Ascorbic acid and catecholamine secretion from cultured chromaffin cells.

Abstract

Ascorbic acid was found to be secreted from cultured bovine adrenal chromaffin cells coincidentally with catecholamines using a variety of secretagogues, including veratridine, nicotine, acetylcholine, and potassium chloride. Secretion of ascorbic acid was measured by preloading the cells for 3 h with (R)-[14C]ascorbic acid and then quantitating release of the label. The newly transported ascorbic acid constituted nearly 90% of total cellular ascorbic acid. Although chromaffin granules are known to contain endogenous ascorbic acid, only 16% of the total labeled ascorbic acid taken up by cultured cells was localized to the chromaffin granule fraction. Under the same isolation conditions, 95% of the endogenous catecholamines was found in the granule fraction. The secretion of both (R)-[14C] ascorbic acid and catecholamines was calcium dependent. However, only catecholamine secretion was effectively inhibited by the presence in the medium of isethionate, an impermeant ion that inhibits catecholamine secretion from isolated chromaffin granules. Analysis of the ratios of cellular and granular (R)-[14C] ascorbate as well as secreted (R)-[14C]ascorbate and catecholamines revealed that the secreted (R)-[14C]ascorbic acid did not come exclusively from the chromaffin granule fraction but also from a nonsedimenting cytoplasmic compartment. Indeed, for all four agonists, the amounts of (R)-[14C]ascorbic acid secreted were more than 1.5 times greater than could be accounted for by chromaffin granule ascorbate alone. We took these data to indicate that, while ascorbic acid and catecholamines were secreted concomitantly, the sources of the secreted substances were not necessarily identical.