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Volume 17, Number 19, Issue of October 1, 1997 pp. 7359-7371
Copyright ©1997 Society for Neuroscience

Impact of Cytoplasmic Calcium Buffering on the Spatial and Temporal Characteristics of Intercellular Calcium Signals in Astrocytes

Received Feb. 14, 1997; revised June 19, 1997; accepted July 11, 1997.

Zhong Wang¹, Michael Tymianski², Owen T. Jones², and Maiken Nedergaard¹

¹ Departments of Cell Biology and Anatomy and Neurosurgery, New York Medical College, Valhalla, New York 10595, and ² Playfair Neuroscience Unit, University of Toronto, The Toronto Hospital-Western Division, Toronto, Ontario, Canada M5T-2S8

The impact of calcium buffering on the initiation and propagation of mechanically elicited intercellular Ca²⁺ waves was studied using astrocytes loaded with different exogenous, cell membrane-permeant Ca²⁺ chelators and a laser scanning confocal or video fluorescence microscope. Using an ELISA with a novel antibody to BAPTA, we showed that different cell-permeant chelators, when applied at the same concentrations, accumulate to the same degree inside the cells. Loading cultures with BAPTA, a high Ca²⁺ affinity chelator, almost completely blocked calcium wave occurrence. Chelators having lower Ca²⁺ affinities had lesser affects, as shown in their attenuation of both the radius of spread and propagation velocity of the Ca²⁺ wave. The chelators blocked the process of wave propagation, not initiation, because large [Ca²⁺]_i increases elicited in the mechanically stimulated cell were insufficient to trigger the wave in the presence of high Ca²⁺ affinity buffers. Wave attenuation was a function of cytoplasmic Ca²⁺ buffering capacity; i.e., loading increasing concentrations of low Ca²⁺ affinity buffers mimicked the effects of lesser quantities of high-affinity chelators. In chelator-treated astrocytes, changes in calcium wave properties were independent of the Ca²⁺-binding rate constants of the chelators, of chelation of other ions such as Zn²⁺, and of effects on gap junction function. Slowing of the wave could be completely accounted for by the slowing of Ca²⁺ ion diffusion within the cytoplasm of individual astrocytes. The data obtained suggest that alterations in Ca²⁺ buffering may provide a potent mechanism by which the localized spread of astrocytic Ca²⁺ signals is controlled.

Key words: calcium buffering; astrocytes; calcium chelators; BAPTA; calcium waves; cell culture; digital imaging

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