A link of Ca(2+ )to cAMP oscillations in Dictyostelium: the calmodulin antagonist W-7 potentiates cAMP relay and transiently inhibits the acidic Ca(2+)-store

BACKGROUND: During early differentiation of Dictyostelium the attractant cAMP is released periodically to induce aggregation of the cells. Here we pursue the question whether pulsatile cAMP signaling is coupled to a basic Ca(2+)-oscillation. RESULTS: We found that the calmodulin antagonist W-7 transiently enhanced cAMP spikes. We show that W-7 acts on an acidic Ca(2+)-store: it abolished ATP-dependent vesicular acidification, inhibited V-type H(+)ATPase activity more potently than the weaker antagonist W-5 and caused vesicular Ca(2+)-leakage. Concanamycin A, an inhibitor of the V-type H(+)-pump, blocked the Ca(2+)-leakage elicited by W-7 as well as cAMP-oscillations in the presence of W-7. Concanamycin A caused an increase of the cytosolic Ca(2+)-concentration whereas W-7 did not. In case of the latter, Ca(2+ )was secreted by the cells. In accord with our hypothesis that the link from Ca(2+ )to cAMP synthesis is mediated by a Ca(2+)-dependent phospholipase C we found that W-7 was not active in the phospholipase C knockout mutant. CONCLUSION: We conclude that the potentiation of cAMP relay by W-7 is due to a transient inhibition of the acidic Ca(2+)-store. The inhibition of the proton pump by W-7 causes a leakage of Ca(2+ )that indirectly stimulates adenylyl cyclase activity via phospholipase C

Cell communication by periodic cyclic-AMP pulses

At the surface of aggregating cells of the slime mould, Dictyostelium discoideum, two different sites interacting with extracellular cAMP are detectable: binding sites and cyclic-nucleotide phosphodiesterase. Both sites are developmentally regulated. An adequate stimulus for the chemoreceptor system in D. discoideum is the change of cAMP concentration in time, rather than concentration per se: long-term binding of cAMP causes only a short-term response. The system is, consequently, adapted to the recognition of pulses rather than to steady-state concentrations of cAMP. The cells are, nevertheless, able to sense stationary spatial gradients and to respond to them by chemotactic orientation. The possibility is discussed that they do so by transforming spatial concentration changes into temporal ones, using extending pseudopods as sensors

The contractile vacuole in Ca(2+)-regulation in Dictyostelium: its essential function for cAMP-induced Ca(2+)-influx

BACKGROUND: cAMP-induced Ca(2+)-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca(2+)-stores: acidic stores and the endoplasmic reticulum (ER). The acidic stores may comprise the contractile vacuole network (CV), the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca(2+)-store was investigated. RESULTS: Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release Ca(2+). Release of Ca(2+ )was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca(2+)-transport and Ca(2+)-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca(2+)-compartment we wanted to know whether or not it takes part in cAMP-induced Ca(2+)-influx. We made use of the LvsA(-)-mutant expected to display reduced Ca(2+)-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca(2+)-influx into whole cells. CONCLUSION: The contractile vacuoles in Dictyostelium represent a highly efficient acidic Ca(2+)-store that is required for cAMP-induced Ca(2+)-influx

Ca(2+ )regulation in the absence of the iplA gene product in Dictyostelium discoideum

BACKGROUND: Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca(2+ )concentration ([Ca(2+)](i)). The [Ca(2+)](i)-change is composed of liberation of stored Ca(2+ )and extracellular Ca(2+)-entry. The significance of the [Ca(2+)](i)-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca(2+)-buffers in the cytosol indicates that a [Ca(2+)](i)-increase is required for chemotaxis. Yet, the iplA(- )mutant disrupted in a gene bearing similarity to IP(3)-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca(2+)](i)-transient which favours the view that [Ca(2+)](i)-changes are insignificant for chemotaxis. RESULTS: We investigated Ca(2+)-fluxes and the effect of their disturbance on chemotaxis and development of iplA(- )cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca(2+). Chemotaxis was impaired when [Ca(2+)](i)-transients were suppressed by the presence of a Ca(2+)-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca(2+)-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca(2+)-influx and liberation of stored Ca(2+), respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca(2+)-uptake and Ca(2+)-release activated by fatty acids or Ca(2+)-ATPase inhibitors were similar to wild type. Mn(2+)-quenching of fura2 fluorescence allows to study Ca(2+)-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn(2+ )was necessary to observe agonist-induced Mn(2+)-influx. cAMP evoked a [Ca(2+)](i)-elevation when stores were strongly loaded with Ca(2+), again with a similar shift in sensitivity in the mutant. In addition, basal [Ca(2+)](i )was significantly lower in iplA(- )than in wild type amoebae. CONCLUSION: These results support the view that [Ca(2+)](i)-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca(2+)-release from stores to Ca(2+)-entry in Dictyostelium. The iplA(- )strain retains the capacitative Ca(2+)-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure

Cyclic-AMP reception and cell recognition in dictyostelium discoideum

Single cells of the slime mold, Dictyostelium discoideum, aggregate into a multicellular organism in response to cyclic AMP, which they detect by binding to cellsurface receptors. During the aggregation phase, two different responses to cyclic-AMP are observed. First, the cells orientate by chemotaxis towards the source of a concentration gradient which initially is a group of cells forming an aggregation center. Second, the cells relay pulses which are periodically generated by the centers

Understanding calcium dynamics: experiments and theory

Intracellular Calcium is an important messenger in living cells. Calcium dynamics display complex temporal and spatial structures created by the concentration patterns which are characteristic for a nonlinear system operating far from thermodynamic equilibrium. Written as a set of tutorial reviews on both experimental facts and theoretical modelling, this volume is intended as an introduction and modern reference in the field for graduate students and researchers in biophysics, biochemistry and applied mathematics

A plausible role for a membrane-bound cyclic AMP phosphodiesterase in cellular slime mold chemotaxis

1. 1. Kinetics of membrane-bound cyclic AMP phosphodiesterase of the cellular slime mold, Dictyostelium discoideum, were studied under two conditions: in the 27 000 × g sediment of cell homogenates (particle-bound phosphodiesterase) and in cell suspensions using external cyclic AMP as a substrate (cell-bound phosphodiesterase). Both methods revealed non-Michaelian kinetics with interaction coefficients less than 1. 2. 2. The membrane-bound phosphodiesterase has a specificity different from that of the cyclic AMP receptor, also present at the cell surface. 3. 3. The membrane-bound enzyme was solubilized by lithium 3,5-diiodosalicylate and partially purified. In this state the non-linear kinetics were still retained; however, the enzyme was now inhibited by the D. discoideum inhibitor, unlike the cell-bound phosphodiesterase in vivo. This indicates that both enzymes share an inhibitor binding site and that this site is cryptic in the cell-bound state. 4. 4. Production of periodic cyclic AMP pulses by centers, and their relay by other cells, is believed to occur during aggregation. It is suggested that the cell-bound enzyme determines a "time window" significantly smaller than the period of pulsing, and optimizes stimulation of the cyclic AMP receptors in chemotaxis and signal relaying