Rac-1-mediated O-2(-) secretion requires Ca2+ influx in neutrophil-like HL-60 cells

Neutrophil-like HL-60 cells reacted to N-formyl-L-Methionyl-L-Leucyl-L-Phen ylalanine (fMLP) with a rise in the intracellular calcium concentration ([C a2+](i)), NADPH oxidase activation, and increased superoxide anion (O-2(-)) production. [Ca2+](i) mobilization and superoxide production were largely dependent on extracellular calcium (Ca-e(2+)) and a capacitative calcium en try. The monomeric G-protein, Rac-1, regulates NADPH oxidase activity. We t ested the effect of removal of Ca-e(2+) on Rac-1 plasma membrane sequestrat ion and activation of NADPH oxidase using immunodetection and a double labe lling fluorescent method. Results showed that Rac-1 activation is mediated via a pertussis toxin (PTX)-sensitive heteromeric G-protein pathway, and th at Rac-1 membrane sequestration was preceded by [Ca2+](i) mobilization foll owing entry of Ca-e(2+). Therefore, we propose that O-2(-) production is de pendent on activation of PTX-sensitive G-proteins and sequestration of Rac- 1 in the plasma membrane, following entry of Ca-e(2+). (C) 2001 Harcourt Pu blishers Ltd.