Abstract
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.
Highlights
Agonist-induced [Ca2ϩ]i1 signaling induces liberation of stored Ca2ϩ and capacitative or noncapacitative Ca2ϩ entry in almost all nonexcitable cells
One hypothesis favors extracellular Ca2ϩ influx to be the sole source for the generation of cAMP-induced [Ca2ϩ]i transients [6], since they were practically undetectable at extracellular Ca2ϩ levels of less than 10 l of cAMP (10 M) [5, 6]
In contrast to the above model, a second hypothesis assigns a crucial function to internal stores as the trigger for the generation of agonist-activated [Ca2ϩ]i transients, with liberation of sequestered Ca2ϩ being followed by capacitative Ca2ϩ entry. cAMP-induced Ca2ϩ influx was observed to be sensitive to agents known to empty storage compartments; it was blocked, for example, by inhibitors of Hϩ and Ca2ϩ pumps present on either the acidic, fatty acid-sensitive [10, 11] or the inositol 1,4,5-trisphosphate-sensitive store [11,12,13,14], so both types of stores were concluded to regulate Ca2ϩ entry via capacitative Ca2ϩ influx [11, 14]
Summary
Agonist-induced [Ca2ϩ]i1 signaling induces liberation of stored Ca2ϩ and capacitative or noncapacitative Ca2ϩ entry in almost all nonexcitable cells (for a review, see Refs. 1–3). Agonist-induced [Ca2ϩ]i1 signaling induces liberation of stored Ca2ϩ and capacitative or noncapacitative Ca2ϩ entry in almost all nonexcitable cells Subsequent activation of plasma membrane Ca2ϩ-ATPases results in efflux, which, decreases in the course of differentiation [8] with a concomitant increase of the level of sequestered Ca2ϩ [9]. In contrast to the above model, a second hypothesis assigns a crucial function to internal stores as the trigger for the generation of agonist-activated [Ca2ϩ]i transients, with liberation of sequestered Ca2ϩ being followed by capacitative Ca2ϩ entry. We found that the classical way to activate capacitative Ca2ϩ entry does not operate in Dictyostelium; inhibition of the internal SERCA Ca2ϩ pump abolished both agonist- and CaCl2-induced Ca2ϩ entry
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