Direct observation of calcium-independent intercellular ATP signaling in astrocytes

Adenosine triphosphate (ATP) is assumed to be involved in the regulation of many extracellular signaling systems including calcium wave propagation. S o far all supportive evidence is indirect, such as monitoring changes in in tracellular calcium on application of extracellular ATP or off-site measure ment of ATP from superfusates. Furthermore, the causal relationships among the various signaling agents are still unclear. A novel chemiluminescence d ynamic imaging method was developed to monitor ATP release from living biol ogical cells. The assay has linear response over 3 orders of magnitude for fixed concentrations of enzyme and cofactors, with a correlation coefficien t of 0.999. The detectability of ATP is down to 10(-8) M at millisecond exp osure times with an intensified charge-coupled device camera. The direct im aging of ATP waves in astrocyte cultures was performed together with Fluo-3 -Ca imaging at millisecond temporal resolution and micrometer-scale spatial resolution. We discovered that extracellular ATP mediates intercellular ca lcium wave propagation, but surprisingly, release and propagation of ATP ar e not calcium dependent. Therefore, ATP rather than Ca or IP3 is the primar y intercellular signaling messenger.