Calcium Homeostasis in Digitonin‐Permeabilized Bovine Chromaffin Cells

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 carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-sensitive and -insensitive pools. The CCCP-sensitive pool--mainly mitochondria--was active when the calcium concentration was greater than 1 microM and was not saturated at 25 microM. The Ca2+ sequestered by the CCCP-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 approximately 1 microM. When the Ca2+ concentration was adjusted to the level in the cytoplasm of resting cells (0.1 microM), the presumed endoplasmic reticulum pool was responsible for approximately 90% of the ATP-stimulated calcium uptake. At a calcium level similar to the acetylcholine-stimulated level in intact cells (5-10 microM), most of the Ca2+ (greater than 95%) went into the CCCP-sensitive pool.

GTP and Ca2+ Modulate the Inositol 1,4,5‐Trisphosphate‐Dependent Ca2+ Release in Streptolysin O‐Permeabilized Bovine Adrenal Chromaffin Cells

“…One explanation may be that the digitonin-permeabilized cells lose the ability to take up additional Ca2+. This interpretation is supported by the minute amounts of 45Ca2+ whose release was induced by 1P3 from a nonmitochondrial intracellular compartment in digitonin-permeabilized chromaffin cells (Kao, 1988). In contrast, the present data demonstrate that the IP3-sensitive Ca2+ compartment plays an important role in intracellular Ca2+ signaling and control of exocytosis within chromaffin cells.…”

“…In digitonin-permeabilized adrenal chromaffin cells, high concentrations of IP3 were required to induce release of Ca2+. Furthermore, a second dose of IP3 failed to induce release of Ca2+, and the Ca2+ released could not be taken up again by the compartment (Stoehr et al, 1986;Kao, 1988). These findings argue against a major contribution of the IP3-sensitive Ca2+ compartment during stimulation of adrenal chromaffin cells.…”

Inositol 1,4,5-Trisphosphate- and Caffeine-Sensitive Ca2+-Storing Organelle in Bovine Adrenal Chromaffin Cells

“…This agrees well with the finding that CCCPsensitive Ca 21 pools become active at above pCa 6 in permeabilized chromaffin cells (14). We also obtained the same results using 1 mM NaN3, another metabolic inhibitor (31) (data not shown).…”

“…6: a and b). The Ca2+-storing organelles in the adrenal medulla have been studied using the uncoupler CCCP or vanadate (nonselective Ca"-ATPase inhibitor), in digitonin-permeabilized cells (14). In organelles of adrenal chromaffin cells, however, the selectivity of these drugs has not been studied in detail.…”

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

“…It has been previously shown that the intracellular nonmitochondrial Ca 2~pool has high affinity and that the Na + /Ca 2+ exchanger has low affinity for handling Ca2~ (Kao, 1988;Kao and Cheung, 1990). The Na~/ Ca2~exchanger is important in removing Ca2~from the cytosol, when the [Ca 2+ I, is increased after stimulation, and therefore controls the time for cessation of secretion.…”