Complex Actions of Ionomycin in Cultured Cerebellar Astrocytes Affecting Both Calcium-Induced Calcium Release and Store-Operated Calcium Entry

Abstract

The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca²⁺ in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca²⁺ into the cytosol, a number of studies have demonstrated that the mechanism of action is likely to be more complex, involving modulation of intrinsic Ca²⁺ signaling pathways. In the present study we assessed the effect of ionomycin on primary cultures of murine cerebellar astrocytes. Ionomycin concentrations ranging from 0.1 to 10 μM triggered a biphasic increase in cytosolic Ca²⁺, consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca²⁺, the peak phase persisted. The peak amplitude could be lowered significantly by application of dantrolene, demonstrating involvement of Ca²⁺-induced Ca²⁺-release (CICR). The plateau phase was markedly reduced when store-operated Ca²⁺-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca²⁺ stores in astrocytes, causing release of Ca²⁺ into the cytosol, which in turn triggers further depletion of the stores through CICR and subsequently activates SOCE. This mechanistic action of ionomycin is important to keep in mind when employing it as a pharmacological tool.