CALCIUM-DEPENDENT NEUTROPHIL SECRETION - CHARACTERIZATION AND REGULATION BY ANNEXINS

To gain direct access to the secretory machinery and study the regulat ion, mechanisms, and effecters of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies t hat permeabilized neutrophils secrete in response to calcium alone, bu t we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 mu M Ca2+ and reaches a plateau between 1.0 and 60 to 80 mu M. When stimulated with >80 mu M Ca2+, secretion is two-to th reefold greater than at lower [Ca2+], suggesting that two distinct mec hanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells i s 80% that of f-met-leu-phe-stimulated intact cells, indicating that t he essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization cause s leakage of 100% of annexins V and VI, but 41% of annexin I and 12% o f annexin III are retained. Immunofluorescence microscopy revealed tha t retained annexins I and III are associated with granule membranes. A ddition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mecha nism of secretion.