Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Abstract

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca 2+ , ATP, and proteins and allows micromolar Ca 2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 Μ M ) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [ 3 H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [ 3 H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [ 3 H]norepinephrine uptake was inhibited by 1 Μ M reserpine, 30 m M NH 4 + , or 1 Μ M carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [ 3 H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H + electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules within digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 Μ M or greater. Catecholamine released into the medium by micromolar Ca 2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-Β-hydroxylase. The studies demonstrate that 20 Μ M of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca 2+ , the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.