak-out spectroscopy setup (CALOS) a differentiation between 14NO and 15NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of 14NO/N 2 and 15NO/N 2 were used to calibrate the system. 14 NO 2 - and 15 NO 2 - of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation. The correlation received for both methods for the detection of NO in the gas phase was r = 0.999, p < 0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for 14 NO 2 - 0.3 pmol and 15 NO 2 - 0.5 pmol; correlation 14NO: p < 0.0001, r = 0.975, 15NO: p < 0.0001, r = 0.969). The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.

Computational Modeling of Enzymatic Nitric Oxide Production And Transport In The Microvasculature

Nitric oxide (NO) can be released from multiple enzymatic and non-enzymatic sources to regulate O2 delivery. In this study, we formulated computational kinetic models to predict NO production rates from specific NO synthase (NOS) sources. Based on the analysis of biochemical pathways of neuronal NOS (NOS1) and endothelial NOS (NOS3) catalytic activities and the availability of NOS isoforms and substrates, our model predicted that the NO production rate from vascular endothelium expressing NOS3 is 0.005 – 0.1 μM/s and the rate from the nearby nerve tissues and interstitial mast cells expressing NOS1 is 0.4 – 1.2 μM/s under 100 μM O2 condition. We also calculated [NO] distribution around an arteriole with the predicted NO production rates and the topographical information on NOS isoforms. From our model, the major source determining the distribution of NO was NOS1 in the nerve fibers and mast cells close to arterioles, rather than the endothelial NOS3. Our models also predicted that O2 reduction significantly limits NO formation from NOS1 and thus NO exposure of vascular smooth muscle, opening the possibility that non-enzymatic NO becomes an important source under hypoxia. Moreover, the perivascular [NO] predicted by the models under normoxia was an order of magnitude lower than a number of experimental measurements, suggesting the existence of other enzymatic or non-enzymatic sources of NO in the microvasculature.

Twenty‐Four‐Hour Variation of L‐Arginine/Nitric Oxide/Cyclic Guanosine Monophosphate Pathway Demonstrated by the Mouse Visceral Pain Model

The L‐arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L‐arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light‐dark cycle. Animals were injected s.c. with saline, 2 mg/kg L‐arginine (a NO precursor), 75 mg/kg L‐NG‐nitroarginine methyl ester (L‐NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p‐benzoquinone (PBQ)‐induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L‐arginine or L‐NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time‐dependent differences in PBQ‐induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L‐NAME, methylene blue, or sodium nitroprusside, but not saline or L‐arginine. These results suggest that components of L‐arginine/NO/cGMP pathway exhibit biological time‐dependent effects on visceral nociceptive process.

Nitric Oxide Delivered By Intraerythrocytic SNOHb To Vascular Smooth Muscle: A Theoretical Analysis

Nitric oxide (NO) can be released from multiple enzymatic and non-enzymatic sources to induce vasodilation. We have previously constructed computational models based on the biochemical pathway analysis of constitutive NO synthases in the vasculature and found a large discrepancy between our model predictions and the perivascular NO measurements, suggesting the existence of non-enzymatic or other enzymatic NO sources. S-nitrosohemoglobin (SNOHb) has been hypothesized to carry and release NO-bioactivity to exert an endocrine regulation of vascular tone, but the amount of NO delivered to smooth muscle through this mechanism has not been quantified. In this study, we formulated a multicellular computational model to quantify this NO delivery. Our calculation based on the parameters from published data showed that ~10 pM NO delivered by SNOHb can reach vascular smooth muscle. Whether this amount is capable of solely inducing vasodilation is a subject requiring further investigation. Also, the ~10 pM NO does not account for the paradoxical discrepancy that we have encountered, which suggests that other enzymatic or non-enzymatic sources of NO may exist in the vasculature. We also examined various model parameters and our simulations showed that the NO delivery to smooth muscle by intraerythrocytic SNOHb strongly depends on the concentration and half-life of SNOHb and the erythrocytic membrane permeability to NO.

Dietary nitrate increases gastric mucosal blood flow and mucosal defense

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.

Modulation of nitric oxide bioactivity by plant haemoglobins

Nitric oxide (NO) is a highly reactive signalling molecule that has numerous targets in plants. Both enzymatic and non-enzymatic synthesis of NO has been detected in several plant species, and NO functions have been characterized during diverse physiological processes such as plant growth, development, and resistance to biotic and abiotic stresses. This wide variety of effects reflects the basic signalling mechanisms that are utilized by virtually all mammalian and plant cells and suggests the necessity of detoxification mechanisms to control the level and functions of NO. During the last two years an increasing number of reports have implicated non-symbiotic haemoglobins as the key enzymatic system for NO scavenging in plants, indicating that the primordial function of haemoglobins may well be to protect against nitrosative stress and to modulate NO signalling functions. The biological relevance of plant haemoglobins during specific conditions of plant growth and stress, and the existence of further enzymatic and non-enzymatic NO scavenging systems, suggest the existence of precise NO modulation mechanisms in plants, as observed for different NO sources.

Nitric oxide formation in the oropharyngeal tract: possible influence of cigarette smoking

Cigarette smoking reduces the level of nitric oxide (NO) in exhaled air by an unknown mechanism. The view that part of the effect of cigarette smoking on NO production should occur in the oropharyngeal tract is supported by several studies. We have therefore compared smokers and non-smokers regarding non-enzymatic formation of NO from nitrite in the oral cavity since this is a primary candidate target for cigarette smoke. We have also looked at NO synthase-dependent NO formation in the mucosa of the oropharyngeal tract as an alternative target for the inhibitory effect induced by cigarette smoke. Smokers exhaled 67% lower levels of NO than controls ( p < 0.01, n = 15 each group). We could not detect any significant difference in salivary nitrite, nitrate or ascorbate between smokers and non-smokers. Mouthwash with the antibacterial agent chlorhexidine reduced salivary nitrite (−65%) and exhaled NO levels (−10%) similarly in the two groups. Immunohistochemical techniques revealed dense expression of inducible (but not endothelial or neuronal) NO synthase in the squamous epithelium of non-inflamed tonsillar and gingival tissue biopsies. In the same biopsies, significant Ca 2+-independent citrulline-forming activity was detected. We found no difference between smoking and non-smoking subjects regarding NO-synthase expression and in vitro activity. In another group of non-smoking subjects ( n = 10), spraying the oropharyngeal tract with the NO-synthase inhibitor N G-monomethyl- l-arginine (250 mg) significantly reduced exhaled NO levels for at least 30 min (−18%, p < 0.01). Our data suggest that cigarette smoking does not affect non-enzymatic NO formation from nitrite in saliva. However, NO is also formed by inducible NO synthase in the squamous epithelium of the normal oropharyngeal tract. We suggest that cigarette smoking may down-regulate enzymatic NO formation in the oropharyngeal compartment as well as in the bronchial compartment.