Nitrite effect on ammonium and nitrite oxidizing processes in a nitrifying sludge

Nitrite accumulation can be undesirable in nitrifying reactors used for the biological elimination of nitrogen from wastewaters because the ammonium oxidation process was seen to be inhibited. There is a need to better understand the effects of nitrite on both ammonium and nitrite oxidizing processes. In this paper, the effect of nitrite on the nitrifying activity of a sludge produced in steady-state nitrification was evaluated in batch cultures. At 25 mg N/l of added nitrite, nitrification was successfully carried out. Addition of higher nitrite concentrations to nitrifying cultures (100 and 200 mg N/l) provoked inhibitory effects on the nitrification respiratory process. Nitrite at 100 and 200 mg N/l induced a significant decrease in the values for nitrate yield (−20% and −34%, respectively) and specific rate of nitrate formation (−26% and −67%, respectively), while the ammonium consumption efficiency kept high and the specific rate of ammonium oxidation did not significantly change. This showed that the nitrite oxidizing process was more sensitive to the presence of nitrite than the ammonium oxidizing process. These results showed that as a consequence of nitrite accumulation in nitrification systems, the activity of the nitrite oxidizing bacteria could be more inhibited than that of the ammonium oxidizing bacteria, provoking a higher accumulation of nitrite in the medium.     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Time-course of changes in plasma nitric oxide following lipopolysaccharide and turpentine injection in rats

The effect of lipopolysaccharide (LPS) and turpentine on nitric oxide (NO) production were investigated in rats. Because of short half-life of NO in biological fluids, the plasma nitrite and nitrate concentrations (two stabile metabolites of NO) were measured based on Griess reaction, which is indirect assay for NO production. Injection of LPS at an intraperitoneal dose of 50 μg/kg caused a 3,5-fold increase in plasma nitrite within 3 h and nitrite levels remained significantly elevated 6, 12, and 24 h after endotoxin treatment with LPS. However, injection of turpentine at an intramuscular dose of 20 μl/rat did not alter plasma nitrite concentration at selected times after turpentine treatment (7, 10, 14, and 24 h postinjection). These results further support the hypothesis that NO is involved in pathogenesis of febrile response due to LPS in rats. Because turpentine did not change concentration of NO in plasma, the role of NO, as mediator/modulator, in development of turpentine fever appears to be controversial and needs further experimental verification.     

Thiols enhance NO formation from nitrate photolysis

Nitrate is generally considered an inert oxidative breakdown product of nitric oxide (NO). Whereas it has been shown that limited amounts of NO are produced during the photolysis of nitrate in aqueous solution, the photochemistry of nitrate in biological matrices such as plasma is unknown. We hypothesized that thiols, which are ubiquitously present in biological systems, may significantly enhance NO-quantum yields from nitrate photolysis. Exposure of fresh human plasma to high-intensity UV-light resulted in NO-formation (19 +/- 3 nmol/l/min) as measured by gas phase chemiluminescence, and this signal was almost completely abolished by the removal of plasma N-oxides (2 +/- 1 nmol/l/min). Reconstitution of NOx-depleted plasma samples with a physiological concentration of nitrate, but not nitrite, restored photolytic NO-generation to values comparable to na?ve plasma. Addition of the thiol-reducing agent, dithiothreitol or the sulfhydryl-bearing amino acid, L-cysteine increased NO-formation above control levels. Thiol-blockade by either N-ethylmaleimide (NEM) or mercuric chloride (HgCl2) reduced basal NO formation from 19 +/- 3 to 7 +/- 2 and 4 +/- 1 nmol/l/min, respectively. Exposure of plasma to UV-light increased NO-adduct concentrations from 18 +/- 5 to 1662 +/- 658 nmol/l. Collectively, our results show that thiols facilitate photolytic conversion of nitrate to NO and NO-adducts such as S-nitrosothiols. This may lead to substantial overestimation of the latter when photolysis-based methodologies are used for their determination. Whether this novel reaction channel also has in vivo relevance remains to be investigated.     

An improved method to measure nitrate/nitrite with an NO-selective electrochemical sensor.

Nitric oxide (NO) produced from NO synthase(s) (NOS) is an important cell signaling molecule in physiology and pathophysiology. It remains challenging, however, to measure NO accurately and reproducibly in many cell types producing relatively low levels of NO from the enzymes such as endothelial NO synthase (eNOS). In the present study, we describe a very sensitive and convenient analytical method that affords measurement of 1 to 2 nM concentration of NO(x) (nitrite plus nitrate) in culture media. In the present study, we used an ultra-sensitive NO-selective electrochemical sensor (AmiNO700) in combination with a highly efficient nitrate conversion method, which coupled the nitrate reductase step with the glucose-6-phosphate dehydrogenase system. An aliquot of conditioned culture media was first treated with nitrate reductase, NADPH, glucose-6-phosphate dehydrogenase and glucose-6-phosphate to convert nitrate to nitrite quantitatively. The nitrite (that is present originally plus the reduced nitrate) was then reduced to equimolar NO in an acidic iodide bath while NO was being detected by the sensor. With this analytical method, we can quantitatively and reliably measure basal and stimulated NO release from cultured endothelial cells. We believe this improved assay should be useful in measuring a wide range of NO levels, especially the low but physiologically relevant levels, in many cell types.     

Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats

Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68 m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1 h later (p<0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1 h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastric mucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p<0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was significantly higher in the BPSDL animals compared to controls, and nitrate administration rescued the WIRS-induced gastric injury in BPSDL rats. In conclusion, this study suggests that stress promotes salivary nitrate secretion and nitrite formation, which may play important roles in gastric protection against stress-induced injury via the nitrate-dependent NO pathway.     

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C₈ column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C₁₈ column (50×4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Role of brain nitric oxide in the thermoregulation of broiler chicks

Nitric oxide (NO) plays a key role in body temperature (Tb) regulation of mammals, acting on the brain to stimulate heat loss. Regarding birds, the putative participation of NO in the maintenance of Tb in thermoneutrality or during heat stress and the site of its action (periphery or brain) is unknown. Thus, we tested if NO participates in the maintenance of chicks' Tb in those conditions. We investigated the effect of intramuscular (im; 25, 50, 100 mg/kg) or intracerebroventricular (icv; 22.5, 45, 90, 180 µg/animal) injections of the non selective NO synthase inhibitor L-NAME on Tb of 5-day-old chicks at thermoneutral zone (TNZ; 31–32 °C) and under heat stress (37 °C for 5–6 h). We also verified plasma and diencephalic nitrite/nitrate levels in non-injected chicks under both conditions. At TNZ, 100 mg/kg (im) or 45, 90, 180 µg (icv) of L-NAME decreased Tb. A significant correlation between Tb and diencephalic, but not plasma, nitrite/nitrate levels was observed. Heat stress-induced hyperthermia was inhibited by all tested doses of L-NAME (im and icv). Tb was correlated neither with plasma nor with diencephalic nitrite/nitrate levels during heat stress. These results indicate the involvement of brain NO in the maintenance of Tb of chicks, an opposite action of that observed in mammals, and may modulate hyperthermia.     

Both physical fitness and acute exercise regulate nitric oxide formation in healthy humans

Jungersten, Lennart, Anneli Ambring, Björn Wall, andÅke Wennmalm. Both physical fitness and acute exerciseregulate nitric oxide formation in healthy humans. J. Appl. Physiol. 82(3): 760-764, 1997.We analyzednitrate, a major stable end product of nitric oxide (NO) metabolism invivo in plasma and urine from groups of healthy subjects with differentworking capacities. Resting plasma nitrate was higher in athleticsubjects than in nonathletic controls [45 ± 2 vs. 34 ± 2 (SE) µM; P < 0.01]. In other subjects, both the resting plasma nitrate level(r = 0.53; P < 0.01) and the urinary excretionof nitrate at rest (r = 0.46; P < 0.01) correlated to thesubjects' peak work rates, as determined by bicycle ergometry. Twohours of physical exercise elevated plasma nitrate by 18 ± 4 (P < 0.01) and 16 ± 6%(P < 0.01), respectively, in athletes and nonathletes, compared with resting nitrate before exercise. We conclude that physical fitness and formation of NO at restare positively linked to each other. Furthermore, a single session ofexercise elicits an acute elevation of NO formation. The observedpositive relation between physical exercise and NO formation may helpto explain the beneficial effects of physical exercise oncardiovascular health.

     

Studies on the Physiology of Nodule Formation: IX. The Influence of Combined Nitrogen, Glucose, Light Intensity and Day Length on Root-hair Infection in Clover

Small amounts of nitrate or nitrite salts (10 µg N/plant)in the root medium of Trifolium glomeratum or T. repens delayednodulation, prolonged the initial rapid phase of root infectionand slightly stimulated lateral root formation, whereas equivalentquantities of ammonium sulphate or urea did not. Growth of rootsand root hairs was unaffected by any of these substances at10 µg N/plant. Altering the carbohydrate status of the clover seedlings byadding glucose to the root medium, or by changing day lengthor light intensity, influenced neither the stimulation of root-hairinfection nor the delay in nodulation induced by nitrate at10 fig N/plant, except that plants grown in total darkness hadfewer hairs infected when the root medium contained small amountsof nitrate. The nitrogenous compounds at 100 µg to 1,000 µg N/plant generally delayed and decreased nodulation,increased lateral root formation, slowed hair infection, andincreased root growth.     

Determination of nitrate and nitrite by high-performance liquid chromatography in human plasma

A new, accurate, fast and simple method has been implemented by which nitrite and nitrate ions, as stable forms of nitric oxide production were studied. A study of these two ions was carried out by a sensitive and accurate HPLC method with two detectors. The most important advantages of the reported method are: short time of analysis, minimal sample pre-treatment, long life of the analytical column and stable eluent solution. The photodiode array UV-Vis detector detected nitrite and nitrate ions at an absorbance of 212 nm. Much more sensitive electrochemical detection with a WE (glassy carbon) electrode was used for the detection of nitrite ions. An analytical chromatographic column was formed by a sorbent, containing strong base anion-exchange groups bound in Cl(-) form in the hydrophilic hydroxyethyl methacrylate matrix. The anions were analysed in human plasma without deproteinization using 0.02 M sodium perchlorate monohydrate as eluent solution at pH 3.9. At this pH organic substances do not affect the analysis. The retention times for nitrite and nitrate were 3.62 and 3.72 min (by electrochemical detection) and 4.44 min, respectively. The method was linear (r=0.9992, 0.9998, 0.996) within a 1-100 (nitrate), 1-20 micro mol/l (nitrite) concentration range.     

A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite.

Numerous methods are available for measurement of nitrate (NO(-)(3)). However, these assays can either be time consuming or require specialized equipment (e.g., nitrate reductase, chemiluminescent detector). We have developed a method for simultaneous evaluation of nitrate and nitrite concentrations in a microtiter plate format. The principle of this assay is reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction. This assay is sensitive to 0.5 microM NO(-)(3) and is useful in a variety of fluids including cell culture media, serum, and plasma. S-Nitrosothiols and L-arginine derivatives were found to be potential interfering agents. However, these compounds are generally minor constituents of biological fluids relative to the concentration of nitrate/nitrite. This report introduces a new, convenient assay for the stable oxidation products of nitrogen oxide chemistry in biological samples.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Monitoring nitric oxide from immobilised denitrifying bacteria, Pseudomonas stutzeri, by the use of chemiluminescence

A chemiluminescence detector was used to measure the production of nitric oxide, NO, from the denitrifying bacteria Pseudomonas stutzeri. NO is an intermediate when P. stutzeri converts nitrate into nitrogen gas. The reaction between NO and ozone is selective and sensitive in generating chemiluminescence. Calibrations were made down to 1 nM, with a signal-to-noise ratio of 3. Bacteria were immobilised in alginate beads. Denitrification experiments were made in an anaerobic non-growth medium by adding nitrate to a certain concentration in the reactor. The bacteria were exposed to nitrate in the concentration range 1 pM–5 mM. The lowest concentration to give a measurable NO response was 100 nM. Received: 16 October 1997 / Received revision: 20 January 1998 / Accepted: 24 January 1998     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Inhaled nitric oxide prevents left ventricular impairment during endotoxemia

We evaluated theeffect of long-term inhalation of nitric oxide (NO) on cardiaccontractility after endotoxemia by using the end-systolicelastance of the left ventricle (LV) as a load-independent contractility index. Chronic instrumentation in 12 pigs included implantation of two pairs of endocardial dimension transducers tomeasure LV volume and a micromanometer to measure LV pressure. One weeklater, the animals were divided into a control group (n = 6) or a NO group(n = 6). All animals receivedintravenous Escherichia coliendotoxin (10 µg · kg¹ · h¹)and equivalent lactated Ringer solution. NO inhalation (20 parts/million) was begun 30 min after the initiation of endotoxemia andwas continued for 24 h. In both groups, tachycardia, pulmonaryhypertension, and systemic hyperdynamic changes were noted. Theend-systolic elastance in the control group was significantly decreasedbeyond 7 h. NO inhalation maintained the end-systolic elastance atbaseline levels and prevented its impairment. These findings indicatethat NO exerts a protective effect on LV contractility in this model of endotoxemia.

     

beta -Adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes

Nitric oxide (NO) has been implicated in endogenous control ofmyocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surfaceof individual neonatal or adult rat ventricular myocytes (n > 6 neonatal and adult cells perexperiment). In beating neonatal myocytes, there was no detectablespontaneous NO release with each contraction. However, norepinephrine(NE; 0.25-1 µM) elicited transient NO release from beatingneonatal (149 ± 11 to 767 ± 83 nM NO) and noncontracting adult(157 ± 13 to 791 ± 89 nM NO) cells. NO was released byadrenergic agonists with the following rank order of potency:isoproterenol(₁₂) > NE (/₁) > dobutamine (₁)  epinephrine(/₁₂) > tertbutylene (₂); NO wasnot released by phenylephrine (). NE-evoked NO release wasreversibly blocked byN^G-monomethyl-L-arginine,trifluoperazine, guanosine5'-O-(2-thiodiphosphate), andnifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 ± 1.6%) and BAY K 8644 (10 µM = 11.9 ± 1%). NO wasalso released by A-23187 (10 µM = 884 ± 88 nM NO), guanosine 5'-O-(3-thiotriphosphate) (1 µM = 334 ± 56 nMNO), and dibutyryl adenosine 3',5'-cyclic monophosphate(10-100 µM = 35 ± 9 to 284 ± 49 nM NO) but not by ATP,bradykinin, carbachol, 8-bromoguanosine 3',5'-cyclicmonophosphate, or shear stress. This first functional demonstration ofa constitutive NO synthase in cardiac myocytes suggests its regulationby a -adrenergic signaling pathway and may provide a novel mechanismfor the coronary artery vasodilatation and enhanced diastolicrelaxation observed with adrenergic stimulation.

     

Microbial transformation of pentachloronitrobenzene under nitrate reducing conditions

The effect of pentachloronitrobenzene (PCNB) on denitrification was assessed with two denitrifying cultures (PCNB-free control and PCNB-acclimated) developed from a contaminated estuarine sediment. PCNB was transformed to pentachloroaniline (PCA) in the PCNB-acclimated culture repeatedly amended with 0.1 μM PCNB, but further dechlorination or degradation of PCA was not observed for almost 1 year. The effect of PCNB on denitrification was also investigated with the PCNB-free control culture. PCNB at an initial concentration of 13 μM was transformed to PCA simultaneously with nitrate reduction but only after the nitrate concentration was at or below 20 mg N/l. PCNB addition at an initial concentration of 13 μM to the control denitrifying culture developed as PCNB-free culture resulted in a transient accumulation of nitric oxide (NO) and nitrous oxide (N₂O). Similarly to the PCNB-acclimated culture, PCNB transformation to PCA started when the nitrate concentration decreased to about 20 mg N/l. A low degree of nitro group removal resulting in the formation of pentachlorobenzene (PeCB) was also observed in the control culture when amended with 13 μM PCNB. Further transformation or degradation of PCA was not observed in all cultures maintained under active nitrate reducing conditions. Based on the results of this study, the presence of nitrate at low concentrations in anoxic/anaerobic soil and sediments is not expected to negatively affect the biotransformation of PCNB to PCA, but dechlorination or degradation of PCA is not expected under active nitrate reducing conditions.