Early Ca2+ signalling events in neutrophils detected by rapid confocal laser scanning

Abstract

Characterization of early Ca2+ signalling events is crucial for understanding the mechanisms which lead to cell signalling. Rapid confocal laser scanning of Fluo3-loaded neutrophils was used to provide spatially resolved cytosolic free Ca2+ measurements from neutrophils stimulated with formyl-Met-Leu-Phe with a time resolution of 12.5 ms. Heterogeneity of the magnitude and timing of the response was observed between individual neutrophils. There was always a delay between contact with the stimulus and onset of the Ca2+ signal, with a minimum delay of 75 ms and a mean delay of 530 ms (n = 150). There was no delay in the cytosolic free Ca2+ rise induced by ionomycin. The earliest Ca2+ event detected following stimulation with formyl-Met-Leu-Phe (1 microM) was a localized rise in cytosolic free Ca2+ from a location within the cell, pointing to Ca2+ store release as the initial event for triggering whole-cell Ca2+ changes in these cells.

Ca2+ and calpain control membrane expansion during rapid cell spreading of neutrophils

“…Cell preparation and probe loading Human neutrophils were isolated from the blood of healthy volunteers as described previously (Pettit and Hallett, 1995) as approved by SMREC ethics board (Cardiff University School of Medicine Research Ethics Committee, Cardiff, UK) with the informed consent of participants. The neutrophils were then suspended in Krebs medium (120 mM NaCl, 4.9 mM KCl 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.3 mM CaCl 2 , 25 mM HEPES and 0.1% BSA, adjusted to pH 7.4 with NaOH).…”

Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists

“…It was thought that this arose from differences in the rates of IP $ generation in different cells, such that the time required to reach a critical number of occupied IP $ Rs on the Ca# + store varied from cell to cell [19]. In order to test this, maximally stimulated neutrophils, in which micro-delays of only 75-1000 ms occur [20], were micro-injected with heparin at different concentrations. At maximal stimulation with f-Met-Leu-Phe it was not possible to inhibit either Ca# + influx or the extent of Ca# + release.…”

Two distinct Ca2+ storage and release sites in human neutrophils

“…These two neutrophil activities are signaled by two signaling mechanisms, the former by immobilization of integrins [2,5] or Fc receptor cross-linking [6], and the latter by occupancy of seven membrane-spanning domain receptors, such as that for formylated peptides [3,4]. Although the release of Ca 2ϩ from intracellularly stored Ca 2ϩ occurs with both sets of stimuli [4,[7][8][9], the mechanism for this is not fully established. It is also not established whether release of Ca 2ϩ by either group of stimuli occurs from the same Ca 2ϩ stores or whether different Ca 2ϩ storage locations occur in neutrophils.…”