Evidence that lithium induces a glutamatergic: Nitric oxide-mediated response in rat brain

Studies have indicated the involvement of a glutamatergic mechanism in lithium (Li⁺) action. Glutamatergic agonists, such as kainic acid, are known to promote the synthesis of nitric oxide (NO) and to increase cGMP, while Li⁺ has displayed a similar, yet unexplained, ability to increase cGMP. NO synthesis is regarded as the principal prodromal event leading to the activation of the guanyl cyclase-cGMP transduction mechanism. In the present study, the involvement of the NO:cGMP pathway in the action of Li⁺ was examined, while the possibility of a glutamatergic mechanism in this response was also investigated. Parameters examined included cortical accumulation of cGMP and the stable oxidative metabolites of NO, viz. NO ₂ ^– and NO ₃ ^–, collectively expressed as NO ₂ ^–. A significant positive correlation was observed in the in vivo cGMP and NO ₂ ^– data throughout all the groups. Chronic treatment of rats with LiCl (0.3% m/m) engendered a significant increase in cGMP levels which was inhibited by the NO-synthase (NOS) inhibitor, N-nitro-l-arginine methyl ester (L-NAME). Acute administration of kainic acid resulted in an increased accumulation of NO ₂ ^–, also prevented by concomitant L-NAME administration. In addition, a synergistic stimulatory response on cortical NO ₂ ^– was observed in the combination of LiCl and kainic acid. Collectively, these data implicate an involvement of a glutamatergic-mediated NO:cGMP transduction mechanism in the action of Li⁺.     

Comparison of the growth rates of dinitrogen fixing subterranean clover swards with those assimilating nitrate ions

Summary Subterranean clover plants were grown as swards (about 2000 plants/m²) under controlled conditions with N provided either by N₂-fixation (NO ₃ ^– withheld) or by assimilation of NO ₃ ^– (NO ₃ ^– supplied). Crop growth rates were measured by dry matter sampling over periods of up to 70 days at PPFD values of 400–1000 mole quanta/m²/s. When NO ₃ ^– was supplied from sowing the swards grew more rapidly than when the swards were not supplied with NO ₃ ^– and plants had to establish an N₂-fixing apparatus. When inter-plant competition was reduced within the sward, a difference in growth rate in favour of NO ₃ ^– -fed plants continued for at least 50 days. When however, a closed canopy was allowed to form, the NO ₃ ^– -fed swards had more dry weight than the N₂-fed swards at the time of canopy closure but thereafter the two swards grew at similar rates at light flux densities of above about 800 mole quanta/m²/s. At light flux densities of about 400 mole quanta/m²/s N₂-fed swards had a growth rate 70–80% of that of NO ₃ ^– -fed plants. NO ₃ ^– -fed plants had a higher organic N content than did N₂-fed plants under all conditions.     

Spatial variability of NO and NO2 flux rates from soil of spruce and beech forest ecosystems

In order to evaluate differences in the magnitude of NO and NO₂ flux rates between soil areas in direct vicinity to tree stems and areas of increasing distance to tree stems, we followed in 1997 at the Höglwald Forest site with a fully automated measuring system a complete annual cycle of NO and NO₂ fluxes from soils of an untreated spruce stand, a limed spruce strand, and a beech stand using at each stand measuring chambers which were installed onto the soils in such a way that they formed a stem to stem gradient. Flux data obtained since the end of 1993 from measuring chambers placed at the interstem areas of the stands, which had been used for the calculation of the long year annual mean of NO and NO₂ flux rates from soils of the stands, are compared to both (a) those obtained from the interstem chambers in 1997 and (b) those from the stem to stem gradient chambers. Daily mean NO fluxes obtained in 1997 were in a range of 0.3 – 280.1 g NO-N m^–2 h^–1 at the untreated spruce stand, 0.5 – 273.2 g NO-N m^–2 h^–1 at the limed spruce stand and 0.5 - 368.8 g NO-N m^–2 h^–1 at the beech stand, respectively. Highest NO emission rates were observed during summer, lowest during winter. Daily mean NO₂ fluxes were in a range of –83.1 – 7.6 g NO₂-N m^–2 h^–1 at the untreated spruce stand, -85.1 – 2.1 g NO₂-N m^–2 h^–1 at the limed spruce stand and –77.9 to –2.0 g NO₂-N m^–2 h^–1 at the beech site, respectively. As had been observed for the years 1994–1996, also in 1997 NO emission rates were highest at the untreated spruce stand and lowest at the beech stand and liming of a spruce stand resulted in a significant decrease in NO emission rates. For NO₂ no marked differences in the magnitude of flux rates were found between the three different stands. Results obtained from the stem to stem gradient experiments revealed that at all stands studied NO emission rates were significantly higher (between 1.6- and 2.6-fold) from soil areas close to the tree stems and decreased – except at the beech stand - with increasing distance from the stems, while for NO₂ deposition no marked differences were found. Including the contribution of soil areas in direct vicinity to the beech stems in the estimation of the annual mean NO source strength revealed that the source strength has been underestimated by 40% in the past.     

The analogous reaction of diSchiff base coordinated copper and Cu2Zn2 superoxide dismutase with nitric oxide

In addition to the well known catalytically accelerated O₂ dismutation, Cu₂Zn₂ Superoxide dismutase (SOD) reversibly reduces NO to NO^– with the consequence of a prolonged half-life of NO. This alternative reactivity was examined in the presence of the intact CuZn enzyme and a diSchiff base copper complex prepared from putrescine and pyridine-2-aldehyde (Cu-PuPy) which is known as a convenient active center analog of the former copper protein. The reaction of this SOD mimick with NO and NO^– was monitored by electronic absorption and electron paramagnetic resonance (EPR) spectroscopy via the formation of nitrosylmyoglobin. Cu-PuPy reacted up to three times faster with NO compared with Cu₂Zn₂ SOD and 15 times faster in comparison with CuSO₄ and copper EDTA. The oxidation rate of NO^– by Cu-PuPy was up to 300% higher compared with the reactivities of CuSO₄ and Cu EDTA. Cu₂Zn₂SOD reacted with NO^– to a neglible extent only. Catalytic characteristics could be observed in the course of the oxidation of NO^– in concentrations between 1 and 20 M copper. Disturbances of the EPR properties suggested a modification of the chemical environment at the copper sites in both the copper complex and the enzyme. As a consequence, no further reactions of the nitrogen monoxides with the respective active centers were seen. In conclusion, Cu-PuPy appears to be an efficient moderator of the biochemical reactivity of nitrogen monoxides attributable to the observed increased half-life of NO.     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Comparison of N losses (NO − 3, N 2O, NO) from surface applied, injected or amended (DCD) pig slurry of an irrigated soil in a Mediterranean climate

Nitrous oxide (N ₂O), nitric oxide (NO), denitrification losses and NO^–₃ leaching from an irrigated sward were quantified under Mediterranean conditions. The effect of injected pig slurry (IPS) with and without the nitrification inhibitor dicyandiamide (DCD) was evaluated and also compared with that of a surface pig slurry application (SPS) and a control treatment (Control) without fertiliser. After application, fluxes of NO and N ₂O peaked from SPS (3.06 mg NO-N m ^–2 d ^–1 and 108 mg N ₂O-N m ^–2 d ^–1) and IPS (3.50 mg NO-N m ^–2 d ^–1 and 105 mg N ₂O-N m ^–2 d ^–1). However, when irrigation was applied, N ₂O and NO emissions declined. The total N ₂O and denitrification losses were slightly large from IPS than from SPS, although the differences were not significant (P < 0.05). Emission of NO was not affected by the method of pig slurry application. DCD inhibited nitrification during the first 20–30 days and reduced N ₂O and NO emissions from pig slurry by at least 46% and 37%, respectively. Considering the 215 days following pig slurry application, the emission factor of N ₂O based on N fertiliser was 1.60% (SPS), 2.95% (IPS), and 0.50% (IPS + DCD). The emission factor for NO was 0.14% (SPS), 0.12% (IPS), and 0.02% (IPS + DCD). Environmental conditions of the crop favoured the denitrification process as the most important source of N ₂O during the experimental period. The differences in the denitrification rate between treatments could be explained by the pattern of water soluble carbon (WSC), that was the highest value in injected pig slurry (with and without DCD). Due to low drainage (5% of water applied), leaching losses of NO₃^– were lower than those of denitrification from the upper soil layer (0–10 cm) in all treatments and especially with IPS + DCD, where the nitrification inhibitor was very efficient in reducing leaching losses.     

Mechanism of nitrite oxidation and oxidoreductase systems inNitrobacter agilis

Chemiosmotic coupling mechanisms operate in the electron transfer reactions from: nitrite to O₂, NO₂ ^– to NAD⁺, ascorbate to O₂, NADH to O₂, and NADH to NO₃ ^–. The enzyme systems catalyzing these reactions are named NO₂ ^–:O₂ oxidoreductase, ATP-dependent NO₂ ^–:NAD⁺ oxidoreductase, ascorbate:O₂ oxidoreductase, NADH:O₂ oxidoreductase, and NADH:NO₃ ^– oxidoreductase, respectively. All of the oxidoreduction reactions are exergonic with the exception of the ATP-dependent NO₂ ^–:NAD⁺ oxidoreductase system, which involves reversed electron flow against the thermodynamic gradients. The mechanism for nitrite oxidation was found to be quite different from that of ascorbate oxidation; both systems were insensitive, however, to rotenone, amytal, antimycin A, and 2-n-heptyl 4-hydroxyquinolineN-oxide. These compounds, on the other hand, severely inhibited the electron transfer reactions catalyzed by NADH:O₂ oxidoreductase, NADH:NO₃ ^– oxidoreductase, and the ATP-dependent NO₂ ^–:NAD⁺ oxidoreductase, indicating a common pathway of electron transport in these oxidoreductase systems. Cyanide inhibited all systems except the NADH:NO₃ ^– oxidoredctase. The uncoupler carbonyl cyanide-m-chlorophenyl hydrazone strongly inhibited NO₂ ^–:O₂ oxidoreductase and ATP-dependent NO₂ ^–:NAD⁺ oxidoreductase, which indicates the involvement of energy-linked reactions in both systems; the uncoupler caused a marked stimulation of the NADH:O₂ oxidoreductase and NADH:NO₃ ^– oxidoreductase without affecting the ascorbate:O₂ oxidoreductase activities.     

Denitrification kinetics of simulated fish processing wastewater at different ratios of nitrate to biomass

Denitrification was studied in anoxic batch cultures of a simulated fish processing wastewater at 37 r C and pH 7.5, using a denitrifying enrichment culture from fishery wastewater. Different initial nitrate to biomass ratios (So/Xo) were used: nitrate and biomass varied from 7.5 to 94.7 mg NO₃-N l^–1, and from 20 to 4300 mg volatile suspended solids l^–1, respectively. The specific maximum denitrification rate (r _m) and the cell yield (Y _{X / S}) depended on the So/Xo ratio under anoxic conditions: r _m increased from 1.2 to 1584 mg NO₃-N g^–1 VSS h^–1 and Y _{X / S} decreased from 42 to 0.03 mg VSS mg^–1 NO₃-N when So/Xo varied from 5.5 10^{– 3} to 9.3 mg NO₃-N/mg VSS. Nomenclature C_{NO3 – N} nitrate concentration, mg NO₃-N l^–1 K _S saturation constant, mg NO₃-N l^–1 r _m specific maximum denitrification rate, mg NO₃-N g^–1 VSS h^–1 So initial substrate concentration, mg l^–1 t time, h TOC total organic carbon VSS volatile suspended solids x biomass concentration, g VSS l^–1 Xo initial biomass concentration, g VSS l^–1 Y _X/S substrate to biomass cell yield, mg VSS/mg N Greek symbols: _m maximum specific growth rate of the anoxic microbial population, 1 h^–1     

Nitrogen relations of a low nitrate uptake inbred line of white clover (Trifolium repens L.)

The nitrogen relations of an inbred line of white clover (Trifolium repens L.) thought to exhibit an abnormally low capacity for NO₃ ^– uptake (line LNU) were compared with a line regarded as normal with respect to NO₃ ^– uptake (line NNU). Growth, nodulation, N₂ fixation and NO₃ ^– uptake were measured over 7 weeks in flowing solution culture (Experiment 1) by plants dependent for N acquisition on either (i) NO₃ ^– uptake, (ii) NO₃ ^– uptake +N₂ fixation, or (iii) N₂ fixation only. Effects of plant N status on the short-term uptake and translocation of ¹⁵NH₄ ⁺ and ¹⁵NO₃ ^– were also investigated (Experiment 2). Nitrate uptake per plant by –fix/+NO₃ ^– line LNU was 50% of uptake by line NNU over 35 days, and there were significant differences in specific uptake rates of NO₃ ^– between the lines over the first 24 days. The `low NO<sub>3</sub> <sup>–</sup> uptake' phenotype was indistinct under +fix/+NO<sub>3</sub> <sup>–</sup> treatment. Nitrate lowered specific rates of nitrogen fixation by line NNU but had no effect on line LNU. Only low N status line LNU plants had lower short-term rates of NH<sub>4</sub> <sup>+</sup> and NO<sub>3</sub> <sup>–</sup> uptake than line NNU. It is concluded that the `low NO₃ ^– uptake' phenotype of line LNU is inconsistently expressed. Circumstantial evidence points to increased NO₃ ^– efflux and decreased xylem translocation of NO₃ ^– as possible explanations for the lower NO₃ ^– uptake by line LNU.     

Modulation of the Photosynthetic Source:Sink Relationship in Cultures of the Cyanobacterium Nostoc Rivulare

Nostoc rivulare was grown in batch cultures under controlled CO₂ and NO₃ ^– concentrations to modulate the photosynthetic source:sink relationship. Increasing CO₂ supply accelerated the accumulation of chlorophyll (Chl) a, i.e., supplemental CO₂ combined with double concentrations of NO₃ ^– more than doubled the amounts of Chl a relative to those of the original medium. Photosynthetic oxygen evolution and respiratory oxygen uptake were both enhanced by elevated CO₂ and NO₃ ^–. Contents of soluble sugars and starch (total non-structural saccharides) as well as insoluble saccharides (structural fraction) were affected by altering CO₂-NO₃ ^– combinations. Uptake as well as reduction of either NO₃ ^– or NO₂ ^– was inhibited by CO₂ deprivation. Expanding the sink size via increasing NO₃ ^– supply enhanced photosynthesis and thus the sink (NO₃ ^–) acted as a feed forward stimulator of the source (photosynthesis). The regulatory role of nitrate on photosynthesis was most influential in CO₂-deprived cultures since it could enhance photosynthesis to higher levels than CO₂-supplemented, nitrate-free cultures.     

Bacterium-Based NO2− Biosensor for Environmental Applications

A sensitive NO₂⁻ biosensor that is based on bacterial reduction of NO₂⁻ to N₂O and subsequent detection of the N₂O by a built-in electrochemical N₂O sensor was developed. Four different denitrifying organisms lacking NO₃⁻ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO₂⁻ tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO₃⁻ and N₂O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N₂O reductase. The macroscale biosensors constructed exhibited a linear NO₂⁻ response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO₂⁻, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO₂⁻, and interference was observed only with NH₂OH, NO, N₂O, and H₂S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO₂⁻ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO₂⁻ biosensor were constructed and used to measure NO₂⁻ profiles in marine sediment.     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

NO 2 − Efflux and its regulation in cyanobacterium Nostoc MAC

NO ₂ ^– efflux and its regulation have been studied in the cyanobacterium Nostoc MAC. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), sodium azide, p-chloromercuribenzoate (PCMB), and dicyclohexylcarbodiimide (DCCD), a specific inhibitor of bacterial ATPase, inhibited the NO ₂ ^– efflux activity singificantly. No NO ₂ ^– efflux activity was observed under dark-aerobic as well as under dark-anaerobic conditions; however, the addition of ATP resulted in NO ₂ ^– efflux even under dark-aerobic condition. Maximum NO ₂ ^– efflux activity was observed when NO ₃ ^– served as the sole nitrogen source. However, NH ₄ ⁺ ions inhibited the NO ₂ ^– efflux activity when both NO ₃ ^– and NH ₄ ⁺ wer simulatneously available to the cells. The NO ₂ ^– efflux was freed from NH ₄ ⁺ repression by l-methionine-dl-sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Chloramphenicol, a protein synthesis inhibitor, inhibited the derepression of NO ₂ ^– efflux system when NH ₄ ⁺ -incubated cells were transferred to NO ₃ ^– medium. Tungstate-treated cells lacking functional NO ₃ ^– reductase but having NO ₃ ^– uptake activity also lacked NO ₂ ^– efflux activity. These results suggest that (i) NO ₂ ^– efflux in Nostoc MAC is NO ₃ ^– dependent and an energy-dependent process that can be regulated at the levels of NO ₃ ^– uptake and NO ₃ ^– reductase; (ii) NO ₂ ^– efflux system is NH ₄ ⁺ repressible; however, the product of NH ₄ ⁺ assimilation via GS is being required for repression to occur; (iii) de novo protein synthesis is required for derepression of the NO ₂ ^– efflux system; and (iv) the catalytic activity of NO ₂ ^– reductase also seems to play an important role in the regulation of NO ₂ ^– efflux system.     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

Gaseous NO2 as a regulator for ammonia oxidation of Nitrosomonas eutropha

Cells of Nitrosomonas eutropha strain N904 that were denitrifying under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were unable to utilize ammonium (ammonia) as an energy source. The recovery of ammonia oxidation activity was dependent on the presence of NO₂. Anaerobic ammonia oxidation activity was observed in a helium atmosphere supplemented with 25 ppm NO₂ after 20 h. Ammonia oxidation activity was detected after 2–3 days using an oxic atmosphere with 25 ppm NO₂. In contrast, ammonia consumption started after 8–9 days under oxic conditions without the addition of NO₂; in this case, small amounts of NO and NO₂ were detected and their concentrations increased with increasing ammonia oxidation activities. Hardly any ammonia oxidation was detected when nitrogen oxides were removed by intensive aeration. It would seem, therefore, that NO₂ is the master regulatory signal for ammonia oxidation in Nitrosomonas eutropha. Anaerobic ammonia oxidation activity was inhibited by the addition of NO. This inhibition was partly compensated by either increasing the NO₂ concentration or by using 2,3-dimercapto-1-propane-sulfonic acid as a NO binding substrate. DMPS was inhibitory to nitrification under oxic conditions, while increased amounts of NO or NO₂ led to increased oxidation activities.     

Effects of light quality,CO2 tensions and NO 3 +− concentrations on the inorganic nitrogen metabolism of Chlamydomonas reinhardii

The blue light dependent utilization of nitrate by green algae under common air and high irradiances, besides its assimilatory nature, is associated with the release of NO₂ ^– and NH₄ ⁺ to the culture medium. If the CO₂ content of the sparging air was increased up to 2%, previously excreted NO₂ ^– and NH₄ ⁺ were rapidly assimilated. When under air and high irradiances the cell density in the culture reached values corresponding to 25 g Ch 1.ml^-1, no further growth was observed and the highest values of NO₃ ^– consumption and NO₂ ^– and NH₄ ⁺ release were attained. Besides low CO₂ tensions, increasing NO₃ ^– concentrations in the medium stimulated the release of NO₃ ^– and NH₄ ⁺. Under CO₂-free air the consumption of NO₃ ^– and the release of NO₂ ^– and NH₄ ⁺ on a total N bases were almost stoichiometric and their rates saturated at much lower irradiances than under air. Under CO₂-free air high rates of NO₂ ^– release were only observed under the blue radiations that were effectively absorbed by photosynthetically active pigments, i.e. 460 nm, but not under 404 and 630 nm radiations. However, the simultaneous illumination of the cells with 404 and 630 nm monochromatic light showed a remarkable synergistic effect on NO₂ ^– release.The results are discussed in terms of the close relationship between C and N metabolism, the photosynthetic reducing power required to convert NO _inf3 ^sup± -N into R – NH₂-N and the blue light activation of nitrate reductase.     

Visualization of NO3?/NO2? Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System

Nitrate (NO₃⁻) and nitrite (NO₂⁻) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO₃⁻/NO₂⁻ exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO₃⁻/NO₂⁻ for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO₃⁻/NO₂⁻ in physiology), by employing NO₃⁻/NO₂⁻-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO₃⁻ and NO₂⁻, and its FRET signal is changed in response to NO₃⁻/NO₂⁻ in the micromolar range. Furthermore, both an increase and decrease in cellular NO₃⁻ concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO₃⁻/NO₂⁻ dynamics in various organisms, including plants and animals.     

Nitrate and chloride ions have different permeation pathways in skeletal muscle fibers ofRana pipiens

Summary The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined.Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K₂SO₄ solutions. External solutions were first changed to ones in which either Rb⁺ or Cs⁺ replaced K⁺ and then to solutions containing either NO ₃ ^– or Cl^– to replace SO ₄ ^2– . The hyperpolarizations produced by Cl^– depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO ₃ ^– were independent of external pH over a pH range from 5.5 to 9.0.In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K₂SO₄ plus Na₂SO₄. KCl or KNO₃ were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K⁺ was replaced by TEA⁺. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl^– or NO ₃ ^– depending on the final equilibrating solution. Cl^– currents during voltage pulses were both external pH and time dependent. By contrast, NO ₃ ^– currents were independent of both external pH and time.The voltage dependence of NO ₃ ^– currents could be fit by constant field equations with aP _{NO 3} of 3.7·10^–6 cm/sec. The voltage dependence of the initial or instantaneous Cl^– currents at pH 7.5 and 9.0 could also be fit by constant field equations with P_Cl of 5.8·10^–6 and 7.9·10^–6 cm/sec, respectively. At pH 5.0, no measurable instantaneous Cl^– currents were found.From these results we conclude that NO ₃ ^– does not pass through the pH, time-dependent Cl^– channels but rather passes through a distinct set of channels. Furthermore, Cl^– ions do not appear to pass through the channels which allow NO ₃ ^– through. Consequently, the measured ratio ofP _Cl/P _{NO 3} based on membrane potential changes to ionic changes made on intact skeletal muscle fibers is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.     

Anthropogenic N deposition and the fate of 15NO 3 − in a northern hardwood ecosystem

Human activity has substantially increased atmospheric NO ₃ ^– deposition in many regions of the Earth, which could lead to the N saturation of terrestrial ecosystems. Sugar maple (Acer saccharum Marsh.) dominated northern hardwood forests in the Upper Great Lakes region may be particularly sensitive to chronic NO ₃ ^– deposition, because relatively moderate experimental increases (three times ambient) have resulted in substantial N leaching over a relatively short duration (5–7 years). Although microbial immobilization is an initial sink (i.e., within 1–2 days) for anthropogenic NO ₃ ^– in this ecosystem, we have an incomplete understanding of the processes controlling the longer-term (i.e., after 1 year) retention and flow of anthropogenic N. Our objectives were to determine: (i) whether chronic NO ₃ ^– additions have altered the N content of major ecosystem pools, and (ii) the longer-term fate of ¹⁵NO ₃ ^– in plots receiving chronic NO ₃ ^– addition. We addressed these objectives using a field experiment in which three northern hardwood plots receive ambient atmospheric N deposition (ca. 0.9 g N m^–2 year^–1) and three plots which receive ambient plus experimental N deposition (3.0 g NO₃ ^–-N m^–2 year^–1). Chronic NO ₃ ^– deposition significantly increased the N concentration and content (g N/m²) of canopy leaves, which contained 72% more N than the control treatment. However, chronic NO ₃ ^– deposition did not significantly alter the biomass, N concentration or N content of any other ecosystem pool. The largest portion of ¹⁵N recovered after 1 year occurred in overstory leaves and branches (10%). In contrast, we recovered virtually none of the isotope in soil organic matter (SOM), indicating that SOM was not a sink for anthropogenic NO ₃ ^– over a 1 year duration. Our results indicate that anthropogenic NO ₃ ^– initially assimilated by the microbial community is released into soil solution where it is subsequently taken up by overstory trees and allocated to the canopy. Anthropogenic N appears to be incorporated into SOM only after it is returned to the forest floor and soil via leaf litter fall. Short- and long-term isotope tracing studies provided very different results and illustrate the need to understand the physiological processes controlling the flow of anthropogenic N in terrestrial ecosystems and the specific time steps over which they operate.