Endogenous Nitrate Research Articles

An Assay for Environmental Nitrate That is Based on its Reduction to Nitrite by Nitrite Reductase Deficient Neurospora Mycelia

A simple, sensitive, reliable and inexpensive assay for environmental nitrate in solution, based on its reduction to nitrite in vivo, by nitrite reductase deficient Neurospora mycelia, is described. Water samples are amended to contain ingredients necessary for the growth of Neurospora except nitrogen, which is provided by the endogenous nitrate. Mycelia are incubated with this amended water sample and allowed to convert the endogenous nitrate to nitrite. The nitrite is then measured colourimetrically.

Functional analysis of two bean nitrate reductase promoters in transgenic tobacco

Transcription of nitrate reductase (NR) genes is regulated by nitrate as well as a variety of other environmental and developmental factors. Two NR genes have previously been characterized in bean (Phaseolus vulgaris). Both genes are nitrate‐ and light‐inducible and their mRNA expression level displays a circadian rhythm. The two genes, NR1 and NR2, show differences in developmental and tissue‐specific expression. In order to deliniate elements in the NR1 and NR2 promoters that are involved in these expression patterns, eight 5′deleted versions of the NR1 promoter and one fragment of the NR2 promoter were cloned in front of the GUS reporter gene and transformed into tobacco. Approximately 50% of the regenerated transformants expressed detectable levels of GUS activity. For the eight NR1 promoter deletion constructs, differences in promoter strength were observed indicating both positive and negative elements in the promoter. For both the NR1 and the NR2 promoter‐GUS constructs no significant nitrate regulation could be detected in the transgenic plants. A possible explanation for this is that elements needed for proper nitrate regulation of the NR1 and NR2 genes are located outside the analysed 3 500 bp and 1 400 bp promoter regions, respectively. A diurnal rhythm regulation similar to that seen for the endogenous nitrate reductase in bean plants, could be found for the GUS mRNA expression in several of the transformants containing the NR1 and NR2 promoter‐GUS constructs.

A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway

One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nial*2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1*3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the transformed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1*2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their 15N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of l-arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [ 15N]nitrite and [ 15N]nitrate were used as internal standards. Endogenous nitrite and [ 15N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [ 15N]nitrate they were reduced to nitrite and [ 15N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [ 15N]nitrite ([ 15N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects ( n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.

Modeling of nitrate disappearance and sludge rising in a settling column system

The determination of different rates of endogenous nitrate respiration due to biomass stratification throughout the depth of a sludge blanket is essential to assess the factors affecting the sludge rising in secondary clarifiers. In this study, an extension of the solids flux theory in conjunction with nitrate mass balance was used to model the fate of nitrate in a sludge settling column. The model-predicted nitrate concentrations agree well with those experimentally determined. A new term, sludge stability index, was introduced to reflect the sludge rising time and the corresponding critical nitrate concentration necessary for causing sludge rising. The model predicted well the observed sludge rising times occurring under batch settling column experiments with initial biomass and nitrate concentration ranging from 2000 to 5000 mg l −1 and 10 to 30 mg-N l −1, respectively. Furthermore, simulation results indicated that both the sludge rising time and CNC are significantly affected by k ENR.

Effects of endogenous nitrate content ofSisymbrium officinaleseeds on germination and dormancy

The nitrate content of seeds of the annual Sisymbrium officinale was raised by weekly nitrate fertilizations of the seeding mother plants. Nitrate content and germination of the seeds produced were positively correlated. Seed lots with different endogenous nitrate contents were buried at 10 cm in sandy loam. At regular intervals seeds were exhumed and germination was tested. Both the initial differences in endogenous nitrate content and the differences of germination rapidly diminished during burial because nitrate rapidly leached from the seeds. The sensitivity of exhumed seeds to applied nitrate strongly varied during this 1-year study. Sensitivity to nitrate was low in December and had increased in January-February probably because of low winter temperatures

Increased Urinary Nitrate Excretion in Inflammatory Bowel Disease

Urinary nitrate is known to be a measure of environmental exposure. Only recently an endogenous metabolic pathway resulting in nitrate synthesis was described. We have determined the concentration of nitrate expressed as nitrate/creatinine ratio in the urine of patients in whom a significant environmental nitrate load was presumed to be absent. A significant elevation of nitrates in patients with inflammatory bowel disease compared with patients with non-inflammatory disorders was observed (0.173 +/- 0.155 vs. 0.037 +/- 0.016 mol/mol creatinine, P < 0.01). Increased nitrate excretion in the urine of patients with inflammatory bowel disease may reflect the endogenous nitrate synthesis induced by the immune activation.

Analysis of bradykinin-induced relaxations in the rat isolated anococcygeus muscle

The present study investigates the mechanism(s) of action of relaxations induced by bradykinin and by electrical field stimulation (EFS) in isolated rat anococcygeus muscle, where contractile tone has been elevated with clonidine. Bradykinin, EFS, and the bradykinin B 1 receptor agonist, des-Arg 9-bradykinin, produced quantitatively and qualitatively similar relaxations. Bradykinin B 1 receptor antagonist, [des-Arg 9, Leu 8]-bradykinin (1 μM), attenuated the relaxation responses of bradykinin B 1 receptor agonist and inhibited bradykinin and EFS-induced relaxation responses. Bradykinin B 2 receptor antagonist, [β-(2-thienyl)-Ala 5,8, D-Phe]-bradykinin (1 μM), significantly inhibited the relaxation responses of bradykinin, EFS, and bradykinin B 1 receptor agonist. Methylene blue (30 μM) and N-methylhydroxylamine (1 mM) significantly inhibited the bradykinin- and EFS-induced relaxation responses. The relaxation responses of bradykinin and EFS were not affected by captopril (5 μM), superoxide dismutase (100 U/ml), and catalase (100 U/ml). Nitric oxide synthase inhibitor, L-N G-nitro-arginine (L-NOARG, 30 μM), significantly inhibited the EFS- and bradykinin-induced relaxation responses. L-arginine (100 μM) reversed the inhibitory effect of L-NOARG on the relaxation responses of EFS and bradykinin. In addition, L-arginine potentiated the relaxation responses of EFS and bradykinin. The data of the present study suggests that bradykinin, similar to EFS, generates an endogenous nitrate, probably nitric oxide, which subsequently activates guanylate cyclase and relaxes the rat anococcygeus muscle.

The influence of L-NG-nitro-arginine on sympathetic nerve induced contraction and noradrenaline release in the rat isolated anococcygeus muscle.

1. The possibility of an interaction between the motor sympathetic and inhibitory non-adrenergic, non-cholinergic (NANC) nerves in the rat anococcygeus was investigated using L-NG-nitro-arginine (L-NOARG), an inhibitor of L-arginine: NO synthase. 2. L-NOARG (50 microM) increased contractions induced by field stimulation (20 s trains; 0.5-40 Hz); overall, the frequency-response curve was displaced six-fold to the left. D-NOARG (50 microM) was without effect. 3. The potentiation produced by L-NOARG was reversed by 200 microM L-, but not D-, arginine. 4. L-NOARG had no effect on contractions induced by exogenous noradrenaline (NA) or on field stimulation-induced overflow of tritium from muscles previously loaded with [3H]-NA. 5. It is concluded that the endogenous nitrate NANC transmitter does not influence release of NA from the sympathetic nerves and the potentiation of contractions induced by field stimulation in the presence of L-NOARG most probably results from removal of the opposing relaxing influence of concomitantly released NANC transmitter.

Variability in light-, gibberellin- and nitrate requirement of Arabidopsis thaliana seeds due to harvest time and conditions of dry storage

Freshly harvested seeds of Arabidopsis thaliana harvested in different seasons showed considerable variability in requirement for light (wild type), gibberellins (GAs) (wild type, GA deficient, ga1-2 mutant) and nitrate. Generally, sensitivity to light was high in seed lots harvested between September and March and significantly lower in those harvested between April and June. Sensitivity to GAs varied in a nearly parallel way. These differences in sensitivity can be attributed to conditions during seed development, like temperature and nitrate availability. Endogenous nitrate levels varied enormously amongst seed lots (about a factor 600) and were weakly correlated to the germination capacity at 10°C after 15 min of red light irradiation. In addition, differences in sensitivity to nitrate may be responsible for the enormous differences in light requirement between seed lots. Other unknown factors may contribute as well. At a germination temperature of 10°C seeds were much more sensitive to GAs and light than at 24°C. The degree of dormancy rapidly declined upon dry storage of the seeds. Loss of dormancy was accelerated with increasing storage temperature; the requirements for light and GAs became less strict. Sensitivity to GAs increased and seeds that initially only responded to continuous irradiation with white fluorescent light, responded to a single red light pulse after 15 months of dry storage at 2°C. It is demonstrated that studies on germination and dormancy of A. thaliana are hampered by the large variability of seed material and the relatively high rate of after-ripening.

Endothelium-derived nitric oxide, endothelin, and platelet vessel wall interaction: alterations in hypercholesterolemia and atherosclerosis.

The endothelium modulates vascular tone by releasing NO, which is a potent vasodilator and inhibitor of platelet aggregation. Thus, the endogenous nitrate has an important protective role in preventing vasospasm and thrombus formation. In addition, the endothelium is a source of contracting factors, such as endothelin-1. Due to its strategical anatomic position, the endothelium is a primary target for injurious stimuli and cardiovascular risk factors. Oxidized LDL reduce the endothelial production of NO and enhance that of endothelin-1. The same pattern of endothelial dysfunction occurs in hypercholesterolemia and in part in atherosclerosis. These alterations of endothelial function may contribute to vasospasm and thrombus formation, which are common events in patients with atherosclerosis.

Endogenous and exogenous nitrates

The endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) or a closely related nitrosothiol derivative. It is formed from the amino acid, L-arginine. NO is rapidly inactivated locally and is instantly destroyed by haemoglobin when released into the blood stream. EDRF-NO as well as NO generated from vasodilator nitrates act by activation of soluble guanylate cyclase, elevating cellular cyclic GMP levels, causing vasodilatation and inhibition of platelet aggregation. Endothelium-dependent vasodilatation is attenuated in hypertension, atherosclerosis and diabetes. This is due to either loss of endothelium or deficient formation of EDRF-NO. In these conditions, therapy with exogenous nitrates may substitute for a failing endogenous mechanism.

Effect of light and its interaction with nitrate and ammonium ions in seed germination of Caesulia axillaris

Seeds of Caesulia axillaris Roxb. displayed an absolute light requirement for germination throughout the period of dry storage at 28°C. The seeds were found to show a gradual increase in percent germination with storage time ‐ reaching a maximum value between 8‐14 months and then a sharp decline. Percent water uptake and photosensitivity were at maximum after a 5‐day imbibition period in the dark in both seedlots studied. Seedlot I, which was only marginally responsive to far‐red light, showed a nearly complete red‐far‐red reversal effect in contrast to seedlot II. The latter also displayed a considerable promotion of germination in far‐red light. Interestingly, a noticeable degree of heterogeneity, besides the one observed in both seedlots with reference to red light, was found to exist in seedlot II for far‐red light. Exogenous application of nitrate and ammonium, at the levels occurring in soil during seed germination/seedling emergence phase of the plant in nature, promoted a considerable proportion of high Ø‐requiring seeds to germinate under irradiation conditions establishing low Ø‐value. The probable ecological implication of this reponse has been discussed. Little correlation was found between the requirement for an exogenous supply of nitrate and the endogenous nitrate level in the seeds in their response to far‐red light.

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans.

Interactions of uptake of malate and nitrate into isolated vacuoles from lettuce leaves

Vacuoles were isolated from leaves of two lettuce (Lactuca sativa L.) genotypes that differed significantly in their nitrate accumulation. The rate of nitrate uptake into the vacuoles did not differ between the genotypes, but the malate-uptake rate did. Fitting a Michaelis-Menten equation, with or without the addition of a linear term, showed that the rates of malate and nitrate uptake can be best described by saturation kinetics for both genotypes. Malate transport across the tonoplast showed a Km value of approx. 40 mM, while the Km value for nitrate uptake was approx. 5 mM. Both malate and nitrate uptake were greatly stimulated by ATP, but not by pyrophosphate. Valinomycin considerably blocked both malate and nitrate uptake while nigericin only slightly affected the rate of malate uptake and had no effect on the nitrate-uptake rate. This indicates that both nitrate and malate transport are driven by the membrane potential, while the pH gradient may play a minor role in malate transport only. The presence of nitrate in the incubation medium inhibited malate uptake (2 mM nitrate caused an inhibition of approx. 50%). In contrast to this, the presence of malate in the incubation medium did not inhibit nitrate uptake. Endogenous nitrate did not affect malate uptake. Thus, we did not find genotypic differences in the uptake pattern which could explain the variation in nitrate accumulation. The possible reason for differences in nitrate accumulation in vivo is discussed.

Differentiation by hydroquinone of relaxations induced by exogenous and endogenous nitrates in non-vascular smooth muscle: role of superoxide anions.

1. The influence of hydroquinone on relaxations induced by nitric oxide (NO), nitrovasodilator drugs, and non-adrenergic, non-cholinergic (NANC) field stimulation has been investigated in three tissues in which endogenous nitrates have been implicated in the NANC response; the mechanism of action of hydroquinone was also studied. 2. In mouse anococcygeus, hydroquinone (10-100 microM) produced a concentration-dependent inhibition of relaxations induced by 15 microM NO. Hydroquinone, 100 microM, which reduced responses to NO by 85%, had no effect on relaxations induced by NANC field stimulation (10 Hz; 20s trains), hydroxylamine (10 microM), sodium nitroprusside (1 microM) or sodium azide (20 microM). 3. In guinea-pig trachea, 100 microM hydroquinone reduced relaxations to 150 microM NO by 75%, but had no effect on those to NANC stimulation (10 Hz; 30 s trains) or sodium azide (5 microM). 4. In rat gastric fundus, 100 microM hydroquinone reduced relaxations to 1 microM NO by 85%, but had no effect on those to NANC stimulation (0.5 Hz; 15 s trains) or sodium azide (2 microM). 5. Superoxide dismutase (SOD; 50 u ml-1) had no effect on relaxations of the mouse anococcygeus in response to 15 microM NO or 10 Hz NANC stimulation. Further, the inhibition of responses to NO by hydroquinone was unaffected in the presence of SOD. 6. Hydroquinone (10-100 microM) failed to generate superoxide anions, as detected by a chemiluminescent assay. However, 100 microM hydroquinone, like SOD (50 u ml-1), produced almost complete inhibition of superoxide anion chemiluminescence induced by xanthine (500 microM): xanthine oxidase (0.07 u ml-1). 7. It is concluded that, in our system, hydroquinone inhibits NO by acting as a free radical scavenger rather than by generating superoxide anions. The ability of hydroquinone to block relaxations to NO, but not NANC stimulation, may suggest that the endogenous nitrate substance released by these NANC nerves may not be free NO, but may be an NO-containing, or NO-generating, molecule.

The influence of L-N G-nitro-arginine on field stimulation induced contractions and acetylcholine release in guinea pig isolated tracheal smooth muscle

The interaction between parasympathetic and inhibitory non-adrenergic, non-cholinergic nerves in tracheal smooth muscle was investigated by determining the effects of the NO-synthase inhibitor L-N G-nitro-arginine (L-NOARG) on contractions and the associated acetylcholine release elicited by field stimulation of the muscle. At frequencies above 2Hz contractile responses to field stimulation were potentiated by L-NOARG (50μM). α-chymotrypsin pre-treatment potentiated contractile responses at all frequencies, but the effects of L-NOARG were unaltered. The effect of L-NOARG on responses to 5Hz electrical stimulation was not mimicked by D-NOARG, was reversed by L-, but not D-arginine and was unaffected by epithelium removal. L-NOARG did not affect responses to exogenous acetylcholine nor the overflow of 3H from tissues previously loaded with [ 3H]-choline. It is therefore concluded that field stimulation of tracheal smooth muscle induces the release of an endogenous nitrate, which, by an inhibitory action on smooth muscle, functionally antagonises the concomitantly released parasympathetic neurotransmitter.

Endothelium, lipoproteins and atherosclerotic vascular disease

The endothelium modulates vascular tone by releasing nitric oxide, which is a potent vasodilator and inhibitor of platelet aggregation. Together with prostacyclin, the endogenous nitrate nitric oxide has an important protective role in preventing vasospasm and thrombus formation. In addition, the endothelium is a source of contracting factors such as endothelin-1, thromboxane A2 and endoperoxides. Due to its strategic anatomical position, the endothelium is a primary target for injurious stimuli and cardiovascular risk factors. Low density lipoproteins reduce endothelium-dependent relaxation and enhance endothelium- dependent contraction. The same pattern of endothelial dysfunction occurs in hypercholesterolaemia and atherosclerosis. These alterations of endothelial function may contribute to vasospasm, ischaemia and thrombus formation, which are common events in patients with atherosclerotic vascular disease.

Cyclic Nucleotide Content of the Rat Anococcygeus During Relaxations Induced by Drugs or by Non-adrenergic, Non-cholinergic Field Stimulation

Low concentrations of sodium nitroprusside (0.2 and 1 microM) relaxed carbachol-induced tone of the rat anococcygeus but did not affect the content of either cGMP or cAMP; higher concentrations (10,100 and 1000 microM) produced greater relaxation (greater than 60%) and a rise in cGMP but not cAMP. In the presence of the cGMP-phosphodiesterase inhibitor M&B 22948 (10 microM), 1 microM sodium nitroprusside produced greater relaxation and a selective increase in cGMP. Forskolin (0.5-250 microM) caused relaxation and a selective increase in cAMP; the concentration-response relationships of the two effects were similar. Non-adrenergic, non-cholinergic (NANC) field stimulation (10 Hz; 20 s trains) reduced tone by 52% but had no effect on cyclic nucleotide content; in the presence of 10 microM M&B 22948 or 1 microM sodium nitroprusside, NANC stimulation produced a greater degree of relaxation and increased cGMP but not cAMP content. The results show that NANC stimulation acts like sodium nitroprusside, causing a selective increase in cGMP, and this supports the proposal that NANC transmission in the rat anococcygeus involves an endogenous nitrate; the possibility that multiple pools of cGMP exist in the anococcygeus is discussed.

Alternating aerobic and anoxic digestion of waste activated sludge

AbstractNitrate generated in situ due to nitrification in an aerobic digester was utilized through endogenous nitrate respiration (ENR). This was done by alternating aerobic/anoxic (A/A) cycles. The destruction of volatile suspended solids (VSS) in the A/A system was the same as compared to a controlled‐aerobic digester. Approximately 33% of total nitrogen was removed in the A/A system of which 27% was due to ENR during anoxic cycles.During each anoxic cycle, nitrate reduction was found to be a zero order reaction with respect to nitrate. The specific ENR rates decreased significantly with digestion time due to changes in VSS. Both pH and alkalinity increased during anoxic cycles resulting in enhanced VSS decay and complete nitrification under subsequent aerobic conditions.The energy savings associated with the A/A system and the use of the specific ENR rates are discussed.