Neuron–Glia Communication via Nitric Oxide Is Essential in Establishing Antennal-Lobe Structure in Manduca sexta

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.     

Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta

The intracellular messenger cGMP (cyclic guanosine monophosphate) has been suggested to play a role in olfactory transduction in both invertebrates and vertebrates, but its cellular location within the olfactory system has remained elusive. We used cGMP immunocytochemistry to determine which antennal cells of the hawkmoth Manduca sexta are cGMP immunoreactive in the absence of pheromone. We then tested which antennal cells increase cGMP levels in response to nitric oxide (NO) and to long pheromonal stimuli, which the male encounters close to a calling female moth. In addition, we used in situ hybridization to determine which antennal cells express NO-sensitive soluble guanylyl cyclase. In response to long pheromonal stimuli with NO donors present, cGMP concentrations change in at least a subpopulation of pheromone-sensitive olfactory receptor neurons. These changes in cGMP concentrations in pheromone-dependent olfactory receptor neurons cannot be mimicked by the addition of NO donors in the absence of pheromone. NO stimulates sensilla chaetica type I and II, but not pheromone-sensitive trichoid sensilla, to high levels of cGMP accumulation as detected by immunocytochemistry. In situ hybridizations show that sensilla chaetica, but not sensilla trichodea, express detectable levels of mRNA coding for soluble guanylyl cyclase. These results suggest that intracellular rises in cGMP concentrations play a role in information processing in a subpopulation of pheromone-sensitive sensilla in Manduca sexta antennae, mediated by an NO-sensitive mechanism, but not an NO-dependent soluble guanylyl cyclase.     

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

A cell-based nitric oxide reporter assay useful for the identification and characterization of modulators of the nitric oxide/guanosine 3',5'-cyclic monophosphate pathway

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.     

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.     

Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.     

The nNos/cGMP mediation of the depressor response to NMDA receptor stimulation in the caudal ventrolateral medulla

Using in vivo voltammetry to directly measure extracellular nitric oxide (NO) levels, our previous studies suggested that the neuronal NO synthase (nNOS) and cyclic guanosine monophosphate (cGMP) signal transducing systems are involved in the cardiovascular responses elicited by activation of N-methyl-D-aspartate (NMDA) receptors in the rostral ventrolateral medulla. In this study, we examined if the depressor responses elicited by activation of NMDA receptors in the caudal ventrolateral medulla (CVLM) also depend on the actions of nNOS and soluble guanylyl cyclase. In anesthetized cats, microinjection of NMDA into the CVLM produced hypotension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 2-amino-5-phosphonopentanoate (a NMDA receptor competitive antagonist), 7-nitroindazole (a nNOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). These findings suggest that NO is also involved in the NMDA-induced depressor responses of the CVLM.     

Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.     

Intrinsic nitric oxide regulates the taste response of the sugar receptor cell in the blowfly, Phormia regina

The taste organ in insects is a hair-shaped taste sensory unit having four functionally differentiated contact chemoreceptor cells. In the blowfly, Phormia regina, cGMP has been suggested to be a second messenger for the sugar receptor cell. Generally, cGMP is produced by membranous or soluble guanylyl cyclase (sGC), which can be activated by nitric oxide (NO). In the present paper, we electrophysiologically showed that an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO), an NO donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7) or an NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) specifically affected the response in the sugar receptor cell, but not in other receptor cells. PTIO, when introduced into the receptor cells in a sensillum aided by sodium deoxycholate (DOC, pH 7.2), depressed the response of sugar receptor cells to sucrose but did not affect those of the salt or water receptor cells. NOC 7, given extracellularly, latently induced the response of sugar receptor cells; and L-NAME, when introduced into the receptor cells, depressed the response of sugar receptor cells. The results clearly suggest that NO, which may be produced by intrinsic NOS in sugar receptor cells, participates in the transduction cascade of these cells in blowfly.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.     

Nitric oxide: an unconventional messenger in the nervous system of an orthopteroid insect

Nitric oxide (NO) is a membrane-permeant messenger molecule generated from the amino acid L-arginine. NO can activate soluble guanylyl cyclase leading to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This paper examines biochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of the locust, a commonly used neurobiological preparation. Biochemical investigations suggest that an identical enzyme is responsible for both NO synthase (NOS) and NADPH-diaphorase activity after tissue fixation. Immunocytochemical staining of an olfactory center in the locust brain shows that NOS-immunoreactivity colocalizes with NADPH-diaphorase at the cellular level. The cytochemical staining of NO donor and target cells in adult animals suggests functions in olfaction, vision, and sensorimotor integration. During development, NO is implicated in axonal outgrowth and synaptogenesis. The cellular distribution of NO-responsive cells in neural circuits reflects potential functions of NO as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger independent of conventional synaptic connectivity.     

The nitric oxide/cyclic GMP pathway in the olfactory processing system of the terrestrial slug Limax marginatus

To examine the distribution of nitric oxide (NO)-generative cells and NO-responsive cells in the tentacles and procerebral lobes (olfactory processing center) of terrestrial slugs, we applied NADPH diaphorase (NADPH-d) histochemistry and NO-induced cyclic GMP (cGMP)-like immunohistochemistry. We found that NADPH-d reactive cells/fibers and cGMP-like immunoreactive cells/fibers were different, but they were localized adjacent to each other, in both the tentacles and the procerebral lobes. Then, we measured the concentration of NO that was generated around the procerebral lobes using an NO sensitive electrode, when the olfactory nerve was electrically stimulated as a replacement for an odorant stimulus. Stimulation of the olfactory nerve evoked an increase in NO concentration at nanomolar levels, suggesting that binding of nanomolar concentrations of NO to the prosthetic heme group activates soluble guanylyl cyclase. Taken together with previously reported physiological data, our results, therefore, showed that the NO/cGMP pathways are involved in slug olfactory processing.     

The interaction between thymine DNA glycosylase and nuclear receptor coactivator 3 is required for the transcriptional activation of nuclear hormone receptors

The cellular counterpart of the “soluble” guanylyl cyclase found in tissue homogenates over 30 years ago is now recognized as the physiological receptor for nitric oxide (NO). The ligand-binding site is a prosthetic haem group that, when occupied by NO, induces a conformational change in the protein that propagates to the catalytic site, triggering conversion of GTP into cGMP. This review focuses on recent research that takes this basic information forward to the beginnings of a quantitative depiction of NO signal transduction, analogous to that achieved for other major transmitters. At its foundation is an explicit enzyme-linked receptor mechanism for NO-activated guanylyl cyclase that replicates all its main properties. In cells, NO signal transduction is subject to additional, activity-dependent modifications, notably through receptor desensitization and changes in the activity of cGMP-hydrolyzing phosphodiesterases. The measurement of these parameters under varying conditions in rat platelets has made it possible to formulate a cellular model of NO-cGMP signaling. The model helps explain cellular responses to NO and their modification by therapeutic agents acting on the guanylyl cyclase or phosphodiesterase limbs of the pathway.     

The contribution of invertebrate study to the biology of nitric oxide

After the identification of nitric oxide (NO) with the endothelium derived-relaxing factor, many signaling mechanisms involving NO were identified through experiments on Mammals. NO activates soluble guanylyl cyclase leading to the formation of cGMP, stimulates the ADP-ribosylation of GAPDH, altering the cell energy production and combines with superoxide, generating cytotoxic peroxynitrite. NO was then progressively established as a major messenger molecule in Mammals. It is implied in the regulation of blood vessel dilatation, immune function, development and neurotransmission in brain and peripheral nervous system. Later, parallel findings were observed in Invertebrates and then, NO appeared as a signaling molecule widely spread throughout the animal kingdom and whose functions were highly conserved during evolution. The purpose of this short review is to highlight the contribution of Invertebrate studies to the knowledge of NO biology.     

Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells

It is generally accepted that G protein-coupled receptors stimulate soluble guanylyl cyclase (sGC)-mediated cGMP production indirectly, by increasing nitric oxide (NO) synthase activity in a calcium- and kinase-dependent manner. Here we show that normal and GH(3) immortalized pituitary cells expressed alpha(1)beta(1)-sGC heterodimer. Activation of adenylyl cyclase by GHRH, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and forskolin increased NO and cGMP levels, and basal and stimulated cGMP production was abolished by inhibition of NO synthase activity. However, activators of adenylyl cyclase were found to enhance this NO-dependent cGMP production even when NO was held constant at basal levels. Receptor-activated cGMP production was mimicked by expression of a constitutive active protein kinase A and was accompanied with phosphorylation of native and recombinant alpha(1)-sGC subunit. Addition of a protein kinase A inhibitor, overexpression of a dominant negative mutant of regulatory protein kinase A subunit, and substitution of Ser(107)-Ser(108) N-terminal residues of alpha(1)-subunit with alanine abolished adenylyl cyclase-dependent cGMP production without affecting basal and NO donor-stimulated cGMP production. These results indicate that phosphorylation of alpha(1)-subunit by protein kinase A enlarges the NO-dependent sGC activity, most likely by stabilizing the NO/alpha(1)beta(1) complex. This is the major pathway by which adenylyl cyclase-coupled receptors stimulate cGMP production.     

Elastokine-mediated up-regulation of MT1-MMP is triggered by nitric oxide in endothelial cells

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)₃ peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI₃-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI₃-kinase p110γ sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI₃-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.     

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum: an immunocytochemical and pharmacological study

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1muM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.