The specificity of the cAMP receptor mediating activation of adenylate cyclase in Dictyostelium discoideum

In Dictyostelium discoideum amoebae, binding of cyclic AMP (cAMP) to surface receptors elicits numerous responses including chemotaxis, cyclic GMP (cGMP) accumulation, and activation of adenylate cyclase. The specificity of the surface cAMP receptor which mediates activation of adenylate cyclase and cAMP secretion was determined by testing the relative effectiveness of a series of 10 cAMP analogs. Each of the 10 analogs elicited cAMP secretion, chemotaxis, and cGMP accumulation in the same dose range. The order of potency for eliciting these responses (cAMP greater than 2'-H-cAMP greater than N1-O-cAMP greater than cAMPS(Sp) greater than 6-Cl-cAMP greater than cAMPN(CH3)2(Sp) greater than 3'-NH-cAMP greater than 8-Br-cAMP greater than cAMPS(Rp) greater than cAMPN(CH3)2(Rp] matches that for binding to the major cell surface cAMP binding sites and differs from that of the cell surface phosphodiesterase and the major intracellular cAMP binding protein.     

Transport and metabolism of N6- and C8-substituted analogs of adenosine 3',5'-cyclic monophosphate and adenosine 3'5'-cyclic phosphorothioate by the isolated perfused rat kidney

The clearance and metabolism of N6-substituted (N6-dimethyl-), C8-substituted (8-bromo-, 8-p-chlorophenylthio- (PCPT-)), and exocyclic oxygen substituted phosphorothioate diastereomers (cAMPS(Sp)) and cAMPS (Rp)) of adenosine 3':5'-monophosphate (cyclic AMP, cAMP) has been studied in an isolated perfused rat kidney. The N6- and C8-substituted analogs of cyclic AMP (10-100 microM) were not cleared as rapidly as exogenous cyclic AMP and were metabolized: N6- and C8-substituted analogs of adenosine accumulated in perfusate and urine. All analogs exhibited net transtubular secretion, i.e. their urinary excretion rate greater than glomerular filtration rate. Probenecid (0.9 mM) included in the perfusate abolished transtubular secretion and inhibited the metabolism of PCPT-cyclic AMP, suggesting that cyclic AMP analogs, like cyclic AMP itself, penetrate the renal cell at the peritubular membrane by an organic acid transport system. The phosphorothioate diastereomers of cyclic AMP: cAMPS(Sp) and cAMPS(Rp) were cleared as rapidly from the perfusate as cyclic AMP, were extensively secreted (urinary excretion/ glomerular filtration greater than or equal to 10) and exhibited no metabolism. The latter analog would seem most suitable as an intracellular agonist for cyclic AMP-mediated phenomena in the rat kidney.     

The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst-Forming Strain of Dictyostelium mucoroides

A mutant MF1 previously isolated from Dictyostelium mucoroides -7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one-to-one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst-inducing substance(s) and cAMP at the aggregation stage.     

Transmethylation inhibitors decrease chemotactic sensitivity and delay cell aggregation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular cyclic AMP (cAMP) induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells respond to a cAMP pulse with a rapid, transient increase of protein carboxyl methylation. The transmethylation inhibitors cycloleucine, L-homocysteine thiolactone, and coformycin decrease chemotactic sensitivity and delay cell aggregation when administered in concentrations which do not influence cAMP binding to cell surface receptors or the activity of total phosphodiesterase. The ability of the drugs to inhibit chemotaxis could be correlated with their capacity to convert the initial transient positive response of carboxyl methylation to cAMP into a negative one. This suggests that both protein O-methyltransferase and protein methylesterase are activated after stimulation of aggregative cells with cAMP, the net effect being a transient, positive response of methylation. In the presence of a sufficiently large dose of inhibitor, methyltransferase is inhibited, whereas methylesterase activity is much less affected, so that a transient negative response of methylation to cAMP is observed. The slow, positive response of carboxyl methylation to cAMP which occurs ca. 2.5 to 5 min after stimulus administration is not affected by inhibitors of transmethylation. These results suggest that methylation reactions are involved in the chemotactic response of D. discoideum cells to cAMP.     

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.     

The possible involvement of oscillatory cAMP signaling in multicellular morphogenesis of the cellular slime molds

The involvement of pulsatile chemoattractant emission and signal relay in aggregation and multicellular morphogenesis of a variety of cellular slime mold species was investigated. The species differ from each other in the developmental stage when pulsatile signaling first becomes evident. In D. discoideum, D. mucoroides, and D. purpureum pulsatile signal emission starts in the preaggregative field. In D. vinaceo-fuscum, D. mexicanum, P. violaceum, and P. pallidum the aggregation centers shifts from continuous to pulsatile secretion of chemoattractant during the aggregation process. In D. minutum pulsatile signaling starts after the completion of aggregation and slightly before the onset of culmination. Tip formation is a consequence of continued attraction of amoebae inside the aggregate to the center of signal emission. The occurrence of pulsatile signaling at an early stage of development is correlated with the capacity of the tip (signaling center) to organize a relatively large number of cells into a single fruiting body. Several lines of evidence suggest that cAMP is probably involved in the coordination of morphogenetic movement in the multicellular stage of all investigated species.     

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.     

Inhibition of cAMP-dependent protein kinase by adenosine cyclic 3'-, 5'-phosphorodithioate, a second cAMP antagonist

A single sulfur substitution for either the axial or the equatorial exocyclic oxygen of adenosine cyclic 3', 5'-phosphate (cAMP) results in diastereometric phosphorothioate analogs of cAMP with agonist versus antagonist properties towards activation of cAMP-dependent protein kinase. Sulfur substitutions for both of the exocyclic oxygens of cAMP results in a dithioate analog of cAMP, adenosine cyclic 3', 5'-phosphorodithioate (cAMPS2), which has antagonist properties. cAMPS2 displaced [3H]cAMP from the binding sites on bovine heart Type II cAMP-dependent protein kinase as demonstrated by equilibrium dialysis experiments with an apparent Kd of 6.3 microM. The addition of 10, 30, or 100 microM cAMPS2 when measuring cAMP-induced activation of pure porcine heart Type II cAMP-dependent protein kinase resulted in a concentration-dependent increase in the amount of cAMP required to produce half-maximal activation (EC50). A plot of the EC50 values as a function of the cAMPS2 concentration resulted in a straight line from which a KI value of 4 microM was derived. cAMPS2 had no significant effect on the degree of cooperativity (n) of cAMP activation of the holoenzyme. These data suggest that the most important structural requirement for the dissociation of the holoenzyme is an equatorial exocyclic oxygen.     

Sensory adaptation of Dictyostelium discoideum cells to chemotactic signals

Postvegetative Dictyostelium discoideum cells react chemotactically to gradients of cAMP, folic acid, and pterin. In the presence of a constant concentration of 10(-5) M cAMP cells move at random. They still are able to respond to superimposed gradients of cAMP, although the response is less efficient than without the high background level of cAMP. Cells which are accommodated to 10(-5) M cAMP do not react to a gradient of cAMP if the mean cAMP concentration is decreasing with time. This indicates the involvement of adaptation in the detection of chemotactic gradients: cells adapt to the mean concentration of chemoattractant and respond to positive deviations from the mean concentration. Cells adapted to high cAMP concentrations react normally to gradients of folic acid or pterin. Adaptation to one of these compounds does not affect the response to the other attractants. This suggests that cAMP, folic acid, and pterin are detected by different receptors, and that adaptation is localized at a step in the transduction process before the signals from these receptors coincide into one pathway. I discuss the implications of adaptation for chemotaxis and cell aggregation.     

Apparent Positive Cooperativity at a Surface cAMP Receptor in Dictyostelium

In the large species of the cellular slime mold Dictyostelium , cell aggregation is regulated by extracellular cAMP. During aggregation, cAMP is released in pulses from cells in the aggregation centers and these rhythmic signals are propagated through the population by a signal relay system. In addition to triggering the relay response, the pulsatile signals also regulate the chemotactic movement of the cells and early cell differentiation. These different cellular responses to exogenous cAMP are thought to be mediated via cAMP receptors, which appear on the cell surface shortly after starvation.
Using a sensitive assay, the equilibrium binding properties of these receptors were analyzed at low cAMP concentrations. As reported earlier, Scatchard plots of cAMP binding to preaggregative amoebae of D. discoideum strain NP187 in the concentration range 2–500 nM were curvilinear suggesting either receptor heterogeneity or negative cooperative interactions. However, at cAMP concentrations below approximately 1.5 nM, the affinity of the receptors was found to decline as a function of decreasing receptor occupancy. This apparent positive cooperativity was observed with binding sites on crude plasma membranes as well as on intact cells, and it occurred at both 0°C and 22°C. Moreover, apparent positive cooperativity was a property of the receptors on all strains of D. discoideum examined and on one strain of D. purpureum . Unlike preaggregative cells, receptors on postaggregative cells often lacked this property.
The lowest concentration of cAMP pulses that can appreciably stimulate membrane differentiation in strain NP187 was found to be 0.15–1.5 nM. Since similar concentrations of exogenous cAMP have been reported to trigger minimal chemotactic and relay responses in D. discoideum , the apparent positive cooperative behavior of the cAMP receptor might function to generate a steep cellular response threshold.     

ROLE OF CYCLIC AMP IN THE POLARIZED MOVEMENT OF THE MIGRATING PSEUDOPLASMODIUM OF DICTYOSTELIUM DISCOIDEUM

Cyclic AMP is known to act as a chemotactic agent that directs the movement of aggregating Dictyostelium discoideum cells. Its role in the multicellular organization of this organism was studied with special reference to the polarized movement of the migrating pseudoplasmodium (slug). The results showed that the tip of the slug has the ability to function as an aggregation center, and that slug cells are chemotactically sensitive to cyclic AMP. The addition of calcium or magnesium appeared to enhance formation of cell streams, thus facilitating detection of chemotactic response of slug cells, but this addition was not required for the response itself. These indicate that the polar movement of the slug may be principally controlled by cyclic AMP.     

The influence of light and different ATP concentrations on cell aggregation in cyclic AMP sensitive and insensitive species of the cellular slime molds

The influence of light and different concentrations of ATP on cell aggregation in cyclic AMP sensitive (Dictyostelium mucoroides, D. purpureum) and cyclic AMP insensitive species (Polysphondylium violaceum, P. pallidum, D. lacteum) of the cellular slime molds was observed in small and in large amoebal populations.Both light and ATP (optimal concentration:10^-5M) accelerated cell aggregation and increased the number of aggregating centers in large populations. For cyclic AMP sensitive species the effect of ATP in large populations was more pronounced than for the species that do not react to cyclic AMP.A possible explanation for the similar effect of light and ATP has been discussed.     

Correlation between acrasins and spore germination inhibitors in cellular slime molds.

Discadenine,3-(3-amino-3-carboxypropyl)-N6-delta 2-isopentenyladenine, which inhibits spore germination, was previously found in Dictyostelium discoideum. Studies on the distribution of discadenine in different species of cellular slime molds by high-pressure liquid chromatography showed that discadenine is present in D. discoideum, Dictyostelium purpureum, and Dictyostelium mucoroides, but not in Dictyostelium minutum, Polysphondylium violaceum, or Polysphondylium pallidum. Discadenine synthetase, which is involved in biosynthesis of discadenine with N6-delta 2-isopentenyladenine as substrate, was only detected in cells of the former three species. In addition, discadenine inhibited spore germination only in these three species. These results clearly demonstrate that discadenine is produced as an inhibitor of spore germination in the species of cellular slime molds in which the acrasin is cyclic adenosine 5'-monophosphate (AMP). This means that there is a structural and biochemical correlation between the spore germination inhibitor and the acrasin, since 5'-AMP, a direct precursor in discadenine biosynthesis, can be derived from cyclic AMP by hydrolysis with cyclic AMP phosphodiesterase.     

Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.     

Chemotaxis and binding of cyclic AMP in cellular slime molds.

To obtain more information about how cyclic AMP mediates cell aggregation as found in some species of the cellular slime molds, we determined the maximal binding activity of cyclic AMP in different species under various environmental conditions. The binding of cyclic AMP is limited to amoebae using this cyclic nucleotide as chemotactic agent. Maximal binding activity proved to coincide with a maximal chemotactic response and to be related to the length of the period between the vegetative and the aggregative phase. Of the species studied, Dictyostelium discoideum has the highest cellular density of cyclic AMP receptors and is the most sensitive to cyclic AMP as attractant. At 15 degrees C, aggregation begins later, chemotaxis takes effect over a greater distance, and the maximal binding activity is higher than 22 degrees C. The number of cyclic AMP receptors is independent of temperature. The delay in the onset of aggregation and the increased chemotactic response in darkness is not due to a change in the maximal binding activity. The binding of cyclic AMP and its inactivation is discussed in the light of cell aggregation.     

Analysis of the monocyte chemotactic response to lysophosphatidylcholine: role of lysophospholipase C

Previously, we reported that lysophosphatidylcholine (lyso-PtdCho), a component of oxidized low-density lipoprotein, was a monocyte chemoattractant (M.T. Quinn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2805-2809). Monocyte chemotaxis was also stimulated by lyso-platelet activating factor but not by platelet activating factor itself. In the present studies, we used other analogs of lyso-PtdCho to determine structural and metabolic features required for chemotactic activity. Although both D- and L-lyso-PtdCho stimulated chemotaxis, suggesting a lack of stereospecificity, studies using propanediol and ethanediol analogs of lyso-PtdCho suggested that a free hydroxyl moiety or an ester-linked fatty acid vicinal to the phosphocholine group of the lysophospholipid was required for the expression of activity. Incubation of [3H]choline-labeled lyso-PtdCho with monocytes resulted in the formation of labeled PtdCho, glycerophosphocholine (GPC), phosphocholine, and free choline, while resident peritoneal macrophages, cells which we show do not respond chemotactically to lyso-PtdCho, metabolized the labeled substrate to generate only labeled PtdCho and GPC; no labeled phosphocholine was found, suggesting a possible role for lysophospholipase C activity in the monocyte chemotactic response. Although monoacylglycerol, the product of lysophospholipase C hydrolysis of lyso-PtdCho, was not chemotactic for monocytes, diacylglycerol demonstrated chemotactic activity, suggesting that the subsequent acylation to diacylglycerol may be involved in the monocyte chemotactic response to lyso-PtdCho. Indeed, monocytes incorporated [3H]glycerol from [3H]glycerol-labeled lyso-PtdCho into di- and triacylglycerol. Based on these results, a model is proposed whereby the monocyte chemotactic response to lyso-PtdCho involves a sequence of metabolic steps which includes hydrolysis of lyso-PtdCho to monoacylglycerol and phosphocholine by lysophospholipase C followed by acylation of monoacylglycerol to diacylglycerol. Diacylglycerol would then act as an intracellular second messenger that could activate or facilitate the chemotactic response.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.     

Chemotaxis and binding of cyclic AMP in cellular slime molds

To obtain more information about how cyclic AMP mediates cell aggregation as found in some species of the cellular slime molds, we determined the maximal binding activity of cyclic AMP min different species under various environmental conditions. The binding of cyclic AMP is limited to amoebae using this cyclic nucleotide as chemotactic agent. Maximal binding activity proved to coincide with a maximal chemotactic response and to be related to the lenght of the period between the vegetative and the aggregative phase. Of the species studied, Dictyostelium discoideum has the highest cellular density of cyclic AMP receptors and is the most sensitive to cyclic AMP as attractant.At 15°C, aggregation begins later, chemotaxis takes effect over a greater distance, and the maximal binding activity is higher than at 22°C. The number of cyclic AMP receptors is independent of temperature. The delay in the onset of aggregation and the increased chemotactic response in darkness is not due to a change in the maximal binding activity. The binding of cyclic AMP and its inactivation is discussed in the light of cell aggregation.