cAMP-independent oscillations of adenylate cyclase in Dictyostelium discoideum

In starved Dictyostelium discoideum amoebae, extracellular cAMP appears to regulate adenylate cyclase activity such that synthesis of cAMP is rhythmic. Here we report that periodic modulation of adenylate cyclase also occurs via a cAMP-independent mechanism. This was demonstrated using cells which have high levels of adenylate cyclase activity, as measured in cell extracts, but which do not express this enzymic potential when intact. Such cells still rhythmically modify their adenylate cyclase activity and both the periodicity and the amplitude of the oscillations are similar to those seen in cells actively synthesizing cAMP. The phenomenon is observed using both wild-type cells and certain aggregation-minus mutants. The results implicate a mechanism which is cAMP independent in the regulation of adenylate cyclase as well as in the synchrony of the cell population.     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.     

Cytochemical localization of adenylate cyclase and 3',5'-nucleotide phosphodiesterase in Dictyostelium.

Cytochemical localizations of adenylate cyclase and 3′,5′-nucleotide phosphodiesterase were performed on aggregating Dictytostelium discoideum myxamoebae. The adenylate cyclase reaction product was localized on the inner surface of the plasma membrane. The phosphodiesterase reaction product was localized on the outer surface of the plasma membrane. Differences in enzyme activity were noted according to the state of cell (isolated or aggregated) and according to the cell position in larger aggregates. Heavy precipitation indicative of adenylate cyclase activity was not observed in isolated amoebae, but was often observed in streams and in some cells of aggregates. The precipitation indicative of phosphodiesterase activity could be found in isolated amoebae and in peripheral cells of aggregates.     

Conditions that elevate intracellular cyclic AMP levels promote spore formation in Dictyostelium

We have been using sporogenous mutants of Dictyostelium discoideum strain V12M2 to study regulation of cell fate during terminal differentiation of spores and stalk cells. Analyses of intracellular cAMP accumulation, cAMP secretion, cAMP binding to cell surface receptors, and chemotactic sensitivity to exogenous cAMP during aggregation showed that all of these functions were identical in V12M2 and HB200, a sporogenous mutant. We used several methods of altering intracellular cAMP levels in HB200 cells to test the hypothesis that intracellular cAMP levels affect cell fate. First, HB200 amoebae were treated with 5 mM caffeine for 4 h during growth, washed, and allowed to develop in the absence of caffeine. Treated cells had normal levels of intracellular cAMP and adenylate cyclase activities at the beginning of differentiation; by 6 h development, they contained two to three times more intracellular cAMP and two times more GTP-dependent adenylate cyclase activity than untreated cells. However, their level of basal Mn++-dependent adenylate cyclase activity was the same as untreated controls. Thus, treatment of growing HB200 amoebae with caffeine for only 4 h leads to hyperinduction of a GTP-dependent regulator (or inhibition of a negative regulator) of adenylate cyclase during subsequent differentiation, without induction of basal activity. The fraction of amoebae forming spores increased twofold when HB200 amoebae were treated with caffeine during growth. Spore (but not stalk cell) differentiation by such treated cells was blocked by inhibitors of cAMP accumulation. Second, cells grown on nutrient agar accumulated higher levels of intracellular cAMP and formed more spores in vitro than cells grown in shaken suspension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine and its derivatives inhibit the cAMP signaling response in Dictyostelium discoideum

In developmentally competent Dictyostelium discoideum amoebae, binding of cAMP to high-affinity surface receptors produces a rapid activation of adenylate cyclase which adapts within minutes. The result is a transient increase in intracellular cAMP which is rapidly secreted. Adenosine inhibited this cAMP signaling response with an apparent Ki of 300 microM. The apparent Ki's for 2'-O-methyladenosine and 2-chloroadenosine were approximately 30 and 100 microM, respectively. Inhibition by adenosine was rapid, reversible, and depended on the cAMP stimulus concentration. In addition, the adaptation of the cAMP signaling response was blocked by adenosine. As has been previously reported, adenosine inhibits cAMP binding to intact cells. Under the same developmental conditions as in the perfusion studies, we find the binding inhibition depends on both the cAMP and adenosine concentrations, with an apparent Ki of 100 microM. The apparent Ki's for 2'-O-methyl- and 2-chloroadenosine were approximately 8 and 35 microM, respectively. However, with cells developed for short times and with an axenic strain, inhibition was independent of cAMP concentration or cells showed mixed-type binding inhibition. The effect of adenosine on the cAMP signaling response is consistent with the reported effects of adenosine on other cAMP-mediated processes such as chemotaxis and the increase in intracellular cGMP, and may provide an explanation for the reported inhibition of center formation.     

A stage-specific inhibitor of adenylate cyclase in Dictostelium discoideum

A sudden increase in adenylate cyclase activity occurs during the chemotaxis and aggregation of Dictyostelium discoideum. Preincubation of extracts from the pre-aggregation stage, in which adenylate cyclase activity was low, with post-aggregation stages, in which the increase in activity occurred, resulted in the demonstration of a heat-stable inhibitor of adenylate cyclase (ACI) that was present only during the early stages of development. Cellular fractionation studies showed that ACI was present in both the 100 000 g pellet and supernatant fractions. The inhibitor was not inactivated by proteases or protease inhibitors. A heat-treated preparation of the inhibitor was dialysable. The effect of ACI was dependent upon a pre-incubation treatment, with notable inhibition occurring only after a 20 min pre-incubation period. The apparent inhibition was not artifactual, due to the degradation of the substrate, ATP, or to the loss of the reaction product, cAMP. Additionally, the inhibitor was specific for adenylate cyclase, as it had no effect on the activity of several other enzymes, including cAMP phosphodiesterase.     

Effects of amino acids and glucose on adenylate cyclase and cell differentiation of Dictyostelium discoideum.

Starvation triggers the differentiation of Dictyostelium discoideum amoebas to aggregation competence. To determine more precisely the nature of the starvation signal, the ability of various components of the growth medium to inhibit differentiation was examined. Changes in adenylate cyclase (the enzyme which generates the cAMP pulses basic to the differentiation process), various physiological and biochemical markers of developing cells, and the ability of amoebas to form specific intercellular contacts were monitored. We show that amino acid mixtures inhibit cell differentiation by preventing the increase of adenylate cyclase activity which normally occurs during the early hours of starvation. High concentrations of glucose also inhibit the differentiation process but at a later stage: The rise in adenylate cyclase still occurs when cells are starved in the presence of sugar, but the enzyme does not appear to function in vivo. Exogenously generated cAMP pulses are not able to bypass the block exerted by amino acids but can bypass the block exerted by glucose. Results support the hypothesis that the presence of amino acids inhibits adenylate cyclase synthesis, while the presence of 3% glucose blocks endogenous activation of adenylate cyclase, perhaps as a consequence of high osmotic pressure.     

Mechanisms of muscarinic modulation of protein phosphorylation in intact ventricles

Muscarinic agonists inhibit cyclic AMP (cAMP)-induced phosphorylation of the cardiac protein phospholamban. The mechanism of this muscarinic inhibition of phosphorylation of phospholamban appears to occur at more than one level in the series of reactions comprising the adenylate cyclase, cAMP-dependent protein kinase system. Muscarinic agonists attenuate hormone and drug stimulation of cardiac adenylate cyclase. This results in reduced tissue levels of cAMP and diminished phosphorylation of cardiac proteins and consequent inhibition of biochemical and inotropic effects of drugs that act via cAMP. The mechanism of muscarinic inhibition of adenylate cyclase is only partially understood, but probably involves the inhibitory guanine nucleotide-binding regulatory protein. In addition to the inhibition of adenylate cyclase, muscarinic agonists appear to be able to inhibit the effects of cAMP. The mechanism for this second effect of muscarinic agonists is unknown.     

Interferon-gamma down-regulates adenosine 2b receptor-mediated signaling and short circuit current in the intestinal epithelia by inhibiting the expression of adenylate cyclase

Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.     

Calcium ion effects on cyclic adenosine 3′:5′-monophosphate bindings to the plasma membrane of Dictyostelium discoideum

The study of cell surface cyclic adenosine 3′:5′-monophosphate binding to Dictyostelium discoideum amoebae indicates that Ca²⁺ increases the number of binding sites without significantly affecting their affinity constant(s). The effects of the ion are observed immmediately (within 4 s after addition) and appear to be readily reversible. Ca²⁺ effects are observed at various temperatures and pH values and are not blocked by the presence of various metabolic inhibitors. Increases, and decreases, in the apparent number of cyclic nucleotide binding sites could also be effected by concanavalin A treatments which respectively stimulate, and inhibit cell differentiation.     

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.     

Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP gamma S and GDP beta S on cAMP binding are reduced; the stimulatory effect of cAMP on GTP gamma S binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum.     

A Model Based on Receptor Desensitization for Cyclic AMP Signaling in Dictyostelium Cells

We analyze a model based on receptor modification for the cAMP signaling system that controls aggregation of the slime mold Dictyostelium discoideum after starvation. The model takes into account both the desensitization of the cAMP receptor by reversible phosphorylation and the activation of adenylate cyclase that follows binding of extracellular cAMP to the unmodified receptor. The dynamics of the signaling system is studied in terms of three variables, namely, intracellular and extracellular cAMP, and the fraction of receptor in active state. Using parameter values collected from experimental studies on cAMP signaling and receptor phosphorylation, we show that the model accounts qualitatively and, in a large measure, quantitatively for the various modes of dynamic behavior observed in the experiments: (a) autonomous oscillations of cAMP, (b) relay of suprathreshold cAMP pulses, i.e., excitability, characterized by both an absolute and a relative refractory period, and (c) adaptation to constant cAMP stimuli. A two-variable version of the model is used to demonstrate the link between excitability and oscillations by phase plane analysis. The response of the model to repetitive stimulation allows comprehension, in terms of receptor desensitization, of the role of periodic signaling in Dictyostelium and, more generally, the function of pulsatile patterns of hormone secretion.     

The relationship of phosphodiesterase to the developmental cycle of Dictyostelium discoideum.

Determination of the rate of total phosphodiesterase production by Dictyostelium discoideum shows that a dramatic rise in enzyme production occurs after 3 hours of cell starvation. Use of imposed cAMP pulses indicate that this increase is related to the developmental program of the amoebae and is probably due to a stimulation of adenyl cyclase.     

A stage-specific inhibitor of adenylate cyclase in Dictyostelium discoideum

A sudden increase in adenylate cyclase activity occurs during the chemotaxis and aggregation of Dictyostelium discoideum. Preincubation of extracts from the pre-aggregation stage, in which adenylate cyclase activity was low, with post-aggregation stages, in which the increase in activity occurred, resulted in the demonstration of a heat-stable inhibitor of adenylate cyclase (ACI) that was present only during the early stages of development. Cellular fractionation studies showed that ACI was present in both the 100 000 g pellet and supernatant fractions. The inhibitor was not inactivated by proteases or protease inhibitors. A heat-treated preparation of the inhibitor was dialysable. The effect of ACI was dependent upon a pre-incubation treatment, with notable inhibition occurring only after a 20 min pre-incubation period. The apparent inhibition was not artifactual, due to the degradation of the substrate, ATP, or to the loss of the reaction product, cAMP. Additionally, the inhibitor was specific for adenylate cyclase, as it had no effect on the activity of several other enzymes, including cAMP phosphodiesterase.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin

Cholera toxin is known to inhibit lymphocyte activation in vitro, an effect attributed to its ability to activate adenylate cyclase and increase intracellular cyclic adenosine monophosphate. In these studies the effects of both cholera toxin (CT) and its purified binding subunit (CT-B) on lymphocyte proliferation in vitro was examined, using a variety of cell activators. We found that both CT and CT-B inhibited mitogen- and antigen-induced T cell proliferation and anti-IgM-induced B cell proliferation in a dose-dependent manner. However, only CT-inhibited lipopolysaccharide-induced B cell proliferation. Neither CT nor CT-B inhibited antigen uptake and presentation by macrophages. The CT-B preparation used was shown not to activate lymphocyte adenylate cyclase, although CT itself was a strong activator of this enzyme. Both molecules had to bind to the lymphocyte surface in order to inhibit. The time course of inhibition of both CT and CT-B was similar in that either could be added up to 24 hr after culture initiation and still inhibit substantially. The addition of excess human recombinant interleukin 2 to the cultures did not affect the inhibition by CT, and had only a partial affect on inhibition by CT-B. Similarly, CT was able to substantially inhibit recombinant interleukin 2-dependent T lymphoblast proliferation, whereas CT-B had only a small inhibitory effect. Inhibition was not major histocompatibility complex-restricted. We conclude that the binding of CT or CT-B to the lymphocyte surface membrane interferes in some way with the activation mechanism leading to proliferation. The inhibition mediated by CT-B does not involve the stimulation of intracellular adenylate cyclase. CT appears to inhibit both by binding via its B subunit and by activation of adenylate cyclase via its A subunit.     

In vitro proliferation of murine spleen cells: inhibition by monoclonal antibodies to L3T4 and Lyt-2 T cell markers or intracellular cyclic adenosine monophosphate

Proliferation of normal (not immunized intentionally) murine spleen cells was elicited with concanavalin A, supernatant fluid from cultures of EL-4 cells, human recombinant interleukin 2 (IL-2), or a mixture of phorbol ester and calcium ionophore A23187. IL-2-induced proliferation was inhibited by membrane-permeable dibutyryl cyclic adenosine monophosphate (cAMP) or by the adenylate cyclase activator forskolin. Consistent with these observations was the finding that stimulation with IL-2 decreased and forskolin increased the intracellular content of cAMP. IL-2-induced proliferation, as well as that induced by concanavalin A or phorbol-ionophore mixture, was inhibited by monoclonal antibodies specific for L3T4 or Lyt-2 cell surface markers. This inhibition was observed even when antibodies were added several hours after exposure of cells to IL-2. Notably, antibodies did not alter the intracellular content of cAMP. Thus, the experimental data failed to establish a functional linkage between the inhibitory effect of antibodies and the regulatory effect of the adenylate cyclase system. However, our results provide a rational basis for the postulation that antibodies, upon binding to their corresponding ligands, generate a negative signal that interferes with IL-2-induced proliferation. Therefore, L3T4 and Lyt-2 molecules appear to play an important role in the regulation of lymphocyte proliferation.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7