Ascorbic acid and catecholamine release from digitonin-treated chromaffin cells.

The subcellular localization of catecholamines and ascorbic acid in cultured bovine adrenal chromaffin cells was studied by permeabilizing the cells with digitonin, a steroid glycoside. Catecholamine release from permeabilized chromaffin cells was dependent on the free calcium concentration and the temperature of the incubation mixture. By contrast, [14C]ascorbic acid, preloaded into the cells, was released by digitonin treatment in a manner independent of the concentration of free calcium and with only moderate regard to the incubation temperature. The sensitivity of ascorbic acid release to digitonin treatment was identical to that of calcium-dependent catecholamine release. These results thus suggest that ascorbic acid preloaded into the cells may directly efflux from the cell cytoplasm as a result of the permeabilization of the plasma membrane. Dimethylepinephrine, a permanently positively charged catecholamine analog which is known to be excluded from vesicular fractions, was also released by digitonin treatment in a manner independent of calcium. The time course of dimethylepinephrine release was very similar to that of ascorbic acid release. Thus, newly accumulated ascorbic acid in chromaffin cells may be localized to a free pool in the cell cytoplasm rather than in a vesicular compartment.