Hyperosmotic inhibition of calcium signals and exocytosis in rabbit neutrophils

Abstract

Previous studies have shown that hyperosmotic media inhibit secretion in many cells and this has been interpreted to be a direct effect on membrane fusion during exocytosis. This conclusion is complicated, however, by our recent study in neutrophils (Biochim. Biophys. Acta 931: 175-179, 1987) showing that both calcium signals and enzyme release are inhibited by hyperosmolality. In this report, we extend these observations by demonstrating that chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP)-induced enzyme release, secretory granule-plasma membrane fusion, and cytosolic calcium signals are all inhibited by similar increases in medium osmolality. The extent of beta-glucuronidase release is decreased in hyperosmotic media and its dose dependence is shifted to higher FMLP concentrations. Inhibition is rapid, reversible, and independent of osmoticant. Freeze-fracture replicas of quick-frozen neutrophils show that granules of cells stimulated in hyperosmotic media do not undergo fusion nor do they have specialized interactions with the plasma membrane or with membranes of adjacent granules. Calcium signals monitored and quantitated by indo-1 fluorescence during secretion confirmed the presence of three phases the calcium dependence of which we have described previously: 1) an initial peak of calcium that is independent of extracellular calcium and is inhibited 70% at high osmolality; 2) a broad shoulder of elevated calcium levels 30-90 s after stimulation that is dependent on extracellular calcium and is totally blocked at high osmolality; and 3) a plateau of lower but above basal calcium 2-5 min after stimulation that is dependent on extracellular calcium but is relatively unaffected by high osmolality. These results suggest that the FMLP-elicited calcium signal is a composite of multiple signaling events and that hyperosmotic inhibition of secretion, at least in neutrophils, may result from an impaired calcium signal in addition to the direct effect it has on exocytosis.