Michiel M. van Lookeren Campagne

Altered cGMP-phosphodiesterase activity in chemotactic mutants of Dictyostelium discoideum.

Chemotaxis is very important during the whole life cycle of the cellular slime molds. In the vegetative stage the amoebae have to find their bacterial food in the soil which they inhabit. At this time, the amoebae are chemotactic to folic acid and pterin [ 1,2], both of which are excreted by bacteria; therefore it seems probable that this mechanism is used to find food [l]. When the food source is exhausted the amoebae aggregate to form a multicellular slug which then differentiates into a fruiting body. Different species of slime mold use different compounds as the chemoattractant for this aggregation. The best studied system is Dictyostelium discoideum which utilizes pulsatile signals of CAMP [3]; however, chemoattractants from other species have been partially purified [4,5]. of the change during chemotactic stimulation [14,15] which suggests that it may play an important role in the transduction of the signal.

Postaggregative differentiation induction by cyclic AMP in Dictyostelium: Intracellular transduction pathway and requirement for additional stimuli

Cyclic AMP induces postaggregative differentiation in aggregation competent cells of Dictyostelium by interacting with cell surface cAMP receptors. We investigated the transduction pathway of this response and additional requirements for the induction of postaggregative differentiation. Optimal induction of postaggregative gene expression requires that vegetative cells are first exposed to 2-4 hr of nanomolar cAMP pulses, and subsequently for 4-6 hr to steady-state cAMP concentrations in the micromolar range. Cyclic AMP pulses, which are endogenously produced before and during aggregation, induce full responsiveness to cAMP as a morphogen. The transduction pathway from the cell surface cAMP receptor to postaggregative gene expression may involve Ca2+ ions as intracellular messengers. A cAMP-induced increase in intracellular cAMP or cGMP levels is not involved in the transduction pathway.

Specificity of Adenosine Inhibition of cAMP-Induced Responses in Dictyostelium Resembles That of the P Site of Higher Organisms

Adenosine acts as a cyclic AMP antagonist in Dictyostelium discoideum. It inhibits the binding of cyclic AMP to cell surface receptors and the induction of postaggregative differentiation by cyclic AMP. We investigated the nucleoside specificity and dose dependency of both inhibitory effects of adenosine. It was found that adenosine inhibits cyclic AMP binding and cyclic-AMP-induced differentiation with a Ki of about 300 µM. Alterations in the purine moiety of adenosine generally decrease the inhibitory effect of the molecule, whereas alterations in the ribose moiety are tolerated and in most cases even increase the inhibitory effect of the molecule on both cyclic AMP binding and differentiation induction. A strong correlation (r = 0.996, P < 0.01%) between the specificities for adenosine derivatives of these two inhibitory processes is demonstrated. The nucleoside specificity for the inhibition of cyclic AMP action in D. discoideum resembles that of the P site of higher organisms. In contrast to effects mediated by the P site of higher organisms, the effects of adenosine mediated by the Dictyostelium receptor cannot be prevented by inhibiting adenosine uptake; this makes it very likely that the adenosine receptor, which is involved in the effects of adenosine on cyclic AMP binding and differentiation induction, is located at the cell surface.

Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in Dictyostelium

Abstract Chemoattractants induce a transient accumulation of cGMP levels in Dictyostelium . Intracellular cGMP levels reach a peak at 10 s and prestimulated cGMP levels are recovered at about 30 s. Intracellular and extracellular cGMP levels were detected simultaneously after stimulation of D. lacteum cells with monapterin and of D. discoideum cells with cAMP. In both species about 20% of the intracellularly accumulated cGMP was secreted. All slime mold species investigated so far contain an intracellular phosphodiesterase specific for cGMP. A mutant of D. discoideum which does not contain this cGMP-specific enzyme shows a strongly retarded decline of intracellular cGMP levels. Secretion of cGMP is in this mutant not sufficient to explain the decline of cGMP levels which indicates the involvement of nonspecific phosphodiesterase in intracellular cGMP regulation. These results show multiple degradation pathways of intracellularly accumulated cGMP. In wild-type cells about 20% is secreted, 10–20% is hydrolyzed intracellularly by non-specific phos-phodiesterase, while the majority (60–70%) is hydrolyzed intracellularly by a cGMP-specific phos-phodiesterase. The relationships of intracellular regulation of cGMP and cAMP levels are discussed.

Cyclic AMP induces a transient alkalinization in Dictyostelium

In a wide range of cell types, stimulus‐response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 min after stimulation, pHi starts increasing from pHi∼7.45 to pHi∼7.60, as is revealed independently by two different pH null‐point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pHi is accomplished by the DES‐sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.

Regulation of diacylglycerol kinase in the transition from quiescence to proliferation in Dictyostelium discoideum

Diacylglycerol kinase and phosphatidylinositol kinase were examined in stationary phase D. discoideum amoeba induced to synchronously proliferate by dilution into fresh medium. Membrane bound diacylglycerol kinase activity showed a rapid and transitory 3-5 fold increase in the preproliferative interphase while phosphatidylinositol kinase activity was kept quite constant during the same period. The changes in diacylglycerol kinase activity seem to be due to a translocation of the enzyme from the soluble to the particulate cell compartments.

The developmental regulation of phosphatidylinositol kinase in Dictyostelium discoideum

Phosphatidylinositol kinase was examined in Dictyostelium discoideum since this organism offers molecular and genetic advantages to study the role of phosphatidylinositol metabolism during cell growth and development. D. discoideum homogenates phosphorylated phosphatidylinositol to form phosphatidylinositol 4‐phosphate in a reaction which was found to be linear with time and cell concentration. Optimal activity was obtained in the presence of 1 mM MgCl2 and pH 7.6 and has an apparent K m for ATP of about 250 μM. Changes in phosphatidylinositol kinase were examined during D. discoideum development. Activity increased about 2‐fold, 4 h after removal of the food source, to decline to almost no activity at late aggregation. During slug formation the activity increased about 15‐fold and remained constant during further development. These results suggest a role for D. discoideum phosphatidylinositol kinase during development.

Ca2+- or phorbol ester- dependent effect of ATP on a subpopulation of cAMP cell-surface receptors in membranes from D. discoideum. A role for protein kinase C.

Abstract D. discoideum cells contain surface receptors for the chemoattractant cAMP which are composed of fast and slowly dissociating binding sites with half-lifes of respectively about 1 s and 15 s (Van Haastert and De Wit, J. Biol. Chem. 259 , 13321–13328). In membranes prepared by shearing the cells through a Nucleopore filter, ATP has no effect on cAMP-binding at equilibrium, but the number of slowly dissociating sites is increased about 2-fold by ATP while their apparent affinity and off-rate are not altered by ATP. The effect of ATP is stimulated about 3-fold by Ca2+ with a half maximal effect at 100 μM Ca2+. The tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases this Ca2+-sensitivity of the ATP effect to about 0.2 μM Ca2+. These data suggest that a specific subpopulation of cAMP receptors in membranes from D. discoideum is altered by the action of protein kinase C.

The developmental regulation of L-ornithine decarboxylase in Dictyostelium discoideum and its induction by cAMP.

By the use of an in vivo assay, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) is shown to be developmentally regulated in Dictyostelium discoideum. High levels of cAMP can induce ornithine decarboxylase activity in preaggregative cells kept in shaking suspension, under similar conditions as where other markers for development can also be induced. This induction by cAMP is solely dependent on the total amount of cAMP to which the cells have been exposed, and not on the manner of cAMP addition. Induction of ornithine decarboxylase activity, when measured in vitro, is caused by both an increase in total enzyme activity and by a proportional increase in activity of the high-affinity form for the cofactor pyridoxal phosphate. When measured in vivo, an additional regulatory mechanism seems to be involved. Kinetic studies with the competitive inhibitor putrescine suggest that in cAMP-stimulated cells the low affinity form of the enzyme may also be active in vivo.

Adaptation of Dictyostelium Discoideum Cells to Chemotactic Signals

The cellular slime mold Dictyostelium discoideum lives in the soil where it feeds on bacteria. Exhaustion of the food supply induces cell aggregation. Subsequently, cells differentiate to two cell types; spores embedded in a slime droplet on top of a tubular stalk of vacuolized cells. Cell aggregation is mediated by Chemotaxis. Upon starvation some cells start to secrete a chemoattractant which has been identified as cAMP (1). Extracellular cAMP induces two responses, which are both mediated by cell surface receptors. First, cAMP activates adenylate cyclase; the produced cAMP is secreted and may trigger other cells, thus relaying the signal. Second, cAMP induces a chemotactic response by which cells move in the direction of the cAMP source. The combined effects of cAMP relay and Chemotaxis may lead to the accumulation of as many as 100,000 cells in a central collecting point derived from an area of about 1 cm2 (see 2,3 for recent reviews).