Complex Actions of Ionomycin in Cultured Cerebellar Astrocytes Affecting Both Calcium-Induced Calcium Release and Store-Operated Calcium Entry
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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.
KeywordsIonomycin Store-operated Ca2+-entry Ca2+-induced Ca2+-release
The Lundbeck and the Hørslev Foundations are cordially acknowledged for providing financial support to L.K.B. We are grateful to Avi Ring (Norwegian Defense Research Establishment, Oslo, Norway) for providing technical and scientific advice and laboratory technician Heidi Nielsen is acknowledged for providing technical expertise.
- 9.Hertz L, Juurlink B, Hertz E, Fosmark H, Schousboe A (1989) Preparation of primary cultures of mouse (rat) astrocytes. In: Shahar A, De Vellis J, Vernadakis A, Haber B (eds) Dissection and tissue culture manual of the nervous system. Alan R. Liss, New York, pp 105–108Google Scholar
- 10.Hertz L, Juurlink BHJ, Fosmark H, Schousboe A (1982) Astrocytes in primary cultures. In: Pfeiffer SE (ed) Neuroscience approached through cell culture. CRC Press, Boca Raton, pp 175–186Google Scholar
- 16.Takemura H, Hughes AR, Thastrup O, Putney JW (1989) Activation of calcium entry by the tumor promoter, thapsigargin, in parotid acinar cells. Evidence that an intra-cellular calcium pool, and not an inositol phosphate, regulates calcium fluxes at the plasma membrane. J Biol Chem 264:12266–12271PubMedGoogle Scholar