Effects of Osmolality and Ionic Strength on Secretion from Adrenal Chromaffin Cells Permeabilized with Digitonin

Abstract

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low‐molecular‐weight maltodextrin fraction (average size a tetrasaccharide), one‐half maximal inhibition occurred at 900–1,000 mOs. Prior treatment of permeabilized cells with Ca2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half‐maximal inhibition occurred at 600–700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potasium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca2+ concentration required for half‐maximal stimulated secretion from approximately 1.2 μM. Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions. The experiments place limits on the possible osmotic mechanisms that could be involved in exocytosis.

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

“…The digitonin permeabilization experiments were performed as described by Holz and Senter (1986). In brief, cells were permeabilized in a solution containing 139 mM potassium glutamate, 20 mM PIPES, 5 mM EGTA, 2 mM Mg 2ϩ , and 20 M digitonin, pH 6.6, for 6 min.…”

“…To determine whether insulin release in vivo depends on salt concentration, we permeabilized Min6B1 cells with digitonin and incubated the cells with increasing concentrations of potassium glutamate as described previously for chromaffin granule secretion (Holz and Senter, 1986). The cells were then stimulated with 10 M calcium to initiate the fusion of predocked insulin granules.…”