The dynamics of exocytosis in human neutrophils

Abstract

We have investigated the dynamics of exocytosis in single human neutrophils. The increase of membrane area associated with granule fusion was followed by time-resolved patch-clamp capacitance measurements. Intracellular application of 20 μM guanosine-5'-O(3-thiotriphosphate) (GTPγS) in the presence of 2.5 mM ATP stimulated exocytosis and led to an increase of membrane capacitance from 3.0 to ~8.4 pF corresponding to a 540 μm2 increase of membrane area. This capacitance change is very close to the value expected from morphological data if all primary and secondary granules fuse with the plasma membrane. High resolution measurements revealed stepwise capacitance changes corresponding to the fusion of individual granules. GTPγS-stimulated exocytosis did not require pretreatment with cytochalasin B and the amplitude was independent of the intracellular-free calcium concentration between 10 nM and ~2.5 μM. In the absence of GTPγS elevation of intracellular-free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTPγS started after a lag phase of 2-7 min and was usually complete within 5-20 min. The time course was affected by the intracellular ATP and calcium concentration. Exocytosis was markedly accelerated by pretreatment with cytochalasin B. Our results demonstrate that the final steps leading to primary and secondary granule fusion are controlled by a guanine nucleotide-binding protein and do not require an elevation of intracellular calcium. Calcium and other factors are, however, involved in the regulation having pronounced effects on the dynamics of exocytosis.