1-3-dihydroxy-4-4-5-5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole and 1-1-diethyl-2-hydroxy-2-nitrosohydrazine

NO is a highly diffusible and reactive gas produced in the nervous system, which acts as a neuronal signal mediating physiological or pathological mechanisms. NO can modulate the activity of neurotransmitter receptors and ion channels, including NMDA and GABA(A) receptors. In the present work, we examined whether GABA(C) receptor function can also be regulated by NO.. Homomeric ρ1 GABA(C) receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of the NO donor DEA. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis were used to determine the protein residues involved in the actions of NO.. GABAρ1 receptor responses were significantly enhanced in a dose-dependent, fast and reversible manner by DEA and the specific NO scavenger CPTIO prevented these potentiating effects. The ρ1 subunits contain only three cysteine residues, two extracellular at the Cys-loop (C177 and C191) and one intracellular (C364). Mutations of C177 and C191 render the ρ1 GABA receptors non-functional, but C364 can be safely exchanged by alanine (C364A). NEM, N-ethyl maleimide and (2-aminoethyl) methanethiosulfonate prevented the effects of DEA on GABAρ1 receptors. Meanwhile, the potentiating effects of DEA on mutant GABAρ1(C364A) receptors were similar to those observed on wild-type receptors.. Our results suggest that the function of GABA(C) receptors can be enhanced by NO acting at the extracellular Cys-loop.

Topics: Animals; Benzoates; Ethyl Methanesulfonate; Ethylmaleimide; gamma-Aminobutyric Acid; Hydrazines; Imidazoles; Nitric Oxide; Nitric Oxide Donors; Oocytes; Receptors, GABA; S-Nitrosoglutathione; Xenopus laevis

Although the mast cell is a source of nitric oxide (NO), the effect of NO on human mast cells has not been defined. This study investigated if exogenous NO could affect human mast cell activation.. Effects of different NO donors on immunoglobulin E (IgE)-dependent activation of human-cultured mast cells (HCMC) derived from precursors in buffy coat were investigated by measuring histamine release. Intracellular NO in HCMC was monitored with confocal microscopy using the fluorescent NO indicator 4-amino-5-methylamino-2', 7'-difluorofluorescein.. Diethylamine NONOate (DEA/NO) and MAHMA NONOate (NOC-9), both have rapid NO release rates, only inhibited anti-IgE-induced histamine release when added to HCMC at the time of activation. NO donors with slower NO release kinetics were ineffective even after 30 min incubation. Confocal microscopy revealed that the effectiveness of NO donors was dependent on the availability of adequate NO inside HCMC during activation. The inhibitory action of DEA/NO was diminished by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl but potentiated by the anti-oxidant, N-acetylcysteine (NAC). Furthermore, co-incubation with NAC allowed previously ineffective NO donors to suppress HCMC activation and thus suggested that NAC could increase the availability of NO from NO donors.. Our results demonstrated that NO was able to modulate human mast cell activation but only when enough NO was present at the time of cell activation. Our findings explain the controversy over the effectiveness of NO on mast cell degranulation and supports the possibility that NO donors could be beneficial for treating allergic inflammation.

Topics: Acetylcysteine; Anti-Allergic Agents; Anti-Inflammatory Agents; Antibodies; Antioxidants; Benzoates; Cell Degranulation; Cells, Cultured; Dose-Response Relationship, Drug; Histamine Release; Humans; Hydrazines; Imidazoles; Immunoglobulin E; Kinetics; Mast Cells; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; S-Nitroso-N-Acetylpenicillamine; Superoxide Dismutase

To understand the intrinsic operation of spinal networks generating locomotion, we need to not only characterize the constituent neurons and their connectivity, but also determine the role of intrinsic modulation in shaping the final motor output. We have focused on the effects of nitric oxide (NO) on the locomotor frequency and the underlying synaptic mechanisms in the lamprey spinal cord. To identify the source of NO, we used NADPH-diaphorase histochemistry and nNOS immunocytochemistry. Gray matter and sensory neurons were positively labeled using both methods. Preparations preincubated with NO synthase inhibitors displayed slower locomotor frequency that increased upon washout of the inhibitors, suggesting that NO is an endogenous neuromodulator in the spinal cord. Application of NO donors increased the locomotor frequency that was blocked by an NO scavenger and partially reduced by an inhibitor of sGC. To analyze the synaptic modulation underlying the NO-induced increase of the locomotor frequency we performed intracellular recordings from motoneurons and interneurons. The NO-induced increase in locomotor frequency was associated with a decrease in the midcycle inhibition and an increase in on-cycle excitation. To determine the site of action of NO, we examined the effect of NO donors on miniature PSCs. NO increased both the frequency and amplitude of mEPSCs while it only decreased the frequency of mIPSCs, suggesting the increased excitation is mediated by both presynaptic and postsynaptic mechanisms, while the decrease in inhibition involves only presynaptic mechanisms. Our results demonstrate a significant role of NO in adult vertebrate motor control which, via modulation of both excitatory and inhibitory transmission, increases the locomotor burst frequency.

Topics: Action Potentials; Animals; Benzoates; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Hydrazines; Imidazoles; In Vitro Techniques; Lampreys; Locomotion; NADPH Dehydrogenase; Neuroglia; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Patch-Clamp Techniques; Sodium Channel Blockers; Spinal Cord; Synaptic Transmission; Tetrodotoxin

The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli's salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated (P<0.01) by the HNO scavenger l-cysteine, whereas the sensitivity to GTN and DEA/NO was decreased (P<0.01) by the NO. scavenger carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide]. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one impaired responses to GTN>or=AS>>DEA/NO. Pretreatment with 10, 30, and 100 micromol/L of GTN for 60 minutes induced a 4- (P<0.05), 13- (P<0.01), and 48-fold (P<0.01) decrease in sensitivity to GTN, demonstrating tolerance development. In contrast, pretreatment with AS or DEA/NO (10, 30, and 100 micromol/L) did not alter their subsequent vasorelaxation. All of the nitrovasodilators (30 micromol/L) displayed a similar time course of vasorelaxation and cGMP accumulation over a 60-minute period. Unlike vasorelaxation, the magnitude of peak cGMP accumulation differed substantially: DEA/NO>>AS>GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation (P<0.01) of GTN-mediated relaxation, which was negated by the protein kinase G inhibitor guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,Rp-isomer, triethylammonium salt. In conclusion, vascular tolerance does not develop to HNO, nor does cross-tolerance between HNO and GTN occur. Thus, HNO donors may have therapeutic advantages over traditional nitrovasodilators.

Topics: Animals; Aorta, Thoracic; Benzoates; Cyclic GMP-Dependent Protein Kinases; Cysteine; Drug Tolerance; Enzyme Inhibitors; Free Radical Scavengers; Hydrazines; Imidazoles; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitrogen Oxides; Nitroglycerin; Oxadiazoles; Quinoxalines; Rats; Rats, Inbred WKY; Time Factors; Vasodilation

The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation.

Topics: Animals; Benzoates; Cell Hypoxia; Cell Line; Cobalt; Coculture Techniques; Deferoxamine; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Glutathione; Guanylate Cyclase; Hydrazines; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Inflammation; Kidney Tubules, Proximal; Macrophage Activation; Macrophages; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Oxides; Nitroso Compounds; Nuclear Proteins; Oxadiazoles; Oxazines; S-Nitrosoglutathione; Spermine; Swine; Transcription Factors

The aim of the present study was to investigate whether the activation of muscarinic receptors is a preliminary step to the endogenous release of nitric oxide modulating nicotinic transmission within the prevertebral ganglia. This work has been performed in vitro in isolated rabbit coeliac ganglion. The electrical activity of the ganglionic neurons was recorded using intracellular recording techniques. When a train of pulses of supramaximal intensity was applied to the splanchnic nerves, gradual depression of fast nicotinic transmission occurred: the pulses do not systematically elicit action potentials, but very often elicit excitatory postsynaptic potentials only. The use of pharmacological agents that interfere with the nitric oxide pathway such as L-arginine (precursor of nitric oxide) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (nitric oxide scavenger) demonstrated that nitric oxide modulates this depression phenomenon by facilitating or inhibiting the nicotinic transmission of the ganglionic neurons. A nitric oxide donor (diethylamine/nitric oxide complex) induced an inhibition of the nicotinic synaptic transmission. In the presence of the muscarinic receptors antagonist atropine, L-arginine and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to modify the nicotinic transmission of the ganglionic neurons but diethylamine/nitric oxide complex was still able to inhibit it. These results demonstrate that in the coeliac ganglion, the activation of muscarinic cholinergic receptors is a prerequisite for the activation of neuronal nitric oxide synthase in preganglionic fibres. The nitric oxide released then exerts a facilitation or an inhibition of the nicotinic transmission of the ganglionic neurons. Atropine triggered a facilitation of the nicotinic transmission when superfused alone and an inhibition when superfused in the presence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. These results confirm that muscarinic receptors activate the nitric oxide pathway modulating the nicotinic transmission of the prevertebral neurons. Our results also demonstrate that when the nitric oxide pathway is blocked, activation of muscarinic receptors leads to facilitation of the nicotinic transmission. Our study brings new insights concerning the modulation by nitric oxide and by muscarinic receptors of the synaptic transmission within the prevertebral ganglia.

Topics: Animals; Atropine; Benzoates; Drug Synergism; Electric Stimulation; Electrophysiology; Female; Ganglia, Sympathetic; Hydrazines; Imidazoles; Male; Muscarinic Antagonists; Neurons; Nicotine; Nitric Oxide; Nitrogen Oxides; Rabbits; Receptors, Muscarinic; Synaptic Transmission; Time Factors

Soluble guanylate cyclase (sGC), which is found in many cells and tissues, represents the receptor for the intra- and intercellular messenger molecule NO. Superoxide dismutase (SOD), an enzyme involved in the degradation of toxic superoxide radicals, has been proposed as a non-NO activator of sGC. Here we show that SOD stimulated sGC purified from bovine lung up to 10-fold. Activation by SOD was not influenced by the hydroxyl radical scavengers mannitol and DMSO. In contrast, the presence of the NO scavengers oxyhaemoglobin and 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide, as well as the O2(-)-generating system xanthine oxidase/hypoxanthine, led to inhibition of SOD-stimulated cGMP production. NO-insensitive sGC mutants were not influenced either by SOD or by xanthine oxidase. We have previously shown that sGC was stimulated by NO present in the normal atmosphere. Here we show that the SOD effect depended on the NO concentration from the atmosphere, as the stimulation of sGC by defined NO gases (0, 120, 330 and 1000 parts per billion NO) was potentiated by SOD. NO stimulation of sGC and its potentiation by SOD were inhibited by oxyhaemoglobin to identical levels. We conclude that the SOD-mediated stimulation of sGC is due to the elimination of superoxide, thereby preventing its reaction with NO to form peroxynitrite.

Topics: Animals; Benzoates; Cattle; Enzyme Activation; Free Radical Scavengers; Guanylate Cyclase; Hydrazines; Imidazoles; Kinetics; Lung; Mannitol; Nitric Oxide; Nitrogen Oxides; Oxyhemoglobins; Superoxide Dismutase