Mitochondria Exert a Negative Feedback on the Propagation of Intracellular Ca ²⁺ Waves in Rat Cortical Astrocytes

Abstract

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca ²⁺ uptake and physiological Ca ²⁺ signaling in rat cortical astrocytes. A rise in cytosolic Ca ²⁺ ([Ca ²⁺] _cyt), resulting from mobilization of ER Ca ²⁺ stores was followed by a rise in mitochondrial Ca ²⁺ ([Ca ²⁺] _m, monitored using rhod-2). Whereas [Ca ²⁺] _cyt recovered within ∼1 min, the time to recovery for [Ca ²⁺] _m was ∼30 min. Dissipating the mitochondrial membrane potential (Δψ _m, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca ²⁺ uptake and slowed the rate of decay of [Ca ²⁺] _cyt transients, suggesting that mitochondrial Ca ²⁺ uptake plays a significant role in the clearance of physiological [Ca ²⁺] _cyt loads in astrocytes. Ca ²⁺ signals in these cells initiated either by receptor-mediated ER Ca ²⁺ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 ± 11.2 μm/s ( n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca ²⁺ uptake into mitochondria as the Ca ²⁺ wave traveled across the cell. Collapse of Δψ _m to prevent mitochondrial Ca ²⁺ uptake significantly increased the rate of propagation of the Ca ²⁺ waves by 50%. Taken together, these data suggest that cytosolic Ca ²⁺ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca ²⁺ signals.