noc-7 and linsidomine

Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca(2+)](i)). Moreover, these NO-induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca(2+)](i) that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO-induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process.

Topics: Animals; Calcium; Calcium Signaling; Cell Membrane; Cells, Cultured; Cyclic ADP-Ribose; Ganglia, Invertebrate; Gastropoda; Growth Cones; Guanylate Cyclase; Hydrazines; Image Processing, Computer-Assisted; Molsidomine; Neurons; Nitric Oxide; Nitric Oxide Donors; Pseudopodia; Ryanodine; Ryanodine Receptor Calcium Release Channel

The pattern of axonal projections early in the development of the nervous system lacks the precision present in the adult. During a developmental process of refinement, mistargeted projections are eliminated while correct projections are retained. Previous studies suggest that during development nitric oxide (NO) is involved in the elimination of mistargeted retinal axons, whereas brain-derived neurotrophic factor (BDNF) may stabilize retinal axon arbors. It is unclear whether these neuromodulators interact. This study showed that NO induced growth cone collapse and retraction of developing retinal axons. This effect was not attributable to NO-induced neurotoxicity. BDNF protected growth cones and axons from the effects of NO. This effect was specific to BDNF, because neither nerve growth factor (NGF) nor neurotrophin-3 (NT-3) prevented NO-induced growth cone collapse and axon retraction. Exposure to both BDNF and NO, but not either factor alone, stabilized growth cones and axons. Stabilized axons exhibited minimal retraction or extension. This response appears to be a new axon "state" and not simply a partial amelioration of the effect of NO, because lower doses of BDNF or NO allowed axon extension. Furthermore, BDNF/NO-induced growth cone stabilization correlated with the appearance of a cytochalasin D-resistant population of actin filaments. BDNF protection from NO likely was mediated locally at the level of the growth cone, because growth cones or individual filopodia in contact with BDNF-coated beads were protected from NO-induced collapse. These findings suggest a cellular mechanism by which some axonal connections are stabilized and some are eliminated during development.

Topics: Animals; Axons; Brain-Derived Neurotrophic Factor; Cells, Cultured; Chick Embryo; Ganglia, Spinal; Heart; Hydrazines; Molsidomine; Neurons; Neurotrophin 3; Nitric Oxide; Nitric Oxide Donors; Organ Culture Techniques; Retina; Signal Transduction; Visual Pathways

A characteristic physiological property of the neuromuscular junction between giant motor neurones (MoGs) and fast flexor muscles in crayfish is synaptic depression, in which repetitive electrical stimulation of the MoG results in a progressive decrease in excitatory junction potential (EJP) amplitude in flexor muscle fibres. Previous studies have demonstrated that l-arginine (l-Arg) modulates neuromuscular transmission. Since l-Arg is a precursor of nitric oxide (NO), we examined the possibility that NO may be involved in modulating neuromuscular transmission from MoGs to abdominal fast flexor muscles. The effect of a NO-generating compound, NOC7, was similar to that of l-Arg, reversibly decreasing the EJP amplitude mediated by the MoG. While NOC7 reduced the amplitude of the EJP, it induced no significant change in synaptic depression. In contrast, a scavenger of free radical NO, carboxy-PTIO, and an inhibitor of nitric oxide synthase, l-NAME, reversibly increased the EJP amplitude mediated by MoGs. Synaptic depression mediated by repetitive stimulation of MoGs at 1 Hz was partially blocked by bath application of l-NAME. Bath application of a NO scavenger, a NOS inhibitor and NO-generating compounds had no significant effects on the depolarisation of the muscle fibres evoked by local application of l-glutamate. The opposing effects on EJP amplitude of NOC7 and of carboxy-PTIO and l-NAME suggest that endogenous NO presynaptically modulates neuromuscular transmission and that it could play a prominent role at nerve terminals in eliciting MoG-mediated synaptic depression in the crayfish Procambarus clarkii.

Topics: Animals; Arginine; Astacoidea; Benzoates; Enzyme Inhibitors; Female; Glutamic Acid; Hydrazines; Imidazoles; Male; Molsidomine; Motor Neurons; Neuromuscular Junction; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Superoxide Dismutase; Synaptic Transmission

Guanidinosuccinic acid (GSA) is noted for its nitric oxide (NO) mimicking actions such as vasodilatation and activation of the N-methyl-D-aspartate (NMDA) receptor. We have reported that GSA is the product of argininosuccinate (ASA) and some reactive oxygen species, mainly the hydroxyl radical. We tested for GSA synthesis in the presence of NO donors. ASA (1 mM) was incubated with NOR-2, NOC-7 or 3-morpholinosydomine hydrochloride (SIN-1) at 37 degrees C. GSA was determined by HPLC using a cationic resin for separation and phenanthrenequinone as an indicator. Neither NOR-2 or NOC-7 formed GSA. SIN-1, on the other hand, generates NO and the superoxide anion which, in turn, generated peroxynitrite which was then converted to the hydroxyl radical. Incubation of ASA with SIN-1 leads, via this route, to GSA. When ASA was incubated with 1 mM SIN-1, the amount of GSA produced depended on the incubation time and the concentration of ASA. Among the tested SIN-1 concentrations, from 0.5 to 5 mM, GSA synthesis was maximum at 0.5 mM and decreased with increasing concentrations of SIN-1. Carboxy-PTIO, a NO scavenger, completely inhibited GSA synthesis. SOD, a superoxide scavenger, decreased GSA synthesis by 20%, and catalase inhibited GSA synthesis only by 12%; DMSO, a hydroxyl radical scavenger completely inhibited GSA synthesis in the presence of SIN-1. These data suggest that the hydroxyl radical derived from a combination of NO and the superoxide anion generates GSA, a stable NO mimic. Meanwhile, synthesis of GSA by NO produces reactive oxygen and activates the NMDA receptor that generates NO from GSA, suggesting a positive feed back mechanism.

Topics: Argininosuccinic Acid; Benzoates; Chromatography, High Pressure Liquid; Dimethyl Sulfoxide; Feedback; Free Radical Scavengers; Free Radicals; Guanidines; Hydrazines; Hydroxyl Radical; Imidazoles; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; Succinates; Superoxide Dismutase

Nitric oxide and its derivatives have been shown to both activate and inhibit prostaglandin H(2) synthase 1 (PGHS-1). We set out to determine the mechanisms by which different nitrogen oxide derivatives modulate PGHS-1 activity. To this end, we show that 3-morpholinosydnonimine hydrochloride (SIN-1), a compound capable of generating peroxynitrite, activates purified PGHS-1 and also stimulates PGE(2) production in arterial smooth muscle cells in the presence of exogenous arachidonic acid. The effect of SIN-1 in smooth muscle cells was abrogated by superoxide and peroxynitrite inhibitors, which supports the hypothesis that peroxynitrite is an activating species of PGHS-1. Indeed, authentic peroxynitrite also induced PGE(2) production in arachidonic acid-stimulated cells. In contrast, when cells were exposed to the nitric oxide-releasing compound 1-hydroxy-2-oxo-3-[(methylamino)propyl]-3-methyl-1-triazene (NOC-7), PGHS-1 enzyme activity was inhibited in the presence of exogenous arachidonic acid. Finally, in lipid-loaded smooth muscle cells, we demonstrate that SIN-1 stimulates arachidonic acid-induced PGE(2) production; albeit, the extent of activation is reduced compared to that under normal conditions. These results indicate that formation of peroxynitrite is a key intermediary step in PGHS-1 activation. However, other forms of NO(x)() inhibit PGHS-1. These results may have implications in the regulation of vascular function and tone in normal and atherosclerotic arteries.

Topics: Animals; Aorta, Thoracic; Arteriosclerosis; Cells, Cultured; Cyclooxygenase 1; Enzyme Activation; Enzyme Inhibitors; Free Radical Scavengers; Hydrazines; Isoenzymes; Male; Membrane Proteins; Molsidomine; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Penicillamine; Peroxides; Prostaglandin Antagonists; Prostaglandin-Endoperoxide Synthases; Rats; S-Nitroso-N-Acetylpenicillamine; Sheep; Superoxides