Oxide Release In Response Research Articles

Role of Endothelial STAT3 in Cerebrovascular Function and Protection from Ischemic Brain Injury.

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood–brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke.

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μM, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μM RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm2 shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.

Meta-Analysis of Environmental Impacts on Nitrous Oxide Release in Response to N Amendment

Atmospheric nitrous oxide (N2O) accounts for approximately 5% of the global greenhouse effect and destroys stratospheric ozone. Soils are the most important source of N2O, which is produced during nitrification and denitrification. To assess the impact of environmental variables and ecosystems on N2O flux, we performed a meta-analysis comparing N2O flux in N amended and matched control plots in non-agricultural soils. We found that N2O release increased with N amendment in the short term. Although there were few studies in shrubland, this ecosystem showed the greatest response. The N2O response to N amendment was greater in year-round studies and in studies with more measurements, but lower in longer studies. The N2O response was greater at higher latitudes and precipitation rates. We also observed an unexpected 55% decline in the N2O response to N amendment over the 23 years covered by the studies. This pattern may reflect a suppression of the N2O response from long-term N deposition accumulation, particularly in temperate regions. Although short term increases in reactive N entering natural systems may cause positive feedbacks to the release of N2O, this effect may diminish over time in locations with high rates of N deposition.

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm 2 shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell–matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.

Effect of the 894G>T polymorphism of the endothelial nitric oxide synthase on vascular reactivity following maximal dynamic exercise

Considering that the role of nitric oxide as a vasodilator is increased after an acute bout of exercise and that the 894G>T polymorphism of the endothelial nitric oxide synthase seems to reduce the nitric oxide release in response to shear stress, the present study investigated the 894G>T polymorphism in relation to vascular reactivity following maximal dynamic exercise. We studied 110 healthy volunteers (wild-type group 45.5% and polymorphic group 54.5%). The protocol included vascular reactivity assessment at baseline and during reactive hyperemia, before, 10, 60 and 120 min after a maximal cardiopulmonary exercise test. Genomic DNA was extracted from blood samples to determine the 894G>T polymorphism. There were no differences between the wild-type and polymorphic groups concerning anthropometric, metabolic and hemodynamic characteristics. Blood flow, before maximal exercise, was similar between the wild-type and the polymorphic groups. The polymorphic group presented lower vascular reactivity regardless of time (P = 0.019 for group main effect), and posthoc analysis revealed that polymorphic patients had lower values than wild-type only at the 120 min measurement (P = 0.002). Concerning within-group analysis, vascular reactivity increased at 10 min after exercise (P = 0.029) returning to baseline at 120 min (P = 0.005) in the polymorphic group. Patients with the 894G>T polymorphism had lower vascular reactivity after a single bout of exercise.

Pulse of nitric oxide release in response to activation of N-methyl-D-aspartate receptors in the rat striatum: rapid desensitization, inhibition by receptor antagonists, and potentiation by glycine.

Increased activity of glutamate N-methyl-d-aspartate (NMDA) receptors is the dominant mechanism by which nitric oxide (NO.) is generated. By using a selective direct-current amperometry method, we characterized real time NO* release in vivo in response to chemical stimulation of NMDA receptors in the rat striatum. The application of NMDA caused the appearance of a sharp and transient oxidation signal. Concentration-response studies (10-500 microM) indicated an EC(50) of 48 microM. The NMDA-induced amperometric signal was suppressed by focal application of the nitric-oxide synthase inhibitor L-nitro-arginine methyl ester (L-NAME, 100 microM) or D-(-)-2-amino-5-phosphonopentanoic acid (AP-5, 100 microM) or by systemic injection of dizocilpine (1 mg/kg i.p.), drugs that, when given alone, had no effect on baseline oxidation current. Repeated injections of NMDA at short intervals (approximately 80 s) resulted in a progressive reduction of the amperometric signal with a decay half-life of 1.36 min. Sixty min after the last NMDA application the amperometric response was restored to initial levels. Finally, the coapplication of glycine (50 or 100 microM), which, when given alone had no effect, potentiated the NMDA-induced response. Thus, NMDA receptor-mediated activation of striatal NO* system shuts off quickly and undergoes rapid desensitization, suggesting a feedback inhibition of NMDA receptor function. To the extent of NO* release can represent a correlate of NMDA receptor activity, its amperometric detection could be useful to assess in vivo the state of excitatory transmission under physiological, pharmacological, or pathological conditions.

Assessment of the intestinal permeability after a gastrectomy and the oral administration of anticancer drugs in rats: Nitric oxide release in response to gut injury

Although intestinal permeability is known to increase in conditions of stress (such as endotoxemia, septic shock, and ischemia-reperfusion), the mechanism of gut barrier dysfunction during chemotherapy remains to be elucidated. We designed an experiment in which a gastrectomy and anticancer drugs were administered to rats to assess the extent of damage or an increase in permeability of the intestinal mucosa. The rats were separated into the following groups: group A, no operation without an anticancer drug (control rats); group B, a gastric operation; group C, chemotherapy; and group D, a gastric operation followed by chemotherapy. Six rats were placed in each group. The groups that were given the anticancer drug received 45 mg/kg for 7 and 14 days, respectively. Intestinal permeability was determined by an injection of fluorescein isothiocyanate-dextran (FITC-D) intravenously and the measurement of the amount of FITC-D leakage in the luminal lavage. The plasma nitric oxide concentrations were measured spectrophotometrically by the Griess reaction. The cell surface expression of CD44 in the rat small intestine was evaluated immunohistochemically. During the entire experimental period, the FITC-D levels in the anticancer drug that was administrated to rats (groups C and D) were significantly higher than the levels in groups A and B (P <.001). Similarly, the plasma nitric oxide concentration increased significantly not only in groups C and D but also in group B (P <.05). A linear regression analysis revealed a positive significant correlation between FITC-D and the plasma nitric oxide levels (r = 0.617; P <.001). When the anticancer drug was administered, the intestine histologically revealed so-called mucosal atrophy and a decreased expression of CD44 within the intestinal mucosa. The administration of anticancer drugs impairs the gut barrier function, possibly by reducing the cell-cell and cell-matrix interactions. This may contribute to the development of hyperpermeability that is induced by an overexpression of nitric oxide.

Agonist-induced release of nitric oxide during acute exposure to nicotine

While we have shown that acute infusion of nicotine impairs agonist-induced dilatation of resistance arterioles (Am.J.Physiol. 272:H2337–H2342, 1997), no studies have examined the release of nitric oxide in response to these agonists before and during treatment with nicotine. Thus, the first goal of the present study was to examine agonist-induced release of nitric oxide by the hamster cheek pouch microcirculation under control conditions and during acute infusion of nicotine. We measured the release of nitric oxide (Sievers NO analyzer) in response to repeated topical application of acetylcholine (1.0 uμM) and 5′-adenosine diphosphate (ADP; 1.0 μM) during infusion of vehicle and during infusion of nicotine (2.0 μg/kg/min iv for 30 minutes followed by a maintenance dose of 0.35 μg/kg/min). In hamsters treated with vehicle, topical application of acetylcholine and ADP elicited reproducible increases in nitric oxide release. In contrast, in hamsters treated with nicotine, there was a marked inhibition of nitric oxide release in response to acetylcholine and ADP. In a previous study (J.Appl.Physiol. 85:1292–1298, 1998) we found that treatment of the hamster cheek pouch microcirculation with superoxide dismutase restored unpaired agonist-induced vasodilatation during acute infusion of nicotine. Thus, our second goal was to examine whether Superoxide dismutase would restore agonist-induced release of nitric oxide during infusion of nicotine. We found that treatment of the hamster cheek pouch microcirculation with superoxide dismutase prior to infusion of nicotine prevented nicotine-induced impairment of nitric oxide release in response to acetylcholine and ADP. We suggest that nicotine alters dilatation of arterioles via an increased release of superoxide anion and subsequent inactivation of nitric oxide.

Involvement of endothelium/nitric oxide in vasorelaxation induced by purified green tea (−)epicatechin

The present study investigated the involvement of endothelial nitric oxide in relaxation induced by purified green tea (−)epicatechin in rat isolated mesenteric arteries. (−)Epicatechin caused both endothelium-dependent and -independent relaxation. N G-Nitro- l-arginine methyl ester ( l-NAME, 100 μM) and methylene blue (10 μM) significantly attenuated (−)epicatechin-induced relaxation in endothelium-intact tissues. l-Arginine (1 mM) partially antagonized the effect of l-NAME. (−)Epicatechin-induced relaxation was inhibited by Rp-guanosine 3′,5′-cyclic monophosphothioate triethylamine. In contrast, indomethacin and glibenclamide had no effect. (−)Epicatechin (100 μM) significantly increased the tissue content of cyclic GMP and N G-nitro- l-arginine (100 μM) or removal of the endothelium abolished this increase. (−)Epicatechin (100 μM) induced an increase in intracellular Ca 2+ levels in cultured human umbilical vein endothelial cells. Iberiotoxin at 100 nM attenuated (−)epicatechin-induced relaxation in endothelium-intact arteries and this effect was absent in the presence of 100 μM l-NAME. In summary, (−)epicatechin-induced endothelium-dependent relaxation is primarily mediated by nitric oxide and partially through nitric oxide-dependent activation of iberiotoxin-sensitive K + channels. In addition, there may be a causal link between increased Ca 2+ levels and nitric oxide release in response to (−)epicatechin.

Role of nitric oxide in coronary arterial vasomotion and the influence of coronary atherosclerosis and its risks

Healthy coronary vascular endothelium releases nitric oxide to modulate resting and dynamic coronary arterial tone. We studied the impact of atherosclerosis and/or its risks on endothelial nitric oxide release in response to metabolic stimuli by evaluating coronary vasomotor responses to atrial pacing before and after the inhibition of nitric oxide produciton by intracoronary N G-monomethyl-L-arginine (L-NMMA) (20 μmol/min) infusion. The study includes 34 patients (15 with coronary disease, 11 with normal coronary arteries and ≥1 risk factor, and 8 with normal coronary arteries and no risks). Coronary blood flow was derived from Doppler flow velocity (0.018-inch Doppler wire) and coronary diameter. L-NMMA infusion reduced coronary blood flow by 18 ± 16% and coronary diameter by 10 ± 9%. Responses were identical in all subgroups. Coronary blood flow responses to pacing were similar in all subgroups and were unaffected by L-NMMA (11 ± 11 vs 13 ± 9 ml/min; p = 0.26). Epicardial coronary vasodilation to control pacing occurred in patients with normal coronary arteries with (4.0 ± 5.2%, p = 0.01) or without (8.0 ± 5.2%, p = 0.03) risks, but not in patients with coronary disease (2.8 ± 5.9%). L-NMMA abolished pacing-induced epicardial vasodilation in patients without coronary artery disease, producing a 1.8 ± 5.1% response, which was similar in all subgroups. We conclude that microvascular responses to rapid atrial pacing are not mediated by nitric oxide. Flow-mediated epicardial coronary arterial responses may be nitric oxide dependent.

Difference in acetylcholine-induced nitric oxide release of arterial and venous grafts in patients after coronary bypass operations

Objectives: In vivo investigation of nitric oxide release in coronary bypass grafts has not been reported. We studied acetylcholine-induced nitric oxide release in vivo of coronary bypass grafts and vasomotor responses to acetylcholine of grafted coronary arteries in patients after coronary bypass grafting. Methods: We examined 24 internal thoracic artery grafts and 16 saphenous vein grafts in 39 patients. The mean ages of the patients were 65 years for the arterial grafts and 68 years for the venous grafts. Nitric oxide was measured as the plasma nitrite level by the Griess reaction. Before and after intragraft acetylcholine infusion (5 μg), blood was sampled from the distal end of the graft, and angiograms were taken and analyzed by cine-densitometry. Results: The plasma nitrite concentration after stimulation with acetylcholine compared with the control value was 134% ± 52% at 4 minutes ( P = 0.05) and 184% ± 107% at 6 minutes ( P = 0.01) in the arterial grafts; in the venous grafts these values were 101% ± 24% at 4 minutes ( P = 0.96) and 108% ± 36% at 6 minutes ( P = 0.69). Low-dose acetylcholine dilated the coronary arteries supplied by arterial grafts by 6.3% ± 16.6% whereas coronary arteries supplied by venous grafts were reduced by 9.8% ± 11.8% in diameter and the vasoactive responses were different ( P = 0.01). Conclusions: In vivo internal thoracic artery grafts had more endothelium-derived nitric oxide release in response to acetylcholine than did saphenous vein grafts after coronary bypass grafting. (J Thorac Cardiovasc Surg 1998;116:454-9)

The deletion polymorphism of the angiotensin-converting enzyme gene is related to phenotypic differences in human arteries.

We hypothesized that angiotensin-converting enzyme (ACE) insertion/deletion polymorphism may be related to arterial phenotypic differences that could explain the adverse effects of deletion polymorphism. Accordingly, contractile responses to angiotensin I and II (0.1 nmol.1(-1)-1 micromol.1(-1), endothelium-dependent relaxation to methacholine (0.01-100 micromol.1(-1), and the effect of NG-monomethyl-L-arginine (L-NMMA; 100 micromol.1(-1) on phenylephrine (10 micromol.1(-1) induced contraction, were studied in isolated rings of internal mammary arteries obtained from patients undergoing coronary bypass surgery. The results were analysed according to the ACE genotype of the patient (II, n = 8; ID, n = 11; DD, n = 9) as well as the presence/absence of either allele. The arteries from patients with the D allele (ID/DD) displayed a lower sensitivity to methacholine (P < 0.05 vs II), which suggested that the capacity of the endothelium for nitric oxide release in response to stimulation was also lower. By contrast, the increase in phenylephrine-induced contraction, by pre-incubation with L-NMMA, was more pronounced in the group with the DD allele (31 +/- 5%) than with the ID (11 +/- 11%) and II alleles (1 +/- 11%, P < 0.05 vs DD), which suggested a higher level of basal nitric oxide release. Finally, the differences in the responses to angiotensin I and II, which were used to evaluate the vascular conversion of angiotensin I, indicated that the level of angiotensin I conversion was higher in patients with the D allele (ID/DD, P < 0.05 vs II). The findings of this study indicate that ACE insertion/deletion polymorphism is related to arterial phenotypic differences in endothelial function and angiotensin I conversion.

Repeated Intravenous Administration of Liposome-Encapsulated Muramyl Tripeptide-Phosphatidyl-Ethanolamine Diminishes Cytotoxic Potential of Subpopulations of Rat Liver Macrophages

AbstractIn this study we investigated the effects of repeated intravenous (i.v.) administration of liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (lipMTP-PE) on the antitumor functions of rat liver macrophages. Liver macrophage subtractions, differing in cell-size, were isolated by elutriation 24h after the final injection of liposomes and tested for cytotoxicity against radio-labeled C26 adenocarcinoma cells. Prolonged multiple i.v. injections of lipMTP-PE resulted in decreased tumoricidal activity in all but the small-sized subtraction, compared to a single injection.Immunohistochemical staining of liver sections revealed that while the number of liver macrophages increased significantly after multiple lipMTP-PE injections, the expression of the la antigens on these cells decreased. Additionally, multiple lipMTP-PE treatment resulted in a reduced nitric oxide release in response to muramyl dipeptide in vitro, while incubation with another immunomodulator, lipopoly saccharide, resulted...

N-methyl- d-aspartate-induced nitric oxide release: An in vivo microdialysis study

Increasing evidence indicates that nitric oxide acts as an intercellular signal transduction molecule in the nervous system. In particular, in vitro studies have demonstrated that nitric oxide is produced in the cerebellar cortex and is responsible for the increases in cyclic GMP seen in response to glutamate receptor activation. In this study, we have combined the technique of intracerebellar microdialysis with a sensitive assay for nitric oxide oxidation products nitrate and nitrite, to assess nitric oxide release directly in awake, freely moving animals. We have found that infusion of N-methyl- d-aspartate via the microdialysis probe results in a dose-dependent increase in cerebellar nitric oxide release. This increase was prevented by prior administration of an N-methyl- d-aspartate receptor antagonist, or the nitric oxide synthase inhibitor N G-nitroarginine. Both these pretreatments also reduced the basal extracellular nitrite and nitrate levels, suggesting that there is a tonic glutamate-induced nitric oxide production in the cerebellum of awake, freely moving animals. These results provide direct evidence for nitric oxide release in response to N-methyl- d-aspartate receptor activation in the adult cerebellar cortex, in vivo. This new approach, coupling microdialysis with the azo dye detection method of Griess, should thus prove useful for the in vivo study of nitric oxide release from various brain regions in response to pharmacological, physiological or behavioral manipulations.

Nitric Oxide Release in Response to Stimulation of Nonadrenergic, Noncholinergic Nerves

Summary: The nature of the inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter was studied in the canine ileocolonic junction. In a first series of experiments, circular muscle strips were mounted in organ baths filled with aerated Krebs-Ringer solution for isometric tension recording. During a norepinephrine-induced contraction and in the presence of atropine, nitric oxide (NO) induced tetrodotoxin-resistant relaxations that were similar to the NANC relaxations obtained by electrical stimulation and acetylcholine (ACh). Inhibition of the NO biosynthesis by NG-monomethyl-l-arginine (l-NMMA) and NG-nitro-l-arginine (l-NNA) resulted in an increase in basal tension and inhibited the relaxations to electrical stimulation and ACh, but not those to NO. These effects were partly reversed by l-arginine, but not by d-arginine. Hemoglobin also raised the basal tension and reduced the relaxations to electrical stimulation and ACh and abolished those to NO. In a second series of experiments, we demonstrated the release of a labile factor with vasodilator activity upon stimulation of NANC nerves of the canine ileocolonic junction using a superfusion bioassay cascade. The release of this factor was inhibited by tetrodotoxin and l-NNA, whereas it was increased by l-arginine. The biological activity was enhanced by superoxide dismutase and eliminated by hemoglobin. Our results suggest that NO or an NO-releasing substance represents the NANC neurotransmitter in the canine ileocolonic junction.

Nitric Oxide Release in Response to Stimulation of Nonadrenergic, Noncholinergic Nerves

The nature of the inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter was studied in the canine ileocolonic junction. In a first series of experiments, circular muscle strips were mounted in organ baths filled with aerated Krebs-Ringer solution for isometric tension recording. During a norepinephrine-induced contraction and in the presence of atropine, nitric oxide (NO) induced tetrodoxin-resistant relaxations that were similar to the NANC relaxations obtained by electrical stimulation and acetylcholine (ACh).