The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced Cgmp Increase in Caenorhabditis elegans

Adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.     

A Cyclic GMP Signalling Module That Regulates Gliding Motility in a Malaria Parasite

The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3′, 5′-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase δ (PDEδ), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase β (GCβ), showing that in ookinetes GCβ is an important source for cGMP, and that PDEδ is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.     

Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Polysphondylium violaceum is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3′,5′-cyclic monophosphate (cAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total cAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3′,5′-cyclic monophosphate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either cAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both cAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.     

Artefactual Origins of Cyclic AMP in Higher Plant Tissues

A highly sensitive radioimmunoassay has been used to determine the levels of adenosine 3′,5′-cyclic monophosphate (cAMP) in five higher plants (Lactuca sativa, Helianthus annuus, Oryza sativa, Pinus pinaster, Nicotiana tabacum). Particular attention was paid to the three main sources of errors in the characterization of cAMP in plants: presence of interfering substances in plant tissues; possible artefactual formation of cAMP from endogenous ATP during extraction, purification, and assay; and microbial origin of cAMP. In all the tested tissues, the cAMP level was below the detection limit of 0.5 picomole per gram fresh weight, a value much lower than those reported for similar materials of the same species in many previous studies. This result is not in favor of cAMP-dependent regulations in higher plants.     

Cyclic adenosine 3':5'-monophosphate in moss protonema: a comparison of its levels by protein kinase and gilman assays

From the protonema of the moss Funaria hygrometrica (L.) Sibth, a factor indistinguishable from cyclic adenosine 3′:5′-monophosphate (cAMP) has been isolated. The factor stimulated the activity of protein kinase from rabbit skeletal muscle and co-chromatographed with authentic cAMP in two solvent systems. Its ability to stimulate protein kinase activity was completely abolished by 3′:5′-cyclic nucleotide phosphodiesterase, the rate of inactivation being similar to that of authentic cAMP. Based on these properties, this factor is identified as 3′,5′-cAMP. Cyclic AMP could be readily removed from the cells and washing the cells with water reduced the endogenous level of cAMP by 2- to 3-fold. A comparison of cAMP levels by protein kinase and Gilman assays was made. The intracellular levels determined by protein kinase assay were about 7-fold lower than the values obtained by Gilman assay. This discrepancy was due to the presence of unidentified compounds which were completely degraded by 3′:5′-cyclic nucleotide phosphodiesterase. Although these displaced labeled cAMP in the Gilman assay, they did not stimulate the protein kinase activity. The protonema may contain cyclic nucleotides other than cAMP; these will not be detected in the protein kinase assay due to the specificity of this reaction. The crude extracts were found to be unsuitable for assaying cAMP by either method.     

Cyclic-GMP-Dependent Protein Kinase Inhibits the Ras/Mitogen-Activated Protein Kinase Pathway

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser⁴³, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser⁴³ uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser⁴³ was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser⁴³ in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser⁴³ phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser⁴³ and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.     

Failure to Detect Cyclic 3', 5'-Adenosine Monophosphate in Healthy and Crown Gall Tumorous Tissues of Vicia faba

Attempts were made to provide proof for the occurrence of cyclic 3′,5′-adenosine monophosphate in healthy and crown gall tissues of Vicia faba. Although our purified extracts gave positive readings in the Gilman binding assay for cyclic AMP, they were not digested by a specific cyclic 3′,5′-adenosine monophosphate phosphodiesterase from beef heart. The extracts were digested, however, by a partially purified cyclic nucleotide phosphodiesterase from carrot tissue, which attacks both cyclic 2′,3′- and 3′,5′-nucleotides. The data indicate that the substances detected in the V. faba extracts are perhaps cyclic 2′,3′-nucleotides, a possible RNA degradation product.     

Seven-membered cyclic phosphate: synthesis and properties of S-cycloadenosine 2′,5′-cyclic phosphate and cordycepin 2′,5′-cyclic phosphate. Studies of nucleosides and nucleotides. LXIV1). Purine cyclonucleosides 25

8,3′-Anhydro-8-mercapto-9-β-D-xylofuranosyladenine (8,3′-s-cycloadenosine) was phosphorylated with cyanoethyl phosphate and DCC to 5′-phosphate. After 6-amino group was benzoylated, the monophosphate was treated with DCC to give a cyclic phosphate (II). The structure of compound II was elucidated as 8,3′-s-cycloadenosine 2′,5′-cyclic phosphate by UV, NMR and CD spectra, as well as enzymatic hydrolyses. When compound II was desulfurized with Raney nickel, cordycepin 2′,5′-cyclic phosphate (III) was obtained. Although compound III could be obtained from cordycepin 5′-phosphate with DCC, the yield was extremely low.     

Probability Fluxes and Transition Paths in a Markovian Model Describing Complex Subunit Cooperativity in HCN2 Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are voltage-gated tetrameric cation channels that generate electrical rhythmicity in neurons and cardiomyocytes. Activation can be enhanced by the binding of adenosine-3′,5′-cyclic monophosphate (cAMP) to an intracellular cyclic nucleotide binding domain. Based on previously determined rate constants for a complex Markovian model describing the gating of homotetrameric HCN2 channels, we analyzed probability fluxes within this model, including unidirectional probability fluxes and the probability flux along transition paths. The time-dependent probability fluxes quantify the contributions of all 13 transitions of the model to channel activation. The binding of the first, third and fourth ligand evoked robust channel opening whereas the binding of the second ligand obstructed channel opening similar to the empty channel. Analysis of the net probability fluxes in terms of the transition path theory revealed pronounced hysteresis for channel activation and deactivation. These results provide quantitative insight into the complex interaction of the four structurally equal subunits, leading to non-equality in their function.     

Cyclic Guanosine 3′,5′-Monophosphate in the Dimorphic Fungus Mucor racemosus

The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3′,5′-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3′,5′-monophosphate has upon cell morphology.     

Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3′,5′-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3′,5′-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.     

Influence of theophylline on concentrations of cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate of rat brain

The influence of theophylline (2.5–100 mg/kg p.o.) on cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) in brain of Sprague-Dawley rats (0.5–3.0 hr after administration of theophylline) was investigated. It was found that theophylline increases cAMP and cGMP levels when administered in a dose of 25 mg/kg or higher. A significant decrease of cGMP level was observed after administration of 10 mg/kg. The results of this study suggest that the influence of theophylline on cyclic nucleotide levels of rat brain is the result of two factors: (a) inhibitory properties of theophylline on cAMP and cGMP phosphodiesterases and (b) competition of theophylline with adenosine.     

Studies on the kinetic effects of cyclic nucleotides dependent glutamate dehydrogenase

Stopped-flow spectrophotometric studies of the reductive amination of L-ketoglutarate by L-glutamate dehydrogenase showed a biphase time course, which consisted of a rapid first phase lasting 60–100 msec and a slow final phase in which the rate of coenzyme oxidation increased until the coenzyme was depleted. The effects of 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP) on the time course of both phases were established. The results showed that in the concentration ranges used the cyclic nucleotides accelerate the catalytic reaction. The effect of cAMP was more pronounced as compared to cGMP. In all cases this influence was most clearly expressed in the first phase. Using an Arrhenius plot the activation parameters were calculated. The experiments with cAMP and cGMP at different molar ratios showed that a specific cAMP binding may occur.     

Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3′,5′-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels not preactivated by voltage. We identified a conformational flip of the channel as an intermediate state following the ligand binding and quantified it kinetically. Globally fitting the time courses of ligand binding and unbinding revealed modest cooperativity among the subunits in the conformational flip. The intensity of this cooperativity, however, was only moderate compared to channels preactivated by hyperpolarizing voltage. These data provide kinetic information about conformational changes proceeding in nonactivated HCN2 channels when cAMP binds. Moreover, our approach bears potential for analyzing the function of any other membrane receptor if a potent fluorescent ligand is available.     

Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line

The stimulation of IP₃ production by muscarinic agonists causes both intracellular Ca²⁺ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na⁺ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na⁺ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na⁺ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent K _D of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca²⁺ influx and electrical excitability. In N1E-115 cells, Ca²⁺ entry by this pathway is necessary to refill the IP₃-sensitive intracellular Ca²⁺ pool during repeated stimulation and CNG channels may play a similar role in other neurons.     

The Arabidopsis Wall Associated Kinase-Like 10 Gene Encodes a Functional Guanylyl Cyclase and Is Co-Expressed with Pathogen Defense Related Genes

Background

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants.

Principal Findings

We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10_431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors.

Conclusions

We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP.     

On the presence of adenosine 3′, 5′ -cyclic monophosphate in moss (Funariahygrometrica)

Partially purified nucleotide fraction of moss containing [14C]-labelled putative adenosine 3′, 5′ -cyclic monophosphate (cAMP) and marker authentic [3H] -cAMP was characterized by chemical deamination and also by the enzymatic hydrolysis with beef heart cyclic nucleotide phosphodiesterase. A significant conversion of marker authentic [3H] -cAMP into [3H] -inosine 3′, 5′ -cyclic monophosphate (cIMP) and [3H] -5′ adenosine monophosphate was observed by respective treatments. In contrast, the [14C] -labelled putative cAMP from control and theophylline-treated moss tissue was insensitive to chemical deamination and enzymatic hydrolysis. Apparently, the [14C] -labelled product which comigrates with authentic [3H] -cAMP does not represent true cAMP. Both the methods employed for characterization of the labelled putative cAMP were sensitive enough to detect picomole quantities of authentic [3H] -cAMP. Lack of detectability of prelabelled [14C] -cAMP in our preparations implies that the tissue may contain authentic cyclic AMP below the picomole levels. Thus, the attributed physiological role to adenosine 3′, 5′ -cyclic monophosphate in moss tissue appears somewhat skeptical.     

Cyclic AMP and higher plants

Despite the evidence in support, the extent of which is outlined in this review, the occurrence of cyclic AMP in tissues of higher plants has been doubted by a number of previous reviewers. Recent MS and other evidence vindicates earlier identification of an adenosine nucleotide from plant tissues as adenosine 3′:5′-cyclic monophosphate. The additional demonstration of 3′: 5′-cyclic nucleotide phosphodiesterases in higher plants, together with adenylate cyclase, a specific cyclic AMP binding protein, and calmodulin, means that plants possess all the necessary components for a functional cyclic AMP-regulated system. Whether such a system does function in plants is considered as are also the reported physiological effects of exogenously supplied cyclic AMP on plant tissues.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.