Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal.
Using single-cell ratio imaging of Fura-2-loaded neutrophils, we demonstrate that the heterogeneity and asynchrony of the oxidase response originates from variability in the timing and magnitude of the cytosolic free Ca2+ signal. The Ca2+ signals from individual cells could be classified into four types: (a) type 1, a transient rise in Ca2+ occurring within 6 s; (b) type 2, an oscillating cytosolic free Ca2+; (c) type 3, a latent Ca2+ transient significantly delayed (21-56 s); and (d) type 4, no significant Ca2+ rise. These response types accounted for approximately 41%, 15%, 26% and 18% of the population respectively for stimulation with 1 microM f-met-leu-phe peptide (n = 27) and 52.5%, 15%, 11.5% and 21% respectively for 0.1 microM f-met-leu-phe peptide (n = 52). The oxidase in neutrophils in which the cytosolic free Ca2+ concentration rose to greater than 250 nM always became activated. In the presence of extracellular Ca2+, cytosolic Ca2+ rose uniformly throughout the cell, whereas in the absence of extracellular Ca2+, a localised Ca2+ 'cloud' was observed in approximately 30% of cells. A localised activation of the oxidase accompanied the presence of the Ca2+ 'cloud' when the 250 nM Ca2+ threshold was exceeded. The data presented here therefore demonstrate a tight coupling in individual neutrophils between an elevation in cytosolic free Ca2+ above a threshold of 250 nM and activation of the oxidase.
Hallett
M B
MB
Department of Surgery, University of Wales College of Medicine, Cardiff, UK.
Davies
E V
EV
Campbell
A K
AK
D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005562:Formazans / Q000032:analysis; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D009580:Nitroblue Tetrazolium; D010088:Oxidoreductases / Q000378:metabolism*; D013481:Superoxides / Q000032:analysis
D005562:Formazans; D013481:Superoxides; D009580:Nitroblue Tetrazolium; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010088:Oxidoreductases; D002118:Calcium