cyclic-gmp and peroxynitric-acid

Following its release from nitric oxide synthase, nitric oxide seldom perfuses the cytosol; rather this reactive mediator quickly interacts with available target molecules proximate to its site of release. Within the cell, virtually every component, low-molecular-weight oxidants and reductants, proteins, lipids, sugars, and nucleic acids can be modified by nitrogen oxides thus acting as potential targets for reactive nitrogen oxides. Adducts formed by nitrogen oxides often modulate the cellular activities of the target molecules, and these modified molecules may be differentially metabolized or localized. The formation of nitrogen oxide adducts can be a reversible process, and the reactive nitrogen species released may be specifically oxidized or reduced during the process. Recently, numerous studies have demonstrated that reversible nitration of cellular proteins acts to transduce molecular signals regulating such diverse processes as muscle contraction, neurotransmission, protein metabolism, and apoptosis. The vast numbers of molecules that undergo biologically relevant interactions with nitrogen oxides imply that the cellular concentration of nitrosated and nitrated species may effectively comprise a reserve or cellular store. Potentially, these nitroso reserves function as critical components of the overall redox status of the intracellular environs. Understanding the dynamic regulation of nitric oxide/nitrogen oxides release from these stores is likely to provide clues important in resolving the complex pathophysiology of poorly understood multifactorial disorders, including neurodegeneration, multiorgan failure, cardiomyopathy, and septic shock.

Topics: Animals; Cyclic GMP; Free Radical Scavengers; Humans; Nitrates; Nitric Oxide; Signal Transduction

Leukocyte accumulation has been shown to be increased in sepsis. Moreover, in inducible nitric oxide synthase (iNOS) knockout mice, a further increase in leukocyte accumulation has been observed during sepsis, suggesting that nitric oxide (NO) may affect leukocyte/endothelial interaction. Accelerated peroxynitrite formation also occurs during sepsis. In the present study, the effect of peroxynitrite or NO on leukocyte adhesion to nitric oxide synthase (NOS)-inhibited or endotoxin-treated endothelium was examined. Bovine aortic endothelial cells were treated with either L-NAME or lipopolysaccharide (LPS) and interferon-gamma for 4 hr and subsequent leukocyte adhesion was measured. Both L-NAME and LPS treatment resulted in increased leukocyte adhesion compared with control. Neither a peroxynitrite donor, SIN-1, nor a direct NO donor, DETA-NO, had any effect on leukocyte adhesion to untreated endothelium. However, when the L-NAME or LPS-treated endothelial cells were treated simultaneously with either SIN-1 or DETA-NO, there was a significant reduction in leukocyte adhesion. Moreover, at the concentrations used in the present study, neither peroxynitrite nor NO showed harmful effects on normal cultured endothelial cells. These data demonstrating inhibition of leukocyte adhesion to endotoxin-treated endothelium suggest that peroxynitrite or NO may exert a beneficial effect during sepsis.

Topics: Animals; Cattle; Cell Adhesion; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Interferon-gamma; Leukocytes; Lipopolysaccharides; Molsidomine; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Triazenes

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing. NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for. NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after. NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not. NO-exposed cells. Cell injury from. NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-. NO-producing cells to. NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.

Topics: Acetylcysteine; Animals; Cell Death; Cell Differentiation; Cell Line; Cell Survival; Cyclic GMP; Hydrazines; Molsidomine; Necrosis; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; Sulfhydryl Compounds

In vivo microdialysis was used to investigate whether nitric oxide (NO) modulates striatal neurotransmitter release in the rat through inducing cyclic GMP formation via soluble guanylate cyclase or formation of peroxynitrite (ONOO(-)). When NO donors, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 1 mM) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1 mM), were retrodialysed for 15 min, acetylcholine (ACh), serotonin (5-HT), glutamate (Glu), gamma-aminobutyric acid (GABA), and taurine levels were significantly increased, whereas those of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) were decreased. Only effects on ACh, 5-HT, and GABA showed calcium dependency. Inhibition of soluble guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 100 and 200 microM) dose-dependently reduced NO donor-evoked increases in ACh, 5-HT, Glu, and GABA levels. Coperfusion of SNAP or NOC-18 with an ONOO(-) scavenger, L-cysteine (10 mM) resulted in enhanced concentrations of Glu and GABA. On the other hand, DA concentrations increased rather than decreased, and no reductions in DOPAC and 5-HIAA occurred. This increase in DA and the potentiation of Glu and GABA were calcium-dependent and prevented by ODQ. Similar to NO, infusions of ONOO(-) (10 or 100 microM) decreased DA, DOPAC, and 5-HIAA. Overall, these results demonstrate that NO increases ACh, 5-HT, Glu, and GABA levels primarily through a cyclic GMP-dependent mechanism. For DA, DOPAC, and 5-HIAA, effects are determined by levels of ONOO(-) stimulated by NO donors. When these are high, they effectively reduce extracellular concentrations through oxidation. When they are low, DA concentrations are increased in a cyclic GMP-dependent manner and may act to facilitate Glu and GABA release further. Thus, changes in brain levels of antioxidants, and the altered ability of NO to stimulate cyclic GMP formation during ageing, or neurodegenerative pathologies, may particularly impact on the functional consequences of NO on striatal dopaminergic and glutamatergic function.

Topics: Animals; Calcium; Corpus Striatum; Cyclic GMP; Dopamine; Dose-Response Relationship, Drug; Free Radical Scavengers; Guanylate Cyclase; Male; Microdialysis; Neurotransmitter Agents; Nitrates; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Wistar

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.

Topics: Animals; Cyclic GMP; Diethylamines; Drug Combinations; Enzyme Inhibitors; Glutathione; In Vitro Techniques; Male; Molsidomine; Myocardial Contraction; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Nucleotides, Cyclic; Oxadiazoles; Oxidation-Reduction; Quinoxalines; Rats; Rats, Wistar; Superoxide Dismutase

Lung inflammation is associated with enhanced expression of proinflammatory cytokines and increased production of nitric oxide (NO) by inducible NO synthase (iNOS). To investigate the possible relationship between cytokine-induced expression of iNOS and epithelial ion channel function, we measured whole-cell current in A549 cells treated with a mixture of cytokines: tumor necrosis factor, interleukin-1 beta, and interferon-gamma for 12 h. Cytokines significantly increased the expression and activity of iNOS, and reduced generation of cGMP in response to stimulation with NO donor S-nitroso-glutathione (GSNO). Patch-clamp studies showed that 100 microM GSNO increased the whole-cell current from 11.2 +/- 1.8 to 19.6 +/- 2.7 pA/pF (n = 16) in control cells, but had no effect in cytokine-treated cells (n = 9). N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of iNOS, restored activation of the current by GSNO in cytokine-treated cells, indicating a crucial role for iNOS in this process. Cells treated with cytokines showed increased levels of peroxynitrite (ONOO(-)), compared with the control, or cells that were treated with the cytokines and 1400W or superoxide dismutase/catalase. Treatment of cells with 100 microM ONOO(-) had no effect on the whole-cell current, but in contrast to untreated cells, subsequent application of GSNO did not activate the current. In conclusion, cytokine-induced expression of iNOS affects activation of the whole-cell current via NO/cGMP pathway, likely by increasing the generation of ONOO(-).

Topics: Cyclic GMP; Cytokines; Enzyme Activation; Glutathione; Humans; Interferon-gamma; Interleukin-1; Ion Channels; Lung; Membrane Potentials; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroso Compounds; Patch-Clamp Techniques; Recombinant Proteins; S-Nitrosoglutathione; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

This paper aimed to study the mechanism of vascular hyporeactivity during severe hemorrhagic shock. Rats were divided into control and shock group. Membrane potential of arteriolar strips was measured with intracellular recording method and membrane potential changes in arteriolar smooth muscle cells (ASMC) were recorded with membrane potential sensitive fluorescent dye (DiBAC4) and confocal microscopy. Hyperpolarization of ASMC membrane appeared at the late stage of shock, which correlated to low vasoreactivity. Glybenclamide, an inhibitor of K(ATP) channel reversed the hyperpolarizing effect. S-nitroso-N-acetylpenicillamine (SNAP), a donor of NO, in a higher concentration (400 mol/l) caused membrane hyperpolarization in control and shock group, which was completely reversed by application of Tiron, a scavenger of O2-. The hyperpolarizing effect of SNAP was decreased by ODQ, glybenclamide and (or) charybdotoxin. It is concluded that hyperpolarization of ASMC leads to vascular hyporeactivity. Peroxynitrite (OONO-) involves in the development of hyperpolarization in severe shock. The production of cGMP and activation of K(ATP) and K(Ca) channel contribute to the hyperpolarizing effect of OONO-*.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Animals; Arginine; Arterioles; Charybdotoxin; Cyclic GMP; Drug Interactions; Female; Free Radical Scavengers; Glyburide; Guanidines; Ion Transport; Male; Membrane Potentials; Mesenteric Arteries; Microscopy, Confocal; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide Donors; Norepinephrine; Oxadiazoles; Penicillamine; Potassium; Potassium Channels; Quinoxalines; Rats; Rats, Sprague-Dawley; Shock, Hemorrhagic; Vasodilator Agents

Nitric oxide (NO) is a well-documented effector molecule in rodent phagocytes but its synthesis in human neutrophils has been controversial. In this study, NO production in human neutrophils activated by chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was measured in the presence of L-arginine (L-Arg) and N(G)-hydroxy-L-arginine (OH-L-Arg), the precursor and intermediate amino acids in NO synthesis, respectively. Incubation of fMLP-activated neutrophils with OH-L-Arg resulted in a production of nitrite, nitrate, and citrulline that was greater than with unstimulated neutrophils but was not inhibited by the NOS inhibitors L-NMMA and L-NIO or the cytochrome P450 inhibitor troleandomycin and was not seen when OH-L-Arg was replaced with L-Arg. This nitrite, nitrate, and citrulline production was not associated with any detectable NO synthesis because no increases in cyclic GMP were observed in the presence of phosphodiesterase inhibitors and in the presence or absence of superoxide dismutase. Moreover, no increases in the formation of the reaction product of NO with superoxide, peroxynitrite, were observed on addition of either OH-L-Arg or L-Arg to activated neutrophils, as assessed either by dihydrorhodamine oxidation or protein nitration. This suggests that, in spite of the production of nitrite, nitrate, and citrulline, commonly used indicators of NO formation, normal human blood neutrophils, are not producing detectable amounts of either NO or peroxynitrite when stimulated with fMLP in the presence of OH-L-Arg.

Topics: Arginine; Cells, Cultured; Citrulline; Cyclic GMP; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitrates; Nitric Oxide; Nitrites; Nitroarginine

We studied the mechanism of nitric oxide (NO) toxicity in cultured rat spinal motoneurons. Treatment with the NO donor NOC-18 (NOC) resulted in slow motoneuron death, ending in apoptosis. The observed motoneuron death was completely prevented by hemoglobin. Treatment with inhibitors of the known intracellular targets of NO, soluble guanylate cyclase, polyADP-ribose polymerase (PARP) and superoxide, did not result in any significant protection against NOC-induced motoneuron death. ATP levels were reduced as soon as 3 h after the start of NOC treatment, suggesting a direct inhibition of cellular energy production. NOC toxicity could be blocked by the general voltage-gated calcium channel blocker cobalt, but not by specific blockers of various subtypes of calcium channels.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Calcium; Cells, Cultured; Cobalt; Cyclic GMP; Electrophysiology; Motor Neurons; Nerve Degeneration; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Rats

1. We report opposite inotropic effects of NO donors in frog cardiac fibres. The negative effect, elicited by either 3-morpholino-sydnonimine (SIN-1) or S-nitroso-N-acetyl-penicillamine (SNAP), involved cyclic GMP (cGMP) production. However, SIN-1, unlike SNAP, could elicit a positive effect, in a superoxide dismutase (SOD)-sensitive manner. SIN-1, unlike SNAP, can release both NO and superoxide anion, the precursors of peroxynitrite (OONO-). The role of these messengers was examined. 2. Catalase did not reduce the positive inotropic effect of SIN-1. Thus, a conversion of superoxide anion into hydrogen peroxide was not involved in this effect. In addition, catalase did not modify the negative effects of SIN-1 plus SOD, or SNAP plus SOD. 3. LY 83583, a superoxide anion generator, elicited a positive inotropic effect, like SIN-1. The effect of LY 83583 was additive to the negative effects of SIN-1 or SNAP, and to the positive effect of SIN-1. Thus, superoxide anion generation, per se, did not account for the positive effect of SIN-1. 4. Authentic peroxynitrite (OONO-), but not mock-OONO- (negative control plus decomposed OONO-), exerted a dramatic positive inotropic effect in cardiac fibres. The effect of OONO- was larger in atrial fibres, as compared with ventricular fibres. 5. The positive effect of OONO- was not additive with that of SIN-1, suggesting a common mechanism of action. In contrast, the effects of either OONO- or SIN-1 were additive with the negative inotropic effect of SNAP. Furthermore, the effect of OONO-, like that of SIN-1, was not antagonized by 1H-[1,2,4]xidiazolo[4, 3-a]quinoxaline-1-one (ODQ; 10 microM), the guanylyl cyclase inhibitor. 6. The positive inotropic effects of SIN-1 and OONO- were not modified by hydroxyl radical scavengers, such as dimethyl-thio-urea (DMTU; 10 mM). 7. The positive inotropic effect of SIN-1 (100 microM) was abolished in sodium-free solutions, a treatment that eliminates the activity of the sodium-calcium exchanger. In contrast, the effect of SIN-1 was unchanged by a potassium channel inhibitor (tetraethyl-ammonium, 20 mM), or a sodium-potassium pump inhibitor (ouabain 10 microM). 8. We conclude that OONO- is a positive inotropic agent in frog cardiac fibres. The generation of OONO- accounts for the positive inotropic effect of SIN-1. OONO- itself was responsible for the positive inotropic effect, and appeared to modulate the activity of the sodium-calcium exchanger.

Topics: Aminoquinolines; Animals; Atrial Function; Catalase; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Heart Atria; Heart Ventricles; Hydroxyl Radical; Molsidomine; Muscle Fibers, Skeletal; Myocardial Contraction; Myocardium; Nitrates; Nitric Oxide Donors; Oxadiazoles; Oxidants; Quinoxalines; Rana esculenta; Sodium; Sodium-Calcium Exchanger; Ventricular Function

Topics: Apoptosis; Cell Division; Cyclic GMP; Free Radical Scavengers; Humans; Neoplasms; Nitrates; Nitric Oxide; Superoxides; Vasodilation

Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.

Topics: Chemotaxis, Leukocyte; Cyclic GMP; Free Radicals; Humans; In Vitro Techniques; Methionine; Neutrophils; Nitrates; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxidants

The gas radical, nitric oxide (NO), is a key signalling molecule in the cardiovascular, nervous and immune systems. Recently it has been found that it is produced by both the osteoblast and osteoclast and that it has major effects in producing osteoclast detachment and exerting a tonic inhibition of bone resorption. This detaching effect is mediated by a rapid increase in cGMP following calcium-triggered e-NOS activation during normal bone resorption. This effect is not reproduced in vitro by 8-bromo-cGMP but is seen with the newer rapidly permeant 8-pCPT-cGMP. However the inhibition of bone resorption by SIN-1 in vitro is not mediated solely by cGMP but depends on other factors still unidentified. Superoxide anions alone produces both osteoclast detachment and inhibition of resorption. Both of these actions may be mediated at least in part by peroxynitrite which has the same effect as NO alone on osteoclast detachment.

Topics: Animals; Animals, Newborn; Bone Resorption; Calcium; Catalase; Cells, Cultured; Cyclic GMP; Enzyme Activation; Hydrogen Peroxide; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Osteoclasts; Rats; Rats, Sprague-Dawley; Superoxides; Thionucleotides

This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO-) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40+/-5% depressed an hour after the reintroduction of O2. O2 uptake by rat left ventricular cardiac muscle, measured by a Clark-type O2 electrode, was also inhibited by 24+/-2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro-L-arginine, exogenous superoxide dismutase, and the ONOO- scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO- with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) plus the superoxide-releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO-, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO-dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO- and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO- formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation.

Topics: Animals; Bradykinin; Cyclic GMP; Free Radical Scavengers; Hydroxyl Radical; Hypoxia; In Vitro Techniques; Kinetics; Mannitol; Mitochondria, Heart; Myocardial Contraction; Nitrates; Nitric Oxide; Nitroarginine; Oxidants; Oxygen Consumption; Penicillamine; Rats; S-Nitroso-N-Acetylpenicillamine; Sodium Cyanide; Superoxide Dismutase

We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K+ channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 (Cell Physiol. 39): C1336-C1342, 1996]. Because NO at high concentration has been shown to react with superoxide (O-2) to form peroxynitrite (OONO-) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L699-L722, 1995 and M. S. Wolin. Microcirculation 3: 1-17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100-200 microM S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of O-2. NO donors, 10 microM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an O-2 donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and O-2.

Topics: Animals; Cell Membrane; Cyclic GMP; Ditiocarb; In Vitro Techniques; Kidney Cortex; Kidney Tubules, Collecting; Membrane Potentials; Nitrates; Nitric Oxide; Nitroprusside; Patch-Clamp Techniques; Penicillamine; Potassium Channels; Potassium Channels, Calcium-Activated; Pyrogallol; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Small-Conductance Calcium-Activated Potassium Channels; Superoxides

Anti-ischemic therapy with organic nitrates is complicated by tolerance. Induction of tolerance is incompletely understood and likely multifactorial. Recently, increased production of reactive oxygen species (ROS) has been investigated, but it has not been clear if this is a direct consequence of the organic nitrate on the vessel or an in vivo adaptation to the drugs. To examine the possibility that nitrates could directly stimulate vascular ROS production, we compared the development of nitrate tolerance with the formation of ROS induced by pentaerithrityltetranitrate (PETN) or nitroglycerin (GTN) in vitro in porcine smooth muscle cells, endothelial cells, washed ex vivo platelets and whole blood. By examining cGMP formation, it was found that 24-hr treatment with GTN but not PETN induced significant nitrate tolerance, which was prevented by parallel treatment with Vit C. Incubation of vascular cells acutely with 0.5 mM GTN doubled the rate of ROS generation, whereas PETN had no such effect. The rate of ROS (peroxynitrite and O2) formation detected by specific spin traps in tolerant smooth muscle cells, treated for 24 hr with 0.01 mM GTN, was substantially higher (30.5 nM/min) than in control cells acutely treated with 0.5 mM GTN (25 nM/min). In contrast to PETN, GTN induces nitrate tolerance and also increases the formation of ROS both in vascular cells and in whole blood. ROS formation is minimally stimulated by PETN comparable to data obtained in Vit C-suppressed GTN tolerance. ROS formation induced by organic nitrates seems to be a key factor in the development of nitrate tolerance.

Topics: Animals; Blood Platelets; Cells, Cultured; Cyclic GMP; Drug Tolerance; Endothelium, Vascular; Hydroxyl Radical; Muscle, Smooth, Vascular; Nitrates; Nitroglycerin; Pentaerythritol Tetranitrate; Reactive Oxygen Species; Spin Trapping; Superoxides; Swine

Peroxynitrite (ONOO-) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO- in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO- on human platelets, a system in which we have previously shown that ONOO- has both pro- and anti-aggregatory effects.. Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO- was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin.. Peroxynitrite (50-400 microM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO- acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma, ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50-100 microM) of ONOO- inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin.. Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO- to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO- in biological systems. We hypothesize that the conversion of ONOO- to NO is the critical factor determining the outcome of ONOO- exposure in vivo.

Topics: Blood Platelets; Calcium; Collagen; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Free Radical Scavengers; Guanylate Cyclase; Humans; Immunohistochemistry; Nitrates; Nitric Oxide; Oxadiazoles; Oxidants; Oxyhemoglobins; P-Selectin; Platelet Activation; Quinoxalines

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Topics: Animals; Benzoates; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Kinetics; Molsidomine; Nitrates; Nitric Oxide; Nitrites; Serum Albumin, Bovine; Swine; Tyrosine

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.

Topics: Acetonitriles; Calcium; Cell Degranulation; Cyclic GMP; Humans; Molsidomine; Morpholines; Neutrophils; Nitrates; Nitric Oxide

Peroxynitrite, formed from nitric oxide and superoxide, may affect neurofilament assembly and cause neurofilament accumulation in motoneurons. This hypothesis may reconcile the mutations of two genes: superoxide dismutase-1 in some patients with familial amyotrophic lateral sclerosis, and the gene for the heavy neurofilament in some patients with sporadic amyotrophic lateral sclerosis previously reported. We found colocalization of superoxide dismutase-1 and nitric oxide synthase in the foci of neurofilament accumulation as 'conglomerates' in upper motor neurons and 'axonal spheroids' in lower motor neurons. In addition, all the specific molecules related to the reactions, including calmodulin, 3', 5'-cyclic guanosine-monophosphate, citrulline, and nitrotyrosine were found strongly immunopositive in the site of neurofilament accumulation. Our data support the view that the neurofilament aggregates are tightly linked with superoxide dismutase-1 and nitric oxide synthase activities. Both enzymes may focally contribute to peroxynitrite formation at light neurofilament, which is rich in both tyrosine and arginine residues and hence considered as the vulnerable site for nitrotyrosine formation. Nitrotyrosine is known to inhibit phosphorylation and if it impairs phosphorylation of neurofilament subunits, either light or heavy, may alter the slow axonal transport culminating in proximo-distal accumulation of NF and slowly progressive motoneuron death.

Topics: Amyotrophic Lateral Sclerosis; Citrulline; Cyclic GMP; Cytoplasm; Humans; Immunohistochemistry; Motor Neurons; Neurofilament Proteins; Nitrates; Nitric Oxide; Nitrogen; Oxidation-Reduction; Superoxide Dismutase; Tyrosine

Here we demonstrate that human keratinocytes possess a Ca2+/calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm2) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[3H]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-L-arginine (L-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm2) of particulate NO synthase. A 5-fold increase in superoxide (O2-) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm2)-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm2) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by L-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO- released by UVB-irradiated human keratinocytes in skin erythema and inflammation.

Topics: Allosteric Regulation; Calcium; Calmodulin; Cell Membrane; Citrulline; Cyclic GMP; Fluorescence Polarization; Guanylate Cyclase; Humans; Keratinocytes; Kinetics; Microsomes; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Reactive Oxygen Species; Temperature; Ultraviolet Rays

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. Since reduced EDRF/NO release from the endothelium is a major key event in the development of atherosclerosis, we investigated the effect of cholesterol on endothelial cell particulate (membrane-bound) NO synthase activity. Low concentrations (up to 0.2 mM) of liposomal cholesterol progressively activated plasma membrane-bound NO synthase. Increasing cholesterol concentration above that which maximally stimulated enzyme activity produced a progressive inhibition with respect to the control value. In time course experiments using endothelial cell plasma membranes enriched with cholesterol, changes in NO production were followed by analogous changes in soluble guanylate cyclase activity (sGC). N-Monomethyl-L-arginine (L-NMMA) (1 mM) inhibited particulate NO synthase activity at all cholesterol concentrations used with subsequent decreases in cGMP production. Egg lecithin liposomes (free of cholesterol) had no effect on NO synthase activity. A three-fold increase in superoxide (O2-) and a 2.5-fold increase in NO formation followed by an eight-fold increase in peroxynitrite (ONOO-) production by cholesterol-treated microsomes isolated from endothelial cells was observed, one which rose further up to eight-fold in the presence of superoxide dismutase (SOD) (10 U/mL). Cholesterol had no effect on Lubrol-PX solubilized membrane-bound NO synthase or on cytosolic (soluble) NO synthase activities of endothelial cells. Cholesterol modulated lipid fluidity of plasma membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH) as indicated by the steady state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius plots of [(ro/r)-1]-1 indicated that the lipid phase separation of the membranes at 26.2 +/- 1.5 degrees was elevated to 34.4 +/- 1.9 degrees in cholesterol-enriched membranes, consistent with a general decrease in membrane fluidity. Cholesterol-enriched plasma membranes treated with egg lecithin liposomes showed a lipid phase separation at 27.5 +/- 1.6 degrees, indicating the reversible effect of cholesterol on membrane lipid fluidity. Arrhenius plots of NO synthase activity exhibited break point at 26.9 +/- 1.8 degrees which rose to 35.6 +/- 2.1 degrees in 0.5 mM cholesterol-treated plasma membranes and decreased to 21.5 +/- 1.4 degrees in plasma membranes treated with 0.2 mM cholesterol. The allosteric properties o

Topics: Amino Acid Oxidoreductases; Animals; Cattle; Cell Membrane; Cholesterol; Citrulline; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Manganese; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Superoxides

Peroxynitrite (ONOO-) is widely recognized as mediator of NO toxicity, but recent studies have indicated that this compound may also have physiological activity and induce vascular relaxation as well as inhibition of platelet aggregation. We found that ONOO- induced a pronounced increase in endothelial cyclic GMP levels, and that this effect was significantly attenuated by pretreatment of the cells with GSH-depleting agents. In the presence of 2 mM GSH, ONOO- stimulated purified soluble guanylyl cyclase with a half-maximally effective concentration of about 20 microM. In contrast to the NO donor 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), ONOO- was completely inactive in the absence of GSH, indicating that thiol-mediated bioactivation of ONOO- is involved in enzyme stimulation. Studies on the reaction between ONOO- and GSH revealed that about 1% of ONOO- was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to be stable in solution, but slowly decomposed in the presence of GSH. GSH-induced decomposition of S-nitrosoglutathione was apparently catalyzed by trace metals and was accompanied by a sustained release of NO and a 40-100-fold increase in its potency to stimulate purified soluble guanylyl cyclase. Our data suggest that the biologic activity of ONOO- involves S-nitrosation of cellular thiols resulting in NO-mediated cyclic GMP accumulation.

Topics: Animals; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Glutathione; Guanylate Cyclase; Nitrates; Nitric Oxide; Nitroso Compounds; S-Nitrosoglutathione; Swine

Peroxynitrite stimulated the synthesis of cyclic GMP by rat aortic smooth muscle in a time- and dose-dependent manner. Peak formation of cyclic GMP occurred at 1 min with 100 microM peroxynitrite and was inhibited by oxyhemoglobin. Peroxynitrite was less potent than nitric oxide in stimulating cyclic GMP synthesis. Peroxynitrite also enhanced endothelial-dependent cyclic GMP synthesis, via generation of a long-lived substance, which was prevented by inhibition of glutathione synthesis. These data show that peroxynitrite stimulates cyclic GMP synthesis, inferring production of low yields of nitric oxide or associated derivatives. Additionally, vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase.

Topics: Animals; Aorta; Cattle; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Glutathione; Guanylate Cyclase; Kinetics; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Oxyhemoglobins; Rats

The inhibition of platelet aggregation by peroxynitrite, a reactive oxygen species derived from the interaction of nitric oxide (NO) and superoxide, was examined in platelet-rich plasma. In this report, we have used a preparation of peroxynitrite that was free of H2O2 and MnO2. As such, peroxynitrite dose-dependently (50-200 microM) inhibited aggregation of human platelets stimulated by ADP (5 microM), collagen (0.5 microgram), thrombin (0.5U/microL) and U46619 (1 microM). In addition, peroxynitrite reversed platelet aggregation induced by collagen, ADP, and thrombin. Peroxynitrite, preincubated with platelet-poor plasma or albumin (7%) for 30 min, did not alter the inhibition of platelet aggregation. This suggested that the inhibitory action of peroxynitrite may be due to nitrosylation of proteins, which by themselves possess activity, rather than conversion to NO or NO donors. Furthermore, we show that peroxynitrite increased the cGMP level only at 200 microM concentrations, further suggesting that the action of peroxynitrite was not completely due to its conversion to NO or NO donors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adult; Blood Platelets; Collagen; Cyclic GMP; Humans; Nitrates; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Spectrophotometry; Thrombin; Thromboxane A2

In this study, we analysed the implication of superoxide (O2-.) and nitric oxide (NO.) free radicals and their resulting product peroxynitrite (ONOO-) in the neuronal death induced by the activation of the glutamatergic receptor of the N-methyl-D-aspartate (NMDA) subtype using cultured cerebellar granule cells. The NOl donor SIN-1 (3-morpholinosydnonimine N-ethylcarbamide), at concentrations which produced a much higher guanylate cyclase activation (i.e. NO. concentration) than NMDA, was not neurotoxic and did not increase the NMDA-induced neuronal death. The absence of involvement of NO. in NMDA-induced neuronal death was confirmed by the ineffectiveness of L-NG-nitroarginine (L-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) experiments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, indicated that NMDA receptor stimulation led to the generation of O2-. from at least 15-30 min. The generation of O2-. by xanthine (XA)-xanthine oxidase (XO) induced a neuronal death similar to that of NMDA. XA-XO-induced neuronal death was suppressed by addition of either superoxide dismutase (SOD) plus catalase (CAT), or DMPO in the incubation medium. In contrast, NMDA-induced neuronal death was widely blocked by DMPO and other spin trap compounds, but not by SOD +/- CAT. XA-XO-induced neuronal death was not potentiated by SIN-1 indicating that ONOO- is not more toxic than O2-. in our neuronal model.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Catalase; Cell Death; Cells, Cultured; Cerebellum; Cyclic GMP; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Mice; Molsidomine; N-Methylaspartate; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Receptors, N-Methyl-D-Aspartate; Superoxide Dismutase; Superoxides; Xanthine Oxidase