REMODELING OF THE PLASMA-MEMBRANE AFTER STIMULATION OF NEUTROPHILS WITH F-MET-LEU-PHE AND DIHYDROCYTOCHALASIN-B - IDENTIFICATION OF MEMBRANE SUBDOMAINS CONTAINING NADPH OXIDASE ACTIVITY

Superoxide (O-2(-)) production by neutrophils stimulated with chemotactic peptides [eg., formylmethionyl-leucyl-phenylalanine (fMLP)] is transient but increases in rate and duration after pretreatment of the cells with dihydrocytochalasin B (dhCB), suggesting a possible role for the plasma membrane and membrane skeleton in the regulation of the O-2(-) generating system. Analysis of plasma membrane isolated from these cells by isopycnic sucrose density gradient sedimentation showed that there were no significant variation in the distribution of plasma membrane markers between control and dhCB-treated cells, whereas a significant redistribution of plasma membrane markers was observed in dhCB + fMLP-treated cells. Instead of sedimenting to 31-35% sucrose, as in the former two groups, plasma membrane markers were broadly distributed over 25-50% sucrose in the dhCB + fMLP-treated cells. In addition, similar to 80% degranulation was achieved in these cells, whereas little granule release (<5%) was observed in control and dhCB-treated cells. Analysis of the gradient fractions for membrane skeletal (actin and fodrin) and NADPH oxidase (cytochrome b and p47-phox) components in dhCB + fMLP-treated cells demonstrated that the distribution of fodrin, actin, cytochrome b, and p47-phox followed the broad distribution of plasma membrane markers, with an overall eightfold increase in membrane-associated actin. Despite the broad redistribution of plasma membrane markers, the distribution of O-2(-) generating activity remained confined to a narrower peak at similar to 50% sucrose. These results demonstrate that a heterogeneous surface membrane of higher density with a differential distribution of proteins and O-2(-) generating activity are created after dhCB + fMLP treatment; however, domain structure is conserved in the new membrane, with only a subfraction of the reorganized plasma membrane containing all of the components necessary for active O-2(-) generation. Our results support a role for plasma membrane lateral organization and participation of the membrane skeleton in the regulation of the O-2(-) generating system.