Dieter Malchow

Amplification of cyclic-AMP signals in aggregating cells of Dictyostelium discoideum

Aggregating cells of the slime mold, Dictyostelium discoideum, respond to cyclic AMP by chemotactic orientation [ 1,2] and by propagating chemotactic signals in the form of waves from one cell to the next [3,6]. It has been proposed that each cell acts as a biochemical signal amplifier [3-81 so that cyclic AMP would play the role of a transmitter in a relay system. In order to investigate the amplifier system quantitatively, methods have been developed to measure signal generation, cyclic-AMP binding to receptors as well as cellular responses to cyclic AMP in stirred cell suspensions [9-l 11. Under these conditions evidence for signal amplification has been obtained [ 1 1 13 ] using the protein-binding assay for cyclic-AMP [ 141. In the present paper we report results obtained by an independent method for measuring cyclic-AMP stimulated cyclic-AMP release: prelabelling of cells with [3H]adenine and chromatographic separation of the labelled compounds from the extracellular fluid after stimulation of the cells by unlabelled cyclic AMP.

Cyclic AMP‐induced phosphorylation in Dictyostelium of a polypeptide comigrating with myosin heavy chains

results in a chemotactic response starting within the first few seconds after cyclic AMP addition [ 1,2]. The mecha- nism of coupling of cyclic AMP receptors to the con- tractile apparatus of the cells is unknown. Candidates for intermediary processes are an increase of the cyclic GMP concentration [3,4], an influx of Ca 2+ [5] and a reaction which results in the decrease of extracellular pH [6]. Here we report on a protein, probably myosin, that acts as a phosphate acceptor in lysates from cyclic AMP-stimulated cells. 2. Methods 2.1.

Cyclic AMP phosphodiesterase interaction with its inhibitor of the slime mold, Dictyostelium discoideum

Abstract Amoebae of the cellular slime mold, Dictyostelium discoideum, release an inhibitor of their cAMP-phosphodiesterase into the culture medium at the end of growth. In this paper evidence is provided (1) that the excreted PD that reacts with the inhibitor is not identical with the bulk of soluble phosphodiesterase present in cell homogenates, (2) that the extracellular PD that reacts with the inhibitor has a Km of 4 × 10−6 M, (3) that kinetic data point to a noncompetitive inhibition by a tight-binding inhibitor, (4) that trypsin inactivates the inhibitor.

Cyclic AMP-induced pH changes in Dictyostelium discoideum and their control by calcium

During differentiation of Dictyostelium discoideum the extracellular pH was measured in unbuffered cell suspensions. 1. 1. Stimulation of the cells by cyclic AMP pulses in the nanomolar range resulted in two successive increases of the extracellular proton concentration. 2. 2. Cyclic AMP analogs were effective in the order of their chemotactic activity, indicating that the pH changes were mediated by cyclic AMP receptors present at the cell surface. 3. 3. Cyclic AMP binding to the receptors was, however, not sufficient to elicit the pH changes; for glutaraldehyde-treated cells which exhibit normal binding did not respond. Moreover, the proton : cyclic AMP ratio was in the order of 3 · 103. 4. 4. The second proton peak was not due to cyclic AMP secretion or subsequent hydrolysis and persisted in the presence of dithiothreitol, an inhibitor of cyclic AMP phosphodiesterases. 5. 5. Removal of extracellular calcium increased the proton peaks, particularly the first one, up to 10-fold. A half maximal response was obtained with 3 · 10-10 M cyclic AMP. 6. 6. Only at cyclic AMP doses higher than 0.1 μM did hydrolysis of the added cyclic AMP contribute significantly to the pH changes.

A high-affinity plasma membrane Ca2+-ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca2+ fluxes.

Chemotactic stimulation of Dictyostelium discoideum induces an uptake of Ca2+ by the cells followed by a release of Ca2+. In this study we investigated the mechanism of Ca2+ release and found that it was inhibited by La3+, Cd2+ and azide. Ca2+ release occurred in the absence of external Na+, indicating that an Na+/Ca2+ exchange was not involved. Plasma membranes contained high- and low-affinity ATPase activities. Apparent K0.5 values were 8 microM for the major Mg2+-ATPase and 1.1 microM for the high-affinity Ca2+-ATPase, respectively. The Mg2+-ATPase activity was inhibited by elevated concentrations of Ca2+, whereas both Ca2+-ATPases were active in the absence of added Mg2+. The activities of the Ca2+-ATPases were not modified by calmodulin. The high-affinity Ca2+-ATPase was competitively inhibited by La3+ and Cd2+; we suggest that this high-affinity enzyme mediates the release of Ca2+ from D. discoideum cells.

Single ion channels in the slime mold Dictyostelium discoideum

Single ion channels with different conductances and gating characteristics were observed in the plasma membrane of the slime mold Dictyostelium discoideum by means of the patch-clamp technique in the cell-attached mode. The predominant channel type shows outward current flow, probably carried by K+ ions. The slope conductance of this channel is 9 pS and its probability to be open increases with depolarization of the membrane. The channel is observed from 1 to 8 h after the beginning of starvation.

Calcium regulates cAMP-induced potassium ion efflux in Dictyostelium discoideum.

The chemoattractant cAMP elicits a transient efflux of K+ in cell suspensions of Dictyostelium discoideum. This cellular response displayed half-maximal activity at about 1 microM cAMP and saturated at 100 microM cAMP, cAMP-stimulated K+ efflux, measured with a K+-sensitive electrode, depended on the extracellular free Ca2+ concentration ([Ca2+]0) and was maximal in the presence of EGTA. Usually more than 90% of the K+ release could be inhibited by the addition of Ca2+. Half-maximal reduction occurred at about 2 microM [Ca2+]0. Inhibition was also observed in the presence of caffeine or A23187, drugs known to elevate the intracellular free Ca2+ concentration ([Ca2+]i). Under conditions where [Ca2+]0 was maintained at a low level, half-maximal inhibition was 1 mM for caffeine and 3 microM for A23187. These results indicate that Cai2+ is involved in the regulation of K+ efflux. Simultaneous measurements of Ca2+ uptake and K+ efflux induced by cAMP as well as free running oscillations of both ions revealed that initiation and termination of Ca2+ uptake slightly preceded those of K+ efflux.