Discovery of Some of the Biological Effects of Nitric Oxide and its Role in Cell Signaling

The role of nitric oxide in cellular signaling in the past 22 years has become one of the most rapidly growing areas in biology with more than 20,000 publications to date. Nitric oxide is a gas and free radical with an unshared electron that can regulate an ever-growing list of biological processes. In many instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis from GTP. However, the list of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate its effect, while other reactions can lead to altered protein structure, function, and/or catalytic capacity. These diverse effects of nitric oxide that are either cyclic GMP dependent or independent can alter and regulate important physiological and biochemical events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an autacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique simple molecule with an array of signaling functions. However, as with any messenger molecule, there can be too little or too much of the substance and pathological events result. Some of the methods to regulate either nitric oxide formation, metabolism, or function have been in clinical use for more than a century as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870's. Current and future research with nitric oxide and cyclic GMP will undoubtedly expand the clinicians' therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide and cyclic GMP formation and metabolism. Such promise and expectations have obviously fueled the interests in these signaling molecules for a growing list of potential therapeutic applications.John S. Dunn Distinguished Chair in Medicine and Physiology, Regental Professor and Chair of Department of Integrative Biology, Pharmacology, and Physiology and Director of the Institute of Molecular Medicine     

Identification of Nitric Oxide-Sensitive and -Insensitive Forms of Cytoplasmic Guanylate Cyclase

Abstract: Cytoplasmic, nitric oxide-activated guanylate cyclases are expressed in many regions of the mammalian brain and are thought to participate in functions as diverse as synaptogenesis and long-term potentiation. In this study, we have characterized cytoplasmic guanylate cyclases in the nervous system of an invertebrate, the American lobster. Cytoplasmic cyclase specific activity is higher in lobster nerve cord than in any other lobster tissue tested, and considerably higher than in typical rat tissues (cerebellum, lung, and liver). However, nitric oxide donors have minimal effects on lobster nerve cord cyclic GMP production, when applied either to intact tissue or to cytoplasmic extracts. Parallel immunocytochemical studies, using an anti-cyclic GMP antibody, reveal that only a small subset of lobster neurons responds to nitric oxide with a significant elevation of cyclic GMP levels. HPLC analysis of nerve cord cytoplasm reveals two chromatographically separable cyclases, a minor nitric oxide-sensitive form whose retention time is identical to that of the conventional mammalian enzyme and a more abundant nitric oxide-insensitive form that appears to be novel. The physiological function and phylogenetic distribution of this nitric oxide-insensitive enzyme, and the signaling mechanisms that regulate its activity, are not known.     

Guanylyl cyclase structure, function and regulation

Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.     

Nitric oxide and sphingolipids: mechanisms of interaction and role in cellular pathophysiology

Nitric oxide is a short-lived messenger with pleiotropic roles in the regulation of cell patho-physiological processes, including survival, death, proliferation and differentiation. Increasing evidence over the last few years has shown that nitric oxide effects in apoptosis, growth and differentiation originate in significant part from its interplay with signalling members of the sphingolipid family. In many cell types belonging to different lineages, nitric oxide and sphingolipids interact in two-way pathways leading to regulation of the activity and expression of enzymes involved in each other's signalling events. These crosstalk signalling events involve various sphingolipids, with key roles for ceramide and sphingosine-1-phosphate, and signal transduction molecules downstream of nitric oxide, with cyclic GMP as a main player. The biological implications of some of these interactions are now being understood. The best-characterised so far, the mutual regulation of sphingomyelinases and endothelial nitric oxide synthase, acts as a tuning system in crucial patho-physiological processes such as inflammation, proliferation and cell death.     

Neurotransmitter release regulated by nitric oxide in PC-12 cells and brain synaptosomes

BACKGROUND: Nitric oxide is a messenger molecule of the nervous system, which is produced by the enzyme nitric oxide synthase, which may regulate cyclic guanosine monophosphate levels and which has been implicated in the control of neurotransmitter release. PC-12 pheochromocytoma cells differentiate to form neuronal cells in culture when they are exposed to nerve growth factor. The levels of cyclic guanosine monophosphate in the cells and their ability to release acetylcholine in response to K(+)-depolarization are both maximal after eight days of treatment with nerve growth factor. We set out to assess a possible role for nitric oxide in the processes that occur in differentiating PC-12 cells. RESULTS: Nitric oxide synthase is first evident in differentiating PC-12 cells eight days after beginning treatment with nerve growth factor, coinciding with the marked increase in K(+)-depolarization-induced release of acetylcholine. The release of both acetylcholine and dopamine in response to K(+)-depolarization is blocked by inhibitors of nitric oxide synthase and by hemoglobin, which binds nitric oxide. Providing l-arginine, a precursor required for nitric oxide synthesis, reverses the effects of the inhibitors. In synaptosomal preparations from the corpus striatum, inhibitors of nitric oxide synthase prevent the release of glutamate in response to the glutamate derivative N-methyl-d-aspartate but not in response to K(+)-depolarization. CONCLUSION: Nitric oxide may mediate the release of acetylcholine and dopamine in response to K(+)-depolarization in PC-12 cells and the release of glutamate in response to N-methyl-d-aspartate in striatal synaptosomes. Nitric oxide synthase expression is induced after eight days of treating PC-12 cells with nerve growth factor, coinciding with a marked enhancement of the release of neurotransmitters in response to K(+)-depolarization.     

Nitric oxide as a regulator of neuronal motility and regeneration in the locust embryo

Nitric oxide (NO) is known as a gaseous messenger in the nervous system. It plays a role in synaptic plasticity, but also in development and regeneration of nervous systems. We have studied the function of NO and its signaling cascade via cyclic GMP in the locust embryo. Its developing nervous system is well suited for pharmacological manipulations in tissue culture. The components of this signaling pathway are localized by histochemical and immunofluorescence techniques. We have analyzed cellular mechanisms of NO action in three examples: 1. in the peripheral nervous system during antennal pioneer axon outgrowth, 2. in the enteric nervous system during migration of neurons forming the midgut nerve plexus, and 3. in the central nervous system during axonal regeneration of serotonergic neurons after axotomy. In each case, internally released NO or NO-induced cGMP synthesis act as permissive signals for the developmental process. Carbon monoxide (CO), as a second gaseous messenger, modulates enteric neuron migration antagonistic to NO.     

The distribution of cGMP in the adrenal gland in the rat, guinea pig and mouse after stimulation with sodium nitroprusside

Nitric oxide (NO) acts as an intercellular messenger molecule in the nervous system. In the adrenal gland sympathetic preganglionic fibers innervating the medulla, as well as intrinsic neural ganglion cells, contain nitric oxide synthase (NOS). Nitric oxide stimulates the soluble enzyme guanylate cyclase forming cyclic GMP (cGMP). Using sodium nitroprusside (SNP) as nitric oxide donor we have studied the putative target cells for nitric oxide in the rat adrenal gland, both in vivo and in vitro. The guinea pig and a few mouse adrenal glands were studied after SNP perfusion for comparison. Our results show that after vascular perfusion with a high concentration (3 mM) of SNP both noradrenaline and adrenaline chromaffin cells express cGMP-like immunoreactivity in all three species. After incubation of rat adrenal slices with SNP primarily the noradrenaline chromaffin cells are cGMP-positive. In contrast, detectable levels of cGMP-like immunoreactivity were not found in neuronal ganglion cells. In the adrenal cortex cGMP-like immunoreactivity was seen in blood vessel walls, in small cells with processes forming a reticular network, at least partly presumably representing endothelial cells, as well as in some presumable nerve terminals. These findings support the view that chromaffin cells, especially the noradrenergic ones and blood vessels, are targets for nitric oxide in the adrenal gland.     

Norepinephrine Increases Cyclic GMP Levels in Cerebellar Cells from Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Cyclic GMP is an important intracellular messenger in the nervous system that may mediate cellular forms of neuronal plasticity. Previous studies show that most neurotransmitters stimulate cyclic GMP levels by the activation of nitric oxide synthase (NOS). In this study, we report that in primary cell cultures from the cerebellum of neuronal NOS knockout mice, norepinephrine stimulates an increase in cyclic GMP content. This increase is seen in both granule cell and astrocyte cultures and is not blocked by inhibitors of NOS or by inhibition of soluble guanylyl cyclase. These results suggest a novel pathway by which norepinephrine enhances cyclic GMP levels in the nervous system.     

植物一氧化氮生物学的研究进展

一氧化氮(NO)是植物中的一种关键的信号分子.在植物中,NO的潜在来源包括一氧化氮合成酶、硝酸还原酶、黄嘌呤氧化还原酶和非酶促途径.NO能促进植物生长,延缓叶片、花和果实衰老,促进休眠和需光种子的萌发,能与植物激素相互作用调节气孔运动,诱导程序性细胞死亡和防御相关基因的表达,并在逆境中作为一种抗氧化剂起作用.NO的细胞内信号反应包括环鸟苷酸、环腺苷二磷酸核糖的产生和细胞质Ca2 浓度的增加,其信号转导途径及其生物化学和细胞学本质还不十分清楚.     

植物一氧化氮生物学的研究进展

一氧化氮 (NO) 是植物中的一种关键的信号分子。在植物中, NO的潜在来源包括一氧化氮合成酶、硝酸还原酶、黄嘌呤氧化还原酶和非酶促途径。NO能促进植物生长, 延缓叶片、花和果实衰老, 促进休眠和需光种子的萌发, 能与植物激素相互作用调节气孔运动, 诱导程序性细胞死亡和防御相关基因的表达, 并在逆境中作为一种抗氧化剂起作用。 NO的细胞内信号反应包括环鸟苷酸、环腺苷二磷酸核糖的产生和细胞质Ca2+浓度的增加, 其信号转导途径及其生物化学和细胞学本质还不十分清楚。     

The role of nitric oxide signaling in the formation of the neuromuscular junction

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.     

Nitric oxide, mitochondrial hyperpolarization, and T cell activation

T lymphocyte activation is associated with nitric oxide (NO) production, which plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both activation and apoptosis of Tlymphocytes, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus induces mitochondrial biogenesis and alters Ca(2+) signaling. Thus, whereas NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Eclosion Hormone Stimulates Cyclic GMP Levels in Manduca sexta Nervous Tissue via Arachidonic Acid Metabolism with Little or No Contribution from the Production of Nitric Oxide

The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone-stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, had no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone-stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.     

Sildenafil Citrate-Restored eNOS and PDE5 Regulation in Sickle Cell Mouse Penis Prevents Priapism Via Control of Oxidative/Nitrosative Stress

Sildenafil citrate revolutionized the practice of sexual medicine upon its federal regulatory agency approval approximately 15 years ago as the prototypical phosphodiesterase type 5 inhibitor indicated for the treatment of male erectile dysfunction. We now provide scientific support for its alternative use in the management of priapism, a clinical disorder of prolonged and uncontrolled penile erection. Sildenafil administered continuously to sickle cell mice, which show a priapism phenotype, reverses oxidative/nitrosative stress effects in the penis, mainly via reversion of uncoupled endothelial nitric oxide synthase to the functional coupled state of the enzyme, which in turn corrects aberrant signaling and function of the nitric oxide/cyclic GMP/protein kinase G/phosphodiesterase type 5 cascade. Priapism tendencies in these mice are reverted partially toward normal neurostimulated erection frequencies and durations after sildenafil treatment in association with normalized cyclic GMP concentration, protein kinase G activity and phosphodiesterase type 5 activity in the penis. Thus, sildenafil exerts pleiotropic effects in the penis that extend to diverse erection disorders.     

Nitric oxide inhibits neutrophil β2 integrin function by inhibiting membrane-associated cyclic GMP synthesis

The aim of this investigation was to identify the mechanism by which nitric oxide inhibits neutrophil β₂ integrin dependent adherence. Isolated rat neutrophils from blood and peritoneal exudates were exposed for 2 min to nitric oxide generated by diethylamine-NO at rates between 1.6 and 138 nmol/min. Exposure to nitric oxide at rates less than 14 nmol/min had no effect on adherence. Exposure to 14 to 56 nmol nitric oxide/min inhibited β₂ integrin dependent adherence to endothelial cells, nylon columns, and fibrinogen-coated plates, but higher concentrations had no significant effect on adherence. Adherence by β₂ integrins could be restored by incubating cells with dithioerythritol, phorbol 12-myristate 13-acetate, or 8-bromo cyclic GMP. Elevations in cellular cyclic GMP concentration were associated with adherence, but this did not occur after cells were exposed to concentrations of nitric oxide that inhibited β₂ integrin-dependent adherence. Elevations in cyclic GMP did occur after cells were incubated with dithioerythritol or phorbol 12-myristate 13-acetate. Concentrations of nitric oxide that inhibited β₂ integrin-dependent adherence also inhibited catalytic activity of membrane associated guanylate cyclase and binding of atrial natriuretic peptide, but were insufficient to activate cytosolic guanylate cyclase. Nitric oxide did not inhibit neutrophil oxidative burst or degranulation, nor effect β₂ integrin expression or adherence that did not depend on β₂ integrins, nor cause oxidative stress identified in terms of cellular glutathione concentration or protein nitrotyrosine. The results indicate that nitric oxide inhibited β₂ integrins in a concentration-dependent fashion by inhibiting cell-surface transduction of signals linked to the activity of membrane-bound guanylate cyclase. The inhibitory effect could be overcome by providing cells with cyclic GMP exogenously or by stimulating cytosolic guanylate cyclase. J. Cell. Physiol. 172:12–24, 1997. © 1997 Wiley-Liss, Inc.     

Geometric considerations of nitric oxide-cyclic GMP signalling in the glomerular neuropil of the locust antennal lobe

Using NADPH-diaphorase staining as a marker for nitric oxide (NO) synthase and an antiserum against cyclic GMP, we recently reported the anatomical distribution of nitric oxide donor and target cells in the antennal lobe, the principal olfactory neuropile of the locust. The most striking NADPH-diaphorase activity in the olfactory pathway is concentrated in a cluster of intensely stained local interneurons innervating the glomeruli. After incubation of tissue in a nitric oxide donor and inhibition of phospodiesterase activity, neurons of this cluster expressed cyclic GMP-immunoreactivity in the cell body and neurites. Here we examine the importance of the arrangement of NO donor and target cells for information processing in the glomeruli. The cellular organization of the NO-cyclic GMP system in olfactory interneurons, and the dendritic branching pattern, suggest that nitric oxide may not only act as intercellular, but also as intracellular messenger molecule in the glomerular neuropile of the antennal lobe. <br>     

cGMP对细胞功能的调控

cGMP作为胞内第二信使,主要通过cGMP依赖的蛋白激酶(cGPK),cGMP门控的离子通道,cGMP调节的环核苷酸磷酸二酯酶以及ADP核糖环化酶等后续途径,参与许多细胞功能的调控.cGMP既可以通过蛋白磷酸化,也通过与蛋白磷酸化不直接相关的信号传递途径来调控各类细胞的特定生理功能.     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Inhibition of Nitric Oxide Synthase Blocks N-Methyl-D-Aspartate-, Quisqualate-, Kainate-, Harmaline-, and Pentylenetetrazole-Dependent Increases in Cerebellar Cyclic GMP In Vivo

The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible "intercellular messenger molecule" acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N-mono-methyl-L-arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D-serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important "messenger molecule" in the cerebellum, mediating the actions of kainate, quisqualate, and N-methyl-D-aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N-methyl-D-aspartate.