Propagation of intercellular calcium waves in retinal astrocytes and Muller cells

Intercellular Ca2+ waves are believed to propagate through networks of glia l cells in culture in one of two ways: by diffusion of IP3 between cells th rough gap junctions or by release of ATP, which functions as an extracellul ar messenger. Experiments were conducted to determine the mechanism of Ca2 wave propagation between glial cells in an intact CNS tissue. Calcium wave s were imaged in the acutely isolated rat retina with the Ca2+ indicator dy e fluo-4. Mechanical stimulation of astrocyte somata evoked Ca2+ waves that propagated through both astrocytes and Muller cells. Octanol (0.5 mM), whi ch blocks coupling between astrocytes and Muller cells, did not reduce prop agation into Muller cells. Purinergic receptor antagonists suramin (100 muM ), PPADS (20-50 muM). and apyrase (80 U/ml), in contrast, substantially red uced wave propagation into Muller cells (wave radii reduced to 16-61% of co ntrol). Suramin also reduced wave propagation from Muller cell to Muller ce ll (51% of control). Purinergic antagonists reduced wave propagation throug h astrocytes to a lesser extent (64-81% of control). Mechanical stimulation evoked the release of ATP, imaged with the luciferin-luciferase biolumines cence assay. Peak ATP concentration at the surface of the retina averaged 7 8 muM at the stimulation site and 6.8 muM at a distance of 100 mum. ATP rel ease propagated outward from the stimulation site with a velocity of 41 mum /sec, somewhat faster than the 28 mum/sec velocity of Ca2+ waves. Ejection of 3 muM ATP onto the retinal surface evoked propagated glial Ca2+ waves. T ogether, these results indicate that Ca2+ waves are propagated through reti nal glial cells by two mechanisms. Waves are propagated through astrocytes principally by diffusion of an internal messenger, whereas waves are propag ated from astrocytes to Muller cells and from Muller cells to other Muller cells primarily by the release of ATP.