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.