Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells

Abstract

The effects of the steroid glycoside digitonin on cultured bovine adrenal chromaffin cells were studied as a way to gain access to the intracellular sites of calcium action in stimulus-secretion coupling. Chromaffin cells treated with digitonin secreted catecholamines by an exocytotic mechanism when exposed to free calcium concentrations of greater than 1 microM. At free calcium concentrations of less than 100 microM, an apparently saturable release of catecholamines was observed with a half-maximal effect (EC50) of 3-4 microM. Catecholamine release at these low calcium concentrations was limited to 10-15% of the cellular content, was partially dependent on the presence of MgATP, and was inhibited by magnesium (EC50 = 6 mM). At calcium concentrations between 0.1 and 10 mM, a second, apparently saturable component of catecholamine release from digitonin-treated chromaffin cells was observed (EC50 = 0.3 mM). This release at the higher calcium concentrations was unaffected by the addition of MgATP, magnesium, antipsychotic drugs known to bind and inactivate calmodulin, or calcium channel blockers, but was temperature-dependent and was inhibited by N-ethylmaleimide (EC50 = 140 microM). Strontium and manganese can also evoke catecholamine secretion from digitonin-treated chromaffin cells. The results suggest that digitonin will be a useful preparation in elucidating the molecular mechanisms of the secretory process.