Isolation and partial characterization of a cyclic GMP-dependent cyclic GMP-specific phosphodiesterase from Dictyostelium discoideum

The cellular slime mold, Dictyostelium discoideum, contains at least two classes of phosphodiesterase activity. One class of enzymes hydrolyses cyclic AMP (cAMP) and cyclic GMP (cGMP) with approximately equal rates. Another enzyme, which is less than 5% of the total activity, specifically hydrolyses cGMP. The cGMP-specific enzyme does not bind to a Con A-Sepharose column, while all the cAMP-hydrolyzing activities are retarded by this column. The cGMP-specific enzyme is activated by low cGMP concentrations (10^?8-10^?6 M); the enzyme has normal Michaelis-Menten kinetics at high substrate concentrations with a K_m of about 3–6 μM. The cGMP-binding sites for activation and for catalysis show different cyclic nucleotide specificity, but they are probably located on one protein with a molecular weight of about 70 000. The enzyme is stable only under specific conditions, and the activation property of the enzyme is lost relatively easy. Irreversible modifications occur at temperatures below 0° and above 30°C, and at pH below 6.0. Several other conditions such as high ion concentrations, temperatures just above 0°C and pH above 8.0 lead to reversibel modifications of enzyme activity.     

Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1?2}\text{s}$) and a class of slow-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15?150}\text{s}$). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase.     

Thermostable extracellular cyclic nucleotide phosphodiesterase of the Physarum polycephalum plasmodium

Cyclic nucleotide phosphodiesterase secreted by the Physarum polycephalum plasmodium was partially purified by ion-exchange chromatography on DEAE cellulose, ultrafiltration, and HPLC. The data obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing showed that the active enzyme in solution exists as a monomer of about 90 kDa with pI 3.6–4.0. The K _m values were 0.9 and 7.7 mM for cAMP and cGMP, respectively, whereas the maximal rates of hydrolysis of these nucleotides were virtually equal and reached several millimoles of hydrolyzed cyclic nucleotide per hour per milligram of enzyme. The partially purified enzyme was highly stable. It was not inactivated by heating at 100°C for 30 min. The enzyme remained active in the presence of 1% sodium dodecyl sulfate; however, it was completely inactivated under these conditions in the presence of β-mercaptoethanol.     

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.     

Independent regulation of the extracellular cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation by exogenous cyclic AMP in Dictyostelium discoideum.

When amoebae of Dictyostelium discoideum, suspended in buffer, were treated with 100 nM pulses of cAMP, the extracellular cAMP phosphodiesterase (ePD) activity increased dramatically and the synthesis of the phosphodiesterase inhibitor (PDI) was repressed. In addition, the time of appearance on the cell surface of contact sites A, membrane-bound cAMP phosphodiesterase, and cAMP binding sites was accelerated by 3–4 hr and the concentration of intracellular cAMP increased ?20-fold. When the concentration of the cAMP pulse was reduced to 1 nM, the effect of the pulses on membrane differentiation and on the cAMP pool was virtually the same, while the effect on the ePD-PDI system was reduced. When cAMP was added to the suspension continuously, the nucleotide had no effect on membrane differentiation and failed to stimulate the intracellular cAMP pool, however, the ePD-PDI system was regulated normally. When the developmental mutant, HC112, was treated with cAMP pulses, membrane differentiation and the level of the cAMP pool were unaffected, while the ePD-PDI system responded to the exogenous cAMP. In another mutant, HC53, membrane differentiation was stimulated by cAMP pulses and this response was accompanied by a sharp increase in the concentration of the cAMP pool. These results suggest that the ePD-PDI system and membrane differentiation are regulated independently by exogenous cAMP and that regulation of the ePD-PDI system does not require activation of the adenylyl cyclase.     

Cyclic AMP levels and turnover during development of the cellular slime mold Dictyostelium discoideum.

Changes in intracellular and extracellular cAMP levels are reported for the cellular slime mold Dictyostelium discoideum during its development on filter supports. Examined were axenically and bacterially grown strain A3 and bacterially grown NC-4. In each case a major peak in cAMP occurred during aggregation. In addition, axenically grown A3 showed minor rises in cAMP at 16 hr and during culmination; in contrast, NC-4 showed no increase at 16 hr but gave a very large increase at culmination. Both cell-associated phosphodiesterase and the extracellular phosphodiesterase present in the top filter were measured throughout development. Both showed activity peaks during aggregation with much lower plateau values thereafter. At aggregation about 80% of the activity per filter was contributed by the cell-associated phosphodiesterase. The rate of cAMP turnover during aggregation was estimated by following the hydrolysis of applied [³H]cAMP. A minimum rate of about 7% turnover/sec was obtained. From this turnover rate a minimum value for the stimulated activity of the adenylate cyclase was estimated as 224 pmoles/min-mg. Although this level is already over threefold greater than the highest value obtained in vitro, other experiments indicate that the in vivo adenylate cyclase activity may exceed 700 pmoles/min-mg.     

Veratridine induces a Ca2+ influx,cyclic GMP formation,and backward swimming inParamecium tetraurelia wildtype cells and Ca2+ current-deficient pawn mutant cells

Summary Veratridine opens voltage-dependent Na⁺ channels in many metazoans. InParamecium, which has voltage-dependent Ca²⁺ channels and a Ca/K action potential, no such Na⁺ channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine toParamecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca²⁺. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba²⁺, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba²⁺ and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca²⁺ influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na⁺ in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca²⁺, Mg²⁺, and Ba²⁺) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca²⁺ channels inParamecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca²⁺ channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier.     

Reduced cAMP secretion in Dictyostelium discoideum mutant HB3

Extracellular cAMP induces the intracellular synthesis and subsequent secretion of cAMP in Dictyostelium discoideum (relay). cAMP relay was strongly diminished in mutant HB3 which shows abnormal development by making very small fruiting bodies. Extracellular cAMP binds to receptors on the surface of mutant cells and induces the rapid activation of adenylate cyclase. Intracellular cAMP rises to a concentration as high as that in wild-type cells but only a very small amount of cAMP is secreted. cAMP secretion in wild-type cells starts immediately after cAMP production, and is proportional to the intracellular cAMP concentration. In the mutant cells cAMP secretion starts a few minutes after cAMP production; by that time most of the intracellular cAMP is already degraded by phosphodiesterase and little cAMP is available for secretion. We conclude that mutant HB3 has a defect in the mechanism by which Dictyostelium cells secrete cAMP.     

An intracellular modulator of the 3'5'-cGMP hydrolysis in growing Dictyostelium discoideum amoebae

A heat-stable and acid-stable macromolecular factor present in the cytosol of growing Dictyostelium discoideum amoebae affects specifically the intracellular cGMP phosphodiesterase. It decreases the V of the enzyme but does not alter its Km. It has no effect on the cAMP or cGMP hydrolysis catalyzed by the intracellular cAMP-cGMP phosphodiesterases or by the extracellular phosphodiesterase. It is also expressed in a mutant (HPX235), defective in the synthesis of the cAMP-cGMP phosphodiesterases but capable of intracellular transduction of the chemotactic signal. This factor is resistant to several nucleases, proteases and phospholipases, and has an apparent molecular weight between 3500-10000. In contrast, the protein phosphodiesterase inhibitor secreted by the amoebae exerts an opposite inhibition on the intracellular phosphodiesterases. These two inhibitory factors may regulate intracellular cGMP hydrolysis during the chemotactic response.     

Increased cyclic AMP content directly correlated with morphological transformation of cells infected with a temperature-sensitive mutant of mouse sarcoma virus

Summary Normal rat kidney cells infected with a cold-sensitive mutant of mouse sarcoma virus [NRK(MSV-1b)] morphologically transform when exposed to adenosine 3′∶5′ cyclic monophosphate (cAMP) at the restrictive temperature. The cAMP-induced morphological changes occur rapidly and are reversible. Agents capable of elevating endogenous levels of cAMP [prostaglandin E₁ (PGE₁) and cholera toxin (CT)] induced morphological transformation of NRK(MSV-1b) cells at the restrictive temperature that was concentration dependent, potentiated by cAMP phosphodiesterase inhibitors, and not prevented by inhibitors of DNA, RNA, and protein synthesis. Prostaglandin E₁ stimulated a transient increase in the intracellular level of cAMP with a concomitant morphological transformation and reversion of cells as cAMP levels decline. The maximum increase is reached by 10 min, followed by a decline to near basal level by 80 min. In contrast, incubation of cells with CT resulted in irreversible morphological transformation and increased levels of cAMP first detectable by 1 hr with maximum levels reached by 24 hr. Heated CT (100°C, 20 min) was without effect. Addition of CT to reverted PGE₁-treated cells resulted in morphological transformation suggesting the existence of discrete receptors in NRK (MSV-1b) cells. This research was supported by Grant BC-207 from the American Cancer Society and Cancer Research Emphasis Grant R01 CA 19714 within the Virus Cancer Program of the National Cancer Institute.     

Arachidonic acid is a chemoattractant for <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> cells

Cyclic AMP (cAMP) is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP₃), Ca²⁺ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca²⁺ concentration by causing the release of Ca²⁺ from intracellular stores and activating influx of extracellular Ca²⁺. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8–9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca²⁺ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycolbis(b-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P₃-receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca²⁺ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Cross-Regulation of Intracellular cGMP and cAMP in Cultured Human Corpus Cavernosum Smooth Muscle Cells

The goal of this study was to assess the potential cross-regulation of cyclic nucleotides in human corpus cavernosum (HCC). Incubation of primary cultures of HCC smooth muscle cells with either the NO donor sodium nitroprusside (SNP, 10 μM) or the phosphodiesterase type 5 (PDE 5) inhibitor sildenafil (50 nM) produced little or no changes in the intracellular cGMP levels. Incubation with both SNP and sildenafil produced marked increases in cGMP. Interestingly, incubation of cells with 10 μM of forskolin or PGE₁ produced significant enhancement of cGMP accumulation. These increases were not further enhanced by the addition of SNP and sildenafil. Kinetic analyses of cGMP hydrolysis by PDE 5 showed that high concentrations of cAMP reversibly inhibited the enzyme with a K_i of 258 ± 54 μM. The increase in cGMP levels in response to cAMP generating agents is not due to assay artifact since cAMP did not cross-react with cGMP antibody. Our data suggest that cAMP up-regulates intracellular levels of cGMP, in part, by inhibition of PDE 5. We also noted that cGMP down-regulates cAMP synthesis via a mechanism requiring G-protein coupling of adenylyl cyclase. These observations may have important implications in the utility of pharmacotherapeutic agents targeting cyclic nucleotide metabolism for the treatment of erectile dysfunction.     

Characterization of revertants of stmF mutants of Dictyostelium discoideum: Evidence that stmF is the structural gene of the cgmp-specific phosphodiesterase

stmF mutants of Dictyostelium discoideum produce long, banded aggregation streams on growth plates and exhibit altered cGMP metabolism. To learn more about the role of cGMP in chemotaxis and the nature of the defect in these mutants, 15 nonstreaming (Stm⁺) revertants of two stmF mutants were isolated and characterized. Fourteen of the revertants continued to show the elevated cAMP-induced cGMP response and very low cGMP-specific phosphodiesterase (cGPD) activity characteristic of their stmF parents. Parasexual genetic analysis revealed that many of these Stm⁺ revertants carried phenotypic suppressors unlinked to stmF. One Stm⁺ revertant, strain HC344, exhibited a low, prolonged cGMP response and relatively high cGPD activity throughout development. To determine whether the elevated cGPD activity in this revertant resulted from increased enzyme production or enhanced enzyme activity, cGPDs were partially purified from the wild-type strain, the stmF parent and revertant HC344, and properties of the enzymes were compared. cGPDs from the stmF mutant and the revertant showed similar differences from the wild-type enzyme in kinetic properties, thermal stability, and sensitivity to certain inhibitors. These results suggest that stmF is the structural gene of the cGPD. In addition, the unusual cGMP response in revertant HC344 appeared to be due to increased production of an altered cGPD.     

Inhibition of cyclic nucleotide phosphodiesterase activity by an endogenous factor.

Cyclic nucleotide phosphodiesterase activity of several tissues of rat is inhibited by an endogenous factor isolated from rat adipocytes following exposure of these cells to agents that raise intracellular cyclic AMP levels. The inhibitory action was demonstrated with varying cAMP concentrations from 0.1-400 muM. Enzyme from 10,000 X g supernatant of epididymal adipose tissue was inhibited approximately 2-3 fold more than the plasma membrane of adipocytes by a given concentration of the feedback regulator. Kinetic analysis of cAMP phosphodiesterase of plasma membrane showed that feedback regulator (8.8 U/ml) inhibited the Vmax 48%. The maximum inhibition of phosphodiesterase by feedback regulator (20 U/ml) was about 80%. The apparent Km for cAMP was increased. The ability of phosphodiesterase from several tissues of rat (10,000 X g supernatant) to hydrolyze cAMP and cGMP was tested. Feedback regulator inhibited cGMP hydrolysis in cardiac muscle and 5 other tissues 23-92% more than it inhibited the hydrolysis of cAMP. The physiological significance of this inhibitory effect can begin to be clarified when the feedback regulator is purified to homogeneity and characterized.     

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.