Nitric Oxide Production In Tissues Research Articles

Pyometra alters uterine aquaporins related with lipopolysaccharide concentrations and antioxidant enzyme activities in bitches

Pyometra is a common life-threatening inflammatory disease with a complex etiopathogenesis that develops during the diestrus stage and can be observed in elderly intact bitches. The present study evaluated five aquaporin (AQP1, AQP2, AQP3, AQP5, and AQP9) transcript abundances and immunolocalization in the uterine tissue, and investigated their relationship with uterine tissue and blood lipopolysaccharide (LPS) concentration, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity, and nitric oxide (NO) production in dogs suffering from pyometra. The study sampled 36 client-owned intact bitches from different breeds, of which 24 cases were diagnosed with pyometra. Twelve of these bitches in the diestrus stage that presented for elective ovariohysterectomy were used as the control group. Blood samples were collected into tubes without anticoagulant for serum progesterone, LPS concentration, and antioxidant activities at the time of diagnosis. Bacteriological and tissue samples from the uteri were collected after the ovariohysterectomy. The tissue samples were used to determine antioxidant activity, and hormone and toxin concentrations. Transcript abundance of uterine AQPs were determined by qPCR, and their presence and localization were determined by by immunohistochemistry. For all pyometra samples, the bacteria isolated from the uterine swabs were Escherichia coli. Compared to the control group, AQP1, AQP2, and AQP5 were downregulated more than 2-fold, whereas AQP9 was upregulated nearly 3-fold and AQP3 was upregulated more than 4-fold in the pyometra affected uteri (P<0.05). Uterine AQP1 was moderately negatively correlated with serum LPS concentration (r=-0.568, P<0.01) and tissue NO production (r=-0.407, P<0.05). AQP5 was positively correlated with serum SOD activity (r=0.485, P<0.05) and negatively correlated with serum LPS concentration (r=-0.512, P<0.05). AQP9 was negatively correlated with tissue SOD and serum GPx activity. This is the first study to identify AQP9 transcript abundance and immunolocalization in canine uterine tissue. Uterine AQP1, AQP2, AQP3, AQP5, and AQP9 transcript abundances were altered in spontaneously developed canine pyometra while AQP transcript abundance was negatively related to serum toxin concentration, NO production, and antioxidant enzyme activity. Further studies should be conducted to determine the role of altered abundances of AQPs transcripts in pyometra pathogenesis.

Nitric Oxide Production in Tissues of 7- and 16-Week-Old Rats under Mobility Restriction

Nitric Oxide Production in Tissues of 7- and 16-Week-Old Rats under Mobility Restriction

The nitric oxide production in tissues of 7- and 16-week-old rats under mobility restriction

The electron paramagnetic resonance method was used to study the intensity of nitric oxide production by analyzing the amount of NO-containing paramagnetic complexes in the heart, liver, and spinal cord tissues of 7- and 16-week-old rats growing under restricted mobility conditions. Nitric oxide production was assessed by the intensity of the EPR signal belonging to the (DETC)2-Fe2+NO complex. The results obtained show that growth under conditions of restricted motility leads to an increase in NO production in all organs under study, with the greatest increase observed in 7-week-old rats. Keywords: nitric oxide, heart, liver, spinal cord, electron paramagnetic resonance.

Acer okamotoanum improves cognition and memory function in Aβ25–35-induced Alzheimer’s mice model

We studied the effect of ethyl acetate (EA) fraction from Acer okamotoanum on cognitive improvement and protective abilities in amyloid beta (Aβ)25–35 peptide-injected Alzheimer’s disease (AD) mice. EA was oral administration at 100 and 200 mg/kg/day during the 14 days. We studied the protective effect of EA against AD on the basis of behavioral tests including T-maze test, Novel object recognition test, and Morris water maze test. Control group injected with Aβ25–35 showed significant impairments in memory function. But the oral administration of EA (EA 100 and EA 200 groups) improved the cognition and memory function. In addition, EA against Aβ25–35 peptide has been shown to inhibit lipid peroxidation levels and nitric oxide production in tissues. Acetylcholinesterase (AChE) was elevated in the brain by Aβ25–35 peptide, whereas administration of EA (EA 100 and EA 200 groups) significantly decreased AChE level. Our results indicated that EA improves learning and long-term memory against Aβ25–35 peptide–caused deficit through attenuation of oxidative stress.

The regulatory effect of endogenous hydrogen sulfide on pulmonary vascular structure and gasotransmitters in rats with high pulmonary blood flow

The study aimed to explore the regulatory effect of endogenous hydrogen sulfide (H 2S), a novel gasotransmitter, on pulmonary vascular structure and gasotransmitters in rats with high pulmonary blood flow. Thirty-two Sprague–Dawley rats were randomly divided into a sham group, shunt group, sham + PPG (propargylglycine, an inhibitor of cystathionine-γ-lyase) group and shunt + PPG group. Rats in the shunt and shunt + PPG groups underwent abdominal aorta–inferior vena cava shunting. Rats in the shunt + PPG and sham + PPG groups were intraperitoneally injected with PPG. After 4 weeks of shunting, mean pulmonary artery pressure (MPAP) and pulmonary vascular structural remodeling (PVSR) were evaluated. H 2S, nitric oxide (NO) and carbon monoxide (CO) contents were measured in lung tissues. Meanwhile, nitric oxide synthase (eNOS), heme oxygenase (HO-1) and proliferative cell nuclear antigen (PCNA) protein expressions and ERK activation were evaluated. After 4 weeks of shunting, rats showed PVSR with increased lung tissue H 2S and NO content but decreased CO content. After the PPG treatment, MPAP further increased and PVSR was aggravated. Meanwhile, PCNA expression and ERK activation were augmented with decreased lung tissue CO and HO-1 protein production but increased lung tissue NO production and eNOS expression. H 2S exerted a protective effect on PVSR, and the inhibition of the NO/NOS pathway and the augmentation of the CO/HO pathway might be involved in the mechanisms by which H 2S regulates PVSR in rats with high pulmonary flow.

Correlation of dynamic infrared imaging with radiologic and pathologic response for patients treated with primary systemic therapy for locally advanced breast cancer

10696 Background: Assessment of response to therapy for locally advanced breast cancer includes serial assessments of physical exam, radiologic imaging, or repeated biopsies. Expense, subjective assessment, and patient risk make these methods impractical. Dynamic infrared imaging (DIRI) utilizes a quantum well infrared photon (QWIP) sensor with software to analyze the emission patterns over time. DIRI can detect biological temperature gradients with sensitivity of 0.009ºC. Tumor-induced local tissue nitric oxide production can increase local capillary blood flow. Anti-tumor therapies have been shown to result in decreased peri-tumoral capillary blood flow. These changes in temperature, detected by serial DIRI imaging, may provide a low cost, non-invasive, easily reproducible objective tool for real-time clinical assessment. Methods: In this prospective pilot study, we are evaluating patients with locally advanced breast cancer using serial DIRI. Primary endpoints include: sensitivity, specificity, PPV, and NPV of DIRI in comparison to pathologic response, concordance of DIRI to physical exam, and concordance of DIRI to standard radiographic evaluation at initial diagnosis and prior to surgery. DIRI results are reported as quantification of changes in the 0.2Hz modulation of temperature over the breast during the course of treatment and measurement of area of average temperature in a region of interest compared between breasts. One hundred patients will be enrolled in this trial. Results: Sixteen patients have been enrolled. Six have proceeded to surgery. All but one patient exhibited evidence of tumor response by physical exam. These findings correlated with response when comparing initial to pre-surgical MRI. In all responding patients, DIRI results revealed a decrease in the number of regions over the breast in which the 0.2Hz frequency dominated. Similarly, DIRI evaluation according to area of average temperature in the region of interest compared between breasts was concordant in patients with response to therapy. Conclusions: Assessment of response by physical exam, MRI, and DIRI were consistent. Preliminary data reveals that serial DIRI imaging can be an effective adjunctive tool. No significant financial relationships to disclose.

ESR study of the nitric oxide production in tissues of animals under an external influence on the functioning of the cardiovascular and nervous systems

Electron spin resonance (ESR) of the ternary (DETC)2-Fe2+-NO complex has been applied to determine the nitric oxide production in tissues of rats and snails. A preliminary ESR study of the NO content in tissues of rats before and after artificially induced acute myocardial infarct was performed. The analysis of the obtained results shows that the nitric oxide production during the first hour after the moment of inducing myocardial infarct decreases. It is also demonstrated that ESR may be useful in the study of the influence of the long-term sensitization of snails on the nitric oxide production in their body. The changes in the NO production after the external influences in both cases are discussed.

ADMA regulates angiogenesis: genetic and metabolic evidence

Endothelium-derived nitric oxide (NO) plays an important role in transducing the effects of angiogenic factors. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We used a murine model of hindlimb ischemia to investigate whether genetic or metabolic changes in ADMA levels could impair angiogenic response in vivo. Hindlimb ischemia was surgically induced in C57BL/6J mice, apo E-deficient mice, or transgenic mice overexpressing dimethylarginine dimethylaminohydrolase (DDAH). Some animals were also treated with the NOS antagonist L-nitro-arginine, or the NO precursor L-arginine. Angiogenesis was quantified in the hindlimb skeletal muscle by capillary/myocyte ratio. Plasma or tissue ADMA levels were measured by HPLC. In normal mice, hindlimb ischemia increased tissue ADMA twofold, and reduced DDAH and NOS expression. This was associated with a reduced NOS activity (by over 80%) three days following surgery. On day seven, a threefold increase in DDAH expression and a fall in tissue ADMA levels were associated with a sevenfold increase in NOS activity, whereas NOS expression did not increase above baseline. In DDAH transgenic mice, the elevation of ADMA and decrement in NOS activity was blunted during hindlimb ischemia. Plasma ADMA levels were increased in apo E-mice (1.79 +/- 0.45 versus 1.07 +/- 0.08 pmol/l; p = 0.008). Capillary index was significantly reduced in apo E-mice up to seven weeks after surgery (0.25 +/- 0.05 versus 0.62 +/- 0.08; p < 0.001). The effect of hypercholesterolemia on capillary index was reversed by L-arginine, and (in wild-type mice) mimicked by administration of the NOS antagonist L-nitro-arginine. In conclusion, metabolic or genetic changes in plasma and tissue ADMA levels affect tissue NO production and angiogenic response to ischemia.

Characterization of the Effects of Oxygen on Xanthine Oxidase-mediated Nitric Oxide Formation

Under anaerobic conditions, xanthine oxidase (XO)-catalyzed nitrite reduction can be an important source of nitric oxide (NO). However, questions remain regarding whether significant XO-mediated NO generation also occurs under aerobic conditions. Therefore, electron paramagnetic resonance, chemiluminescence NO-analyzer, and NO-electrode studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. With substrates xanthine or 2,3-dihydroxybenz-aldehyde that provide electrons to XO at the molybdenum site, the rate of NO production followed Michaelis-Menten kinetics, and oxygen functioned as a competitive inhibitor of nitrite reduction. However, with flavin-adenine dinucleotide site-binding substrate NADH as electron donor, aerobic NO production was maintained at more than 70% of anaerobic levels, and binding of NADH to the flavin-adenine dinucleotide site seemed to prevent oxygen binding. Therefore, under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in tissues. Studies of the pH dependence of NO formation indicated that lower pH values decrease oxygen reduction but greatly increase nitrite reduction, facilitating NO generation. Isotope tracer studies demonstrated that XO-mediated NO formation occurs in normoxic and hypoxic heart tissue. Thus, XO-mediated NO generation occurs under aerobic conditions and is regulated by oxygen tension, pH, nitrite, and reducing substrate concentrations.

Enhanced nitric oxide inactivation and protein nitration by reactive oxygen species in renal insufficiency.

Chronic renal failure (CRF) is associated with oxidative stress which promotes production of reactive carbonyl compounds and lipoperoxides leading to the accumulation of advanced glycation and lipoxidation end products. Reactive oxygen species (ROS) avidly reacts with nitric oxide (NO) producing cytotoxic reactive nitrogen species capable of nitrating proteins and damaging other molecules. This study tested the hypothesis that CRF results in enhanced ROS-mediated NO inactivation and protein nitration which can be ameliorated with antioxidant therapy. Male Sprague Dawley rats were randomized to CRF (5/6 nephrectomy) and sham-operated controls and fed either a regular diet (vitamin E, 40 U/Kg food) or an antioxidant-fortified diet (vitamin E, 5000 U/Kg food) for 6 weeks. Blood pressure, plasma malondialdehyde (MDA), tissue NO synthase (NOS) isoforms, tissue nitrotyrosine (the footprint of NO interaction with ROS), and vascular tissue NO production were determined. CRF resulted in marked elevations of blood pressure, plasma MDA, and tissue nitrotyrosine abundance, but did not change plasma L-arginine level. This was coupled with depressed vascular tissue NO production and reduced immunodetectable NOS proteins in the vascular, renal, and cardiac tissues. Antioxidant therapy ameliorated the CRF-induced hypertension, improved vascular tissue NO production, lowered tissue nitrotyrosine burden, and reversed downregulations of NOS isoforms. In contrast, antioxidant therapy had no effects in the controls. CRF is associated with oxidative stress which promotes NO inactivation by ROS leading to functional NO deficiency, hypertension, and widespread accumulation of protein nitration products. Amelioration of oxidative stress by high-dose vitamin E enhances NO availability, improves hypertension, lowers protein nitration products, and increases NOS expression and vascular NO production in CRF animals.

Inhibition of nitric oxide production in tissues of γ-irradiated mice by phenythoin

Phenytoin diminished production of nitric oxide stimulated by lethal γ-irradiation in mouse liver, intestinal mucosa, lungs, heart, and brain 6 and 24 h postirradiation. This inhibition directly depended on the degree of stimulation of nitric oxide production by γ-irradiation in studied tissues.

Nitric oxide synthase expression and nitric oxide production in human colon carcinoma tissue.

Nitric oxide (NO), the production of which is dependent on NO synthase (NOS), has been shown to contribute to various pathogeneses in cancer. The aim of this study was to determine whether inducible NO synthase (iNOS) is overexpressed in human colon carcinoma tissue, and whether NO is produced in tumor tissue. We investigated iNOS mRNA expression in 24 human colon carcinoma tissue specimens by reverse transcription-polymerase chain reaction (RT-PCR). We then examined the expression of iNOS protein and nitrotyrosine, which indicates NO production in tissue, by immunohistochemistry. The possible immunosuppressive role of NO produced by colon carcinoma cells was analyzed in vitro. Semiquantitative RT-PCR analysis showed that iNOS mRNA expression in carcinoma tissues is elevated significantly compared to that in noncarcinoma tissue. Immunohistochemistry revealed that iNOS and nitrotyrosine are expressed strongly in carcinoma tissues. In vitro experiments showed that the supernatant from a culture of cytokine-treated colon carcinoma cells, which contained high levels of NO, significantly reduced the phytohemagglutinin (PHA)-stimulated, human lymphocyte proliferative response (60% of the control value). In human colon carcinoma tissue, iNOS mRNA, protein, and NO products are overexpressed and may contribute to tumor-related immunosuppression.

Direct detection of tissue nitric oxide in septic mice.

Although nitric oxide (NO) is a central mediator during endotoxin-induced sepsis, direct detection of tissue NO in vivo, has until recently been difficult, and techniques have relied on indirect measurement of bi-products in blood or invasive technology. We have utilized electron paramagnetic resonance (EPR) in conjunction with the spin-trapping technique to detect NO directly, and non-invasively, from the tissue of septic mice. Relative signal intensity arising from NO complexed with iron and diethyldithiocarbamate (DETC) measured directly from the liver and kidney of mice given endotoxin was maximal at 6 hours post endotoxin. We failed to detect an EPR signal from mice given pyrogen-free saline. The quality of the EPR signal obtained (high signal to noise ratio of 15:1) using our experimental set-up and L-band EPR hardware was such that we were able to establish a time course of NO production in tissue following endotoxin, and measurement of NO from other organs (kidney and spleen). Our EPR results probably reflected NO arising from inducible NO-synthase enzymes as a result of endotoxin stimulation. This technique was extended to experiments in which we first implanted an oxygen sensitive material (gloxy) into the liver of mice, and then monitored NO production following endotoxin. Due to the fact that the EPR spectrum from gloxy and that of NO-Fe-(DETC)2 do not overlap, we were able to monitor NO production and pO2 simultaneously in tissue, in real time.

Correlation of in vivo nitric oxide and cGMP with glutamate/glutamine metabolism in the rat striatum

We have examined how the suppression of endogenous production of nitric oxide (NO) in the striatal tissue affects release of glutamate (GLU) and glutamine (GLN) in pentobarbital-anesthetized male Sprague-Dawley rats. For the quantitative measurement of tissue NO production and amino acid release, an in vivo assay system for extracellular nitrite (NO 2 −) and amino acids was employed using an in vivo microdialysis technique. An NO synthase inhibitor ( N G-nitro- l-arginine methyl ester, l-NAME) in concentrations ranging between 4–40 mM was perfused into the rat striatum using the assay system. Tissue NO production was found to be inversely proportional to the l-NAME concentration. l-NAME likewise decreased striatal levels of GLU and GLN. Furthermore, tissue NO production showed a positive correlation with GLU ( R = 0.62, P < 0.02) and GLN ( R = 0.86, P < 0.001) concentrations. Exogenous application of NO and cGMP by intrastriatal perfusion with 0.1–2.5 mM hydroxylamine and 0.4–10 mM 8-bromo-cGMP, respectively, increased striatal GLU release in a dose-related manner. Hydroxylamine reduced GLN release, and 8-bromo-cGMP showed a tendency to decrease GLN. In conclusion, striatal GLU/GLN metabolism is a function of the tissue concentration of NO. Normal endogenous concentration of NO causes GLU to be released at a consistent basal level, and enhanced tissue NO production facilitates GLU release via pathways including cGMP formation. We hypothesize that NO may suppress GLN formation by astrocytes.

Correlation of in vivo nitric oxide and cGMP with glutamate/glutamine metabolism in the rat striatum

We have examined how the suppression of endogenous production of nitric oxide (NO) in the striatal tissue affects release of glutamate (GLU) and glutamine (GLN) in pentobarbital-anesthetized male Sprague-Dawley rats. For the quantitative measurement of tissue NO production and amino acid release, an in vivo assay system for extracellular nitrite (NO 2 − ) and amino acids was employed using an in vivo microdialysis technique. An NO synthase inhibitor ( N G -nitro- l -arginine methyl ester, l -NAME) in concentrations ranging between 4–40 mM was perfused into the rat striatum using the assay system. Tissue NO production was found to be inversely proportional to the l -NAME concentration. l -NAME likewise decreased striatal levels of GLU and GLN. Furthermore, tissue NO production showed a positive correlation with GLU ( R = 0.62, P < 0.02) and GLN ( R = 0.86, P < 0.001) concentrations. Exogenous application of NO and cGMP by intrastriatal perfusion with 0.1–2.5 mM hydroxylamine and 0.4–10 mM 8-bromo-cGMP, respectively, increased striatal GLU release in a dose-related manner. Hydroxylamine reduced GLN release, and 8-bromo-cGMP showed a tendency to decrease GLN. In conclusion, striatal GLU/GLN metabolism is a function of the tissue concentration of NO. Normal endogenous concentration of NO causes GLU to be released at a consistent basal level, and enhanced tissue NO production facilitates GLU release via pathways including cGMP formation. We hypothesize that NO may suppress GLN formation by astrocytes.