CSCDB: the cAMP and cGMP signaling components database

Adenylate cyclases, guanylate cyclases, cyclic nucleotide phosphodiesterases, and cyclic nucleotide-binding proteins constitute the core of cAMP and cGMP signaling components. Using a combination of BLAST and profile search methods, we found that cyclic nucleotide-binding proteins exhibited diverse domain architectures. In addition to the domain architectures involved in the characterized functional groups, a cyclic nucleotide-binding domain was also fused to various domains involved in pyridine nucleotide-disulfide oxidoreductase, acetyltransferase, thioredoxin reductase, glutaminase, rhodanese, ferredoxin, and diguanylate cyclase, implying the versatile functions of cyclic nucleotide-binding proteins. We constructed the CSCDB database to accumulate the components of cAMP and cGMP signaling pathways in the complete genomes. User-friendly interfaces were created for easier browsing, searching, and downloading the data. Besides harboring the sequence itself, each entry provided detailed annotation information, such as sequence features, chromosomal localization, functional domains, transmembrane region, and sequence similarity against several major databases. Currently, CSCDB contains 4234 entries covering 466 organisms, including 35 eukaryotes, 382 bacteria, and 29 archaea. CSCDB can be freely accessible on the web at http://cscdb.com.cn.     

IL-4 induces cAMP and cGMP in human monocytic cells

Human monocytes, preincubated with IFN-gamma respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 - 3 min) and cAMP (20 - 25 min) in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1) IL-4 may stimulate different NOS isoforms in resting and IFN-gamma activated monocytes, and (2) cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca(2+)](i) in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.     

Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions

The past eighteen months have provided much progress in the cyclic nucleotide phosphodiesterase (PDE) field. Six new phosphodiesterase genes have been discovered and characterized. In addition, several new highly specific PDE inhibitors have been developed and approved for clinical use. Finally, new strategies have been employed to determine PDE function in model systems including the use of antisense oligonucleotide and disruption techniques.     

cAMP induces a transient elevation of cGMP levels during early culmination of Dictyostelium minutum

Oscillatory cAMP signals very likely organize the cell movement which leads to fruiting body construction in Dictyostelium minutum [Schaap, P., Konijn, T.M. and Van Haastert, P.J.M.: Proc. Natl. Acad. Sci. USA 81, 2122-2126 (1984)]. Stimulation with cAMP induces a transient elevation of cGMP in cells at the early culmination stage, which peaks at 12-18 s. A half maximal cGMP response is induced by 10(-7) M cAMP and saturation of the response is reached at 10(-5) M cAMP. No cGMP accumulation was induced by stimulation of vegetative or aggregative cells of D. minutum by cAMP. Since the transient increase of cGMP is most likely involved in the transduction of chemotactic signals, our results indicate that cAMP signals organize fruiting body formation by inducing chemotaxis inside the aggregate.     

Endothelin receptor heterogeneity,G-proteins,and signaling via cAMP and cGMP cascades

Summary 1. The endothelins (ETs) are potent vasoactive peptides which are involved in diverse biological processes, such as contraction, neuromodulation, and neurotransmission, as well as in certain pathophysiological conditions including cardiac and renal failure.2. The diversity of action of ETs may be attributed to (i) the existence of a number of receptor subtypes, and (ii) the G-protein-mediated activation of different signal transduction pathways.3. The combined action of these two variables modulates the response, since different receptor subtypes can stimulate and/or inhibit the cAMP and cGMP cascades.     

cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.     

Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.     

A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BK(Ca) (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel alpha subunit. Three isoforms, BK(A), BK(B), and BK(C), which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BK(A) and BK(B) by shifting the voltage dependence of the channel to more negative potentials. In contrast, BK(C) was exclusively stimulated by cAK. BK(C) lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser(1151) and Ser(1154). Mutation of this motif in BK(A) switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BK(A) abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.     

Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets.

Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 10nm prostacyclin (PGI2), 1 microM PGD2, or 1mM N2,2'-O-dibutyryl-cAMP. In contrast, incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC50 of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise cAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise cAMP and those which elevate cGMP.     

Ethanol inhibits dual receptor stimulation of pineal cAMP and cGMP by vasoactive intestinal peptide and phenylephrine

Concurrent activation of vasoactive intestinal peptide and alpha 1-adrenergic receptor resulted in greater than 20-fold increases in pineal cAMP and cGMP accumulation. We now find that an intoxicating level of ethanol (0.2%, 34 mM) inhibits greater than 50% the large increases in pineal cAMP and cGMP produced by concurrent treatment with vasoactive intestinal peptide and phenylephrine. The potency of the various alcohols tested was directly related to their chain length. This inhibition appears to be specific since a five-fold higher concentration of ethanol does not inhibit the stimulation of cAMP and cGMP accumulation produced by concurrent treatment with isoproterenol and phenylephrine. Accordingly, it seems that one mechanism of action of ethanol on neural function may be its ability to selectively inhibit ethanol-sensitive integrative mechanisms which regulate cyclic nucleotides.     

Developmental regulation of the pathways of folate-receptor-mediated stimulation of cAMP and cGMP synthesis in Dictyostelium discoideum

Recently, we demonstrated the presence of multiple folate-binding sites on the cell surface of Dictyostelium discoideum. These sites were divided into two major classes, with different ligand specificities (A and B). Each major class consists of several interconvertible subtypes. In the present report, the ability of 13 folate analogs to activate both adenylate and guanylate cyclase in pre- as well as postaggregative cells is examined. The patterns of correlation between binding and activation data indicate that guanylate cyclase activation is mediated by the B-sites in both developmental stages (P less than 0.001). In postaggregative cells, adenylate cyclase also seems to be activated by the B-sites (P less than 0.001). In contrast, adenylate cyclase activation in preaggregative cells was well correlated with the specificity of A-sites (P less than 0.01). Remarkably, the potencies of activation were less affected by molecular modifications than the binding affinities were, as suggested by a slope of 0.4 in a plot of K0.5 values of activation vs. binding. This observation argues against the existence of a transduction mechanism in which the response is proportional to receptor occupancy. For the B-receptor, however, the degree of receptor occupancy appears to determine the response. The existence of folic acid antagonists is demonstrated, some of which are specific for either A-sites coupled to adenylate cyclase or for B-sites coupled to guanylate cyclase.     

Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.     

The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.     

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.     

Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.