The language of nitric oxide signalling

Nitric oxide (NO) has recently joined the select circle of the ubiquitous molecules of plant signalling networks. Indeed, the last decade has produced a tremendous amount of data that evidence the diversity of physiological situations in which NO is involved in plants and the complexity of NO biology. These data also underline our difficulties in providing simple answers to the cardinal questions of where NO comes from and how the NO message is converted into a physiological response. The identification of NO primary targets and NO-regulated genes provides new opportunities to connect NO biochemistry and NO biology. This review summarises our current understanding of NO signalling, from the generation of the NO message to its execution into a cellular response. The review particularly considers whether and how NO may be responsible for specific signalling in different physiological processes.     

Hedgehog signalling: how to get from Smo to Ci and Gli

The secreted morphogens of the Hedgehog family have important roles in normal development as well as in associated pathologies, including cancer. The Hedgehog signalling pathway has been studied in Drosophila and is thought to be conserved in vertebrates. Hedgehog elicits a signalling response that activates Smoothened (Smo). There is evidence of differences between Drosophila and vertebrates concerning signalling downstream of Smo, as well as in Smo itself. Here, we discuss this evidence and its importance for investigations of the pathway and related biology, as well as for the development of drugs targeting components of the pathway for treatment of associated pathologies.     

The hypothesis of the main role of H2S in coupled sulphide-nitroso signalling pathway

As a part of the nitroso signalling pathway, nitroso-compounds serve as stores and carriers of NO; as part of the sulphide signalling pathway, bound sulfane-sulphur compounds serve as stores and carriers of H2S. Here we hypothesise a coupled sulphide-nitroso signalling pathway, in which H2S plays a main role. H2S releases NO from the endogenous S-nitroso-compounds nitroso-cysteine, nitroso-acetylcysteine and nitroso-albumin. Relaxation of noradrenaline-precontracted aortic rings by H2S is also enhanced in the presence of nitroso-albumin, which may implicate the involvement of the nitroso signalling pathway. Pretreatment of albumin, cysteine, N-acetylcysteine and lipids with H2S results in binding of sulphur to these compounds creating thus new-modified sulphur compounds that release NO from nitroso-compounds directly and/or through released H2S, which suggests sulphide-nitroso signalling pathway participation. This hypothesis is supported by the observation that the pretreatment of noradrenaline-precontracted aortic rings with H2S significantly enhanced relaxation induced by nitroso-glutathione in the absence of H2S. We assume that the NO release from nitroso-compounds directly by H2S or indirectly by the H2S-induced sulphur-bound compounds represents coupled sulphide-nitroso signalling, which may explain some of the numerous biological effects of H2S that are shared with NO.     

Mechanisms for nitric oxide synthesis in plants

The discovery that nitric oxide (NO) acts as a signal fundamentally shifted our understanding of free radicals from toxic by-products of oxidative metabolism to key regulators of cellular functions. This discovery has led to intense investigation into the synthesis of NO in both animals and plants. Nitric oxide synthases (NOS) are the primary sources of NO in animals and are complex, highly regulated enzymes that oxidize arginine to NO and citrulline. Plant NO synthesis, however, appears more complex and includes both nitrite and arginine-dependent mechanisms. The components of the arginine pathway have been elusive as no known orthologues of animal NOS exist in plants. An Arabidopsis gene (AtNOS1) has been identified that is needed for NO synthesis in vivo and has biochemical properties similar to animal cNOS, yet it has no sequence similarity to any known animal NOS. An Atnos1 insertion mutant has been useful for genetic studies of NO regulation and for uncovering new roles for NO signalling. The elucidation of plant NO synthesis promises to yield novel mechanisms that may be applicable to animal systems.     

Differential requirement for NO during ABA-induced stomatal closure in turgid and wilted leaves

Abscisic acid (ABA)-induced stomatal closure is mediated by a complex, guard cell signalling network involving nitric oxide (NO) as a key intermediate. However, there is a lack of information concerning the role of NO in the ABA-enhanced stomatal closure seen in dehydrated plants. The data herein demonstrate that, while nitrate reductase (NR)1-mediated NO generation is required for the ABA-induced closure of stomata in turgid leaves, it is not required for ABA-enhanced stomatal closure under conditions leading to rapid dehydration. The results also show that NO signalling in the guard cells of turgid leaves requires the ABA-signalling pathway to be both capable of function and active. The alignment of this NO signalling with guard cell Ca²⁺-dependent/independent ABA signalling is discussed. The data also highlight a physiological role for NO signalling in turgid leaves and show that stomatal closure during the light-to-dark transition requires NR1-mediated NO generation and signalling.     

Triterpenoid biosynthesis and the transcriptional response elicited by nitric oxide in submerged fermenting Ganoderma lucidum

Ganoderic triterpenoid (GT) is a promising anti-tumour constituent in Ganoderma lucidum. The aim of this study was to investigate induction by and a possible signalling mechanism of nitric oxide (NO) for GT synthesis. Compared to the control, the biomass decreased by 43.5% at 120 h and the GT yield increased by 40.94% at 72 h in the presence of a 5 mM NO donor sodium nitroprusside supplement. The gene expression profiles of G. lucidum in response to NO were investigated by RNA-sequencing. Functional annotation and an enrichment analysis of the differentially expressed genes indicated that NO inhibited mycelial growth probably via the suppression of the glycolysis genes involved in carbohydrate metabolism. NO may function directly as a regulator of gene expression in the mevalonate pathway to induce GT biosynthesis, and the hyper-production of GT in response to NO could also be accomplished by a signalling function involving Ca²⁺ and a reactive oxygen species (ROS) pathway. The results of this study are useful for large-scale GT production and can facilitate further studies on the endogenous signalling pathways involved in the GT biosynthetic pathway.     

NO underlies the muscarinic receptor-mediated inhibition of If in early embryonic heart cells.

BACKGROUND/AIMS: Early embryonic cardiomyocytes beat spontaneously. The hyperpolarization-activated cyclic-nucleotide-modulated current (I(f)) appears to be involved in its modulation as it is highly expressed at this stage. The spontaneous beating of early embryonic heart cells is slowed by acetylcholine (ACh), and our earlier studies identified a key role for nitric oxide (NO) in the regulation of the voltage dependent L-type Ca(2+) current (I(Ca,L)). The aim of the present study was to clarify whether and via which signalling pathway(s) I(f) is regulated upon muscarinic receptor activation in early embryonic (E9.5 to E11.5) cardiomyocytes. METHODS: The whole-cell patch clamp technique in combination with pharmacology and/or knock out mouse models was used to investigate the regulation of I(f). RESULTS: We found that the ACh analogue carbachol (CCh, 10 micromol) led in the majority of cells (68%, n=50) to a significant depression of I(f) by 16.3+/-1.4% (n=34, p<0.01, voltage steps from -35 mV to -110 mV). This cholinergic inhibition was mediated by the NO/cGMP signalling pathway as it was largely reversed by superfusion with the non selective nitric oxide synthase (NOS) inhibitor N(G)-Methyl-L-arginine acetate salt (L-NMMA, 1 mmol), the inhibitor of the soluble guanylyl cyclase (sGC) 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ, 100 micromol) and a selective inhibitor of the phosphodiesterase (PDE) type 2 Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 micromol). Analysis of the muscarinic signalling in embryonic cardiomyocytes harvested from NOS2 (-/-) and NOS3 (-/-) mice revealed that the NOS3 isoform was entirely responsible for the muscarinic receptor-induced NO production. CONCLUSIONS: Muscarinic receptor stimulation depresses I(f) by generating NO via the NOS3 and the cGMP/PDE type 2 signalling pathway in early embryonic cardiomyocytes. This suggests that NO is a key signalling molecule involved in the regulation of chronotropy of early embryonic heart cells.     

The role of astrocytic calcium and TRPV4 channels in neurovascular coupling

Neuronal activity evokes a localised change in cerebral blood flow in a response known as neurovascular coupling (NVC). Although NVC has been widely studied the exact mechanisms that mediate this response remain unclear; in particular the role of astrocytic calcium is controversial. Mathematical modelling can be a useful tool for investigating the contribution of various signalling pathways towards NVC and for analysing the underlying cellular mechanisms. The lumped parameter model of a neurovascular unit with both potassium and nitric oxide (NO) signalling pathways and comprised of neurons, astrocytes, and vascular cells has been extended to include the glutamate induced astrocytic calcium pathway with epoxyeicosatrienoic acid (EET) signalling and the stretch dependent TRPV4 calcium channel on the astrocytic endfoot. Results show that the potassium pathway governs the fast onset of vasodilation while the NO pathway has a delayed response, maintaining dilation longer following neuronal stimulation. Increases in astrocytic calcium concentration via the calcium signalling pathway and/or TRPV4 channel to levels consistent with experimental data are insufficient for inducing either vasodilation or constriction, in contrast to a number of experimental results. It is shown that the astrocyte must depolarise in order to produce a significant potassium flux through the astrocytic BK channel. However astrocytic calcium is shown to strengthen potassium induced NVC by opening the BK channel further, consequently allowing more potassium into the perivascular space. The overall effect is vasodilation with a higher maximal vessel radius.     

Multiple roles for lipids in the Hedgehog signalling pathway

The identification of endogenous sterol derivatives that modulate the Hedgehog (Hh) signalling pathway has begun to suggest testable hypotheses for the cellular biological functions of Patched, and for the lipoprotein association of Hh. Progress in the field of intracellular sterol trafficking has emphasized how tightly the distribution of intracellular sterol is controlled, and suggests that the synthesis of sterol derivatives can be influenced by specific sterol-delivery pathways. The combination of this field with Hh studies will rapidly give us a more sophisticated understanding of both the Hh signal-transduction pathway and the cell biology of sterol metabolism.     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Nitric oxide function and signalling in plant disease resistance

Nitric oxide (NO) is one of only a handful of gaseous signalling molecules. Its discovery as the endothelium-derived relaxing factor (EDRF) by Ignarro revolutionized how NO and cognate reactive nitrogen intermediates, which were previously considered to be toxic molecules, are viewed. NO is now emerging as a key signalling molecule in plants, where it orchestrates a plethora of cellular activities associated with growth, development, and environmental interactions. Prominent among these is its function in plant hypersensitive cell death and disease resistance. While a number of sources for NO biosynthesis have been proposed, robust and biologically relevant routes for NO production largely remain to be defined. To elaborate cell death during an incompatible plant-pathogen interaction NO functions in combination with reactive oxygen intermediates. Furthermore, NO has been shown to regulate the activity of metacaspases, evolutionary conserved proteases that may be intimately associated with pathogen-triggered cell death. NO is also thought to function in multiple modes of plant disease resistance by regulating, through S-nitrosylation, multiple nodes of the salicylic acid (SA) signalling pathway. These findings underscore the key role of NO in plant-pathogen interactions.     

Drugging Wnt signalling in cancer

Aberrant regulation of the Wnt signalling pathway has emerged as a prevalent theme in cancer biology. This chapter summarizes the research that provides a proof of concept for inhibiting Wnt signalling in cancer, the potential means by which this could be achieved, and some recent advances towards this goal. A brief discussion of molecular diagnostics and possible safety concerns is also provided.     

NO是植物应激反应的信号分子

根据NO的性质和可能的产生途径,略述了生物胁迫(病原菌侵害)和干旱胁迫、盐胁迫、极端温度、机械损伤、臭氧和紫外辐射等各种非生物胁迫信号与NO信号分子的偶联及其信号的级联途径,概括了NO可能介导的生物过程,讨论了NO通过其下游信号过程对与细胞的生理影响以及该下游信号过程所涉及到的cGMP、cADPR的产生和NO与其它信号分子(ROS、SA、ABA等)的协同作用,表明胁迫诱导的NO爆发是激发、启动和装备植物细胞的重要信号级联环节,这个环节能使植物细胞处于应激状态,并迅速作出反应,形成一系列适应机制。     

Homeostatic NMDA receptor down-regulation via brain derived neurotrophic factor and nitric oxide-dependent signalling in cortical but not in hippocampal neurons

Nitric oxide (NO) has been proposed to down-regulate NMDA receptors (NMDA-Rs) in a homeostatic manner. However, NMDA-R-dependent NO synthesis also can cause excitotoxic cell death. Using bicuculline-stimulated hippocampal and cortical cell cultures, we have addressed the role of the brain-derived neurotrophic factor-NO pathway in NMDA-R down-regulation. This pathway protected cortical cells from NMDA-induced death and led to NMDA-R inhibition. In contrast, no evidence was gained for the presence of this protective pathway in hippocampal neurons, in which NMDA-induced NO synthesis was confirmed to be toxic. Therefore, opposing effects of NO depended on the activation of different signalling pathways. The pathophysiological relevance of this observation was investigated in synaptosomes and post-synaptic densities isolated from rat hippocampi and cerebral cortices following kainic acid-induced status epilepticus. In cortical, but not in hippocampal synaptosomes, brain-derived neurotrophic factor induced NO synthesis and inhibited NMDA-R currents present in isolated post-synaptic densities. In conclusion, we identified a NO-dependent homeostatic response in the rat cerebral cortex induced by elevated activity. A low performance of this pathway in brain areas including the hippocampus may be related to their selective vulnerability in pathologies such as temporal lobe epilepsy.     

Nitric oxide signalling with a special focus on lipid-derived mediators

The ways in which cells communicate among each other concerns all aspects of biology, from developmental processes to diseases. Nitric oxide (NO) is one of the most remarkable and unusual regulatory molecules. It is a labile free radical gas that is not stored but generated on demand, and has been implicated in an extraordinarily diverse range of physiological and pathophysiological functions. The modulation of cell signalling by free radicals is an emerging area of research that provides insight into the orchestration of cell adaptation to a changing microenvironment. In a multicellular organism this serves to coordinate complex physiological responses, such as inflammation. Cell signalling is also accompanied by rapid remodelling of membrane lipids by activated lipases. The discovery that NO, which does not reversibly interact with membrane receptors like conventional hormones and growth factors, targets enzymes such as phospholipase A2, sphingomyelinases or ceramidases, has stimulated growing interest in the crosstalk between redox and lipid signalling.     

Phosphorylation of the PKG substrate, vasodilator-stimulated phosphoprotein (VASP), in human cultured prostatic stromal cells.

Nitric oxide (NO) is known to regulate contractility and proliferation of cells within the prostate, however, the mechanism by which this occurs is unknown. The cGMP-dependent protein kinase (PKG) signalling pathway may be involved, and recent work has shown that activation of this pathway can be assessed by analysis of phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The aim of the current study is to characterise the expression of VASP in the human prostate and human cultured prostatic stromal cells (HCPSCs), and to investigate whether NO activates PKG in these cells. Our studies revealed that VASP is expressed, and that incubation of HCPSCs with PKG-activating cGMP-analogues or the NO-donor, SNP, caused a significant PKG-dependent increase in VASP serine-239 phosphorylation. In addition, SNP elicited a reduction in intracellular K(+) in a time frame consistent with the phosphorylation of VASP and activation of PKG. These data demonstrate that VASP can be used to assess the NO/cGMP/PKG signalling pathway in HCPSCs. In addition, we demonstrate for the first time that SNP, probably via NO release, leads to phosphorylation of VASP in a manner consistent with PKG activation.     

Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation

The ubiquitous anion nitrite (NO₂⁻) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron-nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO-cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.