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.     

Cyclic nucleotides and development ofMyxococcus xanthus: Analysis of mutants

Approximately 60 developmental mutants ofMyxococcus xanthus M300 were obtained through nitrosoguanidine mutagenesis and placed into three operationally defined categories. Type-I strains exhibited no aggregation or sporulation. Type-II strains were able to aggregate but did not sporulate. A strain classed as a type-III strain was a low-capacity fruiter. Each category displayed defects in cyclic nucleotide behavior that could be predicted from the current model. Most significantly, several aggregationless (type I) mutants lacking cGMP phosphodiesterase aggregated in the presence of externally applied phosphodiesterase. A requirement for cell-cell contact in sporulation has been confirmed. Evidence is presented that suggests the involvement of cAMP phosphodiesterase in sporulation and that sporulation may be a developmental pathway independent of aggregation. These results support a previously published hypothesis of the role of cyclic nucleotides in the development ofM. xanthus.     

cAMP AND cAMP-STIMULATED PROTEIN PHOSPHORYLATION IN ZOOSPORES OF BROWN ALGAE1

Adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) were detected at concentrations of 8–11 and 10–20 pmol · mg^?1 protein, respectively, in zoospores of a brown alga, Undaria pinnatifida (Harvey) Suringer. Cellular levels of these cyclic nucleotides did not substantially change during dark to light transition. cAMP-stimulated protein phosphorylation was found in soluble cell-free extracts of zoospores of Undaria pinnatifida and Laminaria angustata Kjellman.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3',5'-nucleotides, nucleoside 5'-phosphates and methylxanthines

cAMP, dbcAMP, cCMP, cGMP, theophylline and caffeine caused reversible melanosome dispersion within 5 minutes at 10 mM in the dermal melanophores of the black goldfish, Carassius auratus L. cTMP, cUMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-TMP, and 5′-UMP did not produce melanosome dispersion or aggregation in this melanophore system. cAMP was the most effective nucleotide in the induction of melanosome dispersion; at 10 mM, cGMP and at 5 mM, dbcAMP were the least effective of those nucleotides inducing melanosome dispersion. At the 10 mM level dbcAMP required 30 minutes to evoke the same degree of melanosome dispersion as 5 minutes cAMP treatment. Theophylline was more effective than caffeine in eliciting melanosome dispersion. At 1 mM, theophylline and caffeine first induced melanosome dispersion which was followed by aggregation in the course of the 30 minute test period. These reactions suggest both a high melanophore phosphodiesterase activity and competitive inhibition of phosphodiesterase by theophylline and caffeine. Induction of melanosome dispersion by several cyclic 3′,5′-nucleotides suggest multi-nucleotide control of melanosome dispersion. These findings also support a proposed mechanism of prostaglandin induced melanosome dispersion as well as the “second messenger” hypothesis.     

Cyclic nucleotide alterations in mixed leukocyte reaction: a preliminary report

Endogenous cyclic adenosine and guanosine monophosphate (cAMP, cGMP) levels were studied in human peripheral blood lymphocytes during mixed leukocyte reactions (MLR). cAMP level was consistently elevated in one-way MLR, with good correlation to 3H-thymidine uptake in these reactions. In contrast, cGMP level was practically unchanged. Irradiation of reacting cell populations resulted in inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. These results suggest that metabolic alterations in cAMP may be associated with immune reactions of cellular recognition.     

Substrate and effector specificity of a guanosine 3':5'-monophosphate phosphodiesterase from rat liver.

Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

The effect of serum on lipid synthesis and on the expression of oligodendrocyte marker-enzymes in oligodendrocyte-enriched glial cells in culture

Glial cells were isolated from the cerebra of 7-day old rats and the effect of serum on the development of these cells in culture was studied. The activities of the oligodendrocyte marker-enzymes, 2′3′-cyclic nucleotide 3′-phosphodiesterase and glycerol 3-phosphate dehydrogenase and the synthesis of the myelin-associated sulpholipid, sulphatide, were used to monitor the differentiation of these cells in vitro. The results indicate that serum: (i) represses lipogenesis, cholesterogenesis and sulphatide synthesis, (ii) lowers the expression of 2′3′-cyclic nucleotide 3′ phosphodiesterase and glycerol 3-phosphate dehydrogenase but not of lactate dehydrogenase and (iii) thus impairs the differentiation of oligodendrocytes.     

Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation

Melanophores are pigmented cells capable of quick colour changes through coordinated transport of their intracellular pigment granules. We demonstrate the involvement of phosphoinositide 3-kinase (PI3-K) in Xenopus and Labrus aggregation by the use of the PI3-K inhibitor, LY-294002. In Xenopus, wortmannin-insensitive PI3-K was found to be essential for the aggregation, mitogen-activated protein kinase (MAPK) activation and tyrosine phosphorylation of a 280-kDa protein, and for the maintenance of low cyclic adenosine 3′:5′-monophosphate (cAMP) during the aggregated state. Pre-aggregated cells disperse completely to LY-294002 at 50–100 μM, involving a transient elevation in cAMP due to adenylate cyclase (AC) stimulation or to inhibition of cyclic nucleotide phosphodiesterase (PDE). The inactive analogue LY-303511 did not induce dispersion at the same concentrations. PDE4 and/or PDE2 was found to be involved in melanosome aggregation. The similar kinetics of LY-294002 and various PDE inhibitors indicates that the elevation of cAMP might be due to inhibition of PDE. In Labrus melanophores, LY-294002 had a less dramatic effect, probably due to less dependence on PDE in regulation of cAMP levels. In Xenopus aggregation, we suggest that melatonin stimulation of the Mel1c receptor via G_βγ activates PI3-K that, directly or indirectly via MAPK, activates PDE.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.     

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.     

Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system

Concentrations of cAMP (cyclic adenosine 3′,5′-monophosphate) and cGMP (cyclic guanosine 3′,5′-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4–6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or Na Fstimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Cyclic nucleotides, cyclic nucleotide phosphodiesterase, and development in Myxococcus xanthus.

Exogenous cyclic nucleotide phosphodiesterase (PD) accelerated fruiting body (FB) formation and increased territory size of aggregates in Myxococcus xanthus. Both guanosine 3'5'-monophosphate (cGMP) and guanosine 5'-monophosphate (GMP) were antagonistic to the PD effect. Adenosine 3'5'-monophosphate (cAMP) increases FB numbers twofold in the absence but not in the presence of PD. PD induction is not affected by methionine or isoleucine, which inhibit, or by threonine, which stimulates, FB formation. There is an increase and subsequent decrease in cAMP levels during early glycerol-induced microcyst development but 10 mM theophylline or caffeine not only inhibited microcyst development but induced germination in the presence of glycerol. On the basis of these results and the reports of other investigators a tentative model is proposed based on a dual role for cyclic nucleotides in the development in M. xanthus.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.