Receptor-mediated actin assembly in electropermeabilized neutrophils: role of intracellular pH.
Neutrophil activation by a variety of stimuli is accompanied by an intracellular acidification, which has been postulated to mediate actin polymerization (Yuli and Oplatka, Science 1987, 235, 340). This hypothesis was tested using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin staining and flow cytometry, or right angle light scattering to study actin assembly in intact and electrically permeabilized human neutrophils. Intracellular pH was measured fluorimetrically using a pH sensitive dye. In cells stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) at 21 degrees C, actin assembly clearly preceded the intracellular acidification in response to fMLP. Moreover, actin polymerization persisted in cells where intracellular pH was clamped near the resting (unstimulated) level using nigericin/K+. Finally, fMLP induced a significant increase in F-actin content in electropermeabilized neutrophils equilibrated with an extracellular medium containing up to 50 mM HEPES. These observations indicate that fMLP-stimulated F-actin assembly is not mediated by a decrease in intracellular pH and suggest that changes in transmembrane potential and ionic gradients are unlikely to mediate actin polymerization.
Downey
G P
GP
Respiratory Division, Toronto General Hospital, Ontario, Canada.
Grinstein
S
S
D000199:Actins / Q000097:blood*; D000546:Amanitins; D002463:Cell Membrane Permeability; D004560:Electricity; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D046911:Macromolecular Substances; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D012542:Scattering, Radiation
D000199:Actins; D000546:Amanitins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009240:N-Formylmethionine Leucyl-Phenylalanine; C028877:7-nitrobenz-2-oxa-1,3-diazole-phallacidin