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Epidemiological and clinical studies have reported that olive oil reduces the incidence of cardiovascular disease. However, the mechanisms involved in this beneficial effect have not been delineated. The endothelium plays an important role in blood pressure regulation through the release of potent vasodilator and vasoconstrictor agents such as nitric oxide (NO) and endothelin-1 (ET-1), respectively, events that are disrupted in type 2 diabetes. Extra virgin olive oil contains polyphenols, compounds that exert a biological action on endothelial function. This study analyzes the effects of olive oil polyphenols on endothelial dysfunction using an in vitro model that simulates the conditions of type 2 diabetes. Our findings show that high glucose and linoleic and oleic acids decrease endothelial NO synthase phosphorylation, and consequently intracellular NO levels, and increase ET-1 synthesis by ECV304 cells. These effects may be related to the stimulation of reactive oxygen species production in these experimental conditions. Hydroxytyrosol and the polyphenol extract from extra virgin olive oil partially reversed the above events. Moreover, we observed that high glucose and free fatty acids reduced NO and increased ET-1 levels induced by acetylcholine through the modulation of intracellular calcium concentrations and endothelial NO synthase phosphorylation, events also reverted by hydroxytyrosol and polyphenol extract. Thus, our results suggest a protective effect of olive oil polyphenols on endothelial dysfunction induced by hyperglycemia and free fatty acids., (© 2014 Published by Elsevier Ltd.)

Aim: To examine the relevance of hypoxia inducible factor (HIF-1) and nitric oxide (NO) on the preservation of fatty liver against cold ischemia-reperfusion injury (IRI)., Methods: We used an isolated perfused rat liver model and we evaluated HIF-1alpha in steatotic and non-steatotic livers preserved for 24 h at 4 degrees C in University of Wisconsin and IGL-1 solutions, and then subjected to 2 h of normothermic reperfusion. After normoxic reperfusion, liver enzymes, bile production, bromosulfophthalein clearance, as well as HIF-1alpha and NO [endothelial NO synthase (eNOS) activity and nitrites/nitrates] were also measured. Other factors associated with the higher susceptibility of steatotic livers to IRI, such as mitochondrial damage and vascular resistance were evaluated., Results: A significant increase in HIF-1alpha was found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage. Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters. These benefits were enhanced by the addition of trimetazidine (an anti-ischemic drug), which induces NO and eNOS activation, to IGL-1 solution. In normoxic reperfusion, the presence of NO favors HIF-1alpha accumulation, promoting also the activation of other cytoprotective genes, such as heme-oxygenase-1., Conclusion: We found evidence for the role of the HIF-1alpha/NO system in fatty liver preservation, especially when IGL-1 solution is used.

Objectives: To evaluate the presence of oxidative stress and alterations in the levels of two cytoprotective agents, prostaglandin E2 and nitric oxide, in the gastrointestinal tract of aging rats., Methods: The production of superoxide anion, lipid peroxides, levels of superoxide dismutase and catalase, and production of prostaglandin E2 and nitric oxide in the stomach and duodenum of rats were determined at 1.5, 3, 12, 18 and 24 months of age., Key Findings: Oxidative stress was present in the stomach of the old rats (24 months), whereas prostaglandin E2 and nitric oxide production remained stable at 18 and 24 months. In the duodenum, no oxidative stress was observed at 24 months, but at 18 months, an increase in superoxide anion levels was detected. Prostaglandin E2 remained constant in the aged rats but nitric oxide decreased significantly at 24 months., Conclusions: The absence of macroscopic gastric injury throughout the gastrointestinal tract indicates that the oxidative stress in the stomach and the significant decrease of nitric oxide in the duodenum in the old rats are not sufficient to disrupt the mucosal defence network. The results support the notion that the disruption of the mucosal network is essentially regulated by the cytoprotective agents prostaglandin E2 and nitric oxide, and that injury appears only when both substances are concurrently reduced.

In spite of their high oxidisability, long-chain n-3 PUFA protect against CVD. Dietary fatty acids modulate the fatty acid composition of lipoproteins involved in atherosclerosis. We thought that if long-chain n-3 PUFA were able to increase NO production by the aorta, then by its antioxidant activity the NO will prevent lipid peroxidation. However, the beneficial effect of NO in vivo on VLDL + LDL oxidation would only be possible if NO could diffuse to their lipidic core. Rats were fed maize oil- or fish oil as menhaden oil- (MO) rich diets for 8 weeks, to study the effects of MOon aortic NO production, NO diffusion into VLDL + LDL, the extent of oxidation in native VLDL + LDL and their oxidisability ex vivo. Aortic NO production and its alpha-tocopherol content were increased and n-3 PUFA were incorporated into the VLDL + LDL. In spite of the higher peroxidisability and the low alpha-tocopherol in native VLDL + LDL from rats fed MO, native VLDL + LDL from the two groups shared similar electrophoretic patterns, conjugated dienes, thiobarbituric acid-reactive substances, total antioxidant capacity, and NO diffusibility on VLDL + LDL, indicative of an in vivo protection against oxidation. However, these results do not correlate with the ex vivo oxidisability of VLDL+ LDL, as NO is lacking. Thus, the in vivo beneficial effects can be explained by increased a-tocopherol in aorta and by a compensatory effect of NO onVLDL + LDL against the low alpha-tocopherol levels, which may contribute to the anti-atherogenic properties of fish oil.

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), the neuronal blocker tetrodotoxin (TTX, 10(-6) M), or both on spontaneous contractions and on the production of basal NO. Both L-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by L-NAME and was inhibited by 38% by TTX. When tested together, L-NAME in the presence of TTX or TTX in the presence of L-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.

Previous research has described how high cellular metabolism creates an acidic environment in inflammatory cells during respiratory burst. The aim of our work was to describe the acid-base dependence of exudate in superoxide (O2.-) and nitric oxide (NO.) generation by inflammatory cells from a carrageenan-granuloma. Although the carrageenan solution was alkaline (pH 7.74 when equilibrated with air) the exudate showed an acidification that stabilised at around 7 units of pH. A notable hypercapnia, but not hypoxia, was found in the exudate at up to 24 h. The effect of extracellular acidosis on O2.- and NO. production by inflammatory cells was also studied. The maximum O2.- production and the lowest levels of NO. were found at pH 7, which was closer to the pH of the granuloma-pouch. These results suggest that experiments with inflammatory cells ex vivo should be carried out at an identical pH to that found in vivo in order to reproduce the physiological mechanisms of free radical generation during inflammatory processes.

1. Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide ((*)NO) is a vasodilator and polyphenolic compounds induce endothelium-dependent vasorelaxation in vitro. 2. Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine-induced vasorelaxation and whether ingestion of DRW-, quercetin- or catechin-rich diets modifies the (*)NO-cyclic guanosine-3',5'-monophosphate (cyclic GMP) pathway and superoxide anion (O2(.-)) release in aorta in a resting state in rats fed semi-purified diets containing either 35% (v w(-1)) DRW, 0.3% (w w(-1)) quercetin or 0.3% (w w(-1)) catechin for 10 days. 3. (*)NO-mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW-rich diet than in those that received the control diet. 4. Expression of endothelial (*)NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW-, quercetin-, or catechin-rich diets showed higher NOS activity, (*)NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2(.-) production. 5. In summary, diets rich in either DRW, quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of (*)NO production, without modifying O2(.-) generation, thus the bioavailability of (*)NO was increased. The increase in the (*)NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.

We studied the roles of nitrogen monoxide (NO&z.rad;) and peroxynitrite produced by the polymorphonuclear leukocytes (PMNs) isolated from an inflammatory exudate. PMNs were incubated either in a medium with a submicromolar concentration of iron or in a diethylenetriaminepenta-acetic acid (DTPA)-containing medium, and stimulated with phorbol 12-myristate 13-acetate (PMA) to generate free radicals. In both conditions superoxide anion (O(2)(*)(-)), NO&z.rad; and peroxynitrite were produced. In the presence of arachidonic acid, malondialdehyde (MDA) was generated. This MDA was generated in one of two way; the peroxynitrite iron-independent mechanism (40%) and the Fenton reaction, caused by free iron (60%). We also observed that the addition of L-arginine was followed by a 42% reduction in MDA, which can be explained by the antioxidant effect of NO&z.rad;. These results indicate that lipid peroxidation can occur in the absence of iron, through a peroxynitrite-mediated mechanism, and that NO&z.rad; may act as an antioxidant when it is produced in large amounts.

Nitric oxide (NO.) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO. in vivo in an acute model of inflammation and evaluated the interactions between NO. and superoxide anion (O2-.) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 mumol of L-N5-(1-iminoethyl)ornithine (L-NIO group), or 6 mumol of L-arginine (L-arginine group) into the granuloma pouch induced by carrageenan in rats. NO2- plus NO3- (indicative of NO. generation) was 188 nmol in the exudate of the control group, but it decreased in the L-NIO group (P < 0.05) and increased in the L-arginine group (P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion (O2-.) decreased by approximately 46% in the L-arginine group. Furthermore, O2-. was inhibited in PMNs when L-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO. in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of O2-. production for cell-mediated injury.

Superoxide and nitric oxide release by leukocytes has been usually performed after exposure to a particular stimulus. We measured the generation of superoxide and nitric oxide by cells isolated from an inflammatory exudate of rats in either the absence or the presence of a variety of stimuli. Nonstimulated leukocytes generated superoxide radical (1.2 nmol x 10(6) cells(-1)) and nitric oxide (3.8 nmol x 10(6) cells(-1)) after 2 h incubation. When cells were incubated with lipopolysaccharides, opsonized zymosan or phorbol 12-myristate 13-acetate, superoxide level increased while nitric oxide decreased. Phorbol 12-myristate 13-acetate (100 ng/ml) induced a decrease of 0.88 nmol x 10(6) cells(-1) compared with nonstimulated cells, and incubation with N-iminoethyl-L-ornithine increased superoxide production by 0.81 nmol x 10(6) cells(-1). These results provide clear evidence that cells from an inflammatory exudate which are already triggered are able to generate a considerable amount of nitric oxide and in less proportion superoxide, that the measure of nitric oxide must be performed without a further stimulus, and that both molecules react in an equimolar proportions to give peroxynitrite anion.

It has recently been shown that peroxynitrite anion is a powerful oxidant than can initiate lipid peroxidation. As this oxidant is the product of the reaction between nitric oxide and superoxide, we have studied whether cells isolated from an inflammatory exudate can release both radicals simultaneously under physiological conditions. The carrageenin-induced granuloma model in rats was used. Cells from the inflammatory pouch were stimulated with opsonized zymosan in the absence or in the presence of exogenous L-arginine. Nitric oxide production without exogenous L-arginine was detectable after 15 min (0.29 nmol NO2-) and increased with time (1.65 nmol NO2- at 4 h). When nitrite released from cells was expressed as a rate a burst was shown in the first few minutes. Between 0 and 15 min, cells produced NO2- at the following rates: 20 pmol NO2-/1 x 10(6) cells/min without exogenous L-arginine and 83 pmol NO2-/1 x 10(6) cells/min with exogenous L-arginine. Production was further stimulated with opsonized zymosan (92 pmol NO2-/1 x 10(6) cells/min), and inhibited by L-NMMA and L-NIO. The production of superoxide increased for up to 2 h and then stabilized. A significant increase in nitrite was observed in the presence of SOD, whereas L-NIO increased superoxide generation. These results suggest that peroxynitrite anion may be formed by inflammatory cells.