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Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.

We have studied the dynamics of certain key processes near the plasma membrane inside two types of chemically-triggerable living cells using total internal reflection fluorescence microscopy (TIRFM). In TIRFM, a laser beam is incident upon the cell/glass-substrate interface from the glass side at an angle greater than the critical angle for total internal reflection. This creates an exponentially decaying evanescent field in the cell medium (with a characteristic depth of > 100 nm) capable of exciting fluorescence selectively from the membrane-proximal regions at cell/substrate contacts. Various ways of setting up the optics for such a system are discussed, involving the use of either prisms or very high aperture objectives.In one application of TIRFM, the motion of adrenalin-containing secretory granules in the immediate submembrane region of chromaffin cells is examined before and after chemical stimulation that causes the granules to release their contents to the cell exterior.