cyclic-gmp and 1-1-diethyl-2-hydroxy-2-nitrosohydrazine

There is increasing evidence that the permeability of the glomerular filtration barrier (GFB) is partly regulated by a balance between the bioavailability of nitric oxide (NO) and that of reactive oxygen species (ROS). It has been postulated that normal or moderately elevated NO levels protect the GFB from permeability increases, whereas ROS, through reducing the bioavailability of NO, have the opposite effect. We tested the tentative antagonism between NO and ROS on glomerular permeability in anaesthetized Wistar rats, in which the left ureter was cannulated for urine collection while simultaneously blood access was achieved. Rats were systemically infused with either l-NAME or l-NAME together with the superoxide scavenger Tempol, or together with l-arginine or the NO-donor DEA-NONOate, or the cGMP agonist 8-bromo-cGMP. To measure glomerular sieving coefficients (theta, θ) to Ficoll, rats were infused with FITC-Ficoll 70/400 (mol/radius 10-80 Å). Plasma and urine samples were analyzed by high-performance size-exclusion chromatography (HPSEC) for determination of θ for Ficoll repeatedly during up to 2 h. l-NAME increased θ for Ficoll

Topics: Animals; Cyclic GMP; Enzyme Inhibitors; Glomerular Filtration Rate; Hydrazines; Kidney Glomerulus; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Permeability; Rats; Rats, Wistar; Reactive Oxygen Species

This investigation explored the mechanism for inhibition of β2 integrin adhesion molecules when neutrophils are exposed to nitric oxide ((•)NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA, and cells from knock-out mice. Optimal inhibition occurs with exposures to a (•)NO flux of ∼ 28 nmol/min for 2 min or more, which sets up an autocatalytic cascade triggered by activating type 2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400 W), or with cells from mice lacking NOS-2 or the gp91(phox) component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, and protein-disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The autocatalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high (•)NO flux (∼ 112 nmol/min) that inactivates NOX.

Topics: Actins; Animals; CD18 Antigens; Cell Adhesion; Cyclic GMP; Guanylate Cyclase; Humans; Hydrazines; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Nitric Oxide; Nitric Oxide Synthase Type II; RNA, Small Interfering

Spinal motor control networks are regulated by neuromodulatory systems to allow adaptability of movements. The present study aimed to elucidate the role of nitric oxide (NO) in the modulation of mammalian spinal locomotor networks. This was investigated with isolated spinal cord preparations from neonatal mice in which rhythmic locomotor-related activity was induced pharmacologically. Bath application of the NO donor diethylamine NONOate (DEA/NO) decreased the frequency and modulated the amplitude of locomotor-related activity recorded from ventral roots. Removal of endogenous NO with coapplication of a NO scavenger (PTIO) and a nitric oxide synthase (NOS) blocker [nitro-l-arginine methyl ester (l-NAME)] increased the frequency and decreased the amplitude of locomotor-related activity. This demonstrates that endogenously derived NO can modulate both the timing and intensity of locomotor-related activity. The effects of DEA/NO were mimicked by the cGMP analog 8-bromo-cGMP. In addition, the soluble guanylyl cyclase (sGC) inhibitor ODQ blocked the effects of DEA/NO on burst amplitude and frequency, although the frequency effect was only blocked at low concentrations of DEA/NO. This suggests that NO-mediated modulation involves cGMP-dependent pathways. Sources of NO were studied within the lumbar spinal cord during postnatal development (postnatal days 1-12) with NADPH-diaphorase staining. NOS-positive cells in the ventral horn exhibited a rostrocaudal gradient, with more cells in rostral segments. The number of NOS-positive cells was also found to increase during postnatal development. In summary, we have shown that NO, derived from sources within the mammalian spinal cord, modulates the output of spinal motor networks and is therefore likely to contribute to the fine-tuning of locomotor behavior.

Topics: Action Potentials; Animals; Cyclic GMP; Cyclic N-Oxides; Enzyme Inhibitors; Free Radical Scavengers; Hydrazines; Imidazoles; Locomotion; Mice; Mice, Inbred C57BL; Motor Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Spinal Cord

Nitroxyl (HNO) is a redox congener of NO. We now directly compare the antihypertrophic efficacy of HNO and NO donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO scavenger) or CGRP8-37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.

Topics: Animals; Animals, Newborn; Antioxidants; Cardiomegaly; Cardiovascular Agents; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Inhibitors; Gene Expression Regulation; Guanylate Cyclase; Hydrazines; Myocytes, Cardiac; Nitric Oxide Donors; Nitrogen Oxides; Pyrogallol; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems; Soluble Guanylyl Cyclase; Time Factors

Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.

Topics: Camellia sinensis; Cold Temperature; Cyclic GMP; Cyclic N-Oxides; Free Radical Scavengers; Germination; Hydrazines; Imidazoles; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Pollen Tube; Proline; Signal Transduction; Stress, Physiological

Nitric oxide (NO) is an important regulator of Na(+) reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na(+) channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na(+)/K(+)-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na(+)/K(+)-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na(+)/K(+)-ATPase α subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na(+) reabsorption by interfering with the activity of highly selective Na(+) channels and the Na(+)/K(+)-ATPase.

Topics: Amiloride; Animals; Biotinylation; Blotting, Western; Bronchioles; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Epithelial Sodium Channels; Guanylate Cyclase; Hemoglobins; Humans; Hydrazines; Membrane Potentials; Nitric Oxide; Nitric Oxide Donors; Patch-Clamp Techniques; Receptors, Cytoplasmic and Nuclear; Sodium; Sodium Channel Blockers; Sodium-Potassium-Exchanging ATPase; Soluble Guanylyl Cyclase; Sulfhydryl Compounds; Xenopus

In isolated cells, soluble guanylyl cyclase (sGC) activity is regulated by exogenous nitric oxide (NO) via downregulation of expression and posttranslational S-nitrosylation. The aim of this study was to investigate whether such regulatory mechanism impact on endothelium-dependent vasodilation in a newly developed mouse strain carrying an endothelial-specific overexpression of eNOS (eNOS(++)). When compared with transgene negative controls (eNOS(n)), eNOS(++)-mice showed a 3.3-fold higher endothelial-specific aortic eNOS expression, increased vascular cGMP and VASP phosphorylation, a L-nitroarginine (L-NA)-inhibitable decrease in systolic blood pressure, but normal levels of peroxynitrite and nitrotyrosine formation, endothelium-dependent aortic vasodilation and vasodilation to NO donors. Western blot analysis for sGC showed similar protein levels of sGC-α1 and sGC-β1 subunits in eNOS(n) and eNOS(++). In striking contrast, the activity of isolated sGC was strongly decreased in lungs of eNOS(++). Semiquantitative evaluation of sGC-β1-S-nitrosylation demonstrated that this loss of sGC activity is associated with increased nitrosylation of the enzyme in eNOS(++), a difference that disappeared after L-NA-treatment. Our data suggest the existence of a physiologic NO-dependent posttranslational regulation of vascular sGC in mammals involving S-nitrosylation as a key mechanism. Because this mechanism can compensate for reduction in vascular NO bioavailability, it may mask the development of endothelial dysfunction.

Topics: Animals; Aorta; Cell Adhesion Molecules; Cyclic GMP; Guanylate Cyclase; Hydrazines; Lung; Mice; Mice, Inbred C57BL; Microfilament Proteins; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphoproteins; Protein Processing, Post-Translational

We have previously demonstrated that exogenous nitric oxide (NO) inhibited anti-IgE-mediated histamine release from human cultured mast cells. In the current study, we further investigated if syntheses of eicosanoids and cytokines were also suppressed by NO donors and evaluated if activation of soluble guanylyl cyclase (sGC) was an underlying mechanism. The effects of the NO donor diethylamine NONOate (DEA/NO) on IgE-dependent syntheses of eicosanoids (prostaglandin D(2) and cysteinyl leukotrienes) and cytokines (tumor necrosis factor-alpha and interleukin-8) from buffy coat derived human cultured mast cells were examined. The effects of sGC related agents on human mast cell activation were studied by measuring histamine release. DEA/NO (10(-7)-10(-4)M) dose-dependently inhibited anti-IgE induced release of histamine, eicosanoids and cytokines. It could also significantly increase intracellular cyclic guanosine monophosphate (cGMP) but reduce anti-IgE induced activation of ERK1/2, JNK1/2 and NF-kappaB. The inhibition of anti-IgE induced histamine release by DEA/NO was reversed by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10(-7)M) and the cGMP-dependent protein kinase (PKG) inhibitor, Rp-8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS, 10(-5)M). The current study confirmed the inhibitory action of exogenous NO on immunological activation of human mast cells. We also provided evidence for the first time that the activation of the sGC-cGMP-PKG pathways together with the suppression of phosphorylation of MAPKs and NF-kappaB contributed to the mast cell modulating action of NO in human.

Topics: Antibodies, Anti-Idiotypic; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Guanylate Cyclase; Histamine; Humans; Hydrazines; Mast Cells; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Glucocorticoids (GC) remain the first choice of treatment in asthma, but GC therapy is not always effective and is associated with side effects. In a porcine study in our laboratory, simultaneous administration of GC and nitric oxide (NO) attenuated the endotoxin-induced inflammatory response and made GC treatment more effective than inhaled NO or steroids alone. In the present study, we aimed to further investigate the interactions between NO and GC treatment in two murine models of asthma. Inflammation was induced by endotoxin, ovalbumin, or a combination of both. With an animal ventilator and a forced oscillation method (FlexiVent), lung mechanics and airway reactivity to methacholine in response to various treatments were assessed. We also describe histology and glucocorticoid receptor (GR) protein expression in response to inhaled NO treatment [40 ppm NO gas or NO donors sodium nitroprusside (SNP) or diethylamine NONOate (DEA/NO)]. SNP and GC provided protection against bronchoconstriction to a similar degree in the model of severe asthma. When GC-treated mice were given SNP, maximum airway reactivity was further reduced. Similar effects were seen after DEA/NO delivery to GC-treated animals. Using 1-H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor, we found this effect of NO donors to be mediated through a cGMP-independent mechanism. In the severe model, prolonged NO treatment restored or even increased the nuclear levels of GR. In conclusion, in our murine model of severe asthma GC treatment provided protection to only a limited degree against bronchoconstriction, while concomitant treatment with a NO donor was markedly more potent than the use of either NO or GC alone.

Topics: Administration, Inhalation; Animals; Anti-Asthmatic Agents; Asthma; Bronchial Provocation Tests; Bronchoconstriction; Bronchoconstrictor Agents; Cyclic GMP; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Female; Glucocorticoids; Guanylate Cyclase; Hydrazines; Hydrocortisone; Injections, Intraperitoneal; Lipopolysaccharides; Methacholine Chloride; Mice; Mice, Inbred BALB C; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Ovalbumin; Oxadiazoles; Pneumonia; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Receptors, Glucocorticoid; Respiration, Artificial; Respiratory Mechanics; Soluble Guanylyl Cyclase

Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion.. Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development.. sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Topics: Animals; Calcium; Calcium-Binding Proteins; Carbazoles; Cell Hypoxia; Cells, Cultured; Coronary Vessels; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytosol; Edema, Cardiac; Endoplasmic Reticulum; Endothelial Cells; Enzyme Activators; Gap Junctions; Guanylate Cyclase; Homeostasis; Hydrazines; Male; Myocardial Reperfusion Injury; Myosin Light Chains; ortho-Aminobenzoates; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Second Messenger Systems; Soluble Guanylyl Cyclase; Sulfonamides; Time Factors

Common bile duct ligation (CBDL) in the guinea pig is a well-defined model of acalculous cholecystitis. Nitric oxide (NO) mediates smooth muscle relaxation by stimulating the activity of soluble guanylate cyclase. The aim of this study was to determine whether the NO/cyclic guanosine monophosphate pathway plays a role in gallbladder relaxant response after CBDL.. Relaxant response of gallbladder muscle strips from CBDL and sham-operated guinea pigs was studied in vitro. Animals were treated with saline, aminoguanidine or an aminoguanidine + L-arginine combination in vivo. Concentration-response curves of papaverine, diethylamine/NO, YC-1, sildenafil and amrinone were obtained and relaxations in each group were calculated as the percent of the contractions induced by carbachol (10(-6) M).. There was a significant decrease in the gallbladder muscle relaxant responses to these substances in CBDL and aminoguanidine groups compared with sham surgical controls. The decreased relaxant response was reversed by aminoguanidine + L-arginine but not by aminoguanidine alone.. Decreased relaxant responses might be due to the reduced guanylate cyclase enzyme activity, but further studies are required.

Topics: Amrinone; Animals; Arginine; Bile Ducts; Cyclic GMP; Gallbladder; Guanidines; Guinea Pigs; Hydrazines; In Vitro Techniques; Indazoles; Ligation; Male; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Papaverine; Piperazines; Purines; Sildenafil Citrate; Sulfones

In the classical pathway, the opposing activities of guanylyl cyclases (GC) and phosphodiesterases (PDE), and the effect of the cGMP-dependent protein kinase (cGK) on its targets, determine the biological responses to NO signaling. Here we tested the hypothesis that vascular dysfunction may be due to altered expression and activity of these effectors of NO signaling. Every other set of rat second order mesenteric resistance arteries (MA) were ligated, resulting in chronic low flow (LF) in the upstream MA1 and high flow (HF) in the adjacent MA1 without tissue ischemia. eNOS and iNOS were up-regulated in HF and LF MA1, respectively, in the sub-acute phase (four days) of vascular remodeling. The Day4 HF/LF MA1s were under increased control of NO as indicated by reduced sensitivity to the vasoconstrictor phenylephrine and its normalization with the NOS antagonist L-NAME. PDE5 mRNA and protein were also significantly up-regulated in the HF/LF MA1 with no change in sGC or PKG1, an effect that was dependent upon NO synthesis. The PDE5 inhibitor Sildenafil was several-fold more powerful in relaxing the HF/LF MA1s, and pre-treatment with Sildenafil uncovered an increased responsiveness of HF/LF MA1s to the NO donor DEA/NO. We conclude that induction of PDE5 de-sensitizes this systemic resistance artery to sustained NO signaling under chronic HF/LF. Treatment with PDE5 antagonists, in contrast to NO donors, may more specifically and effectively increase blood flow to chronically hypo-perfused tissues.

Topics: Animals; Blotting, Western; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Hydrazines; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphodiesterase 5 Inhibitors; Piperazines; Polymerase Chain Reaction; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones; Vascular Resistance; Vasodilation

Reduction of intracellular calcium ([Ca(2+)](i)) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca(2+) mobilization during SMC contraction influenced NO efficacy.. Isometric contractions by depolarization (high potassium, K(+)) or alpha-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca(2+)](i) (Fura-2) were measured in aortic segments from C57Bl6 mice.. Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K(+) solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca(2+)](i), which was suppressed by sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K(+)-stimulated segments did not parallel a decline of [Ca(2+)](i). However, stimulation (BAY K8644) of L-type Ca(2+) influx diminished, while inhibition (nifedipine, 1-100 nM) augmented the relaxing capacity of NO.. In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca(2+) re-uptake into the SR and was prominent when intracellular Ca(2+) was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca(2+)](i), presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca(2+) influx, and L-type Ca(2+) channel blockers increase the vasodilator efficacy of NO.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Calcium; Calcium Channels, L-Type; Cyclic GMP; Hydrazines; In Vitro Techniques; Intracellular Space; Membrane Potentials; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Vasodilation

Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.

Topics: Adult; alpha-Thalassemia; Anemia, Sickle Cell; CD11a Antigen; CD11b Antigen; Cell Adhesion; Cyclic GMP; Endothelial Cells; Female; Fibronectins; Humans; Hydrazines; Hydroxyurea; Integrin alpha4; Intercellular Adhesion Molecule-1; Male; Middle Aged; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Pyrazoles; Pyridines; Quinoxalines; Sickle Cell Trait

South American (SA) opossum lower esophageal sphincter (LES) circular smooth muscle relaxes by activation of enteric nerves elicited by EFS (electrical field stimulation, 0.5 ms, 48 V, 0.5-8 Hz for 10 s). The identity of the mediator released and the cellular mechanism, however, remain to be fully elucidated. The purpose of this study was to determine the effect of the enzyme soluble guanylate cyclase (cGC) inhibitors, cystamine (100 microM), methylene blue (30 microM), LY 83583 (6-anilino-5,8 quinoledione, 10 microM) and ODQ (H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one, 1 microM) on the relaxations induced by EFS and by exogenous NO (nitric oxide, 0.5 mM) or NO-donors on SA opossum LES smooth muscle strips. EFS caused frequency-dependent relaxations, which were inhibited by NO-synthase inhibitors and abolished by tetrodotoxin. Cystamine did not affect relaxations caused by EFS and NO or NO-donor. Methylene blue also failed to affect EFS-caused relaxations, although it was capable of inhibiting relaxation induced by NO. LY 83583 inhibited relaxations induced by NO, but did not affect those induced by EFS or by SNAP and HXA. ODQ abolished relaxations caused by EFS at lower frequencies and by HXA (hydroxylamine, 10 microM) and SNAP (S-nitroso-N-acetyl penicillamine, 10 microM). Relaxations at higher frequencies of EFS and induced by SNP (sodium nitroprusside, 30 microM) and NO were only reduced by ODQ. These findings indicate that activation of the cGC can be involved in relaxations induced by EFS at lower frequencies, but other mechanisms can be involved at higher frequencies of EFS and caused by SNP or NO.

Topics: Aminoquinolines; Animals; Cyclic GMP; Cysteamine; Electric Stimulation; Esophageal Sphincter, Lower; Female; Guanylate Cyclase; Hydrazines; Hydroxylamine; In Vitro Techniques; Male; Methylene Blue; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Opossums; Oxadiazoles; Penicillamine; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Previously, using brain slices, we reported NO-mediated cGMP synthesis in all cholinergic fibers in the rat neocortex. In order to answer the question whether this property of cholinergic fibers was present before or developed after birth, we investigated properties of NO-responsiveness of cultured cholinergic forebrain neurons. Basal forebrain neurons of E16 fetal rat were cultured. Under the conditions chosen and after one day of culturing, all cells had attained a cholinergic phenotype using choline acetyltransferase or the vesicular acetylcholine transporter molecule as markers. Between 95-99% of the cells also expressed neuronal NOS. In the presence of 1 mM IBMX, a non-selective phosphodiesterase (PDE) inhibitor, 10 microM of the NO donor diethylamine-NONOate (DEANO) increased cGMP synthesis in 80% of the cells. cGMP levels in the cultured forebrain neurons were also increased when cells were stimulated with DEANO in the presence of the selective PDE inhibitors BAY 60-7550 (PDE2), sildenafil (PDE5), or the mixed type inhibitor papaverine (PDE2,5,10). Subpopulations of cells from the basal forebrain expressed mRNA for PDE2, PDE5, and PDE9. Atropine increased cGMP levels in an NO-dependent manner in a small population of cultured forebrain cells in the presence of IBMX. In conclusion, cultured cholinergic basal forebrain neurons present a heterogeneous cell population in the magnitude of their response to NO. NO-responsiveness of the cultured cholinergic neurons is already detectable after one day of culturing and indicates that NO-sensitivity of the cholinergic neurons of the rat basal forebrain is present well before birth.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cells, Cultured; Cholinergic Fibers; Cyclic GMP; Enzyme Inhibitors; Fetus; Hydrazines; Immunohistochemistry; In Situ Hybridization; Microscopy, Fluorescence; Nitric Oxide; Nitric Oxide Donors; Prosencephalon; Rats; Rats, Inbred Lew

The antiatherogenic role of exercise is poorly understood. We examined the swimming exercise-induced vascular mechanisms which enhance the endothelial vasodilator function in apoE(-/-) mice. Male apoE(-/-) mice treated for 9 weeks with a lipid-rich diet were divided into two groups: the exercise group (apoE(-/-) X), which underwent a 9-week swimming protocol (50 min/day; 5days/week) and the sedentary group (apoE(-/-) S). C57BL/6 mice were used as the control group. Atherosclerotic lesions in the aortic roots were significantly reduced in apoE(-/-) X compared to apoE(-/-) S. Relaxation to acetylcholine was improved in apoE(-/-) X as compared to apoE(-/-) S and control mice with E(max) and pD(2) values significantly higher. pD(2) values in response to papaverine were higher in apoE(-/-) X than in the other groups. Relaxation in response to A23187 and DEA-NONOate were similar. These findings suggest that swimming training may increase the sensitivity of relaxation to acetylcholine, which in turn activates acetylcholine-mediated signaling pathways leading to increased NO bioactivity. Swimming may also prolong the signaling actions of NO by stimulating the sensitivity of vascular smooth muscle cells to cyclic nucleotides. These appear to be the key mechanisms underlying the improvement of the NO-cGMP pathway in exercised apoE(-/-) mice.

Topics: Acetylcholine; Animals; Aorta; Apolipoproteins E; Atherosclerosis; Cyclic GMP; Endothelium, Vascular; Hydrazines; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Physical Conditioning, Animal; Swimming; Triglycerides; Vasodilation; Vasodilator Agents

We examined the localization of natriuretic peptide responsive, cyclic guanosine monophosphate producing cells in the guinea pig bladder.. The bladder was removed from male guinea pigs sacrificed by cervical dislocation. The lateral wall of the bladder was cut into strips 2 mm thick. The tissue pieces were incubated in the presence of human atrial natriuretic peptide, rat brain natriuretic peptide and C-type natriuretic peptide or the nitric oxide donor DEANO (diethylamine NONOate or 1,1-diethyl-2-hydroxy-2-nitrosohydrazine) (Sigma). Cyclic guanosine monophosphate immunoreactivity was localized using an antibody against formaldehyde fixed cyclic guanosine monophosphate.. Atrial natriuretic peptide and brain natriuretic peptide stimulated cyclic guanosine monophosphate synthesis in suburothelial interstitial cells, whereas C-type natriuretic peptide was not effective. In contrast, DEANO stimulated cyclic guanosine monophosphate synthesis in urothelial umbrella cells, suburothelial interstitial cells, muscle interstitial cells and neurons. The effect of atrial natriuretic peptide and brain natriuretic peptide was not inhibited by ODQ (1H-[1, 2, 4]oxadiazolo[4-3a]quinoxalin-1-one), an inhibitor of nitric oxide responsive soluble guanylyl cyclase.. To our knowledge our findings show for the first time a localized effect of atrial natriuretic peptide and brain natriuretic peptide to the suburothelial cells of the guinea pig bladder. These cells express the soluble guanylyl cyclase and particulate guanylyl cyclase-A isoforms. The specific physiological role of these cells is not known but it was suggested that they may be involved in the generation or modulation of sensation. The results imply a role for natriuretic peptide-cyclic guanosine monophosphate signaling in the processing of sensory information in the bladder.

Topics: Animals; Cyclic GMP; Guinea Pigs; Hydrazines; Male; Natriuretic Peptides; Nitric Oxide Donors; Tissue Culture Techniques; Urinary Bladder; Urothelium

The radial artery (RA) is used as a spastic coronary bypass graft. This study was designed to investigate the mechanism of vasorelaxant effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO (diethylamine/nitric oxide), a NO-nucleophile adduct, on the human RA. RA segments (n = 25) were obtained from coronary artery bypass grafting patients and were divided into 3-4 mm vascular rings. Using the isolated tissue bath technique, the endothelium-independent vasodilatation function was tested in vitro by the addition of cumulative concentrations of YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) following vasocontraction by phenylephrine in the presence or absence of 10-5 mol/L ODQ (1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one), the selective sGC inhibitor, 10-7 mol/L iberiotoxin, a blocker of Ca2+-activated K+ channels, or 10-5 mol/L ODQ plus 10-7 mol/L iberiotoxin. We also evaluated the effect of YC-1 and DEA/NO on the cGMP levels in vascular rings obtained from human radial artery (n = 6 for each drug). YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) caused the concentration-dependent vasorelaxation in RA rings precontracted with phenylephrine (10-5 mol/L) (n = 20 for each drug). Pre-incubation of RA rings with ODQ, iberiotoxin, or ODQ plus iberiotoxin significantly inhibited the vasorelaxant effect of YC-1, but the inhibitor effect of ODQ plus iberiotoxin was significantly more than that of ODQ and iberiotoxin alone (p < 0.05). The vasorelaxant effect of DEA/NO almost completely abolished in the presence of ODQ and iberiotoxin plus ODQ, but did not significantly change in the presence of iberiotoxin alone (p > 0.05). The pEC50 value of DEA/NO was significantly lower than those for YC-1 (p < 0.01), with no change Emax values in RA rings. In addition, YC-1-stimulated RA rings showed more elevation in cGMP than that of DEA/NO (p < 0.05). These findings indicate that YC-1 is a more potent relaxant than DEA/NO in the human RA. The relaxant effects of YC-1 could be due to the stimulation of the sGC and Ca2+-sensitive K+channels, whereas the relaxant effects of DEA/NO could be completely due to the stimulation of the sGC. YC-1 and DEA/NO may be effective as vasodilator for the short-term treatment of perioperative spasm of coronary bypass grafts.

Topics: Aged; Analysis of Variance; Coronary Artery Bypass; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Female; Humans; Hydrazines; In Vitro Techniques; Indazoles; Male; Middle Aged; Nitric Oxide Donors; Oxadiazoles; Peptides; Phenylephrine; Potassium Channels, Calcium-Activated; Quinoxalines; Radial Artery; Vasoconstrictor Agents; Vasodilation

We investigated the intracellular signalling pathways by which nitric oxide (NO) donors: diethylamine/NO (DEA/NO) and 3-morpholinosydnonimine (SIN-1) regulate the functional response of human neutrophils to activating stimuli.. The phosphorylation and nitration of signalling proteins, cyclic GMP level, neutrophil respiratory burst and adhesive activities and CD11b/CD18 molecule expression on neutrophils in the presence and absence of soluble guanylate cyclase inhibitors were determined.. NO donors showed strong inhibitory effect on activated neutrophils. NO donors nitrated the tyrosine residues in signalling proteins causing a decrease in tyrosine phosphorylation and neutrophils response to activation. Diethylamine/NO employed cyclic GMP as a signalling molecule in its action on neutrophils, whereas peroxynitrite anion donor affected neutrophil functions in a cGMP-independent manner. Moreover, we observed that peroxynitrite anion can overcome the nitric oxide molecule action.. We conclude that each NO donor depending on its concentration and chemical nature may act on different elements of neutrophil signalling pathways capable of inducing distinct neutrophil functions.

Topics: Animals; CD11b Antigen; CD18 Antigens; Cell Adhesion; Cyclic GMP; Guanylate Cyclase; Humans; Hydrazines; Molsidomine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Respiratory Burst; Signal Transduction

NO antagonizes hepatic stellate cell (HSC) contraction, although activated HSC in cirrhosis demonstrate impaired responses to NO. Decreased NO responses in activated HSC and mechanisms by which NO affects activated HSC remain incompletely understood. In normal rat HSC, the NO donor diethylamine NONOate (DEAN) significantly increased cGMP production and reduced serum-induced contraction by 25%. The guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) abolished 50% of DEAN effects, whereas the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) reiterated half the observed DEAN response, suggesting both cGMP-dependent protein kinase G (PKG)-dependent and -independent mechanisms of NO-mediated antagonism of normal HSC contraction. However, NO donors did not increase cGMP production from in vivo activated HSC from bile duct-ligated rats and showed alterations in intracellular Ca(2+) accumulation suggesting defective cGMP-dependent effector pathways. The LX-2 cell line also demonstrated lack of cGMP generation in response to NO and a lack of effect of ODQ and 8-BrcGMP in modulating the NO response. However, cGMP-independent effects in response to NO were maintained in LX-2 and were associated with S-nitrosylation of proteins, an effect reiterated in primary HSC. Adenovirus-based overexpression of PKG significantly attenuated contraction of LX-2 by 25% in response to 8-BrcGMP. In summary, these studies demonstrate that NO affects HSC through cGMP-dependent and -independent pathways. The HSC activation process is associated with maintenance of cGMP-independent actions of NO but defects in cGMP-PKG-dependent NO signaling that are improved by PKG gene delivery in LX-2 cells. Activating targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.

Topics: Adenoviridae; Animals; Calcium Signaling; Cell Line; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Humans; Hydrazines; Hypertension, Portal; Liver; Male; Nitric Oxide; Nitrogen Oxides; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Transduction, Genetic

Nitric oxide (NO) is a major inhibitor in various parts of the gastrointestinal tract. This study was designed to compare the effects of YC-1, NO-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, NO-nucleophile adduct, on sheep sphincters of Oddi (SO).. SO rings were mounted in a tissue bath and tested for changes in isometric tension in response to 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1, 10(-10)-10(-5)M), diethylamine/NO complex (DEA/NO, 10(-8)-10(-4)M). We also evaluated the effect of YC-1 (10(-6) and 10(-5)M) and DEA/NO (10(-5) and 10(-4)M) on the levels cyclic GMP (cGMP) in isolated SO.. YC-1 (10(-10)-10(-5) M) and DEA/NO (10(-8)-10(-4)M) induced concentration-dependent relaxation of isolated SO rings precontracted with carbachol (10(-6)M). The pEC(50) value of DEA/NO was significantly lower than those for YC-1 (p < 0.05), with no change of E(max) values. YC-1 increased cGMP levels more than control, carbachol and DEA/NO groups (p < 0.05).. These results show that YC-1 is a more potent relaxant than DEA/NO and causes more elevation of cGMP levels in isolated SO rings.

Topics: Animals; Cyclic GMP; Hydrazines; In Vitro Techniques; Indazoles; Male; Muscle Relaxation; Neuromuscular Blocking Agents; Nitrogen Oxides; Sheep; Sphincter of Oddi

Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3',5'-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function.

Topics: Animals; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Glutamic Acid; Hydrazines; Membrane Potentials; Mitochondria; Movement; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Oxadiazoles; Prosencephalon; Quinoxalines; Rats; Time Factors

To describe the distribution of interstitial cells (ICs, defined as cells which show an increase in cGMP in response to nitric oxide, NO) in the isolated mouse bladder, and changes in phasic contractile activity after exposure to a NO donor.. The whole bladder was removed from 17 female mice, killed by cervical dislocation. For immunohistochemistry (six mice) the bladder was incubated in carboxygenated Krebs' solution at 36 degrees C, containing 1 mm of the phosphodiesterase inhibitor isobutyl-methyl-xanthine. Individual pieces of tissue were exposed to 100 microm of the NO donor diethylamine NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde and processed for cGMP immunohistochemistry. Bladder pressure was measured in bladders from 11 mice; the bladders were cannulated via the urethra and suspended in a heated chamber containing carboxygenated Tyrode solution at 33-35 degrees C and intravesical pressure recorded. All drugs were added to the solution bathing the abluminal surface.. NO induced an increase in cGMP in cells in the outer layers of the bladder wall, forming two distinct types based on their location; cells lying on the surface of the muscle bundles (surface muscle ICs) and cells within the muscle bundles (intramuscular ICs). Cholinergic nerve fibres were identified by the expression of vesicular acetylcholine transporter and neuronal NO synthase (nNOS). Choline acetyltransferase- and nNOS-positive nerves also had high cGMP levels in response to 100 microm diethylamine NONOate. In vitro exposure of an isolated whole unstimulated bladder to 100 microm diethylamine NONOate had no effect on resting bladder pressure. When whole bladders were exposed to muscarinic stimulation (30-100 nm arecaidine) there was an initial large transient rise in pressure followed by complex phasic changes in pressure. Adding 100 microm diethylamine NONOate abolished this phasic activity. Interestingly, the phasic activity was inhibited midway between the peak and trough of a phasic cycle. Such a pattern of inhibition might reflect the complexity of the phasic activity involving both excitatory and inhibitory components.. These data show the presence of NO/cGMP-sensitive ICs in the outer muscle layers of the mouse bladder. Activating these cells alters the pattern of muscarinic-induced phasic activity. We suggest that the role of the ICs in the outer muscle layers is to generate and modulate phasic activity. If so, then this is the first report of a functional role for ICs in the bladder.

Topics: Animals; Connective Tissue Cells; Cyclic GMP; Female; Hydrazines; Immunohistochemistry; Mice; Muscle Contraction; Nerve Fibers; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Urinary Bladder

Adenosine is an important inhibitory neuromodulator that regulates neuronal excitability. Several studies have shown that nitric oxide induces release of adenosine. Here we investigated the mechanism of this release. We studied the effects of nitric oxide on evoked field excitatory postsynaptic potentials (fEPSPs) recorded in the CA1 area of rat hippocampal slices. The nitric oxide donor 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA/NO; 100 microm) depressed the fEPSP by 77.6 +/- 4.1%. This effect was abolished by the adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 400 nm), indicating that the nitric oxide effect was mediated by adenosine accumulation. The DEA/NO effect was unaltered by the 5'-ectonucleotidase inhibitor alpha,beta-methylene-adenosine 5'-diphosphate (AMP-CP; 100 microm), indicating that extracellular adenosine did not derive from ATP or cAMP release. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ; 5 microm) did not affect nitric oxide depression of the fEPSPs, indicating that nitric oxide-mediated adenosine release was not mediated through a cGMP signaling cascade. This conclusion was confirmed by the observation that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP; 1 mm) reversibly depressed the fEPSP by 24.9 +/- 4.5%, but this effect was not blocked by adenosine antagonists. Adenosine kinase inhibitor 5-iodotubercidin (ITU; 7 microm) occluded the nitric oxide effects by 74%, suggesting that inhibition of adenosine kinase activity contributes to adenosine release. In conclusion, exogenous nitric oxide evokes adenosine release by a cGMP-independent pathway. Intracellular cGMP elevation partially inhibits the fEPSP but not through adenosine release. Although a direct block of adenosine kinase by nitric oxide can not be excluded, the depression of adenosine kinase activity may be due to inhibition by its own substrate adenosine.

Topics: Adenosine; Adenosine Kinase; Animals; Cyclic GMP; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Hippocampus; Hydrazines; Male; Nitric Oxide; Organ Culture Techniques; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Thionucleotides; Xanthines

Vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic cytokines. Ability of VEGF to stimulate formation of superoxide anion in vivo has not been studied. We hypothesized that in vivo expression of recombinant VEGF in the rabbit carotid artery increases production of superoxide anion.. Plaque-forming units (10(9)) of adenovirus-encoding human VEGF165 (AdVEGF) or beta-galactosidase (AdLacZ) were delivered into the lumen of rabbit carotid arteries. Three days after gene delivery, expression of recombinant proteins was detected in endothelium and smooth muscle cells. Endothelium-dependent relaxations to acetylcholine were impaired in AdVEGF-transduced arteries (P<0.01; n=5). Treatment with superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10(-5) mol/L), improved relaxations to acetylcholine (P<0.01; n=5). Western blot analysis demonstrated increased expression of p47(phox) in AdVEGF-transduced arteries (P<0.05; n=8). Lucigenin chemiluminescence showed significantly higher production of superoxide anion in AdVEGF-transduced arteries (P<0.05; n=5 to 10).. Our results suggest that in vivo expression of recombinant VEGF in the vascular endothelium increases local production of superoxide anion. Superoxide anion appears to be an important mediator of vascular effects of VEGF in vivo.

Topics: Animals; Biopterins; Carotid Arteries; Cyclic GMP; Gene Transfer Techniques; GTP Cyclohydrolase; Humans; Hydrazines; Immunohistochemistry; Male; NADPH Oxidases; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrogen Oxides; Phenylephrine; Phosphoproteins; Rabbits; Recombinant Proteins; Superoxides; Vascular Endothelial Growth Factor A; Vasoconstriction; Vasoconstrictor Agents

Amyloid-beta (Abeta), a peptide thought to play a crucial role in Alzheimer's disease (AD), has many targets that, in turn, activate different second-messenger cascades. Interestingly, Abeta has been found to markedly impair hippocampal long-term potentiation (LTP). To identify a new pathway that might be responsible for such impairment, we analyzed the role of the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK)/cAMP-responsive element-binding protein (CREB) cascade because of its involvement in LTP. The use of the NO donor 2-(N,N-dethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO), the sGC stimulator 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine, or the cGMP-analogs 8-bromo-cGMP and 8-(4-chlorophenylthio)-cGMP reversed the Abeta-induced impairment of CA1-LTP through cGK activation. Furthermore, these compounds reestablished the enhancement of CREB phosphorylation occurring during LTP in slices exposed to Abeta. We also found that Abeta blocks the increase in cGMP immunoreactivity occurring immediately after LTP and that DEA/NO counteracts the effect of Abeta. These results strongly suggest that, when modulating hippocampal synaptic plasticity, Abeta downregulates the NO/cGMP/cGK/CREB pathway; thus, enhancement of the NO/cGMP signaling may provide a novel approach to the treatment of AD and other neurodegenerative diseases with elevated production of Abeta.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cyclic AMP Response Element-Binding Protein; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Fluorescent Antibody Technique; Guanylate Cyclase; Hippocampus; Hydrazines; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; Neuronal Plasticity; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Organ Culture Techniques; Peptide Fragments; Phosphorylation; Up-Regulation

Diazeniumdiolates, more commonly referred to as NONOates, have been extremely useful in the investigation of the biological effects of nitric oxide (NO) and related nitrogen oxides. The NONOate Angeli's salt (Na(2)N(2)O(3)) releases nitroxyl (HNO) under physiological conditions and exhibits unique cardiovascular features (i.e., positive inotropy/lusitropy) that may have relevance for pharmacological treatment of heart failure. In the search for new, organic-based compounds that release HNO, we examined isopropylamine NONOate (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO]), which is an adduct of NO and a primary amine. The chemical and pharmacological properties of IPA/NO were compared to those of Angeli's salt and a NO-producing NONOate, DEA/NO (Na[Et(2)NN(O)NO]), which is a secondary amine adduct. Under physiological conditions IPA/NO exhibited all the markers of HNO production (e.g., reductive nitrosylation, thiol reactivity, positive inotropy). These data suggest that primary amine NONOates may be useful as HNO donors in complement to the existing series of secondary amine NONOates, which are well-characterized NO donors.

Topics: Animals; Azo Compounds; Calcitonin Gene-Related Peptide; Cardiovascular System; Cell Survival; Cells, Cultured; Cricetinae; Cricetulus; Cyclic GMP; Dogs; Glutathione; Hemodynamics; Hydrazines; Lethal Dose 50; Male; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Uric Acid

Nitric oxide (NO) is generated in central synapses on activation of N-methyl-D-aspartate (NMDA) receptors and exerts physiological effects by changing cGMP levels. NO has frequently also been claimed to engage a different mechanism, namely the covalent modification of thiol residues (S-nitrosation), and thereby exert a negative feedback on NMDA receptors. Tests of this hypothesis were conducted by recording NMDA receptor-mediated synaptic potentials in the CA1 area of rat hippocampal slices. Manipulations designed to increase or decrease endogenous NO levels had no effect. Addition of exogenous NO using a NONOate donor in concentrations up to 30-fold higher than those needed to evoke maximal cGMP accumulation also had no effect. Nevertheless, in agreement with previous findings, photolysis of a caged NO derivative with UV light led to an enduring block of synaptic NMDA receptors. To address these contradictory results, NMDA receptor-mediated currents were recorded from HEK-293 cells transfected with NR1 and NR2A subunits. As found in slices, photolysis of caged NO inhibited the currents whereas perfusion of NO (up to 5 microM) was ineffective. However, when NO was supplied at a concentration found to be effective when released photolytically (5 microM) and the cells simultaneously exposed to the UV light used for photolysis, NMDA receptor-mediated currents were inhibited. This effect was not observed at more physiological NO concentrations (10 nM range). The results indicate that neither endogenous NO nor exogenous NO in supra-physiological concentration inhibits synaptic NMDA receptors; the combination of high NO concentration and UV light can give an artifactual result.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Electrophysiology; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Hippocampus; Humans; Hydrazines; In Vitro Techniques; Kidney; Male; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Photolysis; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Superoxide Dismutase; Transfection; Uric Acid

Our purpose was to investigate whether the local or systemic factors of pregnancy are associated with inducible nitric oxide synthase (iNOS) mRNA expression and to determine the inhibitory effects of pharmacological agents that increase cGMP levels in rat myometrium. iNOS mRNA expression was determined in uterine tissues from nonpregnant rats and on day 17 of gestation in the pregnant and non-pregnant uterine horns by RT-PCR. In addition, uterine rings from the pregnant and non-pregnant uterine horns were placed in Krebs-Henseleit solution for isometric recordings of spontaneous contractions. Concentration-inhibition relationships to diethylamine/nitric oxide complex, 8-bromo-cGMP, and the selective phosphodiesterase V inhibitor were obtained. Compared to nonpregnant rats, expression of iNOS mRNA in myometrium increased during pregnancy, which was maximal on day 17, followed by a decrease on day 21 of gestation. Expression of iNOS mRNA at day 17 of gestation was greater in pregnant uterine horns than in nonpregnant ones. Maximal inhibition of phosphodiesterase V and increasing cGMP induced similar inhibition of spontaneous contractions in nonpregnant and pregnant uterine horns, while NO induced less inhibition in the former. The results suggest that the local pregnancy factor is needed for signal transduction from NO to soluble guanylate cyclase at a time when maximal expression of iNOS mRNA is evident.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Female; Guanylate Cyclase; Hydrazines; In Vitro Techniques; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Oxides; Pregnancy; Pregnancy, Animal; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Solubility; Time Factors; Uterine Contraction

Vascular reactivity to nitric oxide (NO) is mediated by NO-sensitive soluble guanylyl cyclase (sGC). Since a diminished activity of vascular sGC has been reported in an animal model of type 2 diabetes, the sGC activity was assayed in vitro in internal mammary artery specimens obtained during bypass surgery from patients with and without type 2 diabetes. The sensitivity of sGC to NO, which is dependent on Fe(2+)-containing heme, was measured in vitro using stimulation with diethylamine NONOate (DEA/NO). In addition, the novel cyclic guanosine monophosphate-elevating compound HMR-1766 was used to test the stimulation of the oxidized heme-Fe(3+)-containing form of sGC. Basal activity of sGC and its sensitivity to stimulation by DEA/NO and HMR-1766 were not different between control and type 2 diabetic patients: maximum stimulation by DEA/NO amounted to 475 +/- 67 and 418 +/- 59 pmol. mg(-1). min(-1) in control and type 2 diabetic patients, respectively. The maximum effects of HMR-1766 were 95 +/- 18 (control subjects) and 83 +/- 11 pmol. mg(-1). min(-1) (type 2 diabetic patients). Hypertension, hyperlipidemia, drug treatment with statins, ACE inhibitors, or nitrates had no effect on sGC activity. In conclusion, the present findings do not support the hypothesis that desensitization of sGC contributes to the pathogenesis of diabetic vascular dysfunction in humans.

Topics: Aged; Coronary Disease; Cyclic GMP; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Female; Guanylate Cyclase; Humans; Hydrazines; Male; Mammary Arteries; Middle Aged; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Ventricular Dysfunction, Left

Nonadrenergic noncholinergic (NANC) nerves are known to be nitrergic and to have an important role in the regulation of gastrointestinal motility and function. Cardiac antiarrhythmic therapy in humans is accompanied by a high incidence of gastrointestinal side-effects. We investigated the effect of mexiletine, a class Ib antiarrhythmic drug, on NANC lower oesophageal sphincter relaxation. Mexiletine concentration dependently inhibited the NANC relaxation induced by 30 mM KCl (EC(50)=4.4 x 10(-6) M); the production of 3',5'-cyclic guanosine monophosphate (cGMP) after KCl stimulation was concentration dependently decreased. The relaxation induced by the exogenous nitric oxide (NO) donor, diethylamine NONOate (10(-5) M), was not inhibited by mexiletine, and the cGMP production after diethylamine NONOate application was not altered. Mexiletine did not alter the activity of NO synthase. These findings suggest that mexiletine inhibits NANC relaxation via NO-cGMP pathway modulation, possibly by inhibiting myenteric nitrergic neurotransmission in the lower oesophageal sphincter in rabbits.

Topics: Animals; Anti-Arrhythmia Agents; Cyclic GMP; Dose-Response Relationship, Drug; Esophagogastric Junction; Hydrazines; In Vitro Techniques; Male; Mexiletine; Muscle Relaxation; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrogen Oxides; Rabbits

Calcium entry via the L-type Ca(2+) channel (LTCC) is crucial for excitation-contraction (EC) coupling and activation of Ca(2+)-dependent signal transduction pathways in cardiac myocytes. Both nitric oxide (NO), signaling via cGMP, and acetylcholine, signaling via the muscarinic receptor, have been identified as negative regulators of beta-adrenoreceptor-stimulated LTCC activity in cardiac myocytes.. To examine the potential role of cGMP-dependent protein kinase type I (PKG I) in the inhibitory effects of NO/cGMP and the muscarinic receptor on LTCC activity, we generated transgenic (TG) mice overexpressing PKG I selectively in cardiac myocytes under the control of the alpha-myocin heavy chain promoter. Single LTCC-gating properties were assessed in isolated ventricular myocytes from adult wild-type (WT) and PKG I transgenic (TG) mice.. Basal LTCC activity (peak average current, mean open probability, mean availability) was significantly decreased by the nitric oxide donor DEA-NO (0.1 micromol/l) and the cGMP-analog 8-Br-cGMP (1 mmol/l) in TG but not in WT cardiac myocytes. Conversely, muscarinic (carbachol, 1 micromol/l) stimulation had no significant effect on basal LTCC activity in either WT or TG cardiac myocytes. beta-Adrenergic stimulation with isoproterenol (1 micromol/l) increases single LTCC activity in WT and TG cardiac myocytes to the same extent. The inhibitory effects of DEA-NO and 8-Br-cGMP on isoproterenol activation of the LTCC current were significantly enhanced in TG as compared to WT cardiac myocytes. By contrast, carbachol inhibition of isoproterenol-stimulated single LTCC activity was not enhanced in TG cardiac myocytes.. Transgenic overexpression of PKG I augments NO/cGMP inhibition but not muscarinic inhibition of single LTCC activity, indicating that PKG I is a downstream target for NO/cGMP, but not the muscarinic receptor in adult cardiac myocytes.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium Channels, L-Type; Carbachol; Cardiotonic Agents; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Hydrazines; Isoproterenol; Mice; Mice, Transgenic; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides

Matrix metalloproteinases (MMPs) are synthesized in response to diverse stimuli, including cytokines, growth factors, hormones, and oxidative stress. Here we show that the nitric oxide (NO) donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO) and NO from murine macrophages transcriptionally regulate MMP-13 expression in vascular endothelial cells (BAEC). The cGMP analog, 8-bromo-cGMP (8-Br-cGMP) mimicked the effect of NO, whereas incubation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or the cGMP-dependent protein kinase (PKG) inhibitor phenyl-1,N (2)- etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (PET) reduced the stimulatory effect of DEA-NO on the activation of the MMP-13 promoter. Overexpression of the catalytic subunit of PKG1-alpha resulted in a 5- to 6-fold increase of the MMP-13 regulatory region over control cells. On the other hand, incubation with the mitogen-activated protein/extracellular signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) significantly reduced DEA-NO and 8-Br-cGMP promoter activation and mRNA expression of MMP-13 in transfected BAEC. Moreover, a complex between PKG1-alpha and the G-protein Raf-1, an upstream activator of the extracellular signal-regulated kinase signaling pathway, was detected in cells overexpressing PKG1-alpha or treated either with DEA-NO or 8-Br-cGMP. Thus, we propose that the NO-cGMP-PKG pathway enhances MMP-13 expression by the activation of ERK 1,2. This effect of NO may be important in the context of pathophysiological conditions such as inflammation or atherogenesis [corrected].

Topics: Animals; Cattle; Cells, Cultured; Collagenases; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Gene Expression Regulation, Enzymologic; Hydrazines; Macrophages; Matrix Metalloproteinase 13; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Protein Kinases; Transcription, Genetic

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.

Topics: Apoptosis; Blotting, Western; Cell Division; Cell Survival; Culture Media; Cyclic GMP; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrazines; Immunohistochemistry; Isoenzymes; Neuroblastoma; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrogen Oxides; Phosphorylation; Signal Transduction; Tumor Cells, Cultured

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.

Topics: Animals; Calcium; Cell Nucleus; Cells, Cultured; COS Cells; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Endoplasmic Reticulum; Hydrazines; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitrogen Oxides; Norepinephrine; Protein Isoforms; Recombinant Fusion Proteins; Signal Transduction; Transfection

There is evidence suggesting that nitric oxide (NO) may play an important role in dopamine (DA) cell death. Thus, the aim of this study was to investigate the effects of NO on apoptosis and functionality of DA neurones and glial cells. The experiments were carried out in neuronal-enriched midbrain cultures treated with the NO donor diethylamine-nitric oxide complexed sodium (DEA-NO). DEA-NO, at doses of 25 and 50 microM, exerted neurotrophic effects on dopamine cells, increasing the number of tyrosine hydroxylase positive (TH(+)) cells, TH(+) neurite processes, DA levels and [(3)H]DA uptake. A dose of 25 microM DEA-NO protected DA cells from apoptosis. In addition, it induced de novo TH synthesis and increased intracellular reduced glutathione (GSH) levels, indicating a possible neuroprotective role for GSH. However, in doses ranging from 200 to 400 microM, DEA-NO decreased TH(+) cells, DA levels, [(3)H]DA uptake and the number of mature oligodendrocytes (O1(+) cells). No changes in either the amount or morphology of astrocytes and glial progenitors were detected. A dose- and time-dependent increase in apoptotic cells in the DEA-NO-treated culture was also observed, with a concomitant increase in the proapoptotic Bax protein levels and a reduction in the ratio between Bcl-xL and Bcl-xS proteins. In addition, DEA-NO induced a dose- and time-dependent increase in necrotic cells. 1H-[1,2,4]oxadiazolo[4, 3a]quinoxaline-1-one (ODQ, 0.5 microM), a selective guanylate cyclase inhibitor, did not revert the NO-induced effect on [(3)H]DA uptake. Glia-conditioned medium, obtained from fetal midbrain astrocyte cultures, totally protected neuronal-enriched midbrain cultures from NO-induced apoptosis and rescued [(3)H]DA uptake and TH(+) cell number. In conclusion, our results show that low NO concentrations have neurotrophic effects on DA cells via a cGMP-independent mechanism that may implicate up-regulation of GSH. On the other hand, higher levels of NO induce cell death in both dopamine neurones and mature oligodendrocytes that is totally reverted by soluble factors released from glia.

Topics: Animals; Apoptosis; Cell Division; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Cyclic GMP; Dopamine; Dose-Response Relationship, Drug; Glutathione; Hydrazines; Mesencephalon; Neuroglia; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Rats; Tyrosine 3-Monooxygenase

The present study was designed to evaluate endothelium-dependent relaxation to the calcium ionophore A-23187 in isolated canine saphenous veins. Isometric force recordings and cGMP measurements using isolated veins with and without valves were performed. During contractions to U-46619 (3 x 10(-7) M), endothelium-dependent relaxations to A-23187 (10(-9)-10(-6) M) were significantly reduced in rings with valves compared with rings without valves. Endothelial removal abolished A-23187-induced relaxation. Relaxations to forskolin (FK; 10(-8)-10(-5) M) and diethylaminodiazen-1-ium-1,2-dionate; DEA-NONOate, 10(-9)-10(-5) M) were identical in rings with and without valves. In rings without valves, a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M), and a cyclooxygenase inhibitor, indomethacin (10(-5) M), partially reduced A-23187-induced relaxation. However, in rings with valves, L-NAME had no effect, whereas indomethacin abolished the relaxation to A-23187. A selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 3x10(-6) M), had no effect on the relaxation to A-23187 in either group. In contrast, ODQ abolished the A-23187-induced increase in cGMP levels, suggesting that relaxation to nitric oxide released by A-23187 is independent of increases in cGMP. These results demonstrate that endothelium-dependent relaxation to A-23187 is reduced in regions of veins with valves compared with relaxation in the nonvalvular venous wall. Lower production of nitric oxide in endothelial cells of valvular segments appears to be a mechanism responsible for reduced reactivity to A-23187.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Calcimycin; Calcium; Colforsin; Cyclic GMP; Cyclooxygenase Inhibitors; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Hydrazines; In Vitro Techniques; Indomethacin; Ionophores; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitrogen Oxides; Oxadiazoles; Potassium Chloride; Quinoxalines; Saphenous Vein; Vasoconstrictor Agents; Vasodilation

In previous studies, a strong synergism between low concentrations of hydrogen peroxide and nitric oxide in the inhibition of agonist-induced platelet aggregation has been established and may be due to enhanced formation of cyclic GMP. In this investigation, hydrogen peroxide and NO had no effect on the activity of pure soluble guanylyl cyclase or its activity in platelet lysates and cytosol. H(2)O(2) was found to increase the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), increasing the amount of the 50-kDa form that results from phosphorylation at serine(157). This occurs both in the presence and in the absence of low concentrations of NO, even at submicromolar concentrations of the peroxide, which alone was not inhibitory to platelets. These actions of H(2)O(2) were inhibited to a large extent by an inhibitor of cyclic AMP-dependent protein kinase, even though H(2)O(2) did not increase cyclic AMP. This inhibitor reversed the inhibition of platelets induced by combinations of NO and H(2)O(2) at low concentrations. The results suggest that the action on VASP may be one site of action of H(2)O(2) but that this event alone does not lead to inhibition of platelets; another unspecified action of NO is required to complete the events required for inhibition.

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Blood Platelets; Blood Proteins; Carbazoles; Cell Adhesion Molecules; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytosol; Drug Synergism; Enzyme Inhibitors; Guanylate Cyclase; Humans; Hydrazines; Hydrogen Peroxide; In Vitro Techniques; Indazoles; Indoles; Kinetics; Microfilament Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Phosphoproteins; Phosphoserine; Platelet Aggregation; Platelet Aggregation Inhibitors; Subcellular Fractions; Thrombin

The direct effects of nitric oxide (NO) donors and sulfhydryl-modifying agents on the GABA(A) receptor function were examined by perforated patch, whole-cell and single channel recordings in cultured frog melanotrophs. In amphotericin B-perforated cells incubated with the soluble guanylyl cyclase inhibitors LY 83583 and ODQ (10-4 M each), the NO donor sodium nitroprusside (SNP) (10(-3) M) reversibly increased the current evoked by GABA (5 x 10(-6) M). In the whole-cell configuration, internal application of the oxidizing agent H2O2 (0.05%) potentiated the GABA-evoked current while the reducing agent 2-mercaptoethanol (5 x 10(-3) M) slightly decreased the current amplitude. In inside-out patches, GABA (2 x 10(-7) M) triggered single channel bursts of openings. Incubation with the NO donors SNP or DEA/NO (10(-4) M each) enhanced the open probability of the GABA(A) receptor channel but did not modify the chloride reversal potential and did not affect the conductance states. The oxidizing agents H2O2 (0.05%) or DTNB (10-4 M) mimicked the stimulatory effect of the NO donors on the open probability while the reducing compounds 2-mercaptoethanol (5 x 10(-3) M) or DTT (10(-4) M) markedly attenuated the channel activity. Potentiation of the GABA-induced single channel activity by SNP or H2O2 was blocked by 2-mercaptoethanol. Similarly, the potentiating effect produced by DEA/NO or DTNB on the open probability was reversed by DTT. In outside-out patches, incubation with SNP also significantly enhanced the open probability of single channels activated by GABA (10(-6) M). These data indicate that, in frog pituitary melanotrophs, NO potentiates the GABA-evoked current independently of the cGMP/protein kinase pathway. The effect of NO can be accounted for by S-nitrosylation/oxidation of thiol groups either directly on the GABA(A) receptor subunits or on a regulatory protein tightly associated with the GABA(A) receptor.

Topics: Aminoquinolines; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dithionitrobenzoic Acid; Electric Conductivity; Enzyme Inhibitors; gamma-Aminobutyric Acid; Hydrazines; Ion Channel Gating; Male; Melanocytes; Membrane Potentials; Mercaptoethanol; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Oxadiazoles; Oxidation-Reduction; Patch-Clamp Techniques; Pituitary Gland; Quinoxalines; Rana ridibunda; Receptors, GABA-A; Sulfhydryl Reagents

The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery. The contribution from cGMP-dependent signalling pathways was examined by exposing arteries to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, while the contribution through potassium channels was assessed with different sub-type-selective potassium channel blockers. DEA NONOate (3 nM-10 microM) evoked sustained relaxation in isolated segments of the rat small mesenteric artery contracted with phenylephrine (pEC50=6.7+/-0.2; n=11). The relaxation was attenuated significantly by either ODQ (10 microM; pEC50=5.8+/-0.4; n=7) or charybdotoxin (ChTX; 50 nM; pEC50=6.3+/-0.2; n=4), a peptide blocker of large conductance, calcium-activated potassium channels (BK(Ca)). The inhibitory effects of ODQ and ChTX were additive (pEC50=5.1+/-0.4; n=9). The selective inhibitor of BK(Ca) channels, iberiotoxin (IbTX; 30 nM), and 4-aminopyridine (4-AP; 1 mM), an inhibitor of voltage-gated potassium channels (Kv), failed to modify DEA NONOate-evoked relaxation. However, in the combined presence of both ODQ and either IbTX or 4-AP the relaxation was attenuated significantly (n=3). The blocker of ATP-modulated potassium channels (K(ATP)), glibenclamide (10 microM), and of small conductance calcium-activated potassium channels (SK(Ca)), apamin (30 nM), each failed to affect ODQ-sensitive or -resistant relaxations to DEA NONOate (n=3). In conclusion, relaxation to DEA NONOate in the rat isolated, small mesenteric artery can occur via both cGMP-dependent (ODQ-sensitive) and -independent (ODQ-resistant) mechanisms. However, the contribution made to relaxation by potassium channels appears to be unmasked following pharmacological attenuation of cGMP-dependent signalling pathways. The inhibitory action of ChTX suggests part of the cGMP-insensitive component involves the activation of potassium channels, a suggestion supported by the inhibitory actions of 4-AP and IbTX in the absence of cGMP.

Topics: Analysis of Variance; Animals; Cyclic GMP; Hydrazines; Male; Mesenteric Arteries; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitrogen Oxides; Potassium Channels; Rats; Rats, Wistar; Vasoconstriction; Vasodilator Agents

Nitric oxide (NO) is a putative participant in synaptic plasticity and demonstrations that exogenous NO can elicit the same plastic changes have been taken to support such a role. The experiments, carried out on the CA1 region of rat hippocampal slices, were aimed at testing this interpretation. A major component of tetanus-induced long-term potentiation (LTP) was lost in response to L-nitroarginine, which inhibits NO synthase, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which inhibits NO-sensitive soluble guanylyl cyclase (sGC). At 0.2 Hz afferent fibre stimulation, exogenous NO produced, concentration-dependently, a synaptic depression that reverted on washout to a persistent potentiation that occluded tetanus-induced LTP. The NO concentrations necessary (estimated in the 100-nM range), however, were mostly supramaximal for stimulating hippocampal slice sGC activity. Nevertheless the potentiation, but not the preceding depression, was blocked by ODQ. L-nitroarginine and an NMDA antagonist were similarly effective, indicating mediation by the endogenous NMDA receptor-NO synthase-sGC pathway. At a concentration normally too low to affect synaptic transmission but sufficient to stimulate sGC (estimated to be 50 nM), exogenous NO reversed the effect of L-nitroarginine and caused a potentiation which was blocked by ODQ. At a concentration inducing the depression/potentiation sequence, NO partially inhibited hippocampal slice oxygen consumption. It is concluded that, at physiological levels, exogenous NO can directly elicit a potentiation of synaptic transmission through sGC, provided that the synapses are suitably primed. At higher concentrations, NO inhibits mitochondrial respiration, which can result in an enduring synaptic potentiation due to secondary activation of the endogenous NO-cGMP pathway.

Topics: 2-Amino-5-phosphonovalerate; Adenosine Triphosphate; Animals; Cyanides; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Guanylate Cyclase; Hippocampus; Hydrazines; Long-Term Potentiation; Male; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitrogen Oxides; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Synaptic Transmission

A major receptor for nitric oxide (NO) is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC), but it is not known how this enzyme behaves in cells. In cerebellar cells, NO (from diethylamine NONOate) increased astrocytic cGMP with a potency (EC(50)

Topics: Animals; Blood Platelets; Cells, Cultured; Cerebellum; Cyclic GMP; Guanylate Cyclase; Humans; Hydrazines; Kinetics; Nitric Oxide; Nitrogen Oxides; Phosphoric Diester Hydrolases; Rats; Time Factors

We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs).. The NO donor diethylenetriamineNONOate (10(-5) to 10(-3) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with N(G)-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10(-3) mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO.. NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Aorta; Blood Platelets; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Line; Colforsin; Cyclic GMP; Cyclin A; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Endothelium, Vascular; Gene Expression Regulation, Enzymologic; Genetic Therapy; Guanylate Cyclase; Humans; Hydrazines; Microtubule-Associated Proteins; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrogen Oxides; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Transfection; Tumor Suppressor Proteins

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous system of many vertebrates and invertebrates. The effects of extracellularly applied sodium nitroprusside (SNP) and diethylamine NO (C(2)H(5))(2)N[N(O)NO]-Na(+) (DEA/NO), NO donors, on a glutamate (Glu)-induced K(+) current in identified Onchidium neurons were investigated using voltage clamp and pressure ejection techniques. Bath-applied SNP (10 microM) and DEA/NO (5-10 microM) reduced the Glu-induced K(+) current without affecting the resting membrane conductance and holding current. The Glu-induced K(+) current also was inhibited by the focal application of SNP to the neuron somata. The suppressing effects of NO donors were concentration-dependent and completely reversible. Pretreatment with hemoglobin (50 microM), a nitric oxide scavenger, and 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Glu-induced current. Bath-applied 50 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, or intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) inhibited the Glu-induced current, mimicking the effect of NO donors. These results demonstrate that SNP and DEA/NO inhibit the Glu-induced K(+) current and that the mechanism of NO inhibition of the Glu-induced current involves cGMP-dependent protein kinase.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cell Membrane; Cyclic GMP; Enzyme Inhibitors; Ganglia, Invertebrate; Glutamic Acid; Hydrazines; Mollusca; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Oxadiazoles; Phosphodiesterase Inhibitors; Potassium Channels; Quinoxalines; Vasodilator Agents

Extracellular recording techniques were used to study the effects of the nitric oxide releasing agents diethylamine-NO (DEA-NO) and S-nitroso-N-acetyl-penicillamine (SNAP) on synaptic transmission in the intermediate and medial part of the hyperstriatum ventrale (IMHV), a part of the domestic chick forebrain that is essential for some forms of early learning. The field response evoked by local electrical stimulation was recorded in the IMHV in an in vitro slice preparation. DEA-NO (100-200 mgr) significantly depressed the field response in a concentration dependent and reversible manner. However, the depression produced by perfusion with 400 mgr DEA-NO, was not reversed following washout of the drug. With 400 mgr DEA-NO, NO reaches a maximum concentration of 10 mgr at 2 min of perfusion, and then declines slowly. SNAP (400 mgr) produced an effect similar to 400 mgr DEA-NO. Neither the immediate nor the longer-term depressive effect of NO is mediated by activation of guanylyl cyclase because in the presence of both low and high doses of ODQ, a potent and selective inhibitor of NO-stimulated guanylyl cyclase, NO produced the same depression of the field response. There is evidence however that the IMHV possesses c-GMP responsive elements since direct perfusion of 8-Br-cGMP (1 mM) produced a long-term but not an immediate depression. The long-term depression produced by 400 mgr DEA-NO was eliminated in the presence of either a selective adenosine A(1) receptor antagonist or an ADP-ribosyltransferase inhibitor. It was also possible to prevent the long-term effect in the presence of tetraethyl ammonium a K(+)-channel blocker. These results suggest that the NO may be acting presynaptically in a synergistic fashion with the adenosine A(1) receptor to depress transmitter release.

Topics: Animals; Chickens; Conditioning, Psychological; Cyclic GMP; Evoked Potentials; Guanylate Cyclase; Hydrazines; Memory; Neuronal Plasticity; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Penicillamine; Poly(ADP-ribose) Polymerases; Potassium Channels; Prosencephalon; Receptors, Adrenergic, alpha-1; Synapses; Synaptic Transmission; Tetraethylammonium; Xanthines

Adenosine is a putative endogenous sleep-inducing substance, and nitric oxide has been implicated in arousal and sleep mechanisms. We found that various nitric oxide donors, including diethylamine NONOate (DEA/NO), stimulated large increases in extracellular adenosine in nearly pure cultures of forebrain neurons. The effect of DEA/NO could be blocked by 2-phenyl-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-oxide and could not be mimicked by degraded solutions of DEA/NO or by DEA itself; therefore, it was caused by nitric oxide release on hydrolysis of the parent compound. The accumulation of adenosine was not blocked by probenecid or GMP, suggesting that neither extracellular cAMP nor extracellular AMP was the source, and that adenosine was therefore the most likely species transported across the plasma membrane. To pursue this further, we tested the effect of DEA/NO on cellular ATP and found a significant fall in ATP associated with exposure to nitric oxide. In addition, exposure to DEA/NO nearly completely inhibited adenosine kinase activity. It has been found previously that adenosine kinase is inhibited by its substrate, adenosine. We found that exposure to nitric oxide increased intracellular adenosine to 125 +/- 18% of control values (p < 0.01), consistent with the possibility that in our system the inhibition of adenosine kinase is related to an increase in intracellular adenosine, and that the effect of nitric oxide on extracellular adenosine is significantly potentiated by substrate inhibition of adenosine kinase. Furthermore, nitric oxide-stimulated adenosine accumulation may be important in the regulation of behavioral state.

Topics: Adenosine; Adenosine Kinase; Adenosine Triphosphate; Animals; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Extracellular Space; Fetus; Hydrazines; Hydrolysis; Intracellular Fluid; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Prosencephalon; Rats; Rats, Sprague-Dawley; Sleep; Time Factors

Soluble guanylyl cyclase activity and its stimulation by diethylamineNONOate was measured in aortae from hypertensive TGR(mREN2)27 rats (TGR) and Sprague-Dawley controls. Superoxide dismutase was added in vitro to evaluate the contribution of oxidative breakdown of nitric oxide (NO) by superoxide anions. Expression of soluble guanylyl cyclase was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Basal and stimulated soluble guanylyl cyclase activity was significantly reduced in TGR rats, addition of superoxide dismutase had no effect. Expression of soluble guanylyl cyclase subunits was not different between strains. The independent contribution of hypertension and the overactive renin-angiotensin system to soluble guanylyl cyclase subsensitivity was assessed after normalization of TGR's blood pressure by the Ca(2+)-channel blocker amlodipine or the angiotensin converting enzyme-inhibitor enalapril. Soluble guanylyl cyclase activity in TGR was slightly increased by amlodipine and almost completely restored by enalapril. In conclusion, TGR showed desensitized vascular soluble guanylyl cyclase, depending on their overactive renin-angiotensin system.

Topics: Aging; Amlodipine; Analysis of Variance; Animals; Animals, Genetically Modified; Aorta, Thoracic; Blood Pressure; Calcium Channel Blockers; Cyclic GMP; Dose-Response Relationship, Drug; Enalapril; Guanylate Cyclase; Hydrazines; Hypertension; Male; Nitrogen Oxides; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Solubility

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.

Topics: Animals; Calcium; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; Heart Atria; Heart Rate; Hydrazines; Indoles; Isoquinolines; Male; Milrinone; Models, Biological; Molsidomine; Nitric Oxide Donors; Nitrogen Oxides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation Inhibitors; Pyrroles; Quinazolines; Ryanodine; Sarcoplasmic Reticulum; Signal Transduction; Spectrometry, Fluorescence; Sulfonamides; Superoxide Dismutase; Tetrahydroisoquinolines; Thionucleotides; Time Factors; Vasodilator Agents

Both cGMP-dependent and -independent mechanisms have been implicated in the regulation of vascular tone by NO. We analyzed acetylcholine (ACh)- and NO-induced relaxation in pressurized small arteries and aortic rings from wild-type (wt) and cGMP kinase I-deficient (cGKI(-/-)) mice. Low concentrations of NO and ACh decreased the spontaneous myogenic tone in wt but not in cGKI(-/-) arteries. However, contractions of cGKI(-/-) arteries and aortic rings were reduced by high concentrations (10 micromol/L) of 2-(N:, N-diethylamino)-diazenolate-2-oxide (DEA-NO). Iberiotoxin, a specific blocker of Ca(2+)-activated K(+) (BK(Ca)) channels, only partially prevented the relaxation induced by DEA-NO or ACh in pressurized vessels and aortic rings. DEA-NO increased the activity of BK(Ca) channels only in vascular smooth muscle cells isolated from wt cGKI(+/+) mice. These results suggest that low physiological concentrations of NO decrease vascular tone through activation of cGKI, whereas high concentrations of DEA-NO relax vascular smooth muscle independent of cGKI and BK(Ca). NO-stimulated, cGKI-independent relaxation was antagonized by the inhibition of soluble guanylyl cyclase or cAMP kinase (cAK). DEA-NO increased cGMP to levels that are sufficient to activate cAK. cAMP-dependent relaxation was unperturbed in cGKI(-/-) vessels. In conclusion, low concentrations of NO relax vessels by activation of cGKI, whereas in the absence of cGKI, NO can relax small and large vessels by cGMP-dependent activation of cAK.

Topics: Acetylcholine; Animals; Aorta; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Hydrazines; In Vitro Techniques; Male; Mice; Muscle, Smooth, Vascular; Nitric Oxide; Nitrogen Oxides; Peptides; Vasodilation

Intracellular Ca(2+) mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1beta (IL-1beta) and other pyrogens. The source and mechanism of IL-1beta-induced intracellular Ca(2+) mobilization was investigated using two experimental models. IL-1beta (10 ng/ml) treatment of rat striatal slices preloaded with (45)Ca(2+) elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous (45)Ca(2+) release that was potentiated by l-arginine (300 microm) and counteracted by N-omega-nitro-l-arginine methyl ester (l-NAME) (1 and 3 mm). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mm) and spermine/NO (0.1 and 0.3 mm) mimicked the effect of IL-1beta on Ca(2+) release. IL-1beta stimulated tissue cGMP concentration, and dibutyryl cGMP enhanced Ca(2+) release. The guanyl cyclase inhibitors 1H-[1,2, 4]oxadiazole[4,3-a] quinoxalin-1-one (100 microm) and 6-[phenylamino]-5,8 quinolinedione (50 microm) counteracted Ca(2+) release induced by 2.5 but not 10 ng/ml IL-1beta. Ruthenium red (50 microm) and, to a lesser extent, heparin (3 mg/ml) antagonized IL-1beta-induced Ca(2+) release, and both compounds administered together completely abolished this response. Similar results were obtained in human astrocytoma cells in which IL-1beta elicited a delayed (30 min) increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (402 +/- 71.2% of baseline), which was abolished by 1 mm l-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1beta-evoked Ca(2+) mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca(2+) stores are involved.

Topics: Aminoquinolines; Animals; Arginine; Astrocytoma; Calcium; Corpus Striatum; Cyclic GMP; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Heparin; Humans; Hydrazines; In Vitro Techniques; Interleukin-1; Intracellular Fluid; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Ruthenium Red; Spermine; Tumor Cells, Cultured

1. Sodium nitroprusside (SNP, 10(-9)-3x10(-4) M), diethylamine/NO complex (DEA/NO, 10(-9)-10(-4) M) and spermine/NO complex (SPER/NO, 10(-8)-3x10(-4) M) induced concentration-dependent relaxation of isolated rabbit carotid arteries precontracted with KCl (50 mM) or with histamine (3x10(-6) M). 2. In KCl-precontracted arteries the order of potency was SNP=DEA/NO>SPER/NO, and in histamine-precontracted arteries the order of potency was SNP>DEA/NO>SPER/NO. Relaxations to the three NO donors were significantly higher in histamine-precontracted arteries than in KCl-precontracted arteries. 3. The guanylyl cyclase inhibitor methylene blue (10(-5) M) significantly inhibited relaxations to the three NO donors in histamine-precontracted arteries and, to a lesser extent, in KCl-precontracted arteries. 4. In conclusion, the NO donors SNP, DEA/NO and SPER/NO induce quantitatively different relaxation of rabbit carotid artery. Both, lower relaxant effects in depolarized arteries and inhibition of relaxation by methylene blue indicate the mediation of cGMP formation in the relaxant effects of the three NO donors.

Topics: Animals; Carotid Arteries; Cyclic GMP; Dose-Response Relationship, Drug; Hydrazines; Male; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Potassium Chloride; Rabbits; Spermine; Vasodilator Agents

Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.

Topics: Animals; Antioxidants; Calcium; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Excitatory Amino Acid Agonists; Hydrazines; Intracellular Membranes; Mice; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Rats; Rats, Inbred Strains

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.

Topics: Animals; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Growth Cones; Guanylate Cyclase; Hydrazines; Molsidomine; Mutagens; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Oxadiazoles; Pseudopodia; Quinoxalines; Snails; Sulfhydryl Reagents

The role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of cardiac contractility remains controversial. The present study has examined the effects of high concentrations of the nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine (SNAP) and 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), on cGMP levels and isoproterenol-induced increases in contractility in rat cardiomyocytes before and after selective inhibition of soluble guanylyl cyclase with 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In control myocytes, 100 microm SNAP or 100 microm DEA/NO increased cGMP levels by more than 15-fold at 2 and 6 min and produced marked attenuations of isoproterenol-mediated increases in maximal cell shortening over the same time period. The NO donors had no significant effect on basal cell shortening (in the absence of isoproterenol). Pretreatment of myocytes with 25 microm ODQ for 30 min resulted in a complete blockade of the SNAP- or DEA/NO-induced increases in cGMP with no reversal of negative inotropy. ODQ did not affect basal contractility, basal cGMP levels or isoproterenol-induced increases in cell shortening. Furthermore, myocytes exposed to the cGMP analog, 8-bromo-cGMP (100 microm), did not exhibit significant differences in basal contractility or isoproterenol-induced increases in cell shortening. These results suggest that attenuation of cardiac contractility by NO donors in rat cardiomyocytes occurs by a mechanism independent of increases in cGMP levels.

Topics: Animals; Cardiotonic Agents; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Hydrazines; In Vitro Techniques; Isoproterenol; Male; Myocardial Contraction; Myocardium; Nitric Oxide Donors; Nitrogen Oxides; Oxadiazoles; Oxidation-Reduction; Penicillamine; Quinoxalines; Rats; Rats, Wistar

1. Sodium nitroprusside, S-nitroso-N-acetyl-D,L-penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38 +/- 0.02 microM. 2. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half-life (t1/2) of 3.9 +/- 0.2 min. The t1/2 for SPER/NO was 37 +/- 3 min. SNAP decomposed more slowly (t1/2 = 37 +/- 4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. 3. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 microM and maximal effective concentration between 2.5 and 5 microM. 4. For SNAP, threshold activation was seen at 1 microM, whereas full activation required a higher concentration (500-750 microM). The dose-response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide-deficient SNAP analogue or other NO donors. 5. Although NO-donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

Topics: Animals; Cattle; Cells, Cultured; Chromaffin Cells; Cyclic GMP; Hydrazines; Intracellular Fluid; Kinetics; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroprusside; Penicillamine; Spermine

A major transduction pathway for nitric oxide (NO) is stimulation of soluble guanylyl cyclase and the generation of cyclic GMP (cGMP). In the central nervous system, the NO-cGMP pathway has previously been associated primarily with synapses, particularly glutamatergic synapses. We report here that NO caused a large increase in the levels of cGMP in a central white matter tract devoid of synapses, namely in the rat isolated optic nerve. Cyclic GMP immunohistochemistry indicated that this response was confined to the axons. Accordingly, nerves previously subjected to 1 h of oxygen/glucose deprivation, which leads to irreversible axonal damage, displayed an 80% reduction in their subsequent capacity to generate cGMP in response to NO and a corresponding reduction in the numbers of cGMP-immunostained axons. Protection of the axon cGMP response against this insult was achieved by omission of Ca2 + or Na + from the incubation medium, and by the pharmacological agents tetrodotoxin, lamotrigine, BW619C89 and BW1003C87, all of which protect axonal structure from oxygen/glucose deprivation-induced damage. The results suggest that the NO-cGMP pathway has a hitherto unsuspected function in the optic nerve. Additionally, the expression of NO-stimulated guanylyl cyclase in optic nerve axons provides a simple, sensitive and specific marker of their functional integrity that is likely to be valuable in investigating the mechanisms responsible for axon degeneration in ischaemia and other conditions.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Axons; Biomarkers; Cell Hypoxia; Cell Survival; Cyclic GMP; Glucose; Hydrazines; Immunohistochemistry; In Vitro Techniques; Neuroprotective Agents; Nitric Oxide; Nitrogen Oxides; Optic Nerve; Phosphodiesterase Inhibitors; Piperazines; Pyrimidines; Rats; Rats, Wistar

Conduction in rat peripheral nerve has been monitored following the stimulated release of nitric oxide (NO) from diethylamine-NONOate (DEA-NONOate). Branches of the sciatic nerve were dissected, but left otherwise intact, and propagating signals recorded externally. At levels consistent with inflammation, NO exposure resulted in a complete loss of the compound action potential. Conduction was fully restored on removal of the drug. Most notably, this loss of excitability was dependent on the axonal environment. Removal of the connective tissue sheaths surrounding the nerve bundle, a process that normally enhances drug action, prevented block of signal propagation by nitric oxide. The epineurium seemed not to be required, and the decreased susceptibility to NO appeared to be correlated with a gradual loss of a component of the endoneurium that surrounds individual fibers. Tested on the rat vagus nerve, NO eliminated action potentials in both myelinated and unmyelinated fibers. One chemical mechanism that is consistent with the reversibility of block and the observed lack of effect of 8-Br-cGMP on conduction is the formation of a nitrosothiol through reaction of NO with a sulfhydryl group. In contrast to DEA-NONOate, S-nitrosocysteine, which can both transfer nitrosonium cation (NO+) to another thiol and also release nitric oxide, was effective on both intact and desheathed preparations. It has previously been demonstrated that chemical modification of invertebrate axons by sulfhydryl-reactive compounds induces a slow inactivation of Na+ channels. Nitric oxide block of axonal conduction may contribute to clinical deficits in inflammatory diseases of the nervous system.

Topics: Action Potentials; Animals; Axons; Cyclic GMP; Cysteine; Depression, Chemical; Female; Hydrazines; Myelin Sheath; Neural Conduction; Nitric Oxide; Nitrogen Oxides; Nitroso Compounds; Oxidation-Reduction; Rats; Rats, Inbred Lew; S-Nitrosothiols; Sciatic Nerve; Sodium Channels; Sulfhydryl Compounds; Tetrodotoxin; Vagus Nerve

The mechanism underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide is still not completely understood. The present study was designed to determine the role of soluble guanylate cyclase in the relaxations to a nitric oxide/nucleophile complex, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate). Rings of canine middle cerebral arteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured by radioimmunoassay technique. During contractions to uridine 5'-triphosphate (UTP), DEA-NONOate (10(-10) to 10(-5) M) caused concentration-dependent relaxations. Measurements of cyclic GMP levels in cerebral arterial wall demonstrated that DEA-NONOate is a potent stimulator of guanylate cyclase and subsequent formation of cyclic GMP. Increasing concentrations of a selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), caused concentration-dependent reduction of both cyclic GMP production and relaxations to DEA-NONOate. Interestingly, in the presence of the highest concentration (3 x 10(-6) M) of ODQ, production of cyclic GMP in response to 10(-6) M of DEA-NONOate was abolished, whereas the same concentration of DEA-NONOate caused almost complete relaxation, suggesting that mechanisms independent of cyclic GMP production may mediate relaxing effect of high concentration of a nitric oxide donor. A selective Ca2+-activated potassium channel blocker charybdotoxin (CTX) significantly reduced relaxations to DEA-NONOate resistant to ODQ, supporting the idea that in cerebral arteries nitric oxide may activate potassium channels independently of cyclic GMP. The results of our study suggest that under physiological conditions, guanylate cyclase is a key mediator of cerebral arterial relaxations to nitric oxide. However, under pathological conditions associated with induction of nitric oxide synthase and increased biosynthesis of nitric oxide (e.g., cerebral ischemia, inflammation, sepsis), mechanisms other than formation of cyclic GMP may be activated.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bradykinin; Cerebral Arteries; Charybdotoxin; Colforsin; Cyclic GMP; Dogs; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Hydrazines; In Vitro Techniques; Isometric Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitrogen Oxides; Oxadiazoles; Quinoxalines; Uridine Triphosphate

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP). 2. Using 10 microM of each donor compound, mean+/-s.e.mean rate of NO release ranged from 0.04+/-0.001 nmol min(-1) (for SNP) to 3.15+/-0.29 nmol min(-1) (for cysNO); cyclic GMP accumulation ranged from 0.43+/-0.05 pmol per 10(8) platelets (for SNP) to 2.67+/-0.31 pmol per 10(8) platelets (for cysNO), and inhibition of platelet aggregation ranged from 40+/-6.4% (for SNP) to 90+/-3.8% (for SNAC). 3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r = 0.83; P = 0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r= -0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r = 0.34). 4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P < 0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P < 0.01 vs DEANO, and P < 0.001 vs 8-bromo cyclic GMP). 5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu+) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO. 6. Cyclic GMP accumulation in response to GSNO (10(-9) 10(-5) M) was undetectable following treatment of platelets with ODQ (100 microM), a selective inhibitor of soluble guanylate cyclase. Despi

Topics: Cell Membrane; Cyclic AMP; Cyclic GMP; Glutathione; Humans; Hydrazines; In Vitro Techniques; Nitric Oxide; Nitrogen Oxides; Nitroso Compounds; Oxadiazoles; Phenanthrolines; Platelet Aggregation Inhibitors; Quinoxalines; S-Nitrosoglutathione; Sulfhydryl Compounds

The effect of two nitric oxide (NO) donors, SIN-1 and DEA/NO, as well as of the inactive SIN-1 derivative molsidomin, was studied on locus coeruleus (LC) neurons in a slice preparation using intracellular recordings. In addition, the effect of the guanylate cyclase inhibitor ODQ was analysed. Furthermore, the effect of NO donors on cyclic guanosine monophosphate (GMP) levels in the LC was studied using the indirect immunofluorescence technique, and the expression of soluble guanylyl cyclase with in situ hybridization. In 36 of 66 LC neurons extracellular application of SIN-1 and DEA/NO caused a hyperpolarization and a decrease in apparent input resistance. In almost 20% of neurons SIN-1 increased the firing rate. No effect could be recorded with the brain-inactive SIN-1 derivative molsidomin. The membrane permeable cGMP analogue 8-bromo-cGMP imitated the action of SIN-1. The hyperpolarizing effect of SIN-1 and DEA/NO was attenuated by preincubation with the guanylyl cyclase inhibitor ODQ. The immunohistochemical analysis revealed lack of cGMP immunostaining in non-stimulated slices, whereas SIN-1 dramatically increased this staining in about 40% of the LC neurons, and these neurons were all tyrosine hydroxylase positive, that is noradrenergic. A large proportion of the LC neurons expressed soluble guanylyl cyclase mRNA. The present and previous results suggest that NO, released from a small number of non-noradrenergic neurons in the LC, mainly has an inhibitory influence on many noradrenergic neurons, by upregulating cGMP levels via stimulation of soluble guanylyl cyclase. As nitric oxide synthase is present only in a small number of non-noradrenergic neurons (Xu et al., 1994), a few neurons may influence a large population of noradrenergic LC neurons, which in turn may control activity in many regions of the central nervous system.

Topics: Animals; Cyclic GMP; Excitatory Postsynaptic Potentials; Fluorescent Antibody Technique, Indirect; Hydrazines; In Situ Hybridization; In Vitro Techniques; Locus Coeruleus; Male; Molsidomine; Neural Pathways; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley

The aim of this investigation was to identify the mechanism by which nitric oxide inhibits neutrophil beta 2 integrin dependent adherence. Isolated rat neutrophils from blood and peritoneal exudates were exposed for 2 min to nitric oxide generated by diethylamine-NO at rates between 1.6 and 138 nmol/min. Exposure to nitric oxide at rates less than 14 nmol/min had no effect on adherence. Exposure to 14 to 56 nmol nitric oxide/min inhibited beta 2 integrin dependent adherence to endothelial cells, nylon columns, and fibrinogen-coated plates, but higher concentrations had no significant effect on adherence. Adherence by beta 2 integrins could be restored by incubating cells with dithioerythritol, phorbol 12-myristate 13-acetate, or 8-bromo cyclic GMP. Elevations in cellular cyclic GMP concentration were associated with adherence, but this did not occur after cells were exposed to concentrations of nitric oxide that inhibited beta 2 integrin-dependent adherence. Elevations in cyclic GMP did occur after cells were incubated with dithioerythritol or phorbol 12-myristate 13-acetate. Concentrations of nitric oxide that inhibited beta 2 integrin-dependent adherence also inhibited catalytic activity of membrane associated guanylate cyclase and binding of atrial natriuretic peptide, but were insufficient to activate cytosolic guanylate cyclase. Nitric oxide did not inhibit neutrophil oxidative burst or degranulation, nor effect beta 2 integrin expression or adherence that did not depend on beta 2 integrins, nor cause oxidative stress identified in terms of cellular glutathione concentration or protein nitrotyrosine. The results indicate that nitric oxide inhibited beta 2 integrins in a concentration-dependent fashion by inhibiting cell-surface transduction of signals linked to the activity of membrane-bound guanylate cyclase. The inhibitory effect could be overcome by providing cells with cyclic GMP exogenously or by stimulating cytosolic guanylate cyclase.

Topics: Animals; Atrial Natriuretic Factor; CD18 Antigens; Cell Adhesion; Cell Degranulation; Cyclic GMP; Glutathione; Guanylate Cyclase; Hydrazines; Male; Neutrophils; Nitric Oxide; Nitrogen Oxides; Protein Kinase C; Rats; Rats, Wistar; Respiratory Burst; Tetradecanoylphorbol Acetate; Tyrosine

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin-1beta and tumor necrosis factor-alpha expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by L-NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by L-NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose-dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine-induced inhibition of AIPase activity is mediated via mechanism other than NO.

Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Cells, Cultured; Cyclic GMP; Dinoprostone; Hydrazines; Interleukin-1; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitrogen Oxides; Nitroprusside; Osteoblasts; Osteocalcin; RNA, Messenger; Tumor Necrosis Factor-alpha

1. We iontophoretically applied NG-nitro-L-arginine (L-NOArg), an inhibitor of nitric oxide synthase (NOS), to cells (n = 77) in area 17 of anaesthetized and paralysed cats while recording single-unit activity extracellularly. In twenty-nine out of seventy-seven cells (38%), compounds altering NO levels affected visual responses. 2. In twenty-five out of twenty-nine cells, L-NOArg non-selectively reduced visually elicited responses and spontaneous activity. These effects were reversed by co-application of L-arginine (L-Arg), which was without effect when applied alone. Application of the NO donor diethylamine-nitric oxide (DEA-NO) produced excitation in three out of eleven cells, all three cells showing suppression by L-NOArg. In ten cells the effect of the soluble analogue of cGMP, 8-bromo-cGMP, was tested. In three of those in which L-NOArg application reduced firing, 8-bromo-cGMP had an excitatory effect. In six out of fifteen cells tested, L-NOArg non-selectively reduced responses to NMDA and alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA). Again, co-application of L-Arg reversed this effect, without enhancing activity beyond control values. 3. In a further subpopulation of ten cells, L-NOArg decreased responses to ACh in five. 4. In four out of twenty-nine cells L-NOArg produced the opposite effect and increased visual responses. This was reversed by co-application of L-Arg. Some cells were also affected by 8-bromo-cGMP and DEA-NO in ways opposite to those described above. It is possible that the variety of effects seen here could also reflect trans-synaptic activation, or changes in local circuit activity. However, the most parsimonious explanation for our data is that NO differentially affects the activity of two populations of cortical cells, in the main causing a non-specific excitation.

Topics: Acetylcholine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Arginine; Cats; Cyclic GMP; Electrophysiology; Enzyme Inhibitors; Excitatory Amino Acids; Hydrazines; Iontophoresis; N-Methylaspartate; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitrogen Oxides; Visual Cortex

Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects of the organic nitrates glyceryl trinitrate (100 mumol/L), pentaerythritol tetranitrate (10 mumol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors S-nitroso-N-acetyl-d,1-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 mumol/L caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8 +/- 6.7% (SNAP) and 80.9 +/- 6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 mumol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5 +/- 14.0%) By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6 +/- 6.4%) A similar effect was observed with a low concentration (1 mumol/L of SNAP and DEA/NO. All increases in CR induce by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 mumol/L). The positive contractile effect of isoprenaline was enhanced by 1 mumol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrate are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of CGMP-dependent protein kinase.

Topics: Animals; Cells, Cultured; Cyclic GMP; Electric Stimulation; Heart Ventricles; Hydrazines; Isosorbide Dinitrate; Male; Myocardial Contraction; Nitrogen Oxides; Nitroglycerin; Penicillamine; Pentaerythritol Tetranitrate; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Vasodilator Agents

Nitric oxide (NO)-nucleophile adducts are compounds of the general structure [XN(O)NO]-, where X is a nucleophile residue, and they release NO spontaneously in aqueous solution. This study determined the effects of two NO-nucleophile adducts [diethylaminodiazen-1-ium-1,2-diolate (DEA-NO) and sperminediazen-1-ium-1,2-diolate (DEA-NO) and sperminediazen-1-ium-1,2-diolate (spermine-NO)] on isometric force and the cytosolic concentrations of cyclic GMP ([cGMP]1), cyclic AMP ([cAMP]i) and calcium in canine tracheal smooth muscle. The ratio of fura-2 emission fluorescence intensities with excitation at 340- and 380-nm wavelengths (F340/ F380) was used as an index of cytosolic [Ca+2] concentration. Both DEA-NO and spermine-NO caused a concentration-dependent and reversible decrease in force (EC50 of 0.13 +/- 0.02 microM and 4.1 +/- 0.3 microM, respectively) and F340/F380, a concentration-dependent increase in [cGMP]i and no change in [cAMP]i. There were no significant differences in the relationship between [cGMP]l and the percentage decrease in force or in the relationship between the percentage decrease in F340/ F380 and the percentage decrease in force between tissues relaxed with DEA-NO or spermine-NO. Oxyhemoglobin increased the EC50 for both DEA-NO (from 0.13 +/- 0.03 microM to 8.1 +/- 0.2 microM) and spermine-NO (from 3.9 +/- 0.3 microM to 81.6 +/- 6.4 microM) and completely scavenged NO released by 0.13 (EC50) and 1 (EC100)microM DEA-NO. These results suggest that both DEA-NO and spermine-NO generate NO extracellularly and relax airway smooth muscle in association with an increase in [cGMP]i and a decrease in cytosolic Ca+2 concentration. NO-nucleophile adducts may serve as vehicles for the controlled delivery of NO into biological systems and thus provide a useful tool by which the physiology of NO can be investigated.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dogs; Dose-Response Relationship, Drug; Female; Hydrazines; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Neuromuscular Agents; Nitric Oxide; Nitrogen Oxides; Oxyhemoglobins; Spermine; Trachea

Nitric oxide (NO.) does not react significantly with thiol groups under physiological conditions, whereas a variety of endogenous NO donor molecules facilitate rapid transfer to thiol of nitrosonium ion (NO+, with one less electron than NO.). Here, nitrosonium donors are shown to decrease the efficacy of evoked neurotransmission while increasing the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). In contrast, pure NO donors have little effect (displaying at most only a slight increase) on the amplitude of evoked EPSCs and frequency of spontaneous mEPSCs in our preparations. These findings may help explain heretofore paradoxical observations that the NO moiety can either increase, decrease, or have no net effect on synaptic activity in various preparations.

Topics: Animals; Aplysia; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Cysteine; Ethylmaleimide; Fetus; Hippocampus; Hydrazines; Kinetics; Neuroglia; Neurons; Nitric Oxide; Nitrogen Oxides; Nitroglycerin; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; S-Nitrosothiols; Synapses; Synaptic Transmission

The mechanisms by which the paint constituent 2-nitropropane (2-NP) exerts genotoxicity and hepatocarcinogenicity are poorly understood. The hypothesis was tested that nitric oxide (NO) is a hepatic metabolic intermediate generated from 2-NP and/or its anionic tautomer propane 2-nitronate (P2N). Incubations of liver microsomes from phenobarbital-pretreated rats or mice with 2-NP or P2N gave spectra with Soret maxima at 448 nm which indicated the presence of a ferrous-NO complex. Levels of 3':5'-cyclic guanosine monophosphate (cGMP) and nitrite were measured by ELISA assay and HPLC, respectively, in freshly isolated mouse hepatocytes. Levels of cGMP generated within 3 h in cells by 2-NP, P2N (5 mM each) or the diethylamine/NO complex [Et2NNO(N==O)]Na (0.6 mM), an NO precursor, were 6, 15 and 34 times, respectively, those seen in control hepatocytes. Production of cGMP following treatment with 2-NP was linear with time of incubation; cGMP generation from P2N reached its peak already after 1 h. cGMP levels observed in incubates with 1-nitropropane and 2-deutero 2-nitropropane (5 mM), 2-NP isomers devoid of genotoxic properties, were significantly lower than those seen in the presence of 2-NP. Inclusion in the incubate of methylene blue, which inhibits NO-mediated reactions, decreased cGMP formation in hepatocytes with [Et2NNO(N==O)]Na, but increased it in cells with 2-NP or P2N. The production of nitrite from 2-NP, P2N or [Et2NNO(N==O)]Na mirrored cGMP formation. The results suggest that 2-NP and its nitronate generate an NO species in cells which may mediate, or contribute to, 2-NP genotoxicity.

Topics: Animals; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Hydrazines; Liver; Male; Methylene Blue; Mice; Mice, Inbred BALB C; Microsomes; Nitric Oxide; Nitrites; Nitrogen Oxides; Nitroparaffins; Propane; Rats; Rats, Sprague-Dawley; Solvents; Spectrophotometry