cyclic-gmp and 2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide

Topics: Animals; Arginine; Cattle; Cryopreservation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic N-Oxides; Heparin; Imidazoles; Male; Nitric Oxide; Sperm Capacitation; Sperm Motility; Spermatozoa; Thionucleotides

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid-β (Aβ) in the brain. Recent findings have revealed that nitric oxide (NO) modulates the processing of amyloid-β precursor protein (APP) and alters Aβ production; however, the previously presented data are contradictory and the underlying molecular mechanisms are still incomplete. Here, using human SH-SY5Y neuroblastoma cells stably transfected with wild-type APPwt695, we found that NO, derived from NO donor sodium nitroprusside (SNP), bi-directionally modulates APP processing in vitro. The data from ELISA and Western blot (WB) tests indicated that SNP at lower concentrations (0.01 and 0.1 μM) inhibits BACE1 expression, thus consequently suppresses APP β-cleavage and decreases Aβ production. In contrast, SNP at higher concentrations (10 and 20 μM) biases the APP processing toward the amyloidogenic pathway as evidenced by an increased BACE1 but a decreased ADAM10 expression, together with an elevated Aβ secretion. This bi-directional modulating activity of SNP on APP processing was completely blocked by specific NO scavenger c-PTIO, indicating NO-dependent mechanisms. Moreover, the anti-amyloidogenic activity of SNP is sGC/cGMP/PKG-dependent as evidenced by its reversal by sGC/PKG inhibitions, whereas the amyloidogenic activity of SNP is peroxynitrite-related and can be reversed by peroxynitrite scavenger uric acid. In summary, these present findings predict a double-edged role of NO in APP processing in vitro. Low (physiological) levels of NO inhibit the amyloidogenic processing of APP, whereas extra-high (pathological) concentrations of NO favor the amyloidogenic pathway of APP processing. This preliminary study may provide further evidence to clarify the molecular roles of NO and NO-related signaling in AD and supply potential molecular targets for AD treatment.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Carbazoles; Cell Line, Tumor; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Humans; Imidazoles; Membrane Proteins; Neuroblastoma; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Peroxynitrous Acid; Signal Transduction; Superoxides; Transfection

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

In many marine animals, adult habitat is selected by lecithotrophic (non-feeding) larvae with a limited lifespan. In generalist species, larvae may increasingly accept sub-optimal habitat over time as energy stores are depleted ('desperate larva' hypothesis). If the fitness cost of suboptimal habitat is too high, larvae of specialists may prolong the searching phase until they encounter a high-quality patch or die ('death before dishonor' hypothesis). In generalists, starvation is hypothesized to lead to a decline in inhibitory nitric oxide (NO) signaling, thereby triggering metamorphosis. Here, we document alternative functions for identified signaling pathways in larvae having 'desperate' versus 'death before dishonor' strategies in lecithotrophic clutches of a habitat specialist, the sea slug Alderia willowi. In an unusual dimorphism, each clutch of A. willowi hatches both non-selective larvae that settle soon after hatching and siblings that delay settlement in the absence of cues from the alga Vaucheria, the sole adult food. Pharmacological manipulation of NO signaling induced metamorphosis in non-selective but not selective stages. However, decreased NO signaling in selective larvae lowered the threshold for response to habitat cues, mimicking the effect of declining energy levels. Manipulation of cGMP or dopamine production induced metamorphosis in selective and non-selective larvae alike, highlighting a distinct role for the NO pathway in the two larval morphs. We propose a model in which NO production (1) links nitrogen metabolism with sensory receptor signaling, and (2) shifts from a regulatory role in 'desperate larva' strategies to a modulatory role in 'death before dishonor' strategies. This study provides new mechanistic insight into how the function of conserved signaling pathways may change in response to selection on larval habitat choice behaviors.

Topics: Adaptation, Biological; Analysis of Variance; Animal Distribution; Animals; Appetitive Behavior; Arginine; Biological Evolution; California; Cyclic GMP; Cyclic N-Oxides; Dopamine; Ecosystem; Gastropoda; Imidazoles; Larva; Levodopa; Linear Models; Metamorphosis, Biological; Models, Biological; NG-Nitroarginine Methyl Ester; Nitric Oxide; Serotonin; Signal Transduction; Stramenopiles

At presynaptic terminals vesicular membranes are fused into plasma membrane upon exocytosis and retrieved by endocytosis. During a sustained high-frequency transmission, exoendocytic coupling is critical for the maintenance of synaptic transmission. Here, we show that this homeostatic coupling is supported by cGMP-dependent protein kinase (PKG) at the calyx of Held. This mechanism starts to operate after hearing onset during the second postnatal week, when PKG expression becomes upregulated in the brainstem. Pharmacological tests with capacitance measurements revealed that presynaptic PKG activity is supported by a retrograde signal cascade mediated by NO that is released by activation of postsynaptic NMDA receptors. Activation of PKG also upregulates phosphatidylinositol-4,5-bisphosphate, thereby accelerating endocytosis. Furthermore, presynaptic PKG activity upregulates synaptic fidelity during high-frequency transmission. We conclude that maturation of the PKG-dependent retrograde signal cascade strengthens the homeostatic plasticity for the maintenance of high-frequency synaptic transmission at the fast glutamatergic synapse.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Biophysics; Brain Stem; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electric Capacitance; Electric Stimulation; Endocytosis; Enzyme Inhibitors; Exocytosis; Gene Expression Regulation, Developmental; Imidazoles; In Vitro Techniques; Membrane Potentials; Nerve Tissue Proteins; Neurons; Patch-Clamp Techniques; Phosphoric Monoester Hydrolases; Rats; Rats, Wistar; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptophysin; Thionucleotides

The airways of patients with cystic fibrosis (CF) exhibit decreased nitric oxide (NO) concentrations, which might affect airway function. The aim of this study was to determine the effects of NO on ion transport in human airway epithelia. Primary cultures of non-CF and CF bronchial and bronchiolar epithelial cells were exposed to the NO donor sodium nitroprusside (SNP), and bioelectric variables were measured in Ussing chambers. Amiloride was added to inhibit the Na(+) channel ENaC, and forskolin and ATP were added successively to stimulate cAMP- and Ca(2+)-dependent Cl(-) secretions, respectively. The involvement of cGMP was assessed by measuring the intracellular cGMP concentration in bronchial cells exposed to SNP and the ion transports in cultures exposed to 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (ODQ), or to 8Z, a cocktail of 8-bromo-cGMP and zaprinast (phosphodiesterase 5 inhibitor). SNP decreased the baseline short-circuit current (I(sc)) and the changes in I(sc) induced by amiloride, forskolin, and ATP in non-CF bronchial and bronchiolar cultures. The mechanism of this inhibition was studied in bronchial cells. SNP increased the intracellular cGMP concentration ([cGMP](i)). The inhibitory effect of SNP was abolished by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger (PTIO) and ODQ and was partly mimicked by increasing [cGMP](i). In CF cultures, SNP did not significantly modify ion transport; in CF bronchial cells, 8Z had no effect; however, SNP increased the [cGMP](i). In conclusion, exogenous NO may reduce transepithelial Na(+) absorption and Cl(-) secretion in human non-CF airway epithelia through a cGMP-dependent pathway. In CF airways, the NO/cGMP pathway appears to exert no effect on transepithelial ion transport.

Topics: Adenosine Triphosphate; Adult; Aged; Amiloride; Bronchi; Chloride Channels; Colforsin; Cyclic GMP; Cyclic N-Oxides; Cystic Fibrosis; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Free Radical Scavengers; Guanylate Cyclase; Humans; Imidazoles; Middle Aged; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Purinones; Quinoxalines; Young Adult

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

The purposes of these studies were to quantify the concentrations of total nitrate and nitrite (NO(x)(-)) cyclic guanosine monophosphate (cGMP), and nitrotyrosine over skin surface in normal weight healthy volunteers (n = 64) compared to overweight/obese subjects (n = 54). A semi-circular plastic tube was taped to the skin along acupuncture points (acupoints), meridian line without acupoint (MWOP), and nonmeridian control and filled with a 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution for 20 min. The concentrations of NO(x)(-), cGMP, and nitrotyrosine in the samples were quantified in a blinded fashion using chemiluminescence and enzyme-linked immunosorbent assay, respectively. In normal weight healthy volunteers, NO(x)(-) and cGMP concentrations were consistently increased over the pericardium meridian (PC) 4-7 compared with nonmeridian areas. NO(x)(-) concentration is enhanced over the bladder meridian (BL) 56-57, but cGMP level is similar between the regions. In overweight/obese subjects, NO(x)(-) contents were increased or tended to be elevated over PC and BL regions. cGMP is paradoxically decreased over PC acupoints and nonmeridian control on the forearm but the decreases were blunted along BL regions on the leg. Nitrotyrosine concentrations are markedly elevated (five- to sixfold) over both PC and BL in all areas of overweight/obese subjects. This is the first evidence showing that nitrotyrosine level is tremendously elevated over skin accompanied by paradoxical changes in nitric oxide (NO)-cGMP concentrations over PC skin region in overweight/obese subject. The results suggest that NO-related oxidant inflammation is systemically enhanced while cGMP generation is impaired over PC skin region but not over BL region in obesity.

Topics: Acupuncture Points; Adult; Cyclic GMP; Cyclic N-Oxides; Female; Humans; Imidazoles; Male; Nitrates; Nitric Oxide; Nitrites; Obesity; Skin; Tyrosine

In goldfish, nitric oxide synthase (NOS) immunoreactivity is present in gonadotropes and extracellular signal-regulated protein kinase (ERK) mediates GnRH stimulation of gonadotropin release and synthesis. In this study, we tested the possible involvement of nitric oxide (NO) and ERK in mediating PACAP-stimulated maturational gonadotropin (GTH-II) release from primary cultures of dispersed goldfish pituitary cells. In static incubation experiments, PACAP-induced GTH-II release was unaffected by two inhibitors of NOS synthase, AGH and 1400W; whereas addition of a NO donor, SNAP, elevated GTH-II secretion. In perifusion experiments, neither NOS inhibitors (AGH, 1400W and 7-Ni) nor NO scavengers (PTIO and rutin hydrate) attenuated the GTH-II response to pulse applications of PACAP. In addition, the GTH-II responses to PACAP and the NO donor SNP were additive while PTIO blocked SNP action. Although dibutyryl cGMP increased GTH-II secretion in static incubation, inhibition of guanylate cyclase (GC), a known down-stream target for NO signaling, did not reduce the GTH-II response to pulse application of PACAP. On the other hand, GTH-II responses to PACAP in perifusion were attenuated in the presence of two inhibitors of ERK kinase (MEK), U 0126 and PD 98059. These results suggest that although increased availability of NO and cGMP can lead to increased GTH-II secretion, MEK/ERK signaling, rather than NOS/NO/GC activation, mediates PACAP action on GTH-II release in goldfish.

Topics: Animals; Butadienes; Cells, Cultured; Cyclic GMP; Cyclic N-Oxides; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Free Radical Scavengers; Goldfish; Gonadotropins; Guanylate Cyclase; Imidazoles; Male; Nitric Oxide; Nitric Oxide Donors; Nitriles; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Gland; Signal Transduction

Nitrate assimilation in plants and related organisms is a highly regulated and conserved pathway in which the enzyme nitrate reductase (NR) occupies a central position. Although some progress has been made in understanding the regulation of the protein, transcriptional regulation of the NR gene (NIA1) is poorly understood. This work describes a mechanism for the ammonium-mediated repression of NIA1. We report the characterization of a mutant defective in the repression of NIA1 and NR in response to ammonium and show that a gene (CYG56) coding for a nitric oxide (NO)-dependent guanylate cyclase (GC) was interrupted in this mutant. NO donors, cGMP analogs, a phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), and a calcium ionophore (A23187) repress the expression of NIA1 in Chlamydomonas reinhardtii wild-type cells and also repress the expression of other ammonium-sensitive genes. In addition, the GC inhibitors LY83,583 (6-anilino-5,8-quinolinedione) and ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one) release cells from ammonium repression. Intracellular NO and cGMP levels were increased in the presence of ammonium in wild-type cells. In the cyg56 mutant, NIA1 transcription was less sensitive to NO donors and A23187, but responded like the wild type to IBMX. Results presented here suggest that CYG56 participates in ammonium-mediated NIA1 repression through a pathway that involves NO, cGMP, and calcium and that similar mechanisms might be occurring in plants.

Topics: Aminoquinolines; Arabidopsis; Arabidopsis Proteins; Calcium; Chlamydomonas reinhardtii; Cyclic GMP; Cyclic N-Oxides; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; Imidazoles; Models, Biological; Molecular Sequence Data; Mutation; NG-Nitroarginine Methyl Ester; Nitrate Reductase; Nitric Oxide; Nitrogen; Oxadiazoles; Quaternary Ammonium Compounds; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Transcription, Genetic

Nitric oxide (NO) acts as a key molecule in many physiological processes in plants. In this study, the roles of NO in mitochondrial respiration were investigated in the calli from wild-type Arabidopsis and NO associated 1 mutant (Atnoa1) which has a reduced endogenous NO level. Long-term exposure of wild-type Arabidopsis callus to sodium nitroprusside (SNP) increased mitochondrial respiration in both cytochrome and alternative pathways. In Atnoa1 callus, the capacity of both the cytochrome pathway and the alternative pathway was lower than that in wild-type callus. Further study indicated that NO enhanced the transcript abundance of genes encoding mitochondrial respiration-chain proteins as well as the protein expression of the NADH-ubiquinone reductase 75 kDa subunit and the alternative oxidase 1/2 in wild-type and Atnoa1 calli. 2-Phenyl-4,4,5,5-tetremethy-limidazolinone-1-oxyl-3-oxide (PTIO), a NO scavenger, inhibited the effects of NO in both calli. Co-incubation of callus with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, also abolished NO effects. The membrane-permeable cGMP analog 8Br-cGMP mimicked NO effects. Moreover, the alternative pathway showed a higher sensitivity to the cellular cGMP changes than the cytochrome pathway did in gene transcription, protein expression and O(2) consumption. Taken together, NO could enhance mitochondrial respiration in both cytochrome and alternative pathways in a cGMP-dependent manner in Arabidopsis.

Topics: Arabidopsis; Arabidopsis Proteins; Cyclic GMP; Cyclic N-Oxides; Electron Transport Chain Complex Proteins; Imidazoles; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 microM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP-PNP (adenyl-5'-yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 microM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC-specific inhibitor LY83583 applied at 50 microM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions.

Topics: Adenosine Triphosphate; Aminoquinolines; Analysis of Variance; Animals; Cell Movement; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Imidazoles; In Vitro Techniques; Leeches; Microglia; Nitric Oxide; Nucleotides; Trauma, Nervous System; Triazines

Asymmetric Ca(2+) signals across the growth cone mediate attractive or repulsive axon guidance depending on the occurrence of Ca(2+)-induced Ca(2+) release (CICR) through ryanodine receptors (RyRs). Although the neuronal isoform of nitric oxide (NO) synthase (nNOS) is highly expressed in developing dorsal root ganglion (DRG) neurons, the role of NO in axon guidance remains essentially unknown. Here we report that the NO-cGMP pathway negatively regulates CICR to control the directional polarity of DRG axon guidance. Intracellular levels of NO and cGMP depend on extracellular substrates: laminin activates the NO-cGMP pathway, whereas the adhesion molecule L1 does not. The activity of NO and cGMP determines the turning direction of growth cones with respect to asymmetric Ca(2+) signals that are produced by photolysing caged Ca(2+). The Ca(2+) signals cause growth cone repulsion on a laminin substrate, which is converted to attraction by pharmacological blockade of the NO-cGMP pathway or genetic deletion of nNOS. Conversely, Ca(2+)-induced growth cone attraction on an L1 substrate is converted to repulsion by increasing NO levels. Such NO-mediated switching of turning direction involves the regulation of CICR through RyRs. Furthermore, growth cone repulsion induced by an extracellular gradient of a physiological cue, neurotrophin-4, is dependent on Ca(2+) signals and converted to attraction by inhibiting the NO-cGMP pathway. These results suggest that, on contact with different adhesive environments, growth cones can change their turning responses to axon guidance cues by modulating CICR via endogenous NO and cGMP.

Topics: Animals; Calcium Signaling; Cell Polarity; Cells, Cultured; Chick Embryo; Cyclic AMP; Cyclic GMP; Cyclic N-Oxides; Cytosol; Egtazic Acid; Enzyme Inhibitors; Free Radical Scavengers; Ganglia, Spinal; Growth Cones; Imidazoles; Lasers; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Ryanodine Receptor Calcium Release Channel; Time Factors

The aim of the present study was to determine the effect of nitric oxide (NO) on the production of cyclic AMP (cAMP) by a human osteoblast cell line (HOS cells) stimulated with hydroxyapatite. Cells were cultured on the HA surfaces with or without the presence of NO donors (SNAP and NAP) for 3 days. The effect of adenylyl cyclase inhibitor (SQ22536), NO scavenger (carboxy PTIO) or endothelial nitric oxide synthase (eNOS) inhibitor (L-NIO), was assessed by adding these to the cultures of HA-stimulated HOS cells with or without the presence of SNAP. Furthermore, HOS cells were pre-treated with anti-human integrin alphaV antibody prior to culturing on HA surfaces with or without the presence of SNAP. The levels of cAMP and cGMP were determined from the 3-day culture supernatants. The results showed that the production of cAMP but not cGMP by HA-stimulated HOS cells was augmented by SNAP. SQ22536 and carboxy PTIO suppressed but L-NIO only partially inhibited the production of cAMP by HA-stimulated HOS cells with or without the presence of exogenous NO. Pre-treatment of the cells with anti-human integrin alphaV antibody suppressed the production of cAMP by HA-stimulated HOS cells with or without the presence of NO. Therefore, the results of the present study suggest that NO may up-regulate the production of cAMP, perhaps, by augmenting adenylyl cyclase activity initiated by the binding between HOS cell-derived integrin alphaV and HA surface.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Antibodies; Cell Line; Cyclic AMP; Cyclic GMP; Cyclic N-Oxides; Durapatite; Free Radical Scavengers; Humans; Imidazoles; Integrin alphaV; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Ornithine; Osteoblasts; S-Nitroso-N-Acetylpenicillamine; Stimulation, Chemical

In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO)-cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration.

Topics: Animals; Axons; Cyclic GMP; Cyclic N-Oxides; Drug Interactions; Embryo, Nonmammalian; Free Radical Scavengers; Ganglia, Invertebrate; Guanylate Cyclase; Imidazoles; Indoles; Locusta migratoria; NADPH Dehydrogenase; Nerve Crush; Nerve Regeneration; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Organ Culture Techniques; Serotonin; Time Factors

Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S-nitroso-acetyl-d-l-penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8-day-old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re-fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re-feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration- and time-dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or LY83583, and mimicked by 8-bromo cyclic guanosine 5'-phosphate (8Br-cGMP) (GMP) or 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol-3 kinase, extracellular-regulated kinases, calmodulin-dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore considered an important mediator of apoptosis and necrosis, as well as boc-aspartyl (OMe) fluoromethylketone (BAF), a caspase inhibitor, also blocked cell death induced by re-feeding the cultures. These findings demonstrate that NO inhibits apoptosis of retinal neurons in a cGMP/protein kinase G (PKG)-dependent way, and strengthens the notion that NO plays an important role during CNS development.

Topics: Adenosine; Aminoquinolines; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Chick Embryo; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Imidazoles; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Penicillamine; Retina; Signal Transduction; Tetrazolium Salts; Thiazoles; Tritium

After a loss against an opponent, the aggressiveness of a male cricket is significantly reduced for up to 30 minutes. This depression of aggressiveness is an important factor in the establishment and maintenance of dominance between individuals. In the present study, we investigated the functional roles of nitric oxide (NO) signaling in the depression of aggressiveness in subordinate male crickets. Pairs of male crickets, pre-injected with various NO-related reagents, were allowed to establish dominant/subordinate relationships in dyadic encounters. Opponents were separated for 15 minutes and then paired again. In second encounters, subordinate crickets pre-injected with PTIO (NO scavenger) showed agonistic behavior towards former dominant opponents. A similar effect was observed in crickets pre-injected with L-NAME (NO synthase inhibitor) or ODQ (soluble guanylate cyclase inhibitor). The effects of the latter two drugs were canceled by co-injection of NOR3 (NO donor) with L-NAME or by co-injection of 8-Br-cGMP (cGMP-analog) with ODQ. Injection of NOR3 alone prolonged the inhibition of agonistic behavior in subordinate crickets from 30 minutes to 3 hours. Our results suggest that the change in agonistic behavior observed in subordinate male crickets is closely linked to NO-mediated cGMP signaling.

Topics: Animals; Cyclic GMP; Cyclic N-Oxides; Enzyme Inhibitors; Female; Free Radical Scavengers; Gryllidae; Imidazoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Social Dominance

In locusts, a central pattern generator underlies the rhythmic movements of the ovipositor valves that serve to drive the abdomen into damp soil in order to lay eggs. We have investigated the role of nitric oxide (NO) in the control of this oviposition digging rhythm. NO increases the frequency of the rhythm by acting via sGC to elevate cGMP, which in turn acts via PKG. Increasing exogenous NO levels using the NO donors SNAP and PAPANONOate increased the cycle frequency of the fictive digging rhythm, as did increasing endogenous NO by bath application of the substrate for NOS, l-arginine. On the other hand, application of the NO scavenger PTIO decreased the cycle frequency, indicating that NO must normally exert a continuous and dynamic role on the central pattern generator underlying the oviposition rhythm. Inhibiting the main molecular target of NO, soluble guanylate cyclase, with ODQ reduced the cycle frequency of the rhythm, suggesting that NO mediated its effects via sGC and cyclic GMP. Further evidence for this was produced by bath application of 8-Br-cGMP, which increased the frequency of the rhythm. Bath application of the generic protein kinase inhibitor and a selective PKG inhibitor, H-7 and KT-5823, respectively, reduced the frequency of the rhythm, suggesting that PKG acted as a target for cGMP. Thus, we conclude that NO plays a key role in regulating the frequency of the central pattern generator controlling rhythmic egg-laying movements in locusts by acting via sGC/cGMP-PKG.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Behavior, Animal; Carbazoles; Cyclic GMP; Cyclic N-Oxides; Female; Grasshoppers; Guanylate Cyclase; Imidazoles; Indoles; Muscles; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Oviposition; Penicillamine; Protein Kinases; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase

Increased expression of glial fibrillary acidic protein (GFAP) represents astroglial activation and gliosis during neurodegeneration. However, the molecular mechanism behind increased expression of GFAP in astrocytes is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of GFAP. Bacterial lipopolysachharides (LPSs) induced the production of NO and the expression of GFAP in mouse primary astrocytes. Either a scavenger of NO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)] or an inhibitor of inducible nitric oxide synthase [l-N6-(I-iminoethyl)-lysine hydrochloride] blocked this induction of GFAP expression. Similarly, other inducers of NO production such as interferon-gamma, interleukin-1beta, human immunodeficiency virus type 1 gp120, fibrillar amyloid beta peptides, and double-stranded RNA (polyinosinic-polycytidilic acid) also induced the expression of GFAP through NO. The role of NO in the expression of GFAP was supported further by increased expression of GFAP by S-nitroso glutathione (GSNO), an NO donor. Interestingly, inhibition of nuclear factor kappaB (NF-kappaB) suppressed LPS- but not GSNO-induced expression of GFAP, suggesting that NO does not require NF-kappaB to induce GFAP and that NF-kappaB functions upstream of NO production. However, inhibition of LPS- and GSNO-induced expression of GFAP either by NS-2028 [a specific inhibitor of guanylate cyclase (GC)] or by KT5823 [a specific inhibitor of cGMP-activated protein kinase (PKG)], and induction of GFAP expression by either 8-Br cGMP (a cell-permeable cGMP analog) or MY-5445 (a specific inhibitor of cGMP phosphodiesterase) suggests that NO induces GFAP via GC-cGMP-PKG. This study illustrates a novel biological role of NO in regulating the expression of GFAP in astrocytes through the GC-cGMP-PKG pathway that may participate in the pathogenesis of neurodegenerative disorders.

Topics: Animals; Animals, Newborn; Astrocytes; Cell Survival; Cells, Cultured; Cerebral Cortex; Corpus Striatum; Cyclic GMP; Cyclic N-Oxides; Cytokines; Dose-Response Relationship, Drug; Drug Interactions; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Fluorescent Antibody Technique; Free Radical Scavengers; Gene Expression; Glial Fibrillary Acidic Protein; HIV Envelope Protein gp120; Imidazoles; Lipopolysaccharides; Lysine; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles; Thionucleotides; Time Factors

Endothelium-independent relaxant activities of N(omega)-hydroxy-L-arginine (L-NOHA) homologues and hydroxylamine, a possible intermediate in nitric oxide (NO) formation, were examined in rat aortic rings. Addition of one -CH(2)- group to the -(CH(2))(x)- chain between the alpha-amino acid and the hydroxyguanidine group (x=4) almost abolished-while deletion of one or two -CH(2)- (x=1 or 2) enhanced-the relaxant activity of L-NOHA homologues. N(omega)-hydroxy-nor-L-arginine- (x=2) and hydroxylamine-induced relaxations were blunted by a NO scavenger and by inhibitors of the guanylyl cyclase pathway, but not by NO synthase or cytochrome P(450) inhibitors (except 7-ethoxyresorufin). However, aortic NO formation was detected (using electron paramagnetic resonance) in the presence of concentrations of these compounds higher than those producing relaxation. These findings support the view that endothelium-independent vasorelaxations induced by both L-NOHA homologues with a required chain length x

Topics: Animals; Aorta, Thoracic; Arginine; Cyclic GMP; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Hydroxylamine; Imidazoles; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Signal Transduction; Vasodilation; Vasodilator Agents

Electrical stimulation of sensory neurons that innervate receptors on the tailfan of crayfish evokes a reflex response of motor neurons that produce movements of the blades of the tailfan, the uropods. We analyzed the modulatory effects of nitric oxide (NO) on the spike frequency of the reflex response. Bath application of L-arginine and SNAP, which elevate endogenous and exogenous NO levels, increased the frequency of the evoked response, whereas the application of L-NAME and PTIO, which reduce NO levels, decreased the frequency of the response. To determine through what pathway and target NO exerted these effects we bath applied ODQ, an inhibitor of soluble guanylyl cyclase (sGC), which decreased the frequency of response, and 8-br-cGMP, which increased the spike frequency of response. To provide further evidence that NO acts via sGC, we elevated NO levels with L-arginine while simultaneously inhibiting sGC with ODQ. This application reduced the response to control levels, indicating that NO in the terminal ganglion of crayfish acts via sGC to modulate cGMP levels, which in turn regulate the responses of the uropod motor neurons.

Topics: Analysis of Variance; Animals; Arginine; Astacoidea; Cyclic GMP; Cyclic N-Oxides; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Female; Free Radical Scavengers; Imidazoles; In Vitro Techniques; Male; Muscles; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Penicillamine; Picolines; Quinoxalines; Reflex; Thionucleotides

The involvement of nitric oxide (NO) in the regulation of goldfish growth hormone (GH) secretion was further characterized using primary cultures of dispersed goldfish pituitary cells. Western blots revealed the presence of an inducible nitric oxide synthase (iNOS)-like protein of approximately 120 kDa in cytosol/plasma membrane extracts. By contrast, brain NOS-immunoreactive proteins of approximately 120-140 kDa were occasionally detected in a cytoskeleton/organelle fraction but were absent from cytosol/plasma membrane extracts. The NO donor sodium nitroprusside (SNP) acutely increased GH secretion but this response was not observed in the presence of either a NO scavenger (PTIO) or a soluble guanylate cyclase inhibitor (ODQ). SNP also significantly increased the levels of cyclic (c)GMP in somatotrope-enriched cell populations. Treatments with 1400W (iNOS inhibitor), PTIO and rutin hydrate (NO scavengers) and ODQ abolished the acute GH-release response to two endogenous gonadotropin-releasing hormones (GnRH). 1400W, rutin hydrate, PTIO and ODQ alone did not significantly alter basal GH secretion. Together, these results establish that an iNOS-like peptide is constitutively present in the pituitary of the goldfish. Furthermore, these data suggest that NO, most likely through the generation of cGMP, is a necessary signal transduction component of GnRH-induced GH secretion.

Topics: Animals; Cells, Cultured; Cyclic GMP; Cyclic N-Oxides; Cytoskeleton; Enzyme Inhibitors; Free Radical Scavengers; Goldfish; Gonadotropin-Releasing Hormone; Growth Hormone; Guanylate Cyclase; Imidazoles; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroprusside; Organelles; Pituitary Gland

Aspirin is known to exert cytoprotection by presently unidentified mechanisms. This study investigates the involvement of nitric oxide (NO) in antioxidant cellular protection induced by aspirin.. A 24-hour incubation with hydrogen peroxide markedly reduced viability of cultured endothelial cells. Preincubation with aspirin (3 to 30 micromol/L) protected endothelial cells from hydrogen peroxide-mediated toxicity and increased viability in a concentration-dependent fashion by up to 95% of control. This effect was specific in that other nonsteroidal anti-inflammatory drugs, such as salicylate or indomethacin, did not alter hydrogen peroxide toxicity. Aspirin-induced endothelial protection was abrogated in the presence of the NO scavenger PTIO (30 micromol/L) and the inhibitor of soluble guanylyl cyclase ODQ (1 micromol/L). Moreover, the l-arginine antagonist L-NMMA (25 micromol/L), but not its D-enantiomer, led to complete inhibition of aspirin-dependent cytoprotection. Correspondingly, aspirin enhanced NO synthase activity (citrulline formation) and intracellular cyclic GMP accumulation in endothelial cells. Protein expression of endothelial NO synthase remained unaffected in the presence of aspirin.. Our data suggest that endothelial NO synthase is a site of action of aspirin and that the NO/cyclic GMP system assumes a crucial function in mediating the cytoprotective action of aspirin.

Topics: Animals; Aspirin; Cattle; Cell Survival; Cells, Cultured; Citrulline; Cyclic GMP; Cyclic N-Oxides; Cytoprotection; Diclofenac; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Hydrogen Peroxide; Imidazoles; Indomethacin; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Swine

We have analyzed the action of nitric oxide on the synaptic inputs of spiking local interneurons that form part of the local circuits in the terminal abdominal ganglion of the crayfish, Pacifastacus leniusculus. Increasing the availability of NO in the ganglion by bath applying the NO donor SNAP, or the substrate for its synthesis, L-arginine, caused a depression of synaptic inputs onto the interneurons evoked by electrically stimulating mechanosensory neurons in nerve 2 of the terminal ganglion. Conversely, reducing the availability of NO by bath application of an NO scavenger, PTIO, and an inhibitor of nitric oxide synthase, L-NAME, increased the amplitude of the evoked potentials. These results suggest that elevated NO concentration causes a depression of the synaptic inputs to spiking local interneurons. To determine whether these effects could be mediated through an NO/cGMP signaling pathway we bath applied a membrane permeable analogue of cGMP, 8-br-cGMP, which decreased the amplitude of the inputs to the interneurons. Bath application of an inhibitor of soluble guanlylyl cyclase, ODQ, produced an increase in the amplitude of the synaptic inputs. Our results suggest that NO causes a depression of synaptic inputs to spiking local interneurons probably by acting through an NO/cGMP signaling pathway. Moreover, application of NO scavengers modulates the inputs to these interneurons, suggesting that NO is continuously providing a powerful and dynamic means of modulating the outputs of local circuits.

Topics: Action Potentials; Animals; Arginine; Astacoidea; Cyclic GMP; Cyclic N-Oxides; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Female; Free Radical Scavengers; Imidazoles; Interneurons; Male; Neurons, Afferent; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Synapses

The nitric oxide (NO)-cGMP signaling pathway is implicated in an increasing number of experimental models of plasticity. Here, in a behavioral analysis using one-trial appetitive associative conditioning, we show that there is an obligatory requirement for this pathway in the formation of long-term memory (LTM). Moreover, we demonstrate that this requirement lasts for a critical period of approximately 5 hr after training. Specifically, we trained intact specimens of the snail Lymnaea stagnalis in a single conditioning trial using a conditioned stimulus, amyl-acetate, paired with a salient unconditioned stimulus, sucrose, for feeding. Long-term associative memory induced by a single associative trial was demonstrated at 24 hr and shown to last at least 14 d after training. Tests for LTM and its dependence on NO were performed routinely 24 hr after training. The critical period when NO was needed for memory formation was established by transiently depleting it from the animals at a series of time points after training by the injection of the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO). By blocking the activity of NO synthase and soluble guanylyl cyclase enzymes after training, we provided further evidence that LTM formation depends on an intact NO-cGMP pathway. An electrophysiological correlate of LTM was also blocked by PTIO, showing that the dependence of LTM on NO is amenable to analysis at the cellular level in vitro. This represents the first demonstration that associative memory formation after single-trial appetitive classical conditioning is dependent on an intact NO-cGMP signaling pathway.

Topics: Animals; Appetitive Behavior; Conditioning, Classical; Cyclic GMP; Cyclic N-Oxides; Drug Administration Schedule; Enzyme Inhibitors; Feeding Behavior; Free Radical Scavengers; Guanylate Cyclase; Imidazoles; Lymnaea; Memory; Neuronal Plasticity; Nitric Oxide; Nitric Oxide Synthase; Pentanols; Receptors, Cytoplasmic and Nuclear; Retention, Psychology; Signal Transduction; Soluble Guanylyl Cyclase; Sucrose; Time Factors

Little is known of the action of nitric oxide (NO) at the synaptic level on identified interneurones in local circuits that process mechanosensory signals. Here, we examine the action of NO in the terminal abdominal ganglion of the crayfish Pacifastacus leniusculus, where it has modulatory effects on the synaptic inputs of 17 identified ascending interneurones mediated by electrical stimulation of a sensory nerve. To analyse the role of NO in the processing of sensory signals, we bath-applied the NO donor SNAP, the NO scavenger PTIO, the nitric oxide synthase (NOS) inhibitor l-NAME, the NOS substrate l-arginine, a cyclic GMP (cGMP) analogue, 8-Br-cGMP, and the soluble guanylate cyclase (sGC) inhibitor ODQ. The effects of these chemicals on the synaptic inputs of the interneurones could be divided into two distinct classes. The NO donor SNAP enhanced the inputs to one class of interneurone (class 1) and depressed those to another (class 2). Neither the inactive isomer NAP nor degassed SNAP had any effect on the inputs to these same classes of interneurone. The NO scavenger PTIO caused the opposite effects to those of the NO donor SNAP, indicating that endogenous NO may have an action in local circuits. Preventing the synthesis of NO using l-NAME had the opposite effect to that of SNAP on each response class of interneurone. Increasing the synthesis of endogenous NO by applying l-arginine led to effects on both response classes of interneurone similar to those of SNAP. Taken together, these results suggested that NO was the active component in mediating the changes in amplitude of the excitatory postsynaptic potentials. Finally, the effects of 8-Br-cGMP were similar to those of the NO donor, indicating the possible involvement of a NO-sensitive guanylate cyclase. This was confirmed by preventing the synthesis of cGMP by sGC using ODQ, which caused the opposite effects to those of 8-Br-cGMP on the two response classes of interneurone. The results indicate that a NO--cGMP signal transduction pathway, in which NO regulates transmitter release from mechanosensory afferents onto intersegmental ascending interneurones, is probably present in the local circuits of the crayfish.

Topics: Animals; Arginine; Astacoidea; Cyclic GMP; Cyclic N-Oxides; Electric Stimulation; Electrophysiology; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Female; Free Radical Scavengers; Ganglia, Invertebrate; Imidazoles; In Vitro Techniques; Interneurons; Male; Mechanoreceptors; Nerve Net; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Penicillamine; Quinoxalines; S-Nitroso-N-Acetylpenicillamine; Synapses; Synaptic Transmission

Several effects of nitric oxide (NO) on the control of L-type calcium current (ICa) and of calcium handling in cardiomyocytes have been described. Cardiomyocytes have been shown to express in different conditions all types of nitric oxide synthases (NOS), but the role of NO in the regulation of calcium current remains controversial. Previously, we have shown in guinea pig ventricular cells a stimulatory effect of NOS inhibitors on ICa. Here we investigate the intracellular mechanisms involved in the putative inhibitory role of NO on basal ICa in ventricular cells. The stimulatory effect of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (1 mM) was present also in calcium transient measurements, but only after a preincubation with L-arginine (L-arg, 0.1 mM). The nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 0.5 mM) increased peak ICa in a similar manner to NOS inhibitors in whole-cell voltage-clamp experiments. Also ODQ (1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one, 0.1 mM), a specific inhibitor of a target of NO, the soluble guanylate cyclase, was able to stimulate ICa. The block of type II phosphodiesterase (cGMP-activated) by EHNA (erythro-9-[2-hydroxy-3-nonylladenine, 30 microM) exerted a similar effect on ICa as PTIO and ODQ. Carbachol (CCh, 1 microM) was able to revert the stimulatory effect on ICa observed with PTIO, ODQ, and EHNA. We propose that the increase of basal ICa in guinea pig cardiomyocytes previously observed with L-NMMA depends on the removal of a tonic NO inhibition. This increase of ICa is mimicked by blocking at different steps the cGMP-cascade activated by NO, suggesting a NO-guanylate cyclase mechanism in the basal control of ventricular calcium current.

Topics: Adenine; Animals; Arginine; Biological Transport; Calcium Channels, L-Type; Carbachol; Cholinergic Agonists; Cyclic GMP; Cyclic N-Oxides; Enzyme Inhibitors; Exonucleases; Free Radical Scavengers; Guinea Pigs; Imidazoles; In Vitro Techniques; Myocardium; Nitric Oxide; omega-N-Methylarginine; Orchiectomy; Oxadiazoles; Quinoxalines; Receptors, Muscarinic

1. In the cerebellar cortex, brief, 8 Hz activation of parallel fibres (PFs) induces a cyclic adenosine 3'5'-monophosphate (cAMP) and protein kinase A (PKA)-dependent form of long-term potentiation between PFs and Purkinje cells. 2. With 10 mM BAPTA in the recording pipette, potentiation evoked by raised frequency stimulation (RFS) to one of two, synaptically independent PF inputs to the same Purkinje cell did not remain input specific but consistently spread to synapses that did not receive RFS, up to the maximum distance tested of 168 microm. 3. LTP at activated and non-activated sites was accompanied by a decrease in paired pulse facilitation (PPF). The PKA inhibitor H-89 blocked both of these effects. Inhibition of nitric oxide synthase (NOS), either by 7-nitro-indazole (7-NI) or N (G)-nitro-L-arginine methyl ester (L-NAME), completely prevented heterosynaptic potentiation and associated reduction in PPF. LTP at distant synapses was selectively prevented by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Inhibition of soluble guanylate cyclase or protein kinase G had no effect on either pathway. 4. Synaptic potentiation at PF-PC synapses, induced by the adenylate cyclase activator forskolin, was also prevented by inhibition of NOS. Forskolin-induced increases in mEPSC frequency were similarly prevented by NOS inhibition and mimicked by the NO donor spermine NONOate. 5. These results are consistent with the notion that heterosynaptic potentiation is of pre-synaptic origin and dependent upon activation of cAMP/PKA and NO. Moreover, they suggest that cAMP/PKA activation stimulates NO production and this diffusible messenger facilitates pre-synaptic transmitter release at synapses within a radius of upwards of 150 microm, through a mechanism that does not involve cGMP.

Topics: Algorithms; Animals; Cerebellum; Colforsin; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic N-Oxides; Electrophysiology; Free Radical Scavengers; Guanylate Cyclase; Imidazoles; In Vitro Techniques; Long-Term Potentiation; Male; Neuronal Plasticity; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Patch-Clamp Techniques; Purkinje Cells; Rats; Rats, Wistar; Synapses

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

Topics: Aminoquinolines; Animals; Antineoplastic Agents; Apyrase; Astrocytes; Caenorhabditis elegans Proteins; Calcium; Calcium Channel Blockers; Cells, Cultured; Chelating Agents; Cyclic GMP; Cyclic N-Oxides; Egtazic Acid; Enzyme Inhibitors; Estrenes; Free Radical Scavengers; GTP-Binding Proteins; Imidazoles; Ionomycin; Ionophores; Neurons; Nicardipine; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Potassium Chloride; Prosencephalon; Pyrrolidinones; Rats; Receptor, Insulin; Ryanodine; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Suramin; Thionucleotides; Type C Phospholipases

NO-Aspirin (NCX-4016) releases nitric oxide (NO) in biological systems through as yet unidentified mechanisms. In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN, 0.1-1 microM) significantly attenuated the cyclic GMP response to a subsequent challenge with both NO-aspirin or GTN. Similarly, NO-aspirin (10-100 microM) was found to induce tolerance to its own cyclic GMP stimulatory action and to that of GTN. In contrast, cyclic GMP stimulation by the spontaneous NO donor SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or NO-aspirin. The observed cross-tolerance between NO-aspirin and GTN cells indicates that bioactivation pathways of organic nitrates, which have been shown to involve cytochrome P450, may also be responsible for NO release from NO-aspirin. Prolonged treatment with NO-aspirin causes down-regulation of the cellular cyclic GMP response, suggesting that tolerance may occur during therapy with NO-aspirin.

Topics: Animals; Aspirin; Cell Line; Cyclic GMP; Cyclic N-Oxides; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Epithelial Cells; Free Radical Scavengers; Imidazoles; Kidney; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Protein Binding; Swine

The organic nitrate pentaerithrityl tetranitrate (PETN) is known to exert long-term antioxidant and antiatherogenic effects by as yet unidentified mechanisms. In porcine aortic endothelial cells, a 24 h incubation with PETN (1-100 microM) or its metabolite pentaerithrityl trinitrate (PETriN) increased levels of the antioxidant protein ferritin up to three-fold over basal, whereas isosorbide dinitrate and isosorbide-5-mononitrate were without significant effect under these conditions. PETriN-induced ferritin expression was blocked by the NO scavenger PTIO but remained unaltered in the presence of ODQ, an inhibitor of soluble guanylyl cyclase. 8-Bromo cyclic GMP and dibutyryl cyclic GMP did not influence basal ferritin synthesis. The iron chelator desferrioxamine abolished ferritin induction by PETriN. Our results show that PETN or its active metabolite PETriN induce ferritin synthesis through NO- and iron-dependent but cyclic GMP-independent pathways. Increased activity of ferritin may contribute to, and at least in part explain, the specific antiatherogenic and antioxidant action of PETN.

Topics: Animals; Antioxidants; Aorta; Cells, Cultured; Cyclic GMP; Cyclic N-Oxides; Deferoxamine; Endothelium, Vascular; Ferritins; Free Radical Scavengers; Gene Expression; Guanylate Cyclase; Imidazoles; Iron Chelating Agents; Isosorbide Dinitrate; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Pentaerythritol Tetranitrate; Spermine; Swine

Incubation with TNF-alpha (50 ng/ml) for 72 hours markedly reduced viability of endothelial cells. A 6-hour preincubation with S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 3-100 microM) protected cells in a concentration-dependent manner and decreased TNF-alpha-mediated toxicity by up to 70%. Cytoprotection by SNAP was completely abolished by the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine and mimicked by 8-bromo cyclic AMP or forskolin. SNAP produced significant increases in cyclic GMP and cyclic AMP, both being abrogated in the presence of the NO scavenger 2-phenyl-4, 4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Moreover, no endothelial protection by SNAP was detected in the presence of the protein kinase A inhibitor KT5720, whereas the protein kinase G inhibitor KT5823 left cytoprotection virtually unaltered. Our results demonstrate a crucial role for cyclic AMP in mediating NO-induced endothelial protection against TNF-alpha, possibly through cyclic GMP-dependent inhibition of cyclic AMP breakdown. NO-dependent endothelial protection may ultimately result from cyclic AMP-induced up-regulation of antioxidant proteins or down-regulation of cytotoxic processes.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclase Inhibitors; Alkaloids; Animals; Carbazoles; Cattle; Cell Line; Cell Survival; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic N-Oxides; Dideoxyadenosine; Endothelium, Vascular; Enzyme Inhibitors; Free Radical Scavengers; Imidazoles; Indoles; Kinetics; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Penicillamine; Pulmonary Artery; Pyrroles; Quinoxalines; S-Nitroso-N-Acetylpenicillamine; Tumor Necrosis Factor-alpha

In the rabbit iris sphincter muscle, sodium nitroprusside (SNP), a nitric oxide (NO) donor, inhibits cholinergic contraction but does not affect tachykinergic contraction in vitro. The objectives of the current study were to clarify the mechanism for the different responsiveness to NO in cholinergic and tachykinergic muscular contractions, and to examine whether the mechanism for NO-induced inhibition of cholinergic muscular contraction is operative in vivo.. Iris sphincter muscle was dissected from the rabbit eye pretreated with or without endotoxin (lipopolysaccharide, LPS) in vivo. Cyclic guanosine monophosphate (cGMP) content in the iris sphincter muscle was determined by radioimmunoassay. The motor activity of the ring-shaped iris sphincter muscle was measured isometrically. Sodium nitroprusside, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO, a scavenger of NO radicals), and 8-bromo cGMP (a permeable cGMP analogue) were administered between the first and second administrations of carbachol and neurokinin A, both of which had caused sustained contraction in the iris sphincter muscle.. Sodium nitroprusside inhibited the contraction of the iris sphincter muscle caused by carbachol but had no effect on the contraction caused by neurokinin A. Application of C-PTIO significantly reduced SNP-induced cGMP accumulation in the muscle, as well as the SNP-induced inhibition of muscular contraction caused by carbachol. Neither carbachol nor neurokinin A influenced SNP-induced cGMP accumulation in the muscle. Induction of 8-bromo-cGMP significantly diminished the muscular contraction caused by carbachol but not that caused by neurokinin A. In vivo pretreatment of the eye with LPS increased, in a time-dependent manner, the cGMP accumulation in the iris sphincter muscle, which was significantly inhibited by pretreatment of NG-nitro-L-arginine methyl ester (an inhibitor of NO synthesis) in vivo.. These results demonstrate that in rabbits the increase in cGMP accumulation induced by NO in the iris sphincter muscle is involved in the cholinergic contraction but not in the tachykinergic contraction, suggesting that different sensitivities to cGMP are essential for the different responsiveness to NO. Furthermore, the results of this study showed that the NO-cGMP pathway is operative in vivo and regulates iris sphincter muscle tone, at least when the eyes are infected with bacteria.

Topics: Animals; Carbachol; Cyclic GMP; Cyclic N-Oxides; Female; Free Radical Scavengers; Imidazoles; Iris; Male; Motor Activity; Muscarinic Agonists; Muscle Contraction; Muscle, Smooth; Neurokinin A; Nitric Oxide; Nitroprusside; Rabbits; Radioimmunoassay

We explored whether endothelium-derived nitric oxide (EDNO) acts in an autocrine or paracrine fashion to activate the soluble guanylate cyclase by measuring the elevations of cGMP levels. Comparisons were made between two culture models in which EDNO can stimulate cGMP synthesis within the same cells (endothelial cells) where it is formed, or in the neighboring cells (smooth muscle cells). The basal amount of EDNO showed no difference in cGMP levels when the endothelial cells (EC) were cultured either alone (iso-culture) or together with the smooth muscle cells (SMC, co-culture). However, cGMP levels were synergistically increased by the BK-stimulated EDNO when EC were co-cultured with SMC. The synergistical increase was significantly inhibited by 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), an extracellular radical scavenger. These findings suggest that the tonic formed basal EDNO has an autocrine effect on cGMP levels of EC themselves. In contrast, BK-stimulated phasic formed EDNO has not only an autocrine effect, but also a paracrine effect on cGMP levels of EC themselves and the adjacent SMC.

Topics: Animals; Antipyrine; Bradykinin; Cattle; Cells, Cultured; Cyclic GMP; Cyclic N-Oxides; Edaravone; Endothelium, Vascular; Free Radical Scavengers; Imidazoles; Muscle, Smooth, Vascular; Nitric Oxide