The dynamics of exocytosis in human neutrophils.

Abstract

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 ~tM guanosine-5'-O(3-thiotriphosphate) (GTP~,S) 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 ~tm 2 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. GTP'tS-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 ~tM. In the absence of GTP~S elevation of intracellular-free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTP~S 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 nucleotidebinding 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.

Rapid biogenesis and sensitization of secretory lysosomes in NK cells mediated by target-cell recognition

“…Highly Ca 2ϩ -buffered pipette solutions with different Ca 2ϩ concentrations ([Ca 2ϩ ] p ) were dialyzed against the cytosol in standard whole-cell patch-clamp mode. to 76 SLs, assuming an average diameter of 500 nm (17), and a specific C m as 10 fF͞ m (2, 18), whereas for mast cells and neutrophils, the estimated granule number is Ͼ1,000 (15,16,19). It is unclear whether the preformed SLs are sufficient or the de novo formation of SLs is necessary for their lytic function.…”

Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils

“…Instead, we observed that amantadine inhibited clathrin reorganization into clathrin-coated pits with subsequent decrease in dual phosphorylation (activation) of p38 MAPK. Many of the cellular responses elicited by PAF are directly influenced by p38 MAPK activation, priming of the respiratory burst, and actin reorganization; however, the effects on intracellular granules, whether due to the specific granules binding to the membrane and increasing the surface expression of CD11b or to the release of proteases from azurophilic granules, is not as well understood and have been proposed to involve multiple mechanisms including MAPKs and changes in cytosolic Ca 2ϩ (18,44,45). Importantly, amantadine does inhibit the PAFinduced activation of p38 MAP kinase but did not affect the PAF-mediated increases in cytosolic Ca 2ϩ , which may be the reason that amantadine only attenuates the PAF-elicited changes in PMN physiology related to the movement or release of granule constituents including both the release of elastase and the increased surface expression of CD11b (7,42,69).…”

Interleukin-8 in gastrointestinal inflammation and malignancy: induction and clinical consequences

“…180 Primary granules (also known as azurophilic granules) are positive for myeloperoxidase and, therefore, have a critical role in the potentiation of the respiratory burst. 211 Furthermore, these granules contain a bevy of antimicrobial compounds such as defensins, lysozyme, and bactericidal/permeability-increasing protein and three serine proteases: cathepsin G, neutrophil elastase, and proteinase 3. 212 These are the first granules formed during PMN differentiation.…”