Intercellular Calcium Signaling in Astrocytes via ATP Release through Connexin Hemichannels*

Abstract

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca²⁺ concentration. We have used patch clamp, dye flux, ATP assay, and Ca²⁺ imaging techniques to show that one mechanism for this intercellular Ca²⁺ signaling in astrocytes is the release of ATP through connexin channels (“hemichannels”) in individual cells. Astrocytes showed low Ca²⁺-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca²⁺ and was inhibited by FFA and Gd³⁺. Mechanical stimulation also evoked release of ATP that was potentiated by low Ca²⁺ and inhibited by FFA and Gd³⁺. Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca²⁺ signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca²⁺ waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd³⁺. Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.