Cytosolic [Ca2+] Transients in Dictyostelium discoideum Depend on the Filling State of Internal Stores and on an Active Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) Ca2+ Pump

Stimulation of Dictyostelium discoideum with cAMP evokes a change of the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ). We analyzed the role of the filling state of Ca 2+ stores for the [Ca 2+ ] transient. Parameters tested were the height of the [Ca 2+ ] i elevation and the percentage of responding amoebae. After loading stores with Ca 2+ , cAMP induced a [Ca 2+ ] i transient in many cells. Without prior loading, cAMP evoked a [Ca 2+ ] i change in a few cells only. This indicates that the [Ca 2+ ] i elevation is not mediated exclusively by Ca 2+ influx but also by Ca 2+ release from stores. Reducing the Ca 2+ content of the stores by EGTA preincubation led to a cAMP-activated [Ca 2+ ] i increase even at low extracellular [Ca 2+ ]. Moreover, the addition of Ca 2+ itself elicited a capacitative [Ca 2+ ] i elevation. This effect was not observed when stores were emptied by the standard technique of inhibiting internal Ca 2+ pumps with 2,5-di-( t -butyl)-1,4-hydroquinone. Therefore, in Dictyostelium , an active internal Ca 2+ -ATPase is absolutely required to allow for Ca 2+ entry. No influence of the filling state of stores on Ca 2+ influx characteristics was found by the Mn 2+ -quenching technique, which monitors the rate of Ca 2+ entry. Both basal and cAMP-activated Mn 2+ influx rates were similar in control cells and cells with empty stores. By contrast, determination of extracellular free Ca 2+ concentration ([Ca 2+ ] e ) changes, which represent the sum of Ca 2+ influx and efflux, revealed a higher rate of [Ca 2+ ] e decrease in EGTA-treated than in control amoebae. We conclude that emptying of Ca 2+ stores does not change the rate of Ca 2+ entry but results in inhibition of the plasma membrane Ca 2+ -ATPase. Furthermore, the activities of the Ca 2+ transport ATPases of the stores are of crucial importance for the regulation of [Ca 2+ ] i changes.