Basal Nitric Oxide Availability Research Articles

1,4-Anhydro-4-seleno-d-talitol (SeTal) protects endothelial function in the mouse aorta by scavenging superoxide radicals under conditions of acute oxidative stress

Hyperglycaemia increases the generation of reactive oxidants in blood vessels and is a major cause of endothelial dysfunction. A water-soluble selenium-containing sugar (1,4-Anhydro-4-seleno-d-talitol, SeTal) has potent antioxidant activity in vitro and is a promising treatment to accelerate wound healing in diabetic mice. One possible mechanism of SeTal action is a direct effect on blood vessels. Therefore, we tested the hypothesis that SeTal prevents endothelial dysfunction by scavenging reactive oxidants in isolated mouse aorta under conditions of acute oxidative stress induced by hyperglycaemia. Aortae were isolated from C57BL/6 male mice and mounted on a wire-myograph to assess vascular function. In the presence of a superoxide radical generator, pyrogallol, 300μM and 1mM of SeTal effectively prevented endothelial dysfunction compared to other selenium-containing compounds. In a second set of ex vivo experiments, mouse aortae were incubated for three days with either normal or high glucose, and co-incubated with SeTal at 37°C in 5% CO2. High glucose significantly reduced the sensitivity to the endothelium-dependent agonist, acetylcholine (ACh), increased superoxide production and decreased basal nitric oxide (NO) availability. SeTal (1mM) co-treatment prevented high glucose-induced endothelial dysfunction and oxidative stress in the mouse aorta. The presence of a cyclooxygenase inhibitor, indomethacin significantly improved the sensitivity to ACh in high glucose-treated aortae, but had no effect in SeTal-treated aortae. Our data show that SeTal has potent antioxidant activity in isolated mouse aortae and prevents high glucose-induced endothelial dysfunction by decreasing superoxide levels, increasing basal NO availability and normalising the contribution of vasoconstrictor prostanoids.

High‐fat Diet Causes Loss of Nitric Oxide Motor Neurons and Impairs Inhibitory Neuromuscular Communication in the Mouse Distal Colon

High‐fat (HF) diet increases the risks of gastrointestinal (GI) motility complications, however, the mechanisms that underlie these complications are still not entirely understood. We tested the hypothesis that HF diet causes oxidative stress, loss of nitric oxide (NO) motor neurons in the myenteric plexus, disrupted inhibitory neuromuscular communication and impaired GI motility.MethodsDistal colon segments from mice fed a control (10 Kcal%) or HF diet (60 Kcal%) were used in vitro. Alexa fluor 546 and 680 conjugated maleimides were used to assess the oxidized/reduced glutathione ratio in myenteric NO neurons. NO neuronal density was assessed using nNOS immunohistochemistry. Basal NO availability was measured using NO induced fluorescence of DAF‐FM diacetate. Electrical field stimulation was applied across varying voltages (60‐120V) and train (200‐1000ms) durations to evoke inhibitory junction potentials (IJPs) in colonic circular smooth muscle cells. Amplitude and area of IJP were examined to assess fast and slow IJPs respectively.ResultsWe observed an increase (17%, p<0.01) in oxidized/reduced glutathione fluorescence ratio in nNOS neurons of HF mice. HF mice had lower (28%, p<0.01) nNOS neurons per ganglionic area than controls. Fluorescence intensity of DAF‐FM diacetate was also lower (20%, p<0.01) in HF mice. HF mice showed a significant loss (52%, p<0.05) in IJP area, when stimulated at higher train duration (800‐1000ms).ConclusionWe conclude that HF diet causes oxidative stress, leading to loss of inhibitory NO neurons, lower NO availability, and disrupted neuromuscular transmission in the mouse distal colon. Supported by NIH (DK094932).

Dissecting the genetic predisposition to albuminuria and endothelial dysfunction in a genetic rat model

The Munich Wistar Frömter (MWF) rat develops progressive spontaneous albuminuria largely attributable to quantitative trait loci on rat chromosome (RNO)6 and RNO8, respectively. We tested the hypothesis whether quantitative trait loci on these chromosomes are linked to both albuminuria and endothelial dysfunction. Experiments were performed in male 12-week-old MWF, Wistar Kyoto (WKY), spontaneously hypertensive rat (SHR) and consomic MWF-6 and MWF-8 rats, in which RNO6 or RNO8 was replaced by the respective SHR chromosome (n = 10 per strain). Vascular function was assessed in aorta and vascular superoxide anion production was determined by confocal microscopy. Acetylcholine potency to induce relaxation was significantly reduced in MWF (6.2 ± 0.1) compared with WKY (7.1 ± 0.1) or SHR (7.3 ± 0.1; P < 0.01). N-nitro-L-arginine methyl ester abolished relaxation to acetylcholine in all three strains, whereas indomethacin exhibited no effect in WKY and MWF. Contractions to noradrenaline and superoxide production were significantly higher in MWF compared with SHR and WKY (P < 0.05, respectively). In consomic MWF-6 and MWF-8 rats albuminuria was markedly suppressed (-85 and -92%, P < 0.005 compared with MWF, respectively). Interestingly, relaxation to acetylcholine and contraction to noradrenaline were restored to normal WKY levels only in MWF-8, but not in MWF-6, due to an increase in stimulated and basal nitric oxide availability, respectively. These data demonstrate the partial genetic independence of albuminuria and endothelial dysfunction in this model. From two known albuminuria quantitative trait loci one locus affects both albuminuria and endothelial dysfunction. Whether this observation is based on a common genetic mechanism related to NO availability warrants further investigation.

Increased Vascular Contractility and Oxidative Stress in β2-Adrenoceptor Knockout Mice: The Role of NADPH Oxidase

Background/Aims: β₂-adrenoceptor (β₂-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal β₂-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional β₂-AR (β₂KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from β₂KO and wild-type mice (WT) were studied. β₂KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between β₂KO and WT mice. Basal NO availability was reduced in aortas from β₂KO mice. Incubation of β₂KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. β₂KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47^phox expression was enhanced in β₂KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of β₂KO mice. This study extends the knowledge of the relevance of the endogenous activity of β₂-AR to the maintenance of the vascular physiology.

Increased basal nitric oxide release despite enhanced free radical production in hypertension

Although in hypertension a defect in stimulated nitric oxide (NO) is well established, little is known about basal NO levels. Thus, we measured directly in vessels from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHR) both basal and stimulated NO production using a novel technique [4,5-diaminofluorescein (DAF-2) fluorescence]. Isolated vessels were exposed to the fluorescent probe DAF-2. After the technique was validated with increasing doses of acetylcholine in the presence and absence of NG-nitro-L-arginine methyl ester (l-NAME), we measured NO production in vessels from WKY rats and SHR in the same experimental setting. Finally, to explore the impact of reactive oxygen species (ROS) on NO release, we analysed the effect of an antioxidant, such as ascorbic acid, on basal and stimulated NO in aortic rings of WKY rats and SHR. Aortic rings from SHR exhibited a higher basal NO production and a lower responsiveness to agonist-induced NO release as compared with those observed in WKY rats. Also in resistance vessels such as mesenteric arteries, basal NO production was higher in hypertension. In hypertensive rats, ascorbic acid was able to further increase basal NO release and recovered the impaired stimulated NO production, whereas no effect was detected in normotensive rats. Our data reveal an increased basal NO availability in hypertension despite the increased production of ROS, suggesting a greater complexity in hypertensive endothelial dysfunction when the analysis is focused on direct NO measurement.

Aprotinin impairs endothelium-dependent relaxation in rat aorta and inhibits nitric oxide release from rat coronary endothelial cells.

Aprotinin, a non-specific serine protease inhibitor, reduces postoperative bleeding after coronary artery surgery. The mechanism of action for this 'blood-sparing' effect of aprotinin is only partially clarified. We therefore aimed to investigate the effect of aprotinin on the release of nitric oxide (NO), a vasodilator and antiaggregant factor, from rat coronary microvascular endothelial cells and on the NO-mediated endothelium-dependent relaxation of rat thoracic aorta. Endothelium-intact and endothelium-denuded thoracic aortic rings from Wistar rats (250-300 g) were suspended in organ chambers. Contractile and relaxant responses in the absence and presence of aprotinin (125, 250 and 500 KIU/ml) were recorded via a mechanotransducer. Coronary microvascular endothelial cells (CMEC) were isolated on a Langendorff system by collagenase perfusion of the hearts from the same rats. Calcium ionophore- (1 microM) induced release of NO from confluent cells was determined spectrophotometrically by measuring its stable metabolites, nitrite and nitrate, via Griess reaction. Aprotinin selectively enhanced phenylephrine-induced contractions in endothelium-intact rat thoracic aortic rings, but not in the endothelium-denuded rings. The use of a nitric oxide synthesis inhibitor Nomega-nitro-L-arginine methyl ester (100 microM) on endothelium-intact rings produced a similar increase in phenylephrine-induced contractions. KCl-induced contractions remained unaltered. Aprotinin inhibited acetylcholine-, calcium ionophore- and L-arginine-induced endothelium-dependent relaxations, but not sodium nitroprusside-induced endothelium-independent relaxation. Aprotinin had no significant effect on basal nitrite-nitrate release from CMEC, while it inhibited calcium ionophore-induced total nitrite accumulation in the supernatants. Aprotinin selectively impairs endothelium-dependent relaxation as well as basal NO availability in rat thoracic aortic rings and inhibits NO release from rat CMEC. This effect of the drug may contribute to its 'blood-sparing' action and may also account for the increase in perioperative restenosis risk observed in clinical practice during aprotinin therapy.

Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP.

Previous studies from our group have shown a deficit in nitric oxide (NO) bioavailability and an excess production of the superoxide anion (O(2)(-)) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the NO/O(2)(-) balance, thereby improving endothelial function. A recombinant adenovirus, Ad/Hu/eNOS, containing the human eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into SHRSP carotid arteries in vivo, using a titre of 2x10(9)-2x10(10) plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. Western blotting confirmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L-NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insignificant increase in NO bioavailability. Our results support our previous findings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a significant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function.