N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Study of dinitrosyl-iron complexes pharmacokinetics and accumulation in depot in rat organs

Protein-bound dinitrosyl-iron complexes (DNIC) in rat whole blood and organs were studied after intravenous injection of this substance with glutathione ligand (DNIC-GH). The effect of DNIC-GH injection on NO level (including NO physiological forms) in hydrophobic areas of rat tissues was also studied in normal physiological blood circulation condition. It has been shown, that after DNIC-GH injection the concentration of protein-bound DNICs in rat whole blood and organs rapidly reached maximum values, and then gradually decreased, that pointed to decomposition of DNIC molecules, coupled with NO release. At the beginning of the experiment the rates of DNIC decay in rat heart and lung were substantially higher, as compared with those in liver and kidney. By spin trappping it has been demonstrated that DNIC-GH, as a source of NO physiological forms (including S-nitrosothiols), in normal physiological blood circulation influence heart more selectively, as compared with the other organs.     

Delayed microcirculatory hyperpermeability following perfusion washout

Recent experimental data have demonstrated improved flap survival following perfusion washout with a synthetic, chemically defined, mammalian plasma. In an effort to define the physiology responsible for the efficacy of perfusion, the method of "labeling" hyperpermeable blood vessels with Monastral blue B in rat epigastric vascular island flaps was utilized. Results confirmed that capillary and venular hyperpermeability is an early and progressive pathophysiologic event in ischemic flap tissue and one which is reversible prior to a critical ischemic period. Perfusion washout with a physiologic, acellular plasma substitute delays the onset of vascular hyperpermeability. This may be a mechanism responsible for improving tissue survival following extended periods of warm ischemia (12 hours). It is implied that stagnant blood and products of hemolysis in the microcirculation may be detrimental to the functional and anatomic integrity of the endothelial wall.     

Nitric oxide modulates oxygen consumption by arteriolar walls in rat skeletal muscle

To study the role of nitric oxide (NO) in regulating oxygen consumption by vessel walls, the oxygen consumption rate of arteriolar walls in rat cremaster muscle was measured in vivo during flow-induced vasodilation and after inhibiting NO synthesis. The oxygen consumption rate of arteriolar walls was calculated based on the intra- and perivascular PO2 values measured by phosphorescence quenching laser microscopy. The perivascular PO2 value of the arterioles during vasodilation was significantly higher than under control conditions, although the intravascular PO2 values under both conditions were approximately the same. Inhibition of NO synthesis, on the other hand, caused a significant increase in arterial blood pressure and a significant decrease in arteriolar diameter. Inhibition of NO synthesis also caused a significant decrease in both the intra- and perivascular PO2 values of the arterioles. Inhibition of NO synthesis increased the oxygen consumption rate of the vessel walls by 42%, whereas enhancement of flow-induced NO release decreased it by 34%. These results suggest that NO plays an important role not only as a regulator of peripheral vascular tone but also as a modulator of tissue oxygenation by reducing oxygen consumption by vessel walls. In addition, enhancement of NO release during exercise may facilitate efficient oxygen supply to the surrounding high metabolic tissue.     

Effect of local laser irradiation on the activity of enzymes of glutamic acid metabolism in rat tissues

In experiments in vivo it was shown that local laser irradiation of the parietal region of the rat head changed the activity of principal enzymes of glutamic acid metabolism both in the brain and in the liver, whereas peripheral irradiation of epigastric region caused changes in the activity of the studied enzymes in the affected site only.     

Vascular endothelial growth factor attenuates leukocyte-endothelium interaction during acute endothelial dysfunction: essential role of endothelium-derived nitric oxide.

Vascular endothelial growth factor (VEGF) is an endothelium-specific secreted protein that induces vasodilation and increases endothelial release of nitric oxide (NO). NO is also reported to modulate leukocyte-endothelium interaction. Therefore, we hypothesized that VEGF might inhibit leukocyte-endothelium interaction via increased release of NO from the vascular endothelium. We used intravital microscopy of the rat mesenteric microcirculation to measure leukocyte-endothelium interactions 2, 4, and 24 h after systemic administration of VEGF to the rat (120 microg/kg, i.v., bolus). Superfusion of the rat mesentery with either 0.5 U/ml thrombin or 50 microM L-NAME consistently increased the number of rolling, adhering, and transmigrated leukocytes (P<0.01 vs. control mesenteries superfused with Krebs-Henseleit buffer). At 4 and 24 h posttreatment, VEGF significantly attenuated thrombin-induced and L-NAME-induced leukocyte rolling, adherence, and transmigration in rat mesenteric venules. In addition, adherence of isolated rat PMNs to thrombin-stimulated mesenteric artery segments in vitro was significantly reduced in mesenteric arteries isolated from VEGF-treated rats (P<0.001 vs. control rats). Direct measurement of NO demonstrated a threefold increase in basal NO release from aortic tissue of rats injected with VEGF, at 4 and 24 h posttreatment (P<0. 01 vs. aortic tissue from control rats). Finally, systemic administration of VEGF to ecNOS-deficient mice failed to inhibit leukocyte-endothelium interactions observed in peri-intestinal venules. We concluded that VEGF is a potent inhibitor of leukocyte-endothelium interaction, and this effect is specifically correlated to augmentation of NO release from the vascular endothelium.--Scalia, R., Booth, G., Lefer, D. J. Vascular endothelial growth factor attenuates leukocyte-endothelium interaction during acute endothelial dysfunction: essential role of endothelium-derived nitric oxide.     

Evaluation of nitric oxide release in the coronary effluent by a novel EPR technique: a study on isolated rat hearts subjected to cold cardioplegia and reperfusion

Aim of this study was to investigate the cardiac release of nitric oxide (NO) before and after cold cardioplegia by a novel electron paramagnetic resonance (EPR) technique. Isolated rat hearts were perfused for 20 min in a Langendorff apparatus and then subjected to 3 hours potassium-hypotermic cardioplegia, followed by 20 min reperfusion. The coronary effluent was collected in a flask containing ferrous-bis-diethyldithiocarbamate as a spin trap of NO. Since the trapping agent was not delivered to the heart with the perfusion medium, we avoided that an abnormal extraction of NO from the tissue could inhibit its biological activity. The EPR signal was well detectable after equilibration (25.6 +/- 3.0 nmol/L +/- S.E.M.) and significantly increased following perfusion with 10 micromol/L serotonin (41.1 +/- 3.2 nmol/L) or 10 micromol/L nitroprusside (43.5 +/- 2.9 nmol/L). The basal level of NO did not change after reperfusion, but serotonin administration was not able to stimulate its release. Serotonin failure to stimulate NO production was not due to a loss of endothelial NO synthase, since its protein expression was not modified after reperfusion. The perfusion pressure increased by 51% after reperfusion and was quite completely restored following serotonin or nitroprusside treatment, with respect to the non-stimulated equilibration condition. Therefore, we suggest that the coronary spasm following a cold cardioplegic arrest is not due to an impaired production of basal NO and that NO-donors can be effective in relaxing vascular smooth muscle cells.     

Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia

The role of nitric oxide (NO) and reactive oxygen species (ROS) in regulating capillary perfusion was studied in the hamster cheek pouch model during normoxia and after 20 min of exposure to 10% O2-90% N2. We measured PO2 by using phosphorescence quenching microscopy and ROS production in systemic blood. Identical experiments were performed after treatment with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) and after the reinfusion of the NO donor 2,2'-(hydroxynitrosohydrazono)bis-etanamine (DETA/NO) after treatment with L-NMMA. Hypoxia caused a significant decrease in the systemic PO2. During normoxia, arteriolar intravascular PO2 decreased progressively from 47.0 +/- 3.5 mmHg in the larger arterioles to 28.0 +/- 2.5 mmHg in the terminal arterioles; conversely, intravascular PO2 was 7-14 mmHg and approximately uniform in all arterioles. Tissue PO2 was 85% of baseline. Hypoxia significantly dilated arterioles, reduced blood flow, and increased capillary perfusion (15%) and ROS (72%) relative to baseline. Administration of L-NMMA during hypoxia further reduced capillary perfusion to 47% of baseline and increased ROS to 34% of baseline, both changes being significant. Tissue PO2 was reduced by 33% versus the hypoxic group. Administration of DETA/NO after L-NMMA caused vasodilation, normalized ROS, and increased capillary perfusion and tissue PO2. These results indicate that during normoxia, oxygen is supplied to the tissue mostly by the arterioles, whereas in hypoxia, oxygen is supplied to tissue by capillaries by a NO concentration-dependent mechanism that controls capillary perfusion and tissue PO2, involving capillary endothelial cell responses to the decrease in lipid peroxide formation controlled by NO availability during low PO2 conditions.     

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.     

Transformations of dinitrosyl iron complexes in an isolated rat heart after introduction of this substance into perfusion medium

The objective of the present research was to study transformations of various physiological NO forms in an isolated rat heart, perfused with the medium containing dinitrosyl iron complexes with glutathione ligand (DNIC-GH). We showed that such aerobic perfusion resulted in accumulation of mostly diamagnetic NO physiological forms (S-nitrosothiols) in myocardial tissue. They were transformed into protein-bound mononuclear dinitrosyl iron complexes during subsequent total ischemia. Meantime, DNIC-GH injection on the onset of ischemia resulted in changes in the state of mitochondrial respiratory chain, characterized by the increase in myocardial concentration of flavosemiquinones.     

Detection of nitrosyl-iron complexes by proton-electron-double-resonance imaging.

The nitrogen monoxide radical (NO*) forms paramagnetic mono- and dinitrosyl-iron complexes in biologic tissues. To establish a noninvasive technique for in vivo NO* imaging, we evaluated the suitability of these complexes as magnetic resonance (MR) contrast agents, making use of the ability of the unpaired electrons of the complexes to enter into dynamic nuclear polarization with water protons and hence produce enhancement on images generated by the technique of proton-electron-double-resonance imaging (PEDRI). Phantom solutions of synthetic nitrosyl-iron complexes (NICs) altered the signal intensity of PEDRI images. The dinitrosyl-iron complex (DNIC) with serum albumin induced a significantly larger signal alteration than the mononitrosyl-iron complex (MNIC) with dithiocarbamate. Exposure of rat liver to sodium nitroprusside (SNP) by ex vivo and in situ perfusion induced a composite X-band electron spin resonance (ESR) spectrum of the isolated liver characteristic of a MNIC and DNIC. On storage of the tissue, the MNIC signal disappeared and the DNIC signal intensity increased. Correspondingly, in cross-sectional PEDRI images taken at room temperature, the SNP-exposed livers initially exhibited a weak signal that strongly increased with time. In conclusion, NICs can be detected using PEDRI and could be exploited for in vivo NO* imaging.     

Occurrence,distribution and possible role of the regulatory peptide endothelin in the nasal mucosa

Nasal blood flow is finely regulated by local release of neurotransmitters, neuropeptides and other bioactive molecules acting via paracrine mechanisms. We have investigated the occurrence and distribution in human nasal mucosa of endothelin, a potent vasoconstrictor peptide, by immunocytochemistry and the effect of systemic administration of endothelin-1 on vascular perfusion of rabbit nasal mucosa by laser Doppler flowmetry. Endothelin-like immunoreactivity was demonstrated within vascular endothelial cells in both developing and mature human mucosa. Nasal epithelial cells and some connective tissue cells, presumed to be macrophages, also displayed specific immunostaining. In rabbits injected with endothelin-1, a potent and prolonged nasal vasoconstriction was observed. It is suggested that endothelin released locally may participate in the regulation of nasal blood flow via paracrine mechanisms. Since endothelin has growth-promoting actions on several cell types, it is also tentatively proposed that this regulatory peptide may play a role during development of the nose.     

Detection of basal NO production in rat tissues using iron–dithiocarbamate complexes

We probe endogenous NO production in WKY rats by trapping NO with iron–dithiocarbamate complexes. The aim was to detect non-stimulated NO production in small organs like kidneys of juvenile rats. The yields of mononitrosyl Fe–dithiocarbamate complexes are small and difficult to quantify in the presence of strong contaminating signals from Cu²⁺–DETC complexes. We evaluate four methods to improve the detection of mononitrosyl Fe–dithiocarbamate adducts: progressive microwave saturation, tissue perfusion, spectral subtraction, and finally, reduction of the tissue with sodium dithionite. While the first three were only moderately useful, reduction was very helpful for quantification of the mononitrosyl Fe–dithiocarbamate yield. The increase in sensitivity allows the detection of non-stimulated NO release in small organs of juvenile rats.     

Inorganic nitrite stimulates pancreatic islet blood flow and insulin secretion

Reactive nitrogen and oxygen species have been proposed to be involved in control of insulin release from the pancreatic β cell. Recent evidence suggests that the supposedly inert anions nitrate and nitrite are metabolized in blood and tissues to form nitric oxide (NO) and other bioactive nitrogen oxides. Here we present evidence for a novel stimulatory role of nitrite in influencing pancreatic islet physiology via a dual mechanism, involving both indirect enhancement (through microcirculation redistribution) and direct insulinotropic effects on the β cell. In rats, intraperitoneal injection of sodium nitrite increased pancreatic islet blood flow by 50% and serum insulin concentrations by 30%, while whole pancreatic blood flow and glycemia remained unaffected. Nitrite also dose dependently enhanced insulin secretion from rat β cells in vitro under nonstimulatory glucose concentrations. This effect was not mimicked by nitrate and was abolished by the guanylyl cyclase (GC) inhibitor ODQ and the NO scavenger cPTIO. It was also mimicked by a cyclic GMP agonist (8-CPT-cGMP) and a classical NO donor (NONOate). Interestingly, a reactive oxygen species scavenger (vitamin E analog, Trolox) abolished the insulin secretion induced by nitrite. We conclude that nitrite exerts dual stimulatory effects on pancreatic islet function, including enhancement of islet blood flow and subsequent insulin secretion in vivo and direct stimulation of insulin release in vitro. The insulinotropic effect of nitrite is cGMP-dependent and involves formation of reactive nitrogen and oxygen species.     

Regulation of Bcl-2 Protein Expression in Human Neuroblastoma SH-SY5Y Cells: Positive and Negative Effects of Protein Kinases C and A, Respectively

Abstract: Nitric oxide (NO), liberated from the photoactive donor Roussin's black salt (RBS), was investigated for its ability to release tritium from [³H]dopamine-loaded rat striatal slices. Our results show that illumination of RBS-pretreated striatal slices caused an increase in basal dopamine release, which was reduced by ∼73% in the presence of oxyhaemoglobin (10 µ M ), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2-bis( o -aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; 10 µ M ) had no effect on the dopamine release stimulated by illumination of RBS-pretreated slices. The concentration of BAPTA-AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 µ M ). Superfusion with zaprinast (10 µ M ) had no effect on RBS-induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro that is independent of calcium.     

Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.     

Effect of uterine blood flow occlusion on shear stress-mediated nitric oxide production and endothelial nitric oxide synthase expression during ovine pregnancy

During normal pregnancy, uterine blood flow (UBF) is increased in association with elevations of endothelial nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Shear stress increases endothelial-derived NO production to reduce vasomotor tone. We hypothesized that decreasing in vivo UBF, and thus shear stress, will decrease NO and/or eNOS levels. In this experiment, one of the main uterine arteries of chronically instrumented late pregnant sheep (125 +/- 1 days' gestation [mean +/- SEM]; n = 15) was occluded for 24 h. Cardiovascular parameters (systemic and uterine arterial pressure, heart rate [HR], and ipsilateral and contralateral UBF) and NO(2)/NO(3) (NO(x)) levels were evaluated. Although UBF measured using Transonic flow probes was reduced unilaterally 41.5% +/- 2.1%, uterine perfusion pressure only fell 12.2% +/- 4.5%. Systemic arterial blood pressure and HR were unaltered. Using radioactive microspheres, ipsilateral UBF was reduced approximately 28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NO(x) levels were reduced 22.1% +/- 6.7% and 22.6% +/- 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial ( approximately 2.5 h) increase in systemic arterial and uterine venous NO(x) levels by 116% +/- 30% and 97% +/- 49%, respectively. Despite a decline in NO(x) levels after 6 h, no significant differences versus preocclusion NO(x) levels were detected by 24 h of occlusion in this experimental group. In contrast, NO(x), UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NO(x).     

Local control of skeletal muscle blood flow during exercise: influence of available oxygen

Reductions in oxygen availability (O(2)) by either reduced arterial O(2) content or reduced perfusion pressure can have profound influences on the circulation, including vasodilation in skeletal muscle vascular beds. The purpose of this review is to put into context the present evidence regarding mechanisms responsible for the local control of blood flow during acute systemic hypoxia and/or local hypoperfusion in contracting muscle. The combination of submaximal exercise and hypoxia produces a "compensatory" vasodilation and augmented blood flow in contracting muscles relative to the same level of exercise under normoxic conditions. A similar compensatory vasodilation is observed in response to local reductions in oxygen availability (i.e., hypoperfusion) during normoxic exercise. Available evidence suggests that nitric oxide (NO) contributes to the compensatory dilator response under each of these conditions, whereas adenosine appears to only play a role during hypoperfusion. During systemic hypoxia the NO-mediated component of the compensatory vasodilation is regulated through a β-adrenergic receptor mechanism at low-intensity exercise, while an additional (not yet identified) source of NO is likely to be engaged as exercise intensity increases during hypoxia. Potential candidates for stimulating and/or interacting with NO at higher exercise intensities include prostaglandins and/or ATP. Conversely, prostaglandins do not appear to play a role in the compensatory vasodilation during exercise with hypoperfusion. Taken together, the data for both hypoxia and hypoperfusion suggest NO is important in the compensatory vasodilation seen when oxygen availability is limited. This is important from a basic biological perspective and also has pathophysiological implications for diseases associated with either hypoxia or hypoperfusion.     

Hemopressin, a hemoglobin fragment, dilates the rat systemic vascular bed through release of nitric oxide

The present study was undertaken to investigate the effects of intravenous (i.v.) administration of rat hemopressin (rHP), 30-1000 microg/kg, on systemic arterial pressure (SAP), cardiac output (CO) and systemic vascular resistance (SVR) in the anesthetized rat. Bolus i.v. injections of rHP produced mild decreases in SAP that were dose-dependent. Since CO was not altered, the decreases in SAP reflect reductions in SVR. The systemic vasodilator response to rHP was not subject to tachyphylaxis. The systemic vasodilator response to rHP was abolished by L-nitro-arginine methylester (L-NAME) but was not altered by meclofenamate. In addition, rHP lacked direct contractile and relaxant activity on isolated rat aortic rings (AA) and pulmonary arterial rings (PA). The present data suggest rHP dilates the rat systemic vascular bed through the endogenous release of nitric oxide (NO) independent of the formation of cyclooxygenase products including prostacyclin. It is possible rHP acts as an endogenous vasodilator substance to regulate local blood flow during clinical states of altered red cell turnover, microvascular disease and hemolysis.