Calcium Homeostasis in Digitonin-Permeabilized Bovine Chromaffin Cells

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Abstract
The regulation of cytosolic calcium was studied in digitonin-permeabilized chromaffin cells. Accumulation of 45Ca2+ by permeabilized cells was measured at various Ca2+ concentrations in the incubation solutions. In the absence of ATP, there was a small (10-15% of total uptake) but significant increase in accumulation of Ca2+ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca2+ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca2+ into carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-sensitive and -insensitive pools. The CCCP-sensitive pool- mainly mitochondria - was active when the calcium concentration was < 1 .mu.M and was not saturated at 25 .mu.M. The Ca2+ sequestered by the CCP-insensitive pool could be inhibited by vanadate and released by inositol trisphosphate, a combination suggesting that this pool was the endoplasmic reticulum. The CCCP-insensitive pool had a high affinity for calcium, with an EC50 of .apprx. 1 .mu.M. When the Ca2+concentration was adjusted to the level in the cytoplasm of resting cells (0.1 .mu.M), the presumed endoplasmic reticulum pool was responsible for .apprx. 90% of the ATP-stimulated calcium uptake. At a calcium level similar to the acetylcholine-stimulated level in intact cells (5-10 .mu.M), most of the Ca2+ (< 95%) went into the CCCP-sensitive pool.