A Rac-cGMP signaling pathway

The small GTPase Rac and the second messenger cGMP (guanosine 3',5'-cyclic monophosphate) are critical regulators of diverse cell functions. When activated by extracellular signals via membrane signaling receptors, Rac executes its functions through engaging downstream effectors such as p21-activated kinase (PAK), a serine/threonine protein kinase. However, the molecular mechanism by which membrane signaling receptors regulate cGMP levels is not known. Here we have uncovered a signaling pathway linking Rac to the increase of cellular cGMP. We show that Rac uses PAK to directly activate transmembrane guanylyl cyclases (GCs), leading to increased cellular cGMP levels. This Rac/PAK/GC/cGMP pathway is involved in platelet-derived growth factor-induced fibroblast cell migration and lamellipodium formation. Our findings connect two important regulators of cellular physiological functions and provide a general mechanism for diverse receptors to modulate physiological responses through elevating cellular cGMP levels.     

Developmental expression of nitric oxide/cyclic GMP synthesizing cells in the nervous system of Drosophila melanogaster

Nitric oxide (NO) is a membrane-permeant signaling molecule which activates soluble guanylyl cyclase and leads to the formation of cyclic GMP (cGMP). The NO/cGMP signaling system is thought to play essential roles during the development of vertebrate and invertebrate animals. Here, we analyzed the cellular expression of this signaling pathway during the development of the Drosophila melanogaster nervous system. Using NADPH diaphorase histochemistry as a marker for NO synthase, we identified several neuronal and glial cell types as potential NO donor cells. To label NO-responsive target cells, we used the detection of cGMP by an immunocytochemical technique. Incubation of tissue in an NO donor induced cGMP immunoreactivity (cGMP-IR) in individual motoneurons, sensory neurons, and groups of interneurons of the brain and ventral nerve cord. A dynamic pattern of the cellular expression of NADPHd staining and cGMP-IR was observed during embryonic, larval, and prepupal phases. The expression of NADPH diaphorase and cGMP-IR in distinct neuronal populations of the larval central nervous system (CNS) indicates a role of NO in transcellular signaling within the CNS and as potential retrograde messenger across the neuromuscular junction. In addition, the presence of NADPH diaphorase-positive imaginal discs containing NO-responsive sensory neurons suggests that a transcellular NO/cGMP messenger system can operate between cells of epithelial and neuronal phenotype. The discrete cellular resolution of donor and NO-responsive target cells in identifiable cell types will facilitate the genetic, pharmacological, and physiological analysis of NO/cGMP signal transduction in the developing nervous system of Drosophila.     

Immortalization of human melanocytes does not alter the de novo properties of nitric oxide to induce cell detachment from extracellular matrix components via cGMP

Nitric oxide (NO) is an important mediator in many (patho)physiological processes including inflammation and skin cancer. A key transducer in NO signaling is the soluble guanylyl cyclase (sGC) that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP). The basic mechanism of NO-cGMP signaling in melanocytic cells is, however, not well elucidated. A setback for such studies is the limited availability of patient-derived melanocytes. Here, we report that immortalized human normal and vitiliginous cell lines generated via cell transfection with human papilloma virus 16 genes E6 and E7 express NO synthase and guanylyl cyclase isoforms and the multidrug resistance-associated proteins 4 and 5 as selective cGMP exporters. Donors of NO (e.g., the NONOate (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NO) and reactive nitrogen oxygen species (RNOS) like 3-morpholino-sydnonimine (SIN-1) as a donor of peroxynitrite as well as YC-1 as a NO-independent sGC stimulator increased intracellular cGMP levels in immortalized melanocytes (up to eightfold over controls), indicating the expression of functional sGC in these cells. PAPA-NO and SIN-1 also reduced the attachment of immortalized melanocytes to extracellular matrix (ECM) components like fibronectin which was dependent on cellular melanin content and cGMP. Such effects on melanoma cells were positively related to metastatic potential and were cGMP independent. Intriguingly, nonpigmented metastatic melanoma cells were more sensitive to exogenous sources of RNOS than of NO. Thus, immortalized melanocytes can be used as a tool for further research on differences in cell signaling between the different melanocytic lineages in particular towards impairment of cell-ECM adhesion by NO or RNOS, which may be important in metastasis and vitiligo pathogenesis.     

Differential expression of genes involved in cGMP-dependent nitric oxide signaling in murine embryonic stem (ES) cells and ES cell-derived cardiomyocytes.

Nitric oxide (NO) performs multiple physiological roles as a biological signaling molecule. The role of NO and cGMP signaling in embryonic stem (ES) cell-derived cardiomyocytes (CM) has been investigated but many questions remain. In this study, we examined the expression of the NO signaling pathway components nitric oxide synthase (NOS-1, 2, 3), soluble guanylyl cyclase (sGCalpha(1) and beta(1)) and protein kinase G (PKG) genes and sGC activity in murine ES cells subjected to differentiation by embryoid body (EB) formation. We found that in undifferentiated ES cells, NOS-1, NOS-3, and sGCbeta(1) were detected while NOS-2, sGCalpha(1), and PKG were very low or undetectable. When ES cells were subjected to differentiation, NOS-1 abruptly decreased within one day, NOS-2 mRNA became detectable after several days, and NOS-3 increased after 7-10 days. Levels of sGCalpha(1), sGCbeta(1), and PKG all increased gradually over a several day time course of differentiation in EB outgrowths. Analysis of sGC activity in cell lysates derived from undifferentiated ES cells revealed that NO could not stimulate cGMP. However, lysates from differentiated EB outgrowths produced abundant cGMP levels after NO stimulation. Purification of ES-cell derived CM revealed that mRNA expression of all the NOS isoforms was very low to absent while sGCalpha(1) and beta(1) subunit mRNAs were abundant and sGC-mediated cGMP production was apparent in this population of cells. These data suggest that cGMP-mediated NO signaling may play a minor role, if any, in undifferentiated ES cells but could be involved in the early differentiation events or physiological processes of ES cells or ES cell-derived lineages.     

Nitric oxide-cyclic GMP signaling in stem cell differentiation

The nitric oxide–cyclic GMP (NO–cGMP) pathway mediates important physiological functions associated with various integrative body systems including the cardiovascular and nervous systems. Furthermore, NO regulates cell growth, survival, apoptosis, proliferation, and differentiation at the cellular level. To understand the significance of the NO–cGMP pathway in development and differentiation, studies have been conducted both in developing embryos and in stem cells. Manipulation of the NO–cGMP pathway, by employing activators and inhibitors as pharmacological probes, and genetic manipulation of NO signaling components have implicated the involvement of this pathway in the regulation of stem cell differentiation. This review focuses on some of the work pertaining to the role of NO–cGMP in the differentiation of stem cells into cells of various lineages, particularly into myocardial cells, and in stem cell-based therapy.     

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: promoter or suppressor of programmed cell death?

Nitric oxide (NO) is a short-lived gaseous free radical that predominantly functions as a messenger and effector molecule. It affects a variety of physiological processes, including programmed cell death (PCD) through cyclic guanosine monophosphate (cGMP)-dependent and-independent pathways. In this field, dominant discoveries are the diverse apoptosis networks in mammalian cells, which involve signals primarily via death receptors (extrinsic pathway) or the mitochondria (intrinsic pathway) that recruit caspases as effector molecules. In plants, PCD shares some similarities with animal cells, but NO is involved in PCD induction via interacting with pathways of phytohormones. NO has both promoting and suppressing effects on cell death, depending on a variety of factors, such as cell type, cellular redox status, and the flux and dose of local NO. In this article, we focus on how NO regulates the apoptotic signal cascade through protein S-nitrosylation and review the recent progress on mechanisms of PCD in both mammalian and plant cells.     

Phosphodiesterase type 5 inhibition improves early memory consolidation of object information

The nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway is assumed to play an important role in processes underlying learning and memory. We used phosphodiesterase type 5 (PDE5) inhibitors to study the role of cGMP in object- and spatial memory. Our results and those reported in other studies indicate that elevated hippocampal cGMP levels are required to improve the memory performance of rodents in object recognition and passive avoidance learning, but not in spatial learning. The timing of treatment modulates the effects on memory and strongly supports a role for cGMP in early stages of memory formation. Alternative explanations for the improved memory performance of PDE5 inhibitors are also discussed. Immunocytochemical studies showed that in vitro slice incubations with PDE5 inhibitors increase NO-stimulated cGMP levels mainly in hippocampal varicose fibers. Reviewing the available data on the localization of the different components of the NO-cGMP signaling pathway, indicates a complex interaction between NO and cGMP, which may be independent of each other. It is discussed that further studies are needed, immunocytochemical and behavioral, to better understand the cGMP-mediated molecular mechanisms underlying memory formation.     

A new Rac/PAK/GC/cGMP signaling pathway

Guanosine 3′,5′-cyclic monophosphate (cGMP) and small GTPase Rac are critical regulators of cell functions. Recently, Rac has been shown to use its downstream effector p21-activated kinase (PAK) to directly activate transmembrane guanylyl cyclases (GCs). This novel Rac/PAK/GC/cGMP signaling pathway bridges Rac and cGMP, and provides a general molecular mechanism for diverse receptors to regulate physiological functions such as cell migration through elevating the cellular cGMP level.     

Involvement of cyclic GMP and protein kinase G in the regulation of apoptosis and survival in neural cells

Our current understanding of nitric oxide (NO), cyclic GMP (cGMP) and protein kinase G (PKG) signaling pathways in the nervous systems has its origins in the early studies conducted on vascular tissues during the late 1970s and early to mid-1980s. The pioneering research into the NO/cGMP/PKG pathway in blood vessels conducted by the laboratories of Drs. Ferid Murad, Louis Ignarro and Robert Furchgott ultimately led to the awarding of the 1998 Nobel Prize in Physiology or Medicine to these three scientists. On the basis of further pioneering studies by Drs. John Garthwaite, Solomon Snyder, Steven Vincent and many other neuroscientists during the late 1980s and throughout the 1990s, it became recognized that NO serves as a neurotransmitter/neuromodulator in the central and peripheral nervous systems and that certain neural cells possess a cGMP signaling pathway similar to that in vascular smooth muscle cells. Although NO (at high concentrations) is toxic and thought to participate in neuronal cell death during stroke and neurodegenerative diseases (e.g. amyotrophic lateral sclerosis, Alzheimer's disease, HIV dementia and Parkinson's disease), recent evidence suggests that NO at low physiological concentrations can act as an antiapoptotic/prosurvival factor in certain neural cells (e.g. PC12 cells, motor neurons and neurons of dorsal root ganglia, hippocampus and sympathetic nerves). The antiapoptotic effects of NO are mediated, in part, by cGMP and a downstream target protein, PKG. Other cGMP-elevating factors (e.g. atrial and brain natriuretic peptides) and direct PKG activator (e.g. 8-bromo-cGMP) also have antiapoptotic effects which have been quantified by the new capillary electrophoresis with laser-induced fluorescence detector technology. Inhibition of soluble guanylyl cyclase and lowering of basal cGMP levels cause apoptosis in unstressed neural cells (NG108-15 and N1E-115 cells). The cGMP/PKG pathway appears to play an essential role in preventing activation of a proapoptotic pathway, thus promoting neural cell survival.     

Sources and targets of nitric oxide signalling in insect nervous systems

Nitric oxide (NO) is a membrane permeant signalling molecule which activates soluble guanylyl cyclase and leads 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 review summarizes neurochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of insects. The anatomical localization of donor and target cells suggests functions in olfaction, vision, and mechanosensation. Behavioural assays have uncovered contributions of NO signalling in oxygen sensing, habituation to chemosensory stimuli, and associative memory formation. During development, NO regulates cell proliferation, axonal outgrowth, and synaptic maturation. The cellular distribution of NO-responsive cells suggests that NO can serve as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger irrespective of conventional synaptic connectivity.     

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.     

Phytochemicals from Flemingia vestita (Fabaceae) and Stephania glabra (Menispermeaceae) alter cGMP concentration in the cestode Raillietina echinobothrida

Cyclic GMP (cGMP) mediates various physiological functions of nitric oxide (NO) synthesized by nitric oxide synthase (NOS). A crude peel extract and purified fraction of Flemingia vestita, as well as a crude rhizome extract of Stephania glabra and fractions were tested with respect to the activity of NOS, NO efflux and cGMP concentration in the cestode Raillietina echinobothrida in order to find out the possible mode of anthelmintic action of these plant-derived components. For comparison purposes, the parasites were also treated with pure genistein, sodium nitroprusside (SNP-a known NO donor), and the reference drug, praziquantel (PZQ). At the time of onset of paralysis in the parasites, a significant increase (32%-87%) in the NOS activity and a two to three fold increase of NO efflux into the incubation medium were observed in the treated parasites in comparison to their respective controls. The cGMP concentration in the treated parasites' tissue was also increased by 44%-103%. However, in the presence of NG-nitro-L-arginine methyl ester, a potent inhibitor of NOS, there was no increase in the cGMP concentration in the parasite tissue. This study indicates that the phytochemicals, in particular genistein and tetrahydropalmatine, from F. vestita and S. glabra, respectively, disturb the downstream signalling pathway of NO, as indicated by the change in cGMP concentration in the parasite tissue.     

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 receptor-like properties of nitric oxide-activated soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO), and so mediates a wide range of effects (e.g. vasodilatation, platelet disaggregation and neural signalling) through the accumulation of cGMP and the engagement of various downstream targets, such as protein kinases and ion channels. Until recently, our understanding of sGC functioning has been derived exclusively from studies of the enzyme in tissue homogenates or in its purified form. Here, NO binds to the haem prosthetic group of sGC, triggering a conformational change and a large increase in catalytic activity. The potency (EC₅₀) of NO appears to be about 100–200 nM. The rate of activation of sGC by NO is rapid (milliseconds) and, in the presence of excess substrate, cGMP is formed at a constant rate; on removal of NO, sGC deactivates slowly (seconds–minutes). Recent investigation of the way that sGC behaves in its natural environment, within cells, has revealed several key differences. For example, the enzyme exhibits a rapidly desensitizing profile of activity; the potency of NO is 45 nM for the minimally-desensitized enzyme but becomes higher with time; deactivation of sGC on removal of NO is 25-fold faster than the fastest estimate for purified sGC. Overall, within cells, sGC behaves in a way that is analogous to the way that classical neurotransmitter receptors operate. The properties of cellular sGC have important implications for the understanding of NO-cGMP signalling. For example, the dynamics of the enzyme means that fluctuations in the rate of NO formation, even on subsecond time scale, will result in closely synchronized sGC activity in neighbouring cells; desensitization of sGC provides an economical way of generating a cellular cGMP signal and, in concert with phosphodiesterases, provides the basis for cGMP signal diversity, allowing different targets (outputs) to be selected from a common input (NO). Thus, despite exhibiting only limited molecular heterogeneity, cellular sGC functions in a way that introduces speed, complexity, and versatility into NO-cGMP signalling pathways.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.