cyclic-gmp and 4-5-diaminofluorescein-diacetate

The source size and density determine the extent of nitric oxide (NO) diffusion which critically influences NO signaling. In the brain, NO released from postsynaptic somas following NMDA-mediated activation of neuronal nitric oxide synthase (nNOS) retrogradely affects smaller presynaptic targets. By contrast, in guinea pig trigeminal motor nucleus (TMN), NO is produced presynaptically by tiny and disperse nNOS-containing terminals that innervate large nNOS-negative motoneurons expressing the soluble guanylyl-cyclase (sGC); consequently, it is uncertain whether endogenous NO supports an anterograde signaling between pre-motor terminals and postsynaptic trigeminal motoneurons. In retrogradely labeled motoneurons, we indirectly monitored NO using triazolofluorescein (DAF-2T) fluorescence, and evaluated sGC activity by confocal cGMP immunofluorescence. Multiple fibers stimulation enhanced NO content and cGMP immunofluorescence into numerous nNOS-negative motoneurons; NOS inhibitors prevented depolarization-induced effects, whereas NO donors mimicked them. Enhance of cGMP immunofluorescence required extracellular Ca(2+), a nNOS-physiological activator, and was prevented by inhibiting sGC, silencing neuronal activity or impeding NO diffusion. In conclusion, NO released presynaptically from multiple cooperative tiny fibers attains concentrations sufficient to activate sGC in many motoneurons despite of the low source/target size ratio and source dispersion; thus, endogenous NO is an effective anterograde neuromodulator. By adjusting nNOS activation, presynaptic Ca(2+) might modulate the NO diffusion field in the TMN.

Topics: Animals; Brain Stem; Calcium Signaling; Central Nervous System; Cyclic GMP; Electrophysiological Phenomena; Enzyme Activation; Fluorescein; Fluorescent Dyes; Guanylate Cyclase; Guinea Pigs; Image Processing, Computer-Assisted; Immunohistochemistry; In Vitro Techniques; Microscopy, Confocal; Motor Neurons; Nerve Fibers; Nitric Oxide; Nitric Oxide Synthase Type I; Receptors, Presynaptic; Recruitment, Neurophysiological; Signal Transduction; Synaptic Transmission; Trigeminal Nerve

In the present study, we quantified the physiological consequences of nitric oxide (NO) on ammonium release in tadpoles of Xenopus laevis. Tadpoles exposed to S-nitro-N-acetylpenicillamine (SNAP), an NO-donor, or L: -arginine, the substrate of NO synthase (NOS), showed a reversible decrease, whereas animals exposed to the NOS inhibitor Nomega-methyl-L: -arginine (L: -NMMA) exhibited an increase in ammonium release. Release of ammonium may be of physiological relevance during stress response of the animal. Handling of tadpoles as well as exposure to hyposmotic environments increased ammonium release. To localize NO synthesizing cells, we used diaminofluorescein-diacetate (DAF-2DA), an NO-sensitive fluorescent dye, and NADPH-diaphorase histochemistry, an indicator for NOS activity. We observed a fluorescence signal as well as NADPH-diaphorase activity in small, solitary cells in the epidermis. Similarly to NADPH-diaphorase histochemistry, silver nitrate staining and rhodamine labelling, markers for mitochondria-rich cells, showed a strong reaction in these cells. These observations indicate that NO (1) inhibits ammonium release, and (2) is endogenously synthesized in mitochondria-rich cells in Xenopus tadpoles. Based on our histochemical results, we speculate that gill epithelium and epidermis work in parallel to release ammonium as epidermal tissue contains mitochondria-rich and NADPH-diaphorase positive cells.

Topics: Animals; Arginine; Cyclic GMP; Enzyme Inhibitors; Epidermis; Fluorescein; Gills; Handling, Psychological; Immunohistochemistry; Indicators and Reagents; Larva; Mitochondria; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; omega-N-Methylarginine; Quaternary Ammonium Compounds; S-Nitroso-N-Acetylpenicillamine; Stress, Physiological; Xenopus laevis

Nitric oxide (NO) controls diverse functions in many cells and organs of animals. It is also produced in plants and has a variety of effects, but little is known about their underlying mechanisms. In the present study, we have discovered a role for NO in the regulation of pollen tube growth, a fast tip-growing cellular system. Pollen tubes must be precisely oriented inside the anatomically complex female ovary in order to deliver sperm. We hypothesized that NO could play a role in this guidance and tested this hypothesis by challenging the growth of pollen tubes with an external NO point source. When a critical concentration was sensed, the growth rate was reduced and the growth axis underwent a subsequent sharp reorientation, after which normal growth was attained. This response was abrogated in the presence of the NO scavenger CPTIO and affected by drugs interfering in the cGMP signaling pathway. The sensitivity threshold of the response was significantly augmented by sildenafil citrate (SC), an inhibitor of cGMP-specific phosphodiesterases in animals. NO distribution inside pollen tubes was investigated using DAF2-DA and was shown to occur mostly in peroxisomes. Peroxisomes are normally excluded from the tip of pollen tubes and little if any NO is found in the cytosol of that region. Our data indicate that the rate and orientation of pollen tube growth is regulated by NO levels at the pollen tube tip and suggest that this NO function is mediated by cGMP.

Topics: 1-Methyl-3-isobutylxanthine; Benzoates; Cyclic GMP; Dose-Response Relationship, Drug; Flowers; Fluorescein; Imidazoles; Lilium; Nitric Oxide; Peroxisomes; Phosphodiesterase Inhibitors; Piperazines; Purines; Sildenafil Citrate; Sulfones