Direct monitoring of ER Ca dynamics reveals that Ca entry induces ER-Ca release in astrocytes.

Excitability in astroglia is controlled by Ca fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsPR) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca-gated Ca release channels is less explored in astroglia. Here we address this process by monitoring Ca dynamics directly in the cytosol and the ER of astroglial cells. Cultured astrocytes exhibited spontaneous and high-K-evoked cytosolic Ca transients, both of them reversibly abolished by external Ca removal, addition of plasma membrane channel blockers or ER Ca depletion with SERCA inhibitors. Resting astrocyte [Ca] averaged 400 μM and maximal stimulation with ATP provoked a complete and reversible ER discharge. Direct monitoring of Ca in the lumen of ER showed that high-K induced a Ca release from the ER, and its amplitude was proportional to the [K]. Furthermore, by combining the low affinity GAP3 indicator targeted to the ER with the high affinity cytosolic Rhod-2, we simultaneously imaged ER- and cytosolic-Ca signals, in astrocytes in culture and in situ. Plasma membrane Ca entry triggered a fast ER Ca release coordinated with an increase in cytosolic Ca. Thus, we identify a Ca-induced Ca-release (CICR) mechanism that is likely to participate in spontaneous astroglial oscillations, providing a graded amplification of the cytosolic Ca signal.