Internal Cyclic AMP Research Articles

Extracellular calcium modulates prereplicative cyclic AMP surges in EGF-stimulated primary neonatal rat hepatocytes.

The cells in nearly pure (96-98%) primary cultures of hepatocytes from neonatal rat liver in high (1.0 mM)-Ca2+, serum-free, synthetic HiWo5Ba2000 medium initiated DNA synthesis and entered mitosis between 11 and 30 h after the addition of 10 ng/ml EGF. During the 10-h prereplicative period, the cultured hepatocytes, like regenerating rat liver cells, generated two large cyclic AMP transients, one peaking between 30 min and 2 h and the other around 6 h. Hepatocytes stimulated by the same concentration of EGF in low (0.02 mM)-Ca2+ medium increased cyclic AMP synthesis as much as the EGF-treated hepatocytes in high-Ca2+ medium, but they released the additional cyclic AMP into the medium and could not generate prereplicative internal cyclic AMP surges, initiate DNA replication, or enter mitosis. These results suggest that one of the ways external Ca2+ controls prereplicative development of hepatocytes is to restrain the release of cyclic AMP and thus enable the cell to accumulate enough internal cyclic AMP to stimulate events required to initiate DNA replication.

Direct modulation by Ca2+–calmodulin of cyclic nucleotide-activated channel of rat olfactory receptor neurons

Olfactory receptor neurons depolarize in response to odorant stimulation of their sensory cilia. One transduction mechanism involves a G-protein-mediated increase in adenylate cyclase activity, raising the internal cyclic AMP concentration to open a cyclic nucleotide-activated cation channel on the plasma membrane. An influx of Ca2+ through this channel, which is permeable to both monovalent and divalent cations, triggers olfactory adaptation. Previous work has indicated that at least part of this Ca(2+)-mediated adaptation resides in the channel itself, but the mechanism remains unclear and controversial. Here we use the cloned channel from rat expressed in a cell line and the native channel from rat olfactory receptor cells to show that Ca2+ reduces the apparent affinity of the channel for cAMP by up to 20-fold in the presence of calmodulin, an abundant protein in olfactory cilia. This decrease in apparent affinity appears to involve a direct interaction between Ca(2+)-calmodulin and the channel, and it can reduce the activation of the channel by cAMP by up to a few hundred-fold, suggesting that it may be a key component of the Ca(2+)-triggered olfactory adaptation.

Co-existence of cationic and chloride components in odorant-induced current of vertebrate olfactory receptor cells.

Odorant stimulation leads to a depolarization of olfactory receptor neurons. A mechanism underlying this transduction, which occurs in the sensory cilia, involves a G-protein-mediated increase in adenylyl cyclase activity, and therefore a rise in internal cyclic AMP and consequent opening of a cAMP-gated cation channel on the plasma membrane. Another mechanism, not as well established, involves the opening of an inositol trisphosphate-activated cation channel on the plasma membrane as a result of phospholipase C activity. In both cases, an influx of cations is thought to generate the depolarizing receptor potential. We now report, however, that the mechanism is actually more complex. The odorant-induced current appears to contain an inward chloride component also, which is triggered by calcium influx through the cation-selective channel. This newly found chloride component can be as large as the cationic component. The co-existence of cationic and chloride components in the odorant response, possibly unique among sensory transduction mechanisms, may serve to reduce variations in the transduction current resulting from changes in external ionic concentrations around the olfactory cilia. Our finding can explain the long-standing puzzle of why removal of most mucosal cations still does not diminish the amplitude of the olfactory receptor cell response.

The ATP‐induced inward current in mouse lacrimal acinar cells is potentiated by isoprenaline and GTP.

1. ATP activates calcium (Ca2+) influx in mouse lacrimal acinar cells in the absence of phosphoinositide hydrolysis. Extracellular ATP (1 mM) activates receptor-operated cation channels, promoting entry of Na+ and Ca2+ (inward current). This Ca2+ influx in turn activates K+ channels resulting in a delayed, outward, current component. The present study uses patch-clamp current recording techniques to investigate the role of beta-adrenoceptor mechanisms, intracellular cyclic AMP and GTP in the regulation of the ATP-induced inward currents. 2. The beta-adrenoceptor agonist, isoprenaline (1 microM), does not increase the resting membrane currents but markedly enhances the ATP-induced inward and outward currents. This effect of isoprenaline is blocked by the beta-adrenoceptor antagonist propranolol. 3. Internal application of cyclic AMP mimics the potentiating effect of isoprenaline. 100 microM-cyclic AMP increases the ATP-induced inward and outward currents to about 200% as compared to control responses. 4. Pre-treatment of the cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), also results in a marked potentiation of the ATP-induced inward currents to 170% as compared to control responses. 5. The ATP-induced inward current responses are not blocked by either the removal of extracellular Ca2+ or by chelation of intracellular Ca2+ (by inclusion of 10 mM-EGTA in the recording pipette). Both protocols did however block the potentiating effect of internal cyclic AMP on the ATP-induced inward current responses. 6. Intracellular ATP (10 mM) reduces the amplitude of the inward currents evoked by external ATP application by about 60% and the currents were no longer potentiated by internal cyclic AMP. 7. Intracellular GTP or GTP-gamma-S (100 microM in the pipette solution) potentiates the current responses to ATP, increasing both the amplitude and duration of the inward currents. 8. In excised inside-out patches, with ATP in the recording pipette (i.e. external ATP), the catalytic subunit of the cyclic AMP-dependent protein kinase activated the cation channels. The effect of the catalytic subunit was readily reversible and abolished by an inhibitor of the protein kinase. 9. External ATP activates Ca2+ influx in lacrimal acinar cells by a mechanism that is distinct from that activated by phosphoinositide-coupled receptors. The effect is mediated by direct activation of cation channels in the cell surface membrane which allow for significant entry of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Mannosyl-phospho-dolichol synthase from Trichoderma reesei is activated by protein kinase dependent phosphorylation in vitro

Preincubation of microsomes from Trichoderma reesei QM 9414 with the catalytic subunit of cyclic AMP dependent protein kinase from bovine heart resulted in increased activity of the mannosylphospho-dolichol synthase (EC 2.4.1.83, ‘Dol-P-Man synthase’) present in the membranes. This effect decreased upon subsequent treatment of the microsomes with alkaline phosphatase. SDS-PAGE of microsomal proteins indicated the presence of a strong band at 32 kDa, which corresponds to the size of yeast Dol-P-Man synthase. Fluorography of microsomal proteins, after incubation with [γ-32P]ATP and protein kinase, revealed the incorporation of 32P into this 32-kDa as well as several other microsomal proteins. Dol-P-Man synthase activity, assayed at various times of growth, correlated with the rise and fall of the cyclic AMP pool in T. reesei. Addition of extracellular dibutyryl cyclic AMP and theophyllin, however, did not elevate the internal cyclic AMP pool in the fungus, and did not cause an effect on Dol-P-Man synthase activity.

Mannosyl-phospho-dolichol synthase fromTrichoderma reeseiis activated by protein kinase dependent phosphorylation in vitro

Abstract Preincubation of microsomes from Trichoderma reesei QM 9414 with the catalytic subunit of cyclic AMP dependent protein kinase from bovine heart resulted in increased activity of the mannosylphospho-dolichol synthase (EC 2.4.1.83, ‘Dol-P-Man synthase’) present in the membranes. This effect decreased upon subsequent treatment of the microsomes with alkaline phosphatase. SDS-PAGE of microsomal proteins indicated the presence of a strong band at 32 kDa, which corresponds to the size of yeast Dol-P-Man synthase. Fluorography of microsomal proteins, after incubation with [γ- 32 P]ATP and protein kinase, revealed the incorporation of 32 P into this 32-kDa as well as several other microsomal proteins. Dol-P-Man synthase activity, assayed at various times of growth, correlated with the rise and fall of the cyclic AMP pool in T. reesei . Addition of extracellular dibutyryl cyclic AMP and theophyllin, however, did not elevate the internal cyclic AMP pool in the fungus, and did not cause an effect on Dol-P-Man synthase activity.

Ca2+ channel currents in rat sensory neurones: interaction between guanine nucleotides, cyclic AMP and Ca2+ channel ligands.

1. The characteristics have been examined of the high threshold calcium channel current in cultured rat dorsal root ganglion (DRG) neurones recorded in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM in the patch pipette). This current, termed IBa, GTP gamma S, was slowly activating and showed little inactivation over 100 ms. 2. External application of forskolin (10 microM) to elevate internal cyclic AMP levels increased the amplitude of IBa, GTP gamma S whereas it had no effect on the control IBa. This cyclic AMP-dependent protein kinase (PKI; 25 microM). 3. The cyclic AMP-dependent phosphorylation induced enhancement of IBa, GTP gamma S was voltage dependent and either did not occur or was observed only transiently at a holding potential (VH) of -30 mV. The forskolin-stimulated enhancement seen at VH -80 mV was lost with a t1/2 of about 1 min when VH was depolarized to -30 mV. Cholera toxin pre-treatment also increased the amplitude of IBa, GTP gamma S at VH -80 mV but not at VH -30 mV. 4. The calcium channel antagonist (-)-202-791 (5 microM) increased the amplitude of IBa, GTP gamma S when applied at VH -80 mV, but either not, or only transiently, at VH -30 mV, as previously observed. This 'agonist' effect of (-)-202-791 was prevented by PKI and was occluded by prior enhancement of IBa, GTP gamma S with forskolin. (-)-202-791 did not increase cyclic AMP levels in DRG neurones. 5. The 'agonist' response of IBa, GTP gamma S to D600 (10 microM) was also occluded by application of forskolin (10 microM) in the patch pipette. Forskolin alone, applied in this manner, increased IBa, GTP gamma S to a similar extent to D600 applied alone. 6. The agonist effect of (+)-202-791 (5 microM) on IBa, GTP gamma S was not prevented by prior enhancement with forskolin, nor was it prevented by PKI. 7. In conclusion, internal GTP gamma S activates G proteins which may interact directly with calcium channels to influence the kinetics of activation and to reduce steady-state inactivation of the channels. There is also an indirect effect on the generation of second messengers such as cyclic AMP. It is likely that forskolin enhances IBa, GTP gamma S by increasing activated Gs coupling to adenylyl cyclase and increasing cyclic AMP generation. The mechanism of action of (-)-202-791 to enhance IBa, GTP gamma S also involves cyclic AMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Potassium and chloride conductances in rat Leydig cells: effects of gonadotrophins and cyclic adenosine monophosphate.

1. The effects of gonadotrophins (luteinizing hormone and human chorionic gonadotrophin) and cyclic AMP on ionic conductances were investigated using the tight-seal whole-cell recording technique in Leydig cells freshly isolated from nature rat testis by enzymatic treatment. 2. In resting cells, the predominant ionic conductance is a voltage-dependent K+ conductance resembling the delayed rectifier K+ conductance of T-lymphocytes. This conductance is characterized by: (1) a time-dependent inactivation for potentials more positive than +20 mV, (2) a reversal potential near -65 mV, (3) a sensitivity to intracellular Cs+, and (4) a sensitivity to extracellular TEA and 4-aminopyridine. 3. A Cl- conductance is also present resembling the Cl- background conductance in squid axons and heart cells. In resting cells, this conductance contributes only a small component of the total outward current obtained with depolarizing pulses. 4. Gonadotrophins (human chorionic gonadotrophin, porcine luteinizing hormone and ovine luteinizing hormone) have little effect on the K+ conductance. They transiently increase a Cl- conductance after a delay of up to 30 s. This response does not occur if the hormones are applied late in the whole-cell recording. Gonadoliberine (GnRH) does not affect the Cl- or K+ conductance. 5. Internal cyclic AMP (100 microM) mimics all these effects while internal application of a GTP-ATP mixture induces a similar response, which is, however, sustained rather than transient. 6. The Cl- conductance was studied quantitatively with a GTP-ATP internal solution. This conductance is activated by depolarizing voltage steps to test potentials of -40 mV or more. Under these conditions, the instantaneous current observed as soon as the depolarizing pulse is applied displays outward rectification and reverses near ECl. During the pulses, a strong inactivation is observed for potentials greater than +40 mV. This conductance is independent of external and internal calcium. 7. It is concluded that the gonadotrophins act through a cyclic AMP-dependent process to activate a Cl- conductance. This conductance is different to the hyperpolarization-activated Cl- conductance and the calcium-activated Cl-conductance also present in the membrane of resting cells.

Cyclic AMP analogs and retinoic acid influence the expression of retinoic acid receptor alpha, beta, and gamma mRNAs in F9 teratocarcinoma cells.

Retinoic acid (RA) receptor alpha (RAR alpha) and RAR gamma steady-state mRNA levels remained relatively constant over time after the addition of RA to F9 teratocarcinoma stem cells. In contrast, the steady-state RAR beta mRNA level started to increase within 12 h after the addition of RA and reached a 20-fold-higher level by 48 h. This RA-associated RAR beta mRNA increase was not prevented by protein synthesis inhibitors but was prevented by the addition of cyclic AMP analogs. In the presence of RA, cyclic AMP analogs also greatly reduced the RAR alpha and RAR gamma mRNA levels, even though cyclic AMP analogs alone did not alter these mRNA levels. The addition of either RA or RA plus cyclic AMP analogs did not result in changes in the three RAR mRNA half-lives. These results suggest that agents which elevate the internal cyclic AMP concentration may also affect the cellular response to RA by altering the expression of the RARs.

Cyclic AMP Analogs and Retinoic Acid Influence the Expression of Retinoic Acid Receptor α, β, and γ mRNAs in F9 Teratocarcinoma Cells

Retinoic acid (RA) receptor alpha (RAR alpha) and RAR gamma steady-state mRNA levels remained relatively constant over time after the addition of RA to F9 teratocarcinoma stem cells. In contrast, the steady-state RAR beta mRNA level started to increase within 12 h after the addition of RA and reached a 20-fold-higher level by 48 h. This RA-associated RAR beta mRNA increase was not prevented by protein synthesis inhibitors but was prevented by the addition of cyclic AMP analogs. In the presence of RA, cyclic AMP analogs also greatly reduced the RAR alpha and RAR gamma mRNA levels, even though cyclic AMP analogs alone did not alter these mRNA levels. The addition of either RA or RA plus cyclic AMP analogs did not result in changes in the three RAR mRNA half-lives. These results suggest that agents which elevate the internal cyclic AMP concentration may also affect the cellular response to RA by altering the expression of the RARs.

An investigation into the mechanisms of inhibition of calcium channel currents in cultured sensory neurones of the rat by guanine nucleotide analogues and (−)− baclofen

1. The mechanism of inhibition of calcium channel currents by the guanine nucleotide analogue guanosine 5'-O-3 thiotriphosphate (GTP-gamma-S) and by the GABAB agonist (-)-baclofen has been studied in cultured dorsal root ganglion neurones of the rat. The inhibition by GTP-gamma-S is particularly characterized by an abolition of the transient component of calcium channel currents carried either by Ba2+ (IBa) or by Ca2+ (ICa). 2. The effect of agents increasing intracellular cyclic AMP levels has been examined. Neither internal cyclic AMP nor forskolin prevented the inhibition of IBa by baclofen. Neither forskolin nor pretreatment of cells with cholera toxin prevented the inhibition of the transient component of IBa by GTP-gamma-S. However, both these treatments increased the amplitude of the sustained IBa in the presence of GTP-gamma-S. The ATP analogue adenosine imido-diphosphate which inhibits many ATP requiring enzymes did not prevent the effect of GTP-gamma-S although it reduced the amplitude of IBa. 3. Baclofen (100 microM) produced a 22 +/- 2% increase in inositol phosphate production in 30 s, whereas the increase produced by bradykinin (1 microM) was 70 +/- 14%. However, unlike baclofen, bradykinin did not inhibit IBa or ICa in these cells. 4. The effect of protein kinase C inhibitors was examined. Polymixin B (20 microM in patch pipette) had no effect on the inhibition of IBa by baclofen or GTP-gamma-S. A higher concentration (100 microM) alone inhibited IBa and no further inhibition by baclofen was observed. Neither H7 (50 microM) nor staurosporine (100 nM), applied extracellularly, prevented the response to GTP-gamma-S. 5. The protein kinase C activator di-octanoyl glycerol (20 microM) did not inhibit IBa. Arachidonic acid (100 microM) also produced no inhibition of IBa. 6. In conclusion we have obtained no evidence that a second messenger system mediates the inhibition of calcium channel currents by GTP-gamma-S or baclofen in dorsal root ganglion neurones. These results support the hypothesis that GABAB receptors are directly coupled to calcium channels by G proteins.

Effect of cyclic AMP-dependent hormones and Ca2+-mobilizing hormones on the Ca2+ influx and polyphosphoinositide metabolism in isolated rat hepatocytes.

The effect of the interaction between the Ca2+-mobilizing hormone adrenaline, used as alpha-adrenergic agonist, and cyclic AMP-dependent hormones, including beta-adrenergic agonists and glucagon, on the initial 45Ca2+ uptake rate and polyphosphoinositide metabolism were investigated in isolated rat hepatocytes. Each hormone alone increased the initial 45Ca2+ uptake rate. When adrenaline was added without inhibitor, it induced a rise in the initial 45Ca2+ uptake rate larger than the sum of the rises elicited by its alpha and beta components singly. Similarly, when adrenaline was used as an alpha-agonist and added together with glucagon, it enhanced the initial 45Ca2+ uptake rate synergistically. Kinetic analysis of the initial 45Ca2+ uptake rate measured at different Ca2+ concentrations suggested that the increased influx elicited by the combination of adrenaline as alpha-adrenergic agonist and glucagon reflects an activation of the rate of Ca2+ transport via a homogeneous population of Ca2+ channels or carriers. Dose-response curves for the alpha-adrenergic action of adrenaline or glucagon applied in the presence of increasing doses of glucagon or adrenaline showed that each hormone increases the maximal response to the other without affecting its ED50. Measurement of polyphosphoinositide hydrolysis and of the inositol phosphates formed in the presence of adrenaline or vasopressin and/or glucagon showed that Ca2+-mobilizing hormones and glucagon had no synergistic effects on inositol 1,4,5-trisphosphate production. It is therefore proposed that the synergistic action of glucagon and Ca2+-mobilizing hormones on Ca2+ influx occurs at a step that takes place close to the Ca2+ channels or carriers themselves. The Ca2+ gating involved might be mainly controlled by two products, one of them arising from the polyphosphoinositide metabolism, and the other from the increase in internal cyclic AMP.

3-O-methylglucose transport in internally dialysed giant axons of Loligo.

1. The transport of the non-metabolized sugar, 3-O-methylglucose, has been studied in the squid axon under conditions where the intracellular environment of the axon is controlled by internal dialysis. 2. Sugar transport is passive, shows saturation kinetics and is asymmetric. At 15 degrees C, the Michaelis and velocity constants for exit are approximately four times those for uptake. The asymmetry of transport is increased by raising the temperature. 3. Sugar uptake is not affected by intracellular sugar levels as high as 100 mM. Sugar exit is, however, reduced by external sugars although the apparent Km for exit is unaffected. 4. The kinetics of sugar exit under exchange conditions are determined by the kinetics of sugar uptake. These results can be accounted for by the asymmetric mobile-carrier and simultaneous-carrier models for transport. 5. Both sugar uptake and exit are reduced in the absence of ATPi. Kinetic analysis of transport under these conditions show that the capacity of the system to transport sugar is unchanged but that the affinity of the system for sugar is reduced. Internal cyclic AMP, AMP, ADP or GTP (2 mM) do not mimic this action of ATP. The hydrolysable analogue of ATP, alpha, beta-methylene-5-ATP (2 mM), (but not the nonhydrolysable analogue beta, gamma-methylene-5-ATP, 2 mM) has an ATP-like action on sugar transport. 6. Transport is unaffected by internal Ca2+ concentrations in the range 4 X 10(-8)--9 X 10(-7) M.

Relationship of internal cyclic AMP levels, rates of protein synthesis and mucor dimorphism.

Changes in the intracellular pools of cyclic AMP and specific rates of protein synthesis have been described as correlates of the yeast-to-hypha conversion in Mucor racemosis. A further examination of the relationship between these physiological parameters and the cellular morphogenesis was conducted in the present study. The levels of intracellular cyclic AMP consistently varied as a function of the cell morphology rather than the CO2 tension, oxygen tension or growth rate. The specific rate of protein synthesis failed to change during a N2-to-air-induced yeast-to-hypha transition. Previously reported changes in this parameter during CO2-to-air- and CO2-to-N2-induced yeast-to-hypha shifts may be a consequence of growth rate changes rather than development per se.

Evidence for a pronounced secretion of cyclic AMP by Tetrahymena.

The unicellular eukaryote Tetrahymena pyriformis secretes significant amounts of cyclic AMP into its external medium. Cells transferred from growth medium into any of the following three different non-nutrient media: (a) 5 mM phosphate buffer containing 47 mM NaCl and 1 mM MgSO4, (b) 10 mM Tris, or (c) 1.3 mM Tris containing 1 mM citrate and 1 mM Ca(OH)2, released to the outside almost 60--80% of the total cyclic AMP produced during 2--5 h of incubation. Tris-citrate-Ca+2 medium was chosen for further experiments because of its minimal nonspecific interference in the cyclic AMP radioimmunoassay. The identity of the secreted material recognized as cyclic AMP by radioimmunoassay was confirmed by demonstrating its almost complete hydrolysis with commerical beef heart phosphodiesterase. Furthermore, the radioimmunoassay-active material in the concentrated medium co-chromatographed on paper with [3H]cyclic AMP, as judged by assay of the eluted material. After resuspending cells in Tris-citrate-Ca2+ medium, the extracellular concentration of cyclic AMP rose steadily over a 5-h period, reaching a level equvalent to approximately 35--50 pmol cyclic AMP/10(6) cells vs. an internal cyclic AMP quantity at 5 h of 8--10 pmol/10(6) cells. After 5 h, the level of extracellular cyclic AMP reached a plateau. There was no degradation or uptake of external cyclic AMP by the cells during this period.

Cyclic AMP and its relation to ganglionic transmission. A combined biochemical and electrophysiological study of the rat superior cervical ganglion in vitro

The rat superior cervical ganglion (SCG) was investigated in vitro by means of gross morphological appearance, postganglionic nerve composition, fresh weight, protein content and cyclic nucleotides (cyclic AMP and cyclic GMP) concentrations. Electrophysiological recordings were performed with surface electrodes from both postganglionic nerve trunks after supramaximal electrical stimulation of preganglionic nerve fibres. Potentials recorded from the internal carotid nerve were different from those of the external carotid nerve in terms of their composition and stimulus to peak amplitude latency. Alpha-adrenergic agonists inhibited the compound action potentials (CAPs) via ganglionic alpha-receptors. The beta-adrenergic receptor in the rat ganglion, which is involved in the generation of cyclic AMP, did not interact with the inhibition of ganglionic transmission. There is no causal relationship between the internal cyclic AMP content and inhibition of CAPs. It is concluded that cyclic AMP plays no role in the inhibition of nerve pulses through the rat ganglion.

Regulation of intracellular adenosine cyclic 3':5'-monophosphate levels in Escherichia coli and Salmonella typhimurium. Evidence for energy-dependent excretion of the cyclic nucleotide.

Sugars and other energy sources were found to lower intracellular concentrations of adenosine 3':5'-monophosphate (cyclic AMP) in strains of Escherichia coli and Salmonella typhimurium which were deficient for cyclic AMP phosphodiesterase. This effect required the presence of the specific transport system responsible for entry of that sugar into the cell and depended on the intracellular catabolic enzymes. Metabolizable sugars were more effective than nonmetabolizable sugars in reducing cellular cyclic AMP levels, and this reduction was blocked partially by uncouplers of oxidative phosphorylation. Electron donors such as lactate and ascorbate plus phenazine methosulfate reduced internal cyclic AMP levels in bacterial membrane vesicles which had been preloaded with the cyclic nucleotide. Uncouplers of oxidative phosphorylation, but not arsenate, blocked the energy-stimulated loss of intravesicular cyclic AMP. Employing intact cells, sugars were shown to have two primary effects on cyclic AMP metabolism: (a) they inhibited net synthesis of the cyclic nucleotide while promoting its degradation, and (b) they stimulated efflux of cyclic AMP into the extracellular fluid. While the former effect was elicited by metabolizable and nonmetabolizable sugars alike, stimulation of cyclic nucleotide excretion was only observed with metabolizable sugars. The results suggest that the extrusion of cyclic AMP from the bacterial cell is energy-dependent and is driven by an energized membrane state.

Effects of progesterone and EDTA on cyclic AMP and phosphodiesterase in Dictyostelium discoideum

WHEN Dictyostelium discoideum amoebae are nutritionally deprived, they undergo a developmental programme which results in the formation of fruiting bodies composed of spore and stalk cells. Early events in this cycle are marked by the aggregation of previously individual amoebae. During this process they become elongated, and establish specific intercellular contacts, resulting in the formation of cell streams1. Orientation of cell movement during this period is thought to be directed by an external gradient of cyclic AMP (ref. 2). This compound elicits a chemotactic response from starved cells but not from growing cells3,4 and can induce a period of coordinated movement in nutritionally deprived cells5. A phosphodiesterase is also excreted by aggregating cells6,7 activity of which modulates the cyclic AMP gradient. Thus, a general model for communication between cells during aggregation is available. Little is known, however, about the regulation of metabolic events which result in the differentiation of growth phase cells into aggregation-competent, cyclic AMP-sensitive cells, and in particular, the influence of cyclic AMP on these processes. Since in other eukaryotes, changes in internal cyclic AMP are thought to mediate the actions of a variety of hormones, and regulate cell growth and development, we investigated the possible intracellular role of this nucleotide in D. discoideum.