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 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 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.     

Human urotensin II increases coronary perfusion pressure in the isolated rat heart: potentiation by nitric oxide synthase and cyclooxygenase inhibition.

Urotensin II (U-II) is a cyclic peptide, recently cloned in man and present in cardiac tissue and arteries. The effects of human U-II (hU-II) on coronary perfusion pressure (CPP) were investigated in isolated rat hearts perfused retrogradely via the aorta at constant flow. hU-II produced a concentration-dependent increase in CPP (pEC50 8.6 +/- 0.3, n = 8), the maximum increase in CPP (12 +/- 4 mmHg) was obtained at 10(-7) M hU-II. At higher concentrations of hU-II CPP fell back towards baseline. Endothelin-1 produced a maximum increase in CPP of 63 +/- 11 mmHg within the concentration-range studied. Addition of the NO synthase inhibitor L N(G)nitro-arginine methyl ester (10(-4) M) and the cyclooxygenase inhibitor, indomethacin (10(-5) M) to the perfusion solution had no effect on the pEC50 value for hU-II, but significantly increased the maximum constriction (to 34 +/- 7 mmHg, n = 8, p < 0.05) and inhibited the later dilator response to hU-II. These results suggest that receptors for hU-II are present in the coronary vasculature and that smooth muscle contraction is modulated by the release of dilator factors, including NO and prostacyclin. Endothelial function is therefore likely to be of primary importance in modulating the coronary vasoconstrictor effects of hU-II in vivo.     

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.     

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.     

The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation

Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent data implicate a critical function for hemoglobin and the erythrocyte in regulating the activity of NO in the vascular compartment. Intravascular hemolysis releases hemoglobin from the red blood cell into plasma (cell-free plasma hemoglobin), which is then able to scavenge endothelium-derived NO 600-fold faster than erythrocytic hemoglobin, thereby disrupting NO homeostasis. This may lead to vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression (ET-1), and end-organ injury, thus suggesting a novel mechanism of disease for hereditary and acquired hemolytic conditions such as sickle cell disease and cardiopulmonary bypass. Furthermore, therapy with NO gas inhalation or infusion of sodium nitrite during hemolysis may attenuate this disruption in vasomotor balance by oxidizing plasma cell-free hemoglobin, thereby preventing the consumption of endogenous NO and the associated pathophysiological changes. In addition to providing an NO scavenging role in the physiological regulation of NO-dependent vasodilation, hemoglobin and the erythrocyte may deliver NO as the hemoglobin deoxygenates. While this process has previously been ascribed to S-nitrosated hemoglobin, recent data from our laboratories suggest that deoxygenated hemoglobin reduces nitrite to NO and vasodilates the human circulation along the physiological oxygen gradient. This newly described role of hemoglobin as a nitrite reductase is discussed in the context of blood flow regulation, oxygen sensing, and nitrite-based therapeutics.     

Crystal structures of the nitrite and nitric oxide complexes of horse heart myoglobin

Nitrite is an important species in the global nitrogen cycle, and the nitrite reductase enzymes convert nitrite to nitric oxide (NO). Recently, it has been shown that hemoglobin and myoglobin catalyze the reduction of nitrite to NO under hypoxic conditions. We have determined the 1.20 A resolution crystal structure of the nitrite adduct of ferric horse heart myoglobin (hh Mb). The ligand is bound to iron in the nitrito form, and the complex is formulated as MbIII(ONO-). The Fe-ONO bond length is 1.94 A, and the O-N-O angle is 113 degrees . In addition, the nitrite ligand is stabilized by hydrogen bonding with the distal His64 residue. We have also determined the 1.30 A resolution crystal structures of hh MbIINO. When hh MbIINO is prepared from the reaction of metMbIII with nitrite/dithionite, the FeNO angle is 144 degrees with a Fe-NO bond length of 1.87 A. However, when prepared from the reaction of NO with reduced MbII, the FeNO angle is 120 degrees with a Fe-NO bond length of 2.13 A. This difference in FeNO conformations as a function of preparative method is reproducible, and suggests a role of the distal pocket in hh MbIINO in stabilizing local FeNO conformational minima.     

Systemic nitric oxide synthase inhibition improves coronary flow reserve to adenosine in patients with significant stenoses

We studied the impact of systemic infusion of the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on coronary flow reserve (CFR) in patients with coronary artery disease (CAD). We have previously demonstrated that CFR to adenosine was significantly increased after systemic infusion of L-NMMA in normal volunteers but not in recently transplanted denervated hearts. At baseline, myocardial blood flow (MBF; ml x min(-1) x g(-1)) was measured at rest and during intravenous administration of adenosine (140 microg x kg(-1) x min(-1)) in 10 controls (47 +/- 5 yr) and 10 CAD patients (58 +/- 8 yr; P < 0.01 vs. controls) using positron emission tomography and (15)O-labeled water. Both MBF measurements were repeated during intravenous infusion of 10 mg/kg L-NMMA. CFR was calculated as the ratio of MBF during adenosine to MBF at rest. CFR was significantly higher in healthy volunteers than in CAD patients and increased significantly after L-NMMA in controls (4.00 +/- 1.10 to 6.15 +/- 1.35; P < 0.0001) and in patients, both in territories subtended by stenotic coronary arteries (>70% luminal diameter; 2.06 +/- 1.13 to 3.21 +/- 1.07; P < 0.01) and in remote segments (3.20 +/- 1.23 to 3.92 +/- 1.62; P < 0.05). In conclusion, CFR can be significantly increased in CAD by a systemic infusion of L-NMMA. Similarly to our previous findings in normal volunteers, this suggests that adenosine-induced hyperemia in CAD patients is constrained by a mechanism that can be relieved by systemic NOS inhibition with L-NMMA.     

Inconsistent role of nitric oxide on lipolysis in isolated rat adipocytes

Though two isoforms of nitric oxide synthase, iNOS and eNOS, were reported in adipocytes, the role of NO in adipose tissue is still ambiguous. The aims of the present study were 1) to follow the effect of bacterial lipopolysaccharide (LPS), on 24 h-lipolysis in rat epididymal adipocyte culture in relation to iNOS stimulation; 2) to compare LPS-induced NO effects with exogenously NO, delivered as S-nitroso-N-acetylpenicillamine (SNAP), and 3) to examine the possible role of NO signaling agonist in lipolysis mediated by the beta(3)-adrenoreceptor agonist. Lipolysis was measured by glycerol and free fatty acid (FFA) production. The medium nitrite levels were used for the indirect estimation of NOS expression. Adipocyte mitochondrial function was assessed by the MTT test. LPS produced a concentration-dependent increase of NO with a decrease of viability at the highest dose. However, LPS did not affect lipolysis. SNAP did not exhibit significant changes in glycerol, FFA or MTT. BRL-37344 and db-cAMP significantly increased nitrite, glycerol and FFA levels. There was a positive correlation between glycerol release and nitrite production. Moreover, BRL-37344 significantly reduced mitochondrial functions. The pretreatment with bupranolol, beta(3)-antagonist, restored all parameters affected by BRL-37344. These results support a concept that NO fulfils multifaceted role of stimulating lipolysis under physiological conditions (beta-agonistic effect) and modulating the same processes during inflammatory (LPS) processes.     

The nature of the nitric oxide complexes of lipoxygenase.

The NO complex of lipoxygenase with EPR signals near g = 4.0 is an S = 3/2 system with D approximately 15 cm-1 similar to Fe2+-EDTA-NO. This may result from antiferromagnetic coupling of axial (D greater than E) high spin ferrous iron to NO. The other NO complex of lipoxygenase, with EPR signals below ge, may result from rhombic high spin ferrous iron coupled to NO with D greater than J. The quenching of both signals by a hydroperoxy derivative of linoleic acid probably represents replacement of NO by an oxygen ligand.     

The origin of sympathetic outflow in heart failure: the roles of angiotensin II and nitric oxide

The regulation of sympathetic nerve activity in chronic heart failure (CHF) has been an area of renewed investigation. Understanding the central mechanisms that are responsible for sympatho-excitation in this disease state may help in reducing the deleterious effects of chronic sympatho-excitation. This review will summarize our understanding of abnormal reflex control of the circulation in CHF. The roles of the arterial baroreflex, the chemoreflex, the cardiac sympathetic afferent reflex and the cardiopulmonary reflex are discussed. New experimental techniques that allow genetic manipulation of substances such as nitric oxide synthase in discrete areas of the brain aid in clarifying the role of NO in the modulation of sympathetic tone in the CHF state. Lastly, clinical implications of this work are discussed.     

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.     

Tonic regulation of vascular tone by nitric oxide and chloride ions in rat isolated small coronary arteries

We have investigated the involvement of Cl(-) in regulating vascular tone in rat isolated coronary arteries mounted on a small vessel myograph. Mechanical removal of the endothelium or inhibition of nitric oxide (NO) synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) led to contraction of rat coronary arteries, and these contractions were sensitive to nicardipine (10(-6) M). This suggests that release of NO tonically inhibits a contractile mechanism that involves voltage-dependent Ca(2+) channels. In arteries contracted with L-NAME, switching the bathing solution to physiological saline solution with a reduced Cl(-) concentration potentiated the contraction. DIDS (5 x 10(-6)-3 x 10(-4) M) caused relaxation of L-NAME-induced tension (IC(50) = 55 +/- 10 microM), providing evidence for a role of Cl(-). SITS (10(-5)-5 x 10(-4) M) did not affect L-NAME-induced tension, suggesting that DIDS is not acting by inhibition of anion exchange. Mechanical removal of the endothelium led to contraction of arteries, which was sensitive to DIDS (IC(50) = 50 +/- 8 microM) and was not affected by SITS. This study suggests that, in rat coronary arteries, NO tonically suppresses a contractile mechanism that involves a Cl(-) conductance.     

Role of nitric oxide in central sympathetic outflow

The gaseous molecule nitric oxide (NO) plays an important role in cardiovascular homeostasis. It plays this role by its action on both the central and peripheral autonomic nervous systems. In this review, the central role of NO in the regulation of sympathetic outflow and subsequent cardiovascular control is examined. After a brief introduction concerning the location of NO synthase (NOS) containing neurons in the central nervous system (CNS), studies that demonstrate the central effect of NO by systemic administration of NO modulators will be presented. The central effects of NO as assessed by intracerebroventricular, intracisternal, or direct injection within the specific central areas is also discussed. Our studies demonstrating specific medullary and hypothalamic sites involved in sympathetic outflow are summarized. The review will be concluded with a discussion of the role of central NO mechanisms in the altered sympathetic outflow in disease states such as hypertension and heart failure.