Related Experiment Videos
Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells
F Valentin1, J Bueb, C Capdeville-Atkinson
1Centre de Recherche Public-Santé, Laboratoire de Neuroimmunologie et Inflammation, L-1150 Luxembourg, Grand-Duché du Luxembourg.
Cell Calcium
|May 16, 2001
Summary
Neutrophil activation by fMLP involves increased intracellular calcium and superoxide production, dependent on extracellular calcium entry. This process requires activation of pertussis toxin-sensitive G-proteins and Rac-1 sequestration in the plasma membrane.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- Neutrophil-like HL-60 cells are a model for studying neutrophil activation.
- N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) is a potent chemoattractant that triggers neutrophil responses.
- NADPH oxidase activation leads to superoxide anion production, a key component of the respiratory burst.
Purpose of the Study:
- To investigate the role of extracellular calcium and G-protein signaling in fMLP-induced NADPH oxidase activation and superoxide production in HL-60 cells.
- To elucidate the mechanism of Rac-1 activation and its relationship with calcium influx.
Main Methods:
- Utilized HL-60 cells stimulated with fMLP.
- Measured intracellular calcium concentration ([Ca2+]i) changes.
- Assessed NADPH oxidase activation and superoxide anion (O2-) production.
- Employed immunodetection and double labeling fluorescent methods to study Rac-1 plasma membrane sequestration.
- Investigated the effect of extracellular calcium removal and pertussis toxin (PTX).
Main Results:
- fMLP induced a rise in [Ca2+]i, NADPH oxidase activation, and O2- production.
- [Ca2+]i mobilization and O2- production were dependent on extracellular calcium (Ca2+]e) and capacitative calcium entry.
- Rac-1 activation was mediated by a PTX-sensitive heteromeric G-protein pathway.
- Rac-1 membrane sequestration preceded [Ca2+]i mobilization following Ca2+ entry.
Conclusions:
- Superoxide production in response to fMLP is dependent on the activation of PTX-sensitive G-proteins.
- Rac-1 sequestration in the plasma membrane, triggered by extracellular calcium entry and subsequent [Ca2+]i mobilization, is crucial for NADPH oxidase activation.
- This study elucidates a signaling cascade involving calcium influx, G-protein activation, and Rac-1 translocation in neutrophil superoxide production.