The effects of folic acid and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart

The aim of this study was to assess the effects of folic acid on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by l-NAME in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180–200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40–120 cmH₂O). Coronary flow and markers of oxidative stress: nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were calculated. The experiments were performed during control conditions and in presence of folic acid (100 μM) alone or folic acid (100 μM) plus l-NAME (30 μM). Control values of coronary flow varied in range from 4.37 ± 0.10 ml/min/g wt at 40 cmH₂O to 12.05 ± 0.42 ml/min/g wt at 120 cmH₂O. Nitrite outflow varied from 1.68 ± 0.17 nmol/min/g wt at 40 cmH₂O to 3.56 ± 0.17 nmol/min/g wt at 120 cmH₂O and was parallel with coronary perfusion pressure-coronary flow curve. Folic acid significantly increased coronary flow (40–120 cmH₂O, 5.63 ± 0.10 ml/min/g wt and 15.2 ± 0.42 ml/min/g wt, respectively) and was accompanied by significant increase in nitrite outflow (2.28 ± 0.29 nmol/min/g wt at 40 cmH₂O to 6.66 ± 0.50 nmol/min/g wt at 120 cmH₂O). In addition, folic acid significantly decreased superoxide anion production especially at upper coronary perfusion pressure values (60% at 120 cmH₂O) and increased index of lipid peroxidation (37.16% at 120 cmH₂O), respectively. Folic acid plus l-NAME did not change control values of coronary flow significantly. However, folic acid plus l-NAME increased nitrite outflow especially at upper coronary perfusion pressure values (43.05% at 120 cmH₂O) and did not change significantly superoxide anion production or index of lipid peroxidation versus control values, respectively. The results clearly showed that on isolated rat hearts at gradually increased constant perfusion pressure, folic acid increased coronary flow, increased nitrite outflow, decreased superoxide anion production, and increased index of lipid peroxidation. These effects were reversed or blocked by l-NAME thus demonstrating mediation or at least participation of NO in the mechanism of the folic acid-induced effects.     

The effects of vitamin C and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart

The aim of this study was to assess the effects of vitamin C (ascorbic acid) on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by N(ω)-nitro-L-arginine monomethyl ester (L-NAME) in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40-120 cm H2O). Coronary flow, nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were determined. The experiments were performed during control conditions and in presence of vitamin C (100 μM) alone or vitamin C (100 μM) + L-NAME (30 μM). Administration of vitamin C induced only increase of nitrite levels, while vitamin C + L-NAME induced significant decrease of coronary flow above autoregulatory range, i.e. especially at higher coronary perfusion pressure (CPP) values, accompanied with similar dynamic in nitrite outflow. Vitamin C + L-NAME also induced significant decrease in TBARS production. The results of our study show no significant effects of vitamin C administration either on ROS levels or on coronary flow in isolated rat heart.     

The effects of nimodipine and L-NAME on coronary flow and oxidative stress parameters in isolated rat heart

The aim of this study was to assess the effects of Ca2+ channel antagonist nimodipine (in concentration which competitive inhibited phosphodiesterase 1--PDE1) on oxidative stress alone or under inhibition of nitric oxide synthase by L-NAME in isolated rat heart. The hearts from male Wistar albino rats (n=18, BM about 200 g, age 8 weeks) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure conditions (CPP, 40-120 cm H2O). The experiments were performed under control conditions, in the presence of Nimodipine (2 microM) or Nimodipine (2 microM) plus L-NAME (30 microM). Coronary flow (CF) varied in the autoregulatory range from 3.7 +/- 0.4 ml/min/g wt at 50 cm H2O to 4.35 +/- 0.79 at 90 cm H2O. Basal nitrite outflow, index of lipid peroxidation (measured as TBARS release) and superoxide anion release (O2-) (at 60 cm H2O) were 0.64 +/- 0.18 nmol/min/g wt, 0.55 +/- 0.13 micromol/min/g wt and 19.72 +/- 3.70 nmol/min/g wt, respectively. Nimodipine induced significant vasodilation at all values of CPP (from 26% at 40 cm H2O to 36% at 120 cm H2O) accompanied with significant decrease of nitrite outflow (from 59% at 40 cm H2O to 40% at 120 cm H2O), significant increase of TBARS above autoregulatory range (about 40%) and significant increase of O2- release (from 186% at 40 cm H2O to 117% at 120 cm H2O). However, perfusion with L-NAME completely reversed the effects of Nimodipine. Nimodipine-induced flow changes were decreased under L-NAME (from 3% at 40 cm H2O to 11% at 120 cm H2O) without changes in the autoregulatory range, accompanied with significantly increased nitrite outflow (from 69% at 40 cm H2O to 36% at 120 cm H2O) and TBARS release (almost 50%), as well as significantly decreased O2- release (from 50% at 40 cm H2O to 43% at 120 cm H20). Our findings show that effect of nimodipine on coronary flow should be significantly influenced by NO, TBARS and O2- release in isolated rat heart.     

The effects of verapamil and its combinations with glutamate and glycine on cardiodynamics,coronary flow and oxidative stress in isolated rat heart

The role of N-methyl-D-aspartate receptor (NMDA-R) in heart is still unclear. For these ionotropic glutamate receptors is characteristic the necessity of both co-agonists, glutamate and glycine, for their activation, which primarily allows influx of calcium. The aim of the present study was to examine the effects of verapamil, as a calcium channel blocker, alone and its combination with glycine and/or glutamate on cardiac function, coronary flow, and oxidative stress in isolated rat heart or to examine the effects of potential activation of NMDA-R in isolated rat heart. The hearts of male Wistar albino rats were excised and perfused according to Langendorff technique, and cardiodynamic parameters and coronary flow were determined during the administration of verapamil and its combinations with glutamate and/or glycine. The oxidative stress biomarkers, including thiobarbituric acid-reactive substances, nitrites, superoxide anion radical, and hydrogen peroxide, were each determined spectrophotometrically from coronary venous effluent. The greatest decline in parameters of cardiac contractility and systolic pressure was in the group that was treated with verapamil only, while minimal changes were observed in group treated with all three tested substances. Also, the largest changes in coronary flow were in the group treated only with verapamil, and at least in the group that received all three tested substances, as well as the largest increase in oxidative stress parameters. Based on the obtained results, it can be concluded that NMDA-R activation allows sufficient influx of calcium to increase myocardial contractility and systolic pressure, as well as short-term increase of oxidative stress.     

Electromechanical effects of menadione on isolated rat heart in relation to oxidative stress

Although Ca2+ overloading has been observed in hepatocytes and in the isolated liver treated with 0.2 mM menadione, it has not been determined if menadione has similar effects on cardiac tissue and, if so, whether Ca2+ overloading leads to cardiac contracture, and if such an event results from plasma membrane peroxidation initiated by oxidative stress. The present study reveals that when the isolated heart is perfused with 0.2 mM menadione for 30 min, it shows Ca2+ overloading, which can not be reversed even after 30 min of drug-free perfusion. The time courses of glutathione, ethane, and LDH release from the hearts do not show a parallel pattern of abnormality between 30 and 60 min, indicating that contractile failure precedes the development of lipid peroxidation or plasma membrane disintegration. The evidence that the plasma membrane of menadione-treated rat cardiac tissue remains intact is supported by the observation that the resting membrane potential of the atrium remains virtually unchanged during the 30 min of drug exposure and then gradually falls (-67 +/- 3.1 vs. -76 +/- 2 mv) only during the last 10 min of the drug washout. Interestingly, even after the atria are treated with menadione for 30 min and followed by washout of 30 min, and have shown calcium overloading, as evidenced by contracture, they are still capable of generating action potentials in response to electrical field stimulation.     

The effects of cyclooxygenase and nitric oxide synthase inhibition on oxidative stress in isolated rat heart

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas still exist about potential impact of these drugs on cardiovascular system. The present study was aimed to estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA], and SC-560) on oxidative stress in isolated rat heart, with special focus on l-arginine/NO system. The hearts of male Wistar albino rats (total number n = 96, each group 12 rats, 8 weeks old, body mass 180–200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40–120 cmH₂O). After control experiments the hearts were perfused with the following drugs: 100 μmol/l ASA (Aspirin), alone or in combination with 30 μmol/l l-NAME, 0.3 μmol/l meloxicam (movalis) with or without 30 μmol/l l-NAME, 3 μmol/l meloxicam (alone or in combination with 30 μmol/l l-NAME), 30 μmol/l l-NAME, and administration of 0.25 μmol/l SC-560. In samples of coronary venous effluent the following oxidative stress markers were measured spectrophotometrically: index of lipid peroxidation (measured as thiobarbituric acid reactive substances [TBARS]), superoxide anion radical release (O₂ ^?), and hydrogen peroxide (H₂O₂). While ASA was found to have an adverse influence on redox balance in coronary circulation, and coronary perfusion, meloxicam and SC-560 do not negatively affect the intact model of the heart. Furthermore, all effects were modulated by NOS inhibition. It seems that interaction between COX and l-arginine/NO system truly exists in coronary circulation, and can be one of the possible causes for achieved effects. That means: those effects induced by different inhibitors of COX are modulated by subsequent inhibition of NOS.     

The effects of training and detraining on memory, neurotrophins and oxidative stress markers in rat brain

In the current investigation we tested how swimming training (T) (8 week, 5 times/week, 2 h/day), and detraining (DT) affects brain functions and oxidative stress markers in rat brain. The free radical concentration, measured by electron paramagnetic resonance, decreased in brain of T and DT rats compared to controls (C). The level of brain-derived neurotrophic factor (BDNF) increased as a result of training, but decreased below the control level after 6 weeks of detraining. In addition, the concentration of nerve growth factor (NGF) also declined with DT. The passive avoidance test was used to assess the memory of rats, and training-induced improvement was observed but the enhancement disappeared with detraining. When the content of mitochondrial electron transport complexes, as a potent free radical generator, was evaluated by the blue native gel method, no significant alterations were observed. The repair of nuclear and mitochondrial 8-oxodeoxyguanosine, as measured by the activity of OGG1, showed no significant difference. Therefore, the results suggest that regular exercise training improves memory, decreases the level of reactive oxygen species, and increase the production of BDNF and NGF. On the other hand, it appears that the beneficial effects of training are reversible in the brain, since detraining down-regulates the neurotrophin level, and memory. It is suggested that exercise training is more likely to beneficially effect the production of reactive oxygen species and the related oxidative damage.     

Catecholamine effects on cardiac remodelling,oxidative stress and fibrosis in experimental heart failure

The aim of the study was to assess the relationships between oxidative stress, cardiac remodelling and fibrosis on an experimental model of heart failure with adrenergic stimulation. Large myocardial infarction (approximately 50% of the left ventricle myocardium) was obtained by ligation of the left coronary artery of normotensive male Wistar rats. Sham animals were submitted to left thoracotomy without coronary ligation. In order to perform cardiac stimulation by catecholamines, mini-osmotic pumps were implanted in animals 10 weeks after surgery to deliver noradrenalin for a 2-week period. At the end of this period, the following investigations were performed: haemodynamics, morphometry, fibrosis quantification, plasma and tissue catecholamine assay and oxidative stress status. Coronary ligation induced dilatation of left ventricle with compensatory hypertrophy of the right ventricle and of the remaining left ventricle myocardium. This remodelling process was associated in non-infarcted myocardium with increased collagen infiltration and increased oxidative stress. Ten weeks after surgery, the chronic administration of noradrenalin for 2 weeks did not increase oxidative stress. Noradrenalin, however, induced inotropic stimulation and myocardial hypertrophy, but to a lesser extent in infarcted rats compared to sham rats. Our results suggest that noradrenalin infusion to levels in excess of those seen post-infarction is associated with fibrosis and oxidative stress. Moreover, noradrenalin in infarcted animals caused additional fibrosis without further increasing oxidative stress. The mechanism of catecholamine-induced fibrosis may thus involve different processes such as ischaemia, increased mechanical stress, cytokines and neurohormones.     

The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart

The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective LOX inhibitor (NDGA) decreased control values of CF only at lower values of CPP but did not change nitrite outflow indicating antioxidant properties of NDGA. In addition, L-NAME decreased the effects induced by NDGA on CF and nitrite outflow indicating the role of NO.     

Increase on the coronary flow induced by dioclein in isolated rat heart

In the present work the effect of dioclein, a new flavonoid from Dioclea grandiflora, was investigated in rat hearts. The experiments were performed using the classic method of Langendorff, where flow, inotropic, chronotropic and electric parameters were analyzed. Bolus administration of Dioclein (1-100 microg) induced a sustained and dose-dependent increase in coronary flow with no modification in inotropic, chronotropic and electrical parameters. The duration of increase in coronary flow induced by dioclein (10 microg) was approximately 4-fold higher than that observed in the presence of sodium nitroprusside (NPS; 10 microg). Besides, the effect of dioclein measured as the area-under-the-curve was approximately 4.5-folds higher than that observed with NPS. Pre-treatment with L-NAME (100 microM) and indomethacin (10 microM) alone did not modify the effect of dioclein (10 microg), suggesting that nitric oxide (NO) and cyclooxygenase-derived factors were not involved. However, association of L-NAME plus indomethacin inhibited the duration of the effect of dioclein (10 microg) without changing its increase in the coronary flow. Furthermore, the absence of alteration in inotropism and chronotropism of the heart associated with its coronary effect suggest that dioclein could be an interesting lead compound for the development of drugs for the treatment coronary heart diseases.     

Protective effects of resveratrol on calcium-induced oxidative stress in rat heart mitochondria

Trans-resveratrol is a nutraceutical with known antioxidant, anti-inflammatory, cardioprotective, and anti-apoptotic properties. The aim of this study was to evaluate the effects of resveratrol on heart mitochondria. Resveratrol significantly decreased Fe²⁺ + ascorbate oxidant system-induced lipid peroxide levels, preserved physiological levels of glutathione, and increased nitric oxide (NO) levels in mitochondria. Under calcium-mediated stress, there was a 2.7-fold increase in the NO levels, and a mild decoupling in the mitochondrial respiratory chain. These results provide a mechanism for and support the beneficial effects of resveratrol under pathological conditions induced by oxidative stress and calcium overload. In addition, these findings underscore the usefulness of resveratrol in the prevention of cardiovascular diseases.     

The effects of moderate-, strenuous- and over-training on oxidative stress markers, DNA repair, and memory, in rat brain

We have tested the hypothesis that training with moderate- (MT), strenuous- (ST), or over- (OT) load can cause alterations in memory, lipid peroxidation, protein oxidation, DNA damage, activity of 8-oxoG-DNA glycosylase (OGG1) and brain-derived neurotrophic factor (BDNF), in rat brain. Rat memory was assessed by a passive avoidance test and the ST and OT group demonstrated improved memory. The content of BDNF was increased only in the OT group. The oxidative damage of lipids and DNA, as measured by thiobarbituric acid reactive substances (TBARS), and 8-hydroxydeoxyguanosine (8-OHdG), did not change significantly with exercise. Similarly, the activity of DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), was not altered with exercise training. On the other hand, the content of reactive carbonyl derivatives (RCDs) decreased in all groups and the decrease reached significance levels in the ST and OT groups. The activity of the proteasome complex increased in the brain of OT. The findings of this study imply that over-training does not induce oxidative stress in the brain and does not cause loss of memory. The improved memory was associated with enhanced BDNF content.     

Fructose 1,6-bisphosphate prevents oxidative stress in the isolated and perfused rat heart

Rat hearts were perfused with the Langendorff technique at constant flux in the presence of the oxidizing agents hydrogen peroxide and diamide. Fructose 1,6-bisphosphate strongly prevented the decline of heart contractility due to the infusion of these oxidizing agents. On the other hand, fructose 1,6-bisphosphate had no effect on the release of total glutathione into the perfusate but prevented the loss of lactate dehydrogenase indicating a protective effect on cell membranes. Comparing the cytosolic and mitochondrial loss of glutathione, fructose 1,6-bisphosphate exerted a beneficial action only on the mitochondrial fraction. Several mechanisms of action have been considered to explain the protective action of frutose 1,6-bisphosphate. In our experimental conditions fructose 1,6-bisphosphate might stimulate its own production giving rise to dihydroxyacetone phosphate, that, after reduction to glycerol 3-phosphate, can permeate the mitochondrial membrane with the final production of energy.     

The relation between fatty acid mobilization and contractility in the isolated,perfused rat heart

The contractility of hearts from normal fed rats is decreased by 70% during perfusion with 50 μM chloroquine, which is a potent inhibitor of endogenous lipolysis. In triacylglycerol-rich hearts, obtained by feeding rats rapeseed-oil, chloroquine depresses lipolysis much less, while contractility was found to be inhibited only 30%. In both groups of hearts the effect of chloroquine was decreased by adding fatty acids, prostaglandin E₁, the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ionophore X-537A or more Ca²⁺ to the perfusion fluid. Norepinephrine and glucagon also stimulate chloroquine-depressed hearts. The conclusion is therefore reached that fatty acids act as Ca²⁺-vehicles in heart cells and that chloroquine, by inhibiting lipolysis, decreases Ca²⁺-transport by lowering unesterified fatty acid levels.     

Differences in oxidative stress markers based on the aetiology of heart failure: Comparison of oxidative stress in patients with and without coronary artery disease

Abstract

Various oxidative stress markers have been measured to evaluate the status of heart failure (HF). However, the relationships between these markers and the aetiology of HF have not been fully investigated. This study compared 8-hydroxy-2′-deoxyguanosine (8-OHdG) and biopyrrins levels in patients with ischemic and non-ischemic HF. Study subjects were divided into a coronary artery disease (CAD) group (n=70), a non-CAD group (n=61) and a control group (n=33). In the CAD group, 8-OHdG and biopyrrins levels increased with the severity of the New York Heart Association (NYHA) functional class and log BNP levels correlated with 8-OHdG and biopyrrins levels. However, non-CAD patients with NYHA class III/IV had significantly lower 8-OHdG levels than CAD patients with NYHA class III/IV and the levels did not correlate with log BNP levels. In the CAD group, 8-OHdG levels reflected the severity of atherosclerosis. These results indicate that the properties of oxidative stress markers should be carefully taken into consideration for the assessment of HF status.     

Vanadium and cadmium in vivo effects in teleost cardiac muscle: metal accumulation and oxidative stress markers

Several biological studies associate vanadium and cadmium with the production of reactive oxygen species (ROS), leading to lipid peroxidation and antioxidant enzymes alterations. The present study aims to analyse and compare the oxidative stress responses induced by an acute intravenous exposure (1 and 7 days) to a sub-lethal concentration (5 mM) of two vanadium solutions, containing different vanadate n-oligomers (n=1-5 or n=10), and a cadmium solution on the cardiac muscle of the marine teleost Halobatrachus didactylus (Lusitanian toadfish). It was observed that vanadium is mainly accumulated in mitochondria (1.33+/-0.26 microM), primarily when this element was administrated as decameric vanadate, than when administrated as metavanadate (432+/-294 nM), while the highest content of cadmium was found in cytosol (365+/-231 nM). Indeed, decavanadate solution promotes stronger increases in mitochondrial antioxidant enzymes activities (catalase: +120%; superoxide dismutase: +140%) than metavanadate solution. On contrary, cadmium increases cytosolic catalase (+111%) and glutathione peroxidases (+50%) activities. It is also observed that vanadate oligomers induce in vitro prooxidant effects in toadfish heart, with stronger effects induced by metavanadate solution. In summary, vanadate and cadmium are differently accumulated in blood and cardiac subcellular fractions and induced different responses in enzymatic antioxidant defence mechanisms. In the present study, it is described for the first time the effects of equal doses of two different metals intravenously injected in the same fish species and upon the same exposure period allowing to understand the mechanisms of vanadate and cadmium toxicity in fish cardiac muscle.     

Coronary flow measurements on the isolated rat heart following experimental coronary artery stenosis]

A stenosing proliferation of the vascular wall was produced by exposing the Ramus descendens of the rat's left coronary artery to continued mechanical stimulation. Two weeks after the operation the hearts were perfused according to a modified Langendorff method, and the flow rate was measured under aerobic conditions and following short-time anoxia. Under either experimental conditions, the flow rate proved to be significantly decreased.