Index Of Nitric Oxide Production Research Articles

Increased nitric oxide production in hypotensive hemodialysis patients.

A subset of patients on long-term hemodialysis have sustained hypotension, defined as a predialysis systolic pressure of < 100 mmHg. To determine the role of nitric oxide (NO), an important vasodilator, in this condition, the authors measured the plasma levels of nitrite (NO2-) and nitrate (NO3-), the known NO metabolites taken as an index of NO production, in 10 hypotensive patients on long-term hemodialysis. None of them had diabetes, cirrhosis of the liver, congestive heart failure, or infection. Fifteen age and gender-matched normotensive patients on hemodialysis were selected as control subjects. Measurements of plasma levels of nitrite and nitrate based on the Greiss reaction were made. There was no significant difference in hematocrit, serum intact parathyroid hormone, total calcium, inorganic phosphorus, albumin, heart rate, cardiac index, or interdialysis weight gain between these two groups. Plasma nitrite and nitrate levels did not correlate with either predialysis serum creatinine or blood urea nitrogen. The mean arterial pressure (MAP) was significantly lower and plasma nitrite and nitrate levels were significantly higher in chronic hypotensive patients than in normotensive patients (MAP: 68.30 +/- 3.24 mmHg vs 95.20 +/- 2.44 mmHg, p < 0.001; plasma nitrite and nitrate: 72.49 +/- 14.41 mumol/L vs 36.42 +/- 5.45 mumol/L, p < 0.05). In addition, MAP from hypotensive and normotensive patients on hemodialysis was inversely correlated with plasma levels of nitrite and nitrate (r = -0.54, p < 0.01). It was concluded that enhanced NO production in this subset of patients on hemodialysis may contribute to their chronic hypotension.

Cerebrospinal fluid and plasma nitrite and nitrate concentrations after head injury in humans

To measure cerebrospinal fluid and plasma nitrite and nitrate concentrations as indicators of nitric oxide production in adults after severe closed-head injury. To determine if there is an association between cerebrospinal fluid and plasma nitrite and nitrate concentrations, and cerebral blood flow, arterio-jugular oxygen content difference, injury severity, and outcome after severe closed-head injury. A prospective, clinical study. Multidisciplinary intensive care unit. Fifteen comatose (Glasgow Coma Scale score of < or = 7) adult patients with severe closed-head injury were studied during the prospective, randomized evaluation of the effect of moderate hypothermia (32 degrees C for 24 hrs) on neurologic outcome after closed-head injury. Seven patients were in the hypothermic group and eight patients were in the normothermic treatment group. None. Patients were examined sequentially, every 12 hrs for 2 days. Intraventricular cerebrospinal fluid was assayed for nitrite and nitrate concentrations. Cerebral blood flow was measured by the 133xenon intravenous method. Simultaneous blood samples were obtained for measurements of arterio-jugular oxygen content difference and plasma nitrite and nitrate concentrations. Cerebral metabolic rate for oxygen was calculated. Cerebrospinal fluid nitrite and nitrate concentrations were highest at 30 to 42 hrs vs. 6 to 18, 18 to 30, and 42 to 54 hrs (26.4 +/- 3.3 vs. 17.3 +/- 2.1, 20.0 +/- 2.2, and 18.8 +/- 2.4 microM, respectively, p < .05). There was no difference over time in plasma nitrite and nitrate concentrations. Cerebral blood flow was increased and arterio-jugular oxygen content difference was reduced at 18 to 30, 30 to 42, and 42 to 54 hrs vs. 6 to 18 hrs (p < .05). At 30 to 42 hrs, cerebrospinal fluid nitrite and nitrate concentrations were 80% higher in patients who died vs. survivors (36.4 +/- 3.2 vs. 20.2 +/- 3.6, p < .05). Using a generalized, multivariate, linear regression model, both plasma nitrite and nitrate concentrations and injury Severity Score independently predicted cerebrospinal fluid nitrite and nitrate concentrations (p < .00001 and p = .0053, respectively). Cerebral blood flow and arterio-jugular oxygen content difference were not associated with cerebrospinal fluid or plasma nitrite and nitrate concentrations using this model. Cerebrospinal fluid nitrite and nitrate concentrations were increased over time in hypothermic vs. normothermic patients. But, where this difference occurred could not be determined by multiple comparisons (p = .03). The hypothermic patients had lower admission Glasgow Coma Scale scores than normothermic patients (p = .04) and tended to have higher injury Severity Scores (p = .09). Increases in cerebrospinal fluid nitrite and nitrate concentrations peaked at 30 to 42 hrs after severe closed-head injury. This increase in cerebrospinal fluid nitrite and nitrate concentrations was greater in nonsurvivors. Also, cerebrospinal fluid and plasma nitrite and nitrate concentrations were associated with injury Severity Score, suggesting that increased nitric oxide production in the brain is associated with injury severity and death. Hypothermia did not prevent the increase in cerebrospinal fluid nitrite and nitrate concentrations. Further study is required to determine the source of this increase in cerebrospinal fluid nitrite and nitrate concentrations and to further define the relationship to outcome and the effect of hypothermia on this process.

Inhibitory effect of peritoneal macrophages on progesterone release from cocultured rat granulosa cells is reversed by dexamethasone: evidence for an action independent of nitric oxide and distal to cyclic adenosine 3',5'-monophosphate generation.

We investigated the effects of cocultured macrophages on progesterone release from granulosa cells obtained at different stages of the rat estrous cycle and determined whether the immunosuppressive actions of the synthetic glucocorticoid can modulate the effects of cocultured macrophages. Basal and hCG-stimulated progesterone release from granulosa cell-only cultures varied according to the stage of the estrous cycle at which granulosa cells were obtained, but macrophages exerted profound inhibitory effects on both basal and hCG-stimulated steroidogenesis at all stages. These inhibitory effects were partially reversed by the addition of dexamethasone (5 x 10(-7) to 5 x 10(-5) M) to the culture medium. Measurement of nitrite accumulation in the media, as an index of nitric oxide production, showed that granulosa cells produce very low levels of this metabolite compared with macrophages, although granulosa/luteal cells from freshly ruptured follicles of estrous rats produced significantly higher concentrations of nitrite compared with granulosa cells obtained from diestrous and proestrous rats. Dexamethasone had only limited effects in suppressing the accumulation of nitrite, showing that the macrophages were synthesizing nitric oxide synthase and suggesting that the action of dexamethasone was independent of nitric oxide. Macrophages also inhibited the steroidogenic response of granulosa cells to forskolin (5 x 10(-6) M to 10(-4) M) and dibutyryl cAMP (1mM and 0.5 mM) and the potentiation of the response by the addition of hCG. However, this inhibition was reversed by the presence of 5 x 10(-6) M dexamethasone, and responses were comparable with those measured from granulosa cell-only cultures. Overall, the results suggest that macrophage inhibition of progesterone synthesis acts at a site distal to the generation of cAMP and that dexamethasone reverses this inhibition.

Evoked Release of Amino Acids and Prostanoids in Spinal Cords of Anesthetized Rats: Changes During Peripheral Inflammation and Hyperalgesia.

C-fiber stimulation of the sciatic nerve in rats with an acute experimental arthritis elicited a greatly enhanced spinal release of several amino acids and prostaglandin PGE(2) (PGE(2)) when compared to control rats. The most dramatic change was in aspartate (Asp) which was not released in the control state but did increase significantly for a period outlasting the stimulation in the animals with knee joint inflammation. Evoked release of Glu, Gly, and PGE(2) also increased in the experimental group. It is proposed that the Asp acts primarily at N-methyl-D-asparate (NMDA) receptors to participate in the generation of hyperalgesia. In a similar model, direct activation of the NMDA receptors evoked spinal release of the same amino acids, including citrulline (an index of nitric oxide production) as well as representative prostanoids. Pretreatment with a NO synthase inhibitor blocked the release of Cit and Glu and reduced PGE(2) release evoked by NMDA, indicating that 1) Glu release is downstream of the actions of NO, and 2) the cyclooxygenase system is not independent of NO production. Evoked release of thromboxane B(2) was not affected by pretreatment with the nitric oxide synthesis inhibitor. Pretreatment with a cyclooxygenase inhibitor antagonized release of both prostanoids, but had little effect on NMDA-evoked increases in amino acids and Cit.

Effect of renal function on serum nitrogen oxide concentrations

Nitric oxide is too short-lived to measure in vivo, but its production can be estimated by measuring its stable oxidation products, nitrites and nitrates, in serum. Renal elimination of these ions has been demonstrated, but the effect of renal function on their concentrations in serum is currently unknown. We evaluated serum and urine nitrates + nitrites as serum nitrogen oxides (sNOx), nitrogen oxide (NOx) clearance, and creatinine clearance in 71 patients on the Intensive Therapy Unit. The correlation between sNOx and plasma creatinine was strong and highly significant (P <0.001). These results suggest that renal function has a significant effect on sNOx concentrations. Studies in which the sNOx concentration is used as an index of nitric oxide production can therefore be interpreted only if renal function has been taken into account.

Interrelation between protein kinase C and nitric oxide in the development of vasospasm after subarachnoid hemorrhage.

This study was undertaken to investigate how protein kinase C (PKC) and nitric oxide (NO) interact to regulate the vascular tone, and how their interaction contributes to the development of vasospasm after subarachnoid hemorrhage (SAH). For these purposes, vasospasm was conducted with a canine model. We investigated the following subjects with arteries from intact animals and those from the SAH model, and compared the results between the two; tension at rest of isometric tension study, the effect of PKC inhibitors and of an inhibitor of NO synthesis on the tension at rest, and levels of guanosine 3',5'-cyclic monophosphate (cGMP) as an indicator of NO production. The tension at rest was enhanced in the artery from the SAH model compared to that from intact animals, and it was PKC-dependent. Arteries from intact animals but not those from the SAH model developed tonic tensions by NO inhibitors, and these tonic tensions were suppressed by PKC inhibitors, and also by cGMP. An enzyme immunoassay revealed a decreased cGMP level in the SAH model. The evidence indicates that NO exerts a negative feedback control on PKC activation. Subarachnoid hemorrhage interferes with this feedback control, resulting in PKC-dependent enhanced vascular tone and vasospasm.

Roles of IL-1 and TNF-alpha in endotoxin-induced activation of nitric oxide synthase in cultured rat brain cells.

In astrocytes and microglia, bacterial lipopolysaccharide (LPS) stimulates production and release of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO). Although IL-1 beta and TNF-alpha are themselves capable of inducing NO synthase (NOS) in glia, the specific factors mediating LPS induction of NOS in brain have not been identified. To determine whether LPS induction of NOS in brain cells is mediated by IL-1 or TNF-alpha, acting alone or in concert, the effects of IL-1-receptor antagonist (IL-1Ra) and of TNF-soluble receptor (TNFsRp55), presented individually and in combination, on LPS-induced NOS activity were tested. In glial-enriched mixed primary cultures of neonatal rat telencephalic cells, LPS (0.1-100 ng/ml), IL-1 beta (0.01-10 nM), and TNF-alpha (0.1-100 nM) each concentration dependently stimulated accumulation of nitrite, an indicator of NO production. Induction of nitrite accumulation by LPS and by IL-1 was blocked by N omega-nitro-L-arginine methyl ester and N omega-monomethyl-L-arginine, indicating that it was mediated by NOS. TNF-alpha alone induced NO production weakly as compared with IL-1, but combined submaximal concentrations of IL-1 beta (1 nM) and TNF-alpha (10 nM) induced NOS synergistically. Furthermore, TNFsRp55 and IL-1Ra each produced a dose-dependent partial inhibition of the NO response to LPS, and the effect of TNFsRp55 was equal to or greater than that of IL-1Ra. TNFsRp55 and IL-1Ra in combination were not significantly more effective than TNF-sRp55 alone. The results indicate that LPS induction of NOS activity in brain cells is mediated in part by both IL-1 beta and TNF-alpha.

Nitric oxide as a challenge for the clinical chemistry laboratory.

Nitric oxide (NO) is an important intra- and intercellular mediator. Although NO can be measured using many different chemical methods, the compound is challenging for a clinical chemistry laboratory, since its half-life in vivo in humans is only a few seconds. Most of the NO is oxidized to nitrite/nitrate, and the concentrations of these anions have been used as quantitative indices of NO production. The simplest and most widely used technique is spectrophotometric measurement of nitrite using the Griess reaction. Nitrate, the main metabolite of NO in blood and urine, must be reduced to nitrite before the colour reaction. Other methods used for measuring nitrite/nitrate in human blood or urine include high-performance liquid chromatography, gas chromatography-mass spectrometry, chemiluminescence, enzymatic assay with nitrate reductase and electron paramagnetic resonance. The reported mean concentrations of nitrite in the blood of healthy adults have varied from non-existent to 4.2 mumol l-1, and those of nitrate from 19.7 to 44 mumol l-1. The technical measurement of nitrite/nitrate is obviously reliable, but there are problematic preanalytical factors. Normal daily food contains more nitrate than that formed from NO, and thus diet-derived nitrate may contribute considerably to the concentration in blood. The problem may to some extent be solved with dietary restrictions, but it is questionable whether the confounding effect of diet-derived nitrate can be totally avoided. Therefore, better methods for measuring the production of NO in vivo would be very welcome.

Nitric oxide-related neural components in the rat small intestine after transplantation.

The changes in nitric oxide (NO)-related neural components in the transplanted small intestine are unknown. In this study, the NO neural component was examined using electrophysiological and NADPH-diaphorase histochemical techniques in a rat small bowel transplantation model. Syngeneic total small bowel transplantation was performed in 26 male Lewis rats using microsurgical techniques. The rats were divided into four groups: an untreated control group and animals at 1 (G1), 2 (G2), and 4 (G4) weeks after transplantation. Jejunal strips were mounted in a superfusion apparatus for examination. In the presence of atropine and guanethidine, the effect of the NO synthesis inhibitor L-NG-nitro-arginine (L-NNA, 100 microM) relaxation mediated by the nonadrenergic, noncholinergic (NANC) neural system was assessed following electrical stimulation at 2Hz. The inhibitory effect of L-NNA on relaxation was taken as an indicator of NO production. The percentage of inhibition in the control group, and in G1, G2, and G4 was 43.30% +/- 6.08% (mean +/- SE), 42.10% +/- 6.69%, 43.62 +/- 10.00%, and 52.46% +/- 6.00%, respectively. Inhibition in G4 was significantly higher than in the other groups (P < 0.01). The percentage of NADPH-diaphorase-positive fibers in the control group, G1, G2, and G4 was 25.06% +/- 4.70% (mean +/- SE), 26.27% +/- 2.17%, 24.73% +/- 2.87%, and 30.76% +/- 3.19%, respectively. Again, G4 showed a significantly higher level than the other groups (P < 0.01). We conclude that increased NO production may play a significant role in maintaining the intrinsic nervous system of the small intestine after transplantation.

Burn Injury Alters Coronary Endothelial Function

This study examined the effects of burn injury on coronary endothelial function and coronary vascular reactivity. Adult rabbits were given a scald burn over 30% of the total body surface area (or sham burn for controls) and resuscitated with Ringer's lactate solution (4 ml/kg/% burn). Subgroups of burned (n= 6) and sham-burned (n= 6) animals were sacrificed at 2, 6, and 24 hr after injury; hearts were harvested and perfused. Changes in coronary perfusion pressure (CPP, mmHg) and coronary vascular resistance (CVR, mmHg/min) were determined at a constant preload and constant coronary flow rate. Changes in coronary endothelial function were determined by the ability of the endothelium to release cGMP as an indicator of nitric oxide production. Compared to values measured in sham burns, CPP and CVR progressively fell during the early postburn period but increased toward values measured in the sham burn group by 24 hr. Cyclic GMP, fmole/ml of coronary perfusate, was significantly lower in burned hearts (27 ± 1) compared to values measured in effluents from sham burn hearts (310 ± 40,P< 0.05). Alterations in coronary effluent cGMP levels after burn injury suggest that thermal injury disrupts coronary endothelial function, likely contributing to postburn changes in cardiac performance.

Inhibition of pleural mesothelial cell collagen synthesis by nitric oxide

The pleural mesothelial cell has a critical role in repairing the mesothelium after injury via its ability to produce connective tissue macromolecules. We have recently shown that proinflammatory cytokines and lipopoly-saccharide induce pleural mesothelial cells to produce nitric oxide. The present study examined the effect of nitric oxide on pleural mesothelial cell protein synthesis. Rat pleural mesothelial cells were exposed to various combinations of tumor necrosis factor, interleukin-1, interferon-γ, and lipopolysaccharide or to the nitric oxide donors: 6-morpholino-sydnonimine, S-nitroso-N-acetyl- d,l-penicillamine, sodium nitroprusside, and spermine-NO adduct for 24–48 h. Nitrate and nitrite (an index of nitric oxide production) and net collagen and noncollagen protein production (uptake of 3H-proline into collagenase-sensitive protein) were then determined. Net collagen production was significantly inhibited by the cytokine-lipopolysaccharide combinations tested. Collagen inhibition paralleled the time course of increased nitric oxide production. The inhibition of collagen production was also significantly reversed by the addition of N G-nitro-L-arginine methyl ester, and was reproduced by the addition of a 5:1 molar excess of Larginine to N G-nitro-L-arginine methyl ester. Additionally, nitric oxide-generating compounds significantly inhibited collagen production in a dose-dependent manner compared to unexposed control cells. Net collagen production was inhibited to a greater degree than noncollagen protein synthesis. These results suggest that nitric oxide may be a significant mediator of PMC collagen production during conditions of significant pleural inflammation.

Aminoguanidine selectively decreases cyclic GMP levels produced by inducible nitric oxide synthase.

Overproduction of nitric oxide (NO) following induction of NO synthase in vascular smooth muscle by endotoxin and certain cytokines contributes to the vasodilation and hyporesponsiveness to vasopressors that characterize the septic circulation. Guanosine 3',5'-cyclic monophosphate (cGMP) mediates the effects of NO in vascular smooth muscle. Vessels from animals treated with endotoxin have elevated cGMP levels compared with control animals. Aminoguanidine has been proposed as a selective inhibitor of the inducible form of NO synthase. This study compares the effects of aminoguanidine on phenylephrine-induced contractions and cGMP levels in thoracic aortic rings from endotoxin treated (20 mg/kg intraperitoneally) with sham-treated (1 ml saline intraperitoneally) rats. Endotoxin-treatment depressed phenylephrine-induced contraction and raised tissue levels of cGMP. Aminoguanidine (100 microM and 1 mM) increased phenylephrine-induced tension and decreased cGMP levels in a dose-dependent manner in intact and endothelium-denuded aortas from endotoxin-treated rats but had no effect on vessels from sham-treated rats. These findings are consistent with the hypothesis that endotoxin treatment causes increased vascular production of endothelium-independent NO, which is associated with a diminished response to vasoconstrictors. Aminoguanidine decreases indices of NO production only after endotoxin treatment, providing further evidence that it is a selective inhibitor of inducible NO synthase.

Oral administration of L-arginine and captopril in rats prevents chronic renal failure by nitric oxide production

The effect of oral supplementation of L-arginine, the substrate of nitric oxide, (1.25 g/liter water) and captopril (15 mg/liter water) was studied in 5/6 nephrectomized rats for a period of three months. N-omega-nitro L-arginine, a nitric oxide synthase inhibitor, was given orally (70 mg/liter water) with or without L-arginine or captopril. The urinary excretion of nitrite (NO2) + nitrate (NO3), the known metabolites of nitric oxide, was taken as an index of nitric oxide production. Chronic renal failure rats were characterized by a low creatinine clearance, high FENa%, proteinuria, hypertension and a low urinary excretion of NO2 + NO3; 0.152 +/- 0.06 (P < 0.001) nmol/micrograms creatinine compared with 0.481 +/- 0.004 (P < 0.001) in normal rats and 0.479 +/- 0.11 (P < 0.001) in untreated sham-operated rats. Both L-arginine and captopril were effective in the normalization of all these parameters. The combination of L-arginine and captopril had no additive effects. The nitric oxide synthase inhibitor significantly diminished the captopril beneficial effect. It is concluded that chronic renal failure in rats is a low nitric oxide production state. The supplementation of L-arginine is shown to overcome this condition. It is suggested that the beneficial effect of captopril on chronic renal failure is through a specific L-arginine--nitric oxide synthase--nitric oxide pathway.

Death of septal cholinergic neurons produced by chronic exposure to glutamate is prevented by the noncompetitive NMDA receptor/channel antagonist, MK‐801: Role of nerve growth factor and nitric oxide

To study the sequence of degenerative events possibly associated with cholinergic cell death in Alzheimer's disease, septal cholinergic neurons derived from rat embryonic brains were exposed to chronic excitotoxic stress by glutamate. Counts of choline acetyltransferase (ChAT)-immunopositive neurons and measurement of ChAT activity revealed that concentrations of glutamate on the order of 70 microM killed 50% of cholinergic neurons after 24 hr of treatment. Neurotoxic effects were not aimed at cholinergic neurons specifically, since other populations of cells present in these cultures were also affected at similar concentrations. The noncompetitive N-methyl-D-aspartate (NMDA) receptor channel antagonist MK-801 (10 microM) abolished acute neuronal swelling and rescued from late degeneration both cholinergic and noncholinergic cells when concentrations of glutamate up to 500 microM were added to the cultures. Protective effects declined progressively with increasing concentrations of the amino acid, even when MK-801 was raised to its highest nontoxic levels, e.g., 50 microM. the kainate/quisqualate receptor antagonist CNQX provided no protection alone or in combination with MK-801. Nerve growth factor (NGF), used in standard culture conditions to stimulate the expression of the cholinergic phenotype, was shown not to influence excitotoxic neurodegenerative changes. Several observations suggested that nitric oxide (NO) may act as an intercellular messenger of NMDA-mediated cell death in septal cultures: 1) Most of the cholinergic neurons contained the NO synthase enzyme as characterized by NADPH-diaphorase (NADPH-d) staining; 2) sodium nitroprusside (SNP) [a chemical with the ability of generating NO] was capable of mimicking some of the aspects of the glutamate-induced degenerative process; 3) the rise in cyclic GMP which was observed in the presence of toxic levels of glutamate and which is usually taken as an index of NO production, was antagonized by MK-801 and by the inhibitor of the NO synthase enzyme, L-NOARG. Yet, the fact that L-NOARG and its congener, L-NAME, were ineffective in preventing glutamate-induced neurodegenerative changes in our culture system did not substantiate our working hypothesis. Altogether these results suggest that glutamate-induced cholinergic neuronal death is the consequence of an overstimulation of NMDA receptors and that neither NGF nor NO plays a key role in the degenerative process.

Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction.

To determine antioxidant vitamin concentrations, lipid peroxidation, and an index of nitric oxide production in patients in the intensive care unit (ICU) with septic shock and relate the findings to the presence of secondary organ failure. A prospective, observational study. A nine-bed ICU in a University teaching hospital. Sixteen consecutive patients with septic shock, defined as: a) clinical evidence of acute infection; b) hypo- or hyperthermia (< 35.6 degrees C or > 38.3 degrees C); c) tachypnea (> 20 breaths/min or being mechanically ventilated); d) tachycardia (> 90 beats/min); e) shock (systolic pressure < 90 mm Hg) or receiving inotropes. Fourteen patients also had secondary organ dysfunction. None. Antioxidant vitamin concentrations were significantly lower in the patients than the reference range obtained from a comparable group of healthy controls. The mean plasma retinol (vitamin A) concentration was 26.5 +/- 19.3 micrograms/dL compared with 73.5 +/- 18.3 micrograms/dL in healthy subjects (p < .01). Additionally, 13 (81%) patients had retinol values below the lower limit of our reference range (< 37.0 micrograms/dL). Tocopherol (vitamin E) plasma concentrations were below the reference range in all patients (< 9.0 mg/L), with a mean value of 3.6 +/- 2.0 mg/L compared with 11.5 +/- 1.3 mg/L in healthy subjects (p < .001). Plasma beta carotene and lycopene concentrations were undetectable (< 15 micrograms/L) in eight (50%) patients, and below our reference range (< 101 micrograms/L and < 154 micrograms/L, respectively) in the remaining patients. In the five patients with three or more dysfunctional secondary organs, plasma thiobarbituric acid-reactive substances were significantly increased (p < .05), suggesting increased lipid peroxidation. Concentrations of thiobarbituric acid-reactive substances correlated negatively with both plasma retinol and plasma tocopherol (r2 = .42, p < .01 and r2 = .48, p < .005, respectively). In the five patients from whom we were able to collect urine, nitrite excretion was increased approximately 400-fold (p < .001). These data indicate decreased antioxidant status in the face of enhanced free radical activity, and suggest potential therapeutic strategies involving antioxidant repletion.

Nitric oxide is important for mouse beta-cell line killing by peritoneal exudate cells obtained from cyclophosphamide treated non-obese diabetic mice.

Macrophages from recent onset non-obese diabetic (NOD) mice showed cytotoxicity against the NOD mouse derived beta-cell line, MIN6N-9a. In this report, we examined whether nitric oxide is associated with beta-cell destruction. Peritoneal exudate cells (PEC), obtained from cyclophosphamide treated NOD mice showed higher cytotoxicity against MIN6N-9a compared to PECs from saline injected NOD mice (P < 0.01). This effect was suppressed in cells incubated with 0.5 mmol/l NG-methyl-L-arginine, a nitric oxide synthase inhibitor (P < 0.001). In addition, the nitrite concentration of the co-culture medium, as an index of nitric oxide production, increased in MIN6N-9a cells co-cultured with peritoneal exudate cells from cyclophosphamide injected NOD mice but not in co-culture with saline injected NOD mice (P < 0.05). Thus, nitric oxide plays an important role in beta-cell line destruction of macrophages obtained from NOD mice.

Cooling effects on nitric oxide production by rabbit ear and femoral arteries during cholinergic stimulation

1. Ear (cutaneous) and femoral (deep) arteries from rabbit were perfused at 37 degrees C and 24 degrees C (cooling) and the production of nitrite, as an index of nitric oxide production, was measured under basal conditions and cholinergic stimulation. 2. In both types of arteries under control conditions, the basal production of nitrite was similar at 24 degrees C and 37 degrees C. Compared with the control conditions, the basal production of nitrite was significantly lower in ear and femoral arteries without endothelium or treated with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) but it was similar in those treated with atropine (10(-6) M). 3. At 37 degrees C, methacholine (10(-7)-10(-5) M) increased the production of nitrite in ear and femoral arteries; this increase persisted during 30-60 min and was practically abolished by L-NAME (10(-4) M), atropine (10(-6) M), or removal of the endothelium. In ear arteries the total nitrite production to activation with methacholine was higher at 24 degrees C than at 37 degrees C due to this production persisted increased for a longer period (> 150 min), whereas in femoral arteries it was lower at 24 degrees C than at 37 degrees C. 4. It is suggested that: (a) the endothelium of rabbit ear and femoral arteries produce nitric oxide under basal conditions, which is increased by cholinergic stimulation, and (b) cooling potentiates endothelial nitric oxide production to cholinergic stimulation in cutaneous arteries, whereas it inhibits this production in deep arteries.

Association between protective efficacy of antibodies to tumor necrosis factor and suppression of nitric oxide production in neonatal rats with fatal infection.

In a rat model of fatal infection caused by Pseudomonas aeruginosa, the circulating level of nitrite/nitrate (NO2-/NO3-), a good indicator for nitric oxide production, was remarkably increased after elevation of circulatory tumor necrosis factor (TNF). Anti-TNF MAb cotreatment was shown to blunt hypoglycemia and hyperlacticemia and was associated with decreased mortality of septic animals. Moreover, anti-TNF MAb significantly reduced not only plasma TNF but also plasma NO2-/NO3- levels. Dexamethasone had a similar effects, and when anti-TNF MAb was used in combination with dexamethasone, the suppression of nitric oxide production and the protective efficacy were more remarkable compared with therapy with either anti-TNF MAb or dexamethasone alone. Our present data suggested that the protective efficacy of anti-TNF MAb may correlate with the suppression of nitric oxide production and also with a modulation in metabolic abnormalities in the septic newborn rats.

Biosynthesis of nitric oxide activates iron regulatory factor in macrophages.

Biosynthesis of nitric oxide (NO) from L-arginine modulates activity of iron-dependent enzymes, including mitochondrial acontiase, an [Fe-S] protein. We examined the effect of NO on the activity of iron regulatory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murine macrophages were activated with interferon-gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG-substituted analogues of L-arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria-free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN-gamma and/or LPS stimulation. Authentic NO gas as well as the NO-generating compound 3-morpholinosydnonimine (SIN-1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post-transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe-S] cluster of IRF mediates iron-dependent regulation.

Nitric oxide synthesis by rat pleural mesothelial cells: induction by cytokines and lipopolysaccharide.

The close proximity of pleural mesothelial cells (PMC) and mononuclear cells during pleural inflammation suggests that leukocyte-derived products (e.g., cytokines) may play an important role in modulating PMC function. The purpose of this study was to determine whether certain cytokines and bacterial products induce PMC to produce nitric oxide (NO). Confluent monolayers of rat PMC were exposed to tumor necrosis factor (TNF), interleukin-1 beta (IL-1), gamma-interferon (IFN), or lipopolysaccharide (LPS) individually and in various double and triple combinations for 6-72 h. Concentrations of nitrite and nitrate were quantified and used as indirect indices of NO production. Nitrite/nitrate accumulation was maximal at 72 h, with most of the increase occurring from 48 to 72 h. Maximal nitrite/nitrate production was observed with triple combinations with the combination of LPS, IL-1, and TNF giving the highest concentration (137.4 +/- 2.8 microM). Nitrite/nitrate production was significantly inhibited by NG-nitro-L-arginine methyl ester, suggesting that nitrite and nitrate were derived from the L-arginine-dependent formation of NO. These data indicate that PMC can be induced to produce large amounts of NO in response to specific combinations of proinflammatory cytokines and LPS.