Effect of low dose L-NAME pretreatment on nitric oxide/reactive oxygen species balance and vasoactivity in L-NAME/salt-induced hypertensive rats.

Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase-dependent reactive oxygen species (ROS) overproduction and decreased nitric oxide (NO) bioavailability lead to vascular dysfunction and development of hypertension. The goal of our study was to analyze an effect of salt diet and NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP), arterial reactivity, NO production, as well as ROS level in adult rats pretreated with low dose of L-NAME (2 mg/kg/day) for three weeks. Higher dose of L-NAME (40 mg/kg/day), or salt diet (8% NaCl), or combination of both were applied for the following four weeks. The administration of L-NAME in low dose had no effect on BP but enhanced the expression of eNOS. Both higher dose of L-NAME and salt diet elevated BP, decreased NOS activity, and impaired the endothelium-dependent arterial relaxation. However, salt diet did not increase ROS production and sympathoadrenergic arterial contractions in low dose L-NAME-pretreated rats. Combination of salt diet with higher dose of L-NAME did not evoke additive decrease of NOS activity, but it caused elevation of conjugated dienes (CD) concentration and NADPH oxidase 2 (Nox-2) protein expression. In conclusion, these findings indicate that chronic low dose of L-NAME treatment has a potential to trigger adapting mechanisms to attenuate some cardiovascular disorders.

Effect of deuterium-depleted water on selected cardiometabolic parameters in fructose-treated rats.

Deuterium-depleted water (DDW) has a lower concentration of deuterium than occurs naturally (less than 145 ppm). While effects of DDW on cancer started to be intensively studied, the effects on cardiovascular system are completely unknown. Thus, we aimed to analyze the effects of DDW (55+/-5 ppm) administration to 12-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) treated with 15 % fructose for 6 weeks. Blood pressure (BP) and selected biochemical parameters were measured together with determination of nitric oxide synthase (NOS) activity and iNOS and eNOS protein expressions in the left ventricle (LV) and aorta. Neither DDW nor fructose had any significant effect on BP in both strains. DDW treatment decreased total cholesterol and triglyceride levels in WKY, but it was not able to prevent increase in the same parameters elevated due to fructose treatment in SHR. Both fructose and DDW increased insulin level in WKY. Fructose did not affect NOS activity either in WKY or SHR. DDW increased NOS activity in LV of both WKY and SHR, while it decreased NOS activity and iNOS expression in the aorta of SHR with or without fructose treatment. In conclusion, DDW treatment significantly modified biochemical parameters in WKY together with NOS activity elevation in the heart. On the other hand, it did not affect biochemical parameters in SHR, but decreased NOS activity elevated due to iNOS upregulation in the aorta.

Effects of provinol and its combinations with clinically used antiasthmatics on airway defense mechanisms in experimental allergic asthma.

Our previous studies show that provinol, a polyphenolic compound, has anti-inflammatory activity during allergic inflammation. In the present study we investigated the effects of provinol and its combinations with clinically used antiasthmatics: budesonide or theophylline on airway defense mechanisms during experimental allergic asthma. Separate groups of guinea pigs were treated during the course of 21-day ovalbumin sensitization with provinol (20 mg/kg/day, p.o.), or budesonide (1 mM by inhalation), or theophylline (10 mg/kg/day, i.p.), and with a half-dose combination of provinol+budesonide or provinol+theophylline. Airways defense mechanisms: cough reflex and specific airway resistance (sRaw) were evaluated in vivo. Tracheal smooth muscle reactivity and mucociliary clearance were examined in vitro. The findings were that provinol caused significant decreases in sRaw and in tracheal smooth muscle contractility, a suppression of cough reflex, and positively modulated ciliary beat frequency. The bronchodilatory and antitussive effects of provinol were comparable with those of budesonide and theophylline. Provinol given as add-on treatment significantly potentiated the effects of budesonide or theophylline, although the doses of each were halved. We conclude that provinol not only has bronchodilatory and antitussive effects, but also potentiates similar effects exerted by budesonide and theophylline.

Short-term administration of alibernet red wine extract failed to affect blood pressure and to improve endothelial function in young normotensive and spontaneously hypertensive rats.

As wine polyphenols were shown to possess many positive effects in mammals, including improvement of vascular function, this study investigated the effect of the Slovak Alibernet red wine extract (AWE) on blood pressure and vascular function in young normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Six weeks old, male, WKY and SHR were treated with AWE for three weeks at the dose of 24.2 mg/kg/day. Blood pressure (BP), determined by tail-cuff plethysmography, was significantly elevated in SHR vs. WKY and AWE failed to affect it. Lipid peroxidation was evaluated by determination of thiobarbituric acid-reactive substances. Vascular function was assessed in rings of the femoral artery using Mulvany-Halpern's myograph. Maximal endothelium-dependent acetylcholine (ACh)-induced relaxation was reduced in control SHR vs. WKY rats by approximately 9.3 %, which was associated with a significant decrease of its NO-independent component. AWE failed to affect maximal ACh-induced relaxation, both its NO-dependent and independent components, compared to controls of the same genotype. AWE however reduced lipid peroxidation in the left ventricle of both WKY and SHR and in the liver of SHR. In conclusion, three-week administration of AWE failed to reduce BP and to improve endothelial function in the femoral arteries of both genotypes investigated.

Effect of natural polyphenols, pycnogenol® on superoxide dismutase and nitric oxide synthase in diabetic rats.

The work is focused on clarifying the impact of diabetes and natural plant polyphenols contained in Pycnogenol® (PYC) on the activity and synthesis of Cu/Zn-SOD and synthesis of nNOS and eNOS in the cerebellum and cerebral cortex in rats with induced diabetes. Rats included in the study (n=38) were divided into three groups: the controls (C), (n=7), untreated diabetics (D) (n=19) and diabetic rats treated with PYC (DP) (n=12). Diabetes significantly decreased synthesis, as well as the activity of Cu/Zn-SOD in both studied parts of the brain. PYC significantly increased the synthesis of Cu/Zn-SOD but had no effect on its activity. Diabetes also reduced the synthesis of nNOS in cerebral cortex and administered PYC elevated its amount to the level of controls. In the cerebellum, diabetes does not affect the synthesis of nNOS and PYC reduces synthesis of NOS. Diabetes as well as PYC had no influence on the synthesis of eNOS in both, the cerebellum and cerebral cortex. PYC modulated level of Cu/Zn-SOD and nNOS in cerebellum and cerebral cortex of diabetic rats, but in a different way.

WITHDRAWN: The efficiency of polyphenolic compounds on allergen induced hyperreactivity of the airways.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.bionut.2010.09.002. The duplicate article has therefore been withdrawn.

A beneficial influence of provinol on the reduction of allergen induced hyperreactivity in guinea pigs.

BACKGROUND: The anti-inflammatory, anti-allergic, antioxidant properties of flavonoids are known in the respiratory tract. We are interested in the role of Provinol during an allergic inflammation of the airway. OBJECTIVES: The aim of this study was to examine the influence of an acute administration of Provinol on tracheal smooth muscle reactivity in guinea pigs and to assess the involvement of nitric oxide in the mechanism of Provinol action. METHODS: This experiment was performed 14 days after the sensitization of animals by ovalbumin. In vivo, the specific airway conductance, as a tracheal smooth muscle reactivity parameter in response to bronchoconstrictor histamine, was evaluated after peroral administration of Provinol alone or together with L-NAME (N(omega)-nitro-L-arginine methyl ester). In vitro, Provinol alone or in combination with L-NAME were added into an organ baths before the supplement of direct bronchoconstrictor histamine, acetylcholine and the allergen ovalbumin in rising concentrations. The amplitude of the tracheal smooth muscle contraction, as a tracheal smooth muscle reactivity parameter in response to histamine, acetylcholine and ovalbumin was evaluated. RESULTS: Our results showed that a Provinol has significant bronchodilatory activities both in vivo and in vitro. CONCLUSION: Provinol alleviated the contraction of tracheal smooth muscle in guinea pigs sumin. Nitric oxide plays an important role in the mechanism of Provinol action (Fig. 2, Ref. 28n.(Fig. 2, Ref. 28).

Red wine polyphenols prevent cyclosporine-induced nephrotoxicity at the level of the intrinsic apoptotic pathway.

Flavonoids, polyphenol derivatives of plant origin, possess a broad range of pharmacological properties. A number of studies have found both pro/anti-apoptotic effects for many of these compounds. For these reasons we investigated whether Provinols flavonoids obtained from red wine, have anti-apoptotic properties. The investigations have been carried out in rats treated with Cyclosporine A (CsA). In particular, four groups of rats have been treated for 21 days with either olive oil (control group), with CsA, with Provinols, or with CsA and Provinols simultaneously. Oxidative stress, systolic blood pressure, body weight, biochemical parameters and different markers of pro/anti-apoptotic pathway were measured. CsA produced an increase of systolic blood pressure, a decrease in body weight, serum creatinine levels, urinary total protein concentration and creatinine clearance. Moreover, CsA induced renal alterations and the translocation of Bax and cytochrome c from cytoplasm to mitochondria and vice versa. These changes activated the caspase cascade pathway, that leads to morphological and biochemical features of apoptosis. Provinols restored morphological and biochemical alterations and prevented nephrotoxicity. In conclusion, this study may augment our current understanding of the controversial pro-/anti-apoptotic properties of flavonoids and their molecular mechanisms.

Red wine polyphenols affect the collagen composition in the aorta after oxidative damage induced by chronic administration of CCl(4).

Increased amount of collagen type I and decreased amount of type III is described in various pathological processes in the vascular wall. Polyphenols were shown to have protective effect on endothelium, decrease blood pressure and prevent oxidative damage induced by various stimuli. Tetrachlormethane (CCl(4)) is a toxic substance with known negative systemic effects induced by free radicals. Chronic administration of CCl(4) for 12 weeks led to an increase of collagen type I and a decrease of type III in the wall of aorta. Parallel administration of red wine polyphenols significantly reduced the increase of collagen type I, at the same time the content of type III rose to the level above controls. After 4 weeks of spontaneous recovery no changes were observed. If polyphenols were administered during the recovery period, there was a decrease of type I and an increase of type III collagen content in the aorta. It can be concluded that polyphenols have a tendency to lower the amount of type I and to increase the proportion of type III collagen in the wall of the aorta. These changes are significant in prevention or in regression of changes induced by chronic oxidative stress. This effect of polyphenols is most likely the result of their influence on MMP-1 and TIMP activities through which they positively influence the collagen types I and III content ratio in the vascular wall in favor of the type III collagen.

Contribution of neuronal nitric oxide (no) synthase to N-acetylcysteine-induced increase of NO synthase activity in the brain of normotensive and hypertensive rats.

The goal of our study was to determine a contribution of nNOS to the increase of brain NO synthase activity induced by chronic N-acetylcysteine (NAC) treatment. Young 4-week-old male Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were subjected to treatment with NAC (1.5 g/kg/day) for 8 weeks. At the end of experiment total NOS activity was determined in the brainstem and cerebellum with and without specific nNOS inhibitor S-methyl-L-thiocitrulline (SMTC, 10(-6) mol/l) by measuring the formation of L-[(3)H] citrulline from L-[(3)H] arginine. Chronic NAC treatment had no effect on blood pressure (BP) of WKY, while it attenuated BP increase in young SHR. Total NOS activity was increased in the brainstem of SHR compared to WKY, but this strain difference was abolished by SMTC. Chronic NAC treatment of SHR increased total NOS activity by 32% in the brainstem and by 67% in the cerebellum. After the incubation of brainstem and cerebellum with SMTC there were no significant differences in NOS activity of NAC-treated rats compared to strain-matched controls. Taken together, nNOS seems to be responsible for the increase of total NOS activity in the brain of SHR. SMTC inhibited 86% and 70% of NAC-induced increase of total NOS activity in the brainstem and cerebellum, respectively. Thus, nNOS is responsible not only for strain differences but also for NAC-induced increase of total NOS activity in the brain.

Regulatory role of nitric oxide on the cardiac Na, K-ATPase in hypertension.

The present study was focused on regulatory role of nitric oxide on functional properties of the cardiac Na, K-ATPase in three various animal models of hypertension: spontaneously hypertensive male rats (SHR) with increased activity of nitric oxide synthase (NOS) by 60 % (Sh1), SHR with decreased activity of NOS by 40 % (Sh2) and rats with hypertension induced by L-NAME (40 mg/kg/day) with depressed activity of NOS by 72 % (LN). Studying the utilization of energy substrate we observed higher Na, K-ATPase activity in the whole concentration range of ATP in Sh1 and decreased activity in Sh2 and LN. Evaluation of kinetic parameters revealed an increase of Vmax value by 37 % in Sh1 and decrease by 30 % in Sh2 and 17 % in LN. The KM value remained unchanged in Sh2 and LN, but was lower by 38 % in Sh1 indicating increased affinity of the ATP binding site, as compared to controls. During the activation with Na+ we observed increased Vmax by 64 % and increased KNa by 106 % in Sh1. In Sh2 we found decreased Vmax by 40 % and increased KNa by 38 %. In LN, the enzyme showed unchanged Vmax with increased KNa by 50 %. The above data indicate a positive role of increased activity of NOS in improved utilization of ATP as well as enhanced binding of Na+ by the cardiac Na, K-ATPase.

Beneficial effects of Provinols: cardiovascular system and kidney.

Red wine polyphenols have been reported to exert beneficial effects in preventing cardiovascular diseases but their molecular mechanisms of hemodynamic effects on functional cardiovascular and renal changes were studied much less. The review is focused on in vitro as well as in vivo effects of red wine extract containing polyphenolic compounds (Provinols) on cardiovascular systems and kidney in relation to the molecular and biochemical mechanisms of these compounds. This review provides the evidence that Provinols is able to produce ex vivo endothelium-dependent relaxation as a result of enhanced NO synthesis. Administration of Provinols partially prevents the development of hypertension during NO deficiency and accelerates the decrease of blood pressure in already established hypertension. The effects of Provinols include prevention and/or attenuation of myocardial fibrosis, reduction of aortic wall thickening and improvement of vascular functions. These functional and structural alterations are associated with significant augmentation of NO production, seen as the increase of NO synthase activity and eNOS protein expression. Moreover, it has been documented that Provinols decreased the oxidative stress within the cardiovascular system and kidney.

The time-dependent effect of Provinols on brain NO synthase activity in L-NAME-induced hypertension.

Red wine polyphenols have been reported to possess beneficial properties for preventing cardiovascular diseases but their neuroprotective effects during chronic L-NAME treatment have not been elucidated. The aim of this study was to analyze a time course of Provinols effects on brain NO synthase activity and oxidative damage in L-NAME-induced hypertension. Male Wistar rats, 12 weeks old, were divided into six groups: control groups, groups treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg/day) for 4 or 7 weeks and groups receiving Provinols (40 mg/kg/day) plus L-NAME for 4 or 7 weeks. At the end of the treatment, marker of membrane oxidative damage - conjugated dienes (CD) in the brain and NO synthase activity in the cerebral cortex, cerebellum and brainstem were determined. L-NAME treatment for 4 or 7 weeks led to the increase in blood pressure, elevation of CD concentration and decrease of NO synthase activity in the brain parts investigated. Provinols partially prevented blood pressure rise and elevation of CD concentration. Comparing to the L-NAME treated group, Provinols increased NO synthase activity after 4 weeks of treatment. However, the prolonged Provinols treatment for 7 weeks had no effect on NO synthase activity decreased by L-NAME treatment. In conclusion, Provinols partially prevents L-NAME induced hypertension via the different mechanisms depending on the duration of treatment. Prevention of oxidative damage in the brain with modulating effect on NO synthase activity is suggested.

Red wine polyphenols prevent endothelial damage induced by CCl4 administration.

It became evident in the present study that carbon tetrachloride (CCl(4)), in addition to its known liver and renal toxicity, causes serious damage to endothelial cells. The preventive effect of red wine on cardiovascular diseases has been documented in a number of human population studies as well as in animal experimental models. In this study, the endothelium protective effect of polyphenolic compounds isolated from red wine was studied in rats administered 0.5 ml of CC(4)/kg body weight intraperitoneally twice a week for 8 weeks. Endothelemia (endothelial cells/10 microl of plasma) was used as the marker of endothelial cell injury in vivo. Chronic CCl(4) treatment for 8 weeks lead to a 3-fold increase of free endothelial cells circulating in the blood when compared to the baseline values (2.5+/-0.3). Parallel oral administration of polyphenols 40 mg/kg/day significantly decreased the endothelemia. Polyphenolic compounds alone did not produce significant changes. Three weeks of spontaneous recovery after the 8-week treatment with CCl(4) did not lead to a marked decrease of endothelemia, but the administration of red wine polyphenols during the 3-week period significantly decreased free endothelial cells in the blood. It can be concluded that long-term administration of CCl(4) may serve as a useful experimental model of endothelial damage. The red wine polyphenolic compounds exert a powerful protective effect on endothelial cells from the injury caused by CCl(4). This effect was documented by decreased endothelemia that corresponded to diminished endothelial cell swelling and detachment evaluated by histology of the vascular intima. The endothelium protective effect may be one of the key factors that contribute to the preventive action of red wine on cardiovascular diseases.

Red wine polyphenols induce vasorelaxation by increased nitric oxide bioactivity.

The aim of the present study was to investigate the mechanism of vasorelaxant responses induced by red wine polyphenolic compounds (Provinol). Rings of rat femoral artery with or without functional endothelium were set up in a myograph for isometric recording and precontracted with phenylephrine (10(-5) M). Provinol in cumulative doses (10(-9) to 10(-3) mg/ml) elicited endothelium- and dose-dependent relaxation of the artery with maximal relaxation of 56 per cent at the concentration of 10(-5) mg/ml. The relaxant responses to Provinol correlated well with the increase of NO synthase activity in the vascular tissue after administration of cumulative doses of Provinol (10(-9) to 10(-3) mg/ml). N(G)-nitro-L-arginine methylester (L-NAME, 3x10(-4) M) significantly attenuated the endothelium-dependent relaxation produced by Provinol. Administration of L-arginine (3x10(-5) M) restored the relaxation inhibited by L-NAME. The relaxant responses of Provinol were abolished in the presence of Ca(2+)-entry blocker, verapamil (10(-6) M). Administration of hydrogen peroxide (H(2)O(2)) abolished acetylcholine (10(-5) M)-induced relaxation of the rat femoral artery, while administration of Provinol (10(2) mg/ml) together with H(2)O(2) helped to maintain the acetylcholine-induced relaxation. Provinol only partially affected the concentration-response curve for the NO donor sodium nitroprusside-induced relaxation in rings without endothelium. In conclusion, Provinol elicited endothelium-dependent relaxation of rat femoral artery by the Ca(2+)-induced increase of NO synthase activity and by protecting NO from degradation.

Changes of sodium and ATP affinities of the renal Na,K-ATPase during and after nitric oxide-deficient hypertension.

The aim of this study was to assess the molecular basis of renal Na,K-ATPase disturbances in response to NO-deficient hypertension induced in rats by NO-synthase inhibition with 40 mg/kg/day N(G)-nitro-L-arginine methyl ester (L-NAME) for four weeks. After 4-week administration of L-NAME, the systolic blood pressure (SBP) increased by 30 %. Three weeks after terminating the treatment, SBP recovered to control value. When activating the Na,K-ATPase with its substrate ATP, a 36 % increase in K(m) and 29 % decrease in V(max) values were observed in NO-deficient rats. During activation with Na+, the V(max) was decreased by 20 % and the K(Na) was increased by 111 %, indicating a profound decrease in the affinity of the Na+-binding site in NO-deficient rats. After spontaneous recovery from hypertension, the V(max) remained at the level as in hypertension for both types of enzyme activation. However, in the presence of lower concentrations of substrate which are of physiological relevance an improvement of the enzyme activity was observed as documented by return of K(m) for ATP to control value. The K(Na) value for Na+ was decreased by 27 % as compared to hypertension, but still exceeded the corresponding value in the control group by 55 % thus resulting in a partial restoration of Na+ affinity of Na,K-ATPase which was depressed as a consequence of NO-dependent hypertension.

Effects of red wine polyphenolic compounds on the cardiovascular system.

Phenolic phytochemicals are widely distributed in the plant kingdom. Regarding the protective effects on organisms, the polyphenol group is the most important. In different experiments, it has been shown that selected polyphenols, mainly flavonoids, possess protective effects on the cardiovascular system, as well as anticancer, antiviral and antiallergic properties. In coronary heart disease, the protective effects include mainly antithrombic, antioxidant, anti-ischaemic and vasorelaxant properties of flavonoids. It has been hypothesised that the phenomenon of a low incidence of coronary heart disease in French people may be partially related to the pharmacological properties of polyphenolic compounds included in red wine. Many epidemiological studies have shown that regular flavonoid intake is associated with reduced risk of cardiovascular diseases. This review article discusses the chemical structure of polyphenols and their beneficial properties in the cardiovascular system. (Fig. 1, Ref 74.).

Short-term NO synthase inhibition and the ATP affinity of cardiac Na,K-ATPase.

It was previously shown that 4 hours lasting inhibition of nitric oxide synthesis by administration of an L-arginine analogue, the A(G)-nitro-L-arginine methyl ester (L-NAME) changed the affinity of the Na-binding site of Na,K-ATPase thus resulting in elevation of enzyme activity especially at higher concentrations of sodium. Using the same experimental model, we focused our attention in the present study to the question of binding of ATP to the enzyme molecule in the left ventricle (LV), ventricular septum (S) and the right ventricle (RV) of the dog heart. Activation of the enzyme by increasing concentrations of ATP revealed a significant increase of the Vmax only in septum (by 38 %). The K(M) increased significantly in septum (by 40 %) and in left ventricle (by 56 %) indicating an altered sensitivity of the ATP-binding site of Na,K-ATPase in the hearts of NO-deficient animals. The alterations of Na,K-ATPase in its ability to bind and hydrolyze ATP are localized to the tissue surrounding the cavity of the left ventricle.

Protective effects of red wine polyphenolic compounds on the cardiovascular system.

Phenolic phytochemicals are widely distributed in the plant kingdom. In terms of protective effects on organisms, the group of polyphenols is the most important. In various experiments, it has been shown that selected polyphenols, mainly flavonoids, confer protective effects on the cardiovascular system and have anti-cancer, antiviral and antiallergic properties. In coronary artery disease, the protective effects are due mainly to antithrombic, antioxidant, anti-ischemic and vasorelaxant properties of flavonoids. Flavonoids are low molecular weight compounds composed of a three-ring structure with various substitutions, which appear to be responsible for the antioxidant and antiproliferative properties. It has been hypothesized that the low incidence of coronary artery disease in the French population may be partially related to the pharmacological properties of polyphenolic compounds present in red wine. Many epidemiological studies have shown that regular flavonoid intake is associated with reduced risk of cardiovascular diseases.

Effect of captopril on cyclic nucleotide concentrations during long-term NO synthase inhibition.

The aim of the present study was to determine the effect of angiotensin-converting enzyme inhibitor captopril on cGMP and cAMP concentration in the left ventricle and aorta after NO synthase inhibition by 4-week-lasting N(G)-nitro-L-arginine-methyl ester (L-NAME) treatment. Five groups of rats were investigated: controls, L-NAME in the dose 20 mg/kg/day (L-NAME 20), L-NAME in the dose 40 mg/kg/day (L-NAME 40), captopril in the dose 100 mg/kg/day, L-NAME 40 mg/kg/day together with captopril 100 mg/kg/day. Captopril completely prevented L-NAME-induced hypertension and LV hypertrophy development. Compared to the controls, cGMP concentration in the L-NAME 20 and L-NAME 40 groups was decreased by 13% and 22%, respectively, in the left ventricle and by 27% and 56% in the aorta, respectively. Captopril did not influence this decrease of cGMP concentration. Cyclic AMP concentration in the aorta of L-NAME 20 group increased by 17%. In the L-NAME 40 group, cAMP concentration increased by 17% in the left ventricle and by 34% in the aorta compared to controls. This increase was enhanced in rats given L-NAME together with captopril. Captopril alone had no effect on cAMP concentration. We conclude that captopril does not affect the concentration of cGMP, however, it has more than the additive effect on the cAMP concentration increase in the cardiovascular system during long-term NO synthase inhibition.