Diamide primes neutrophils for enhanced release of superoxide anion: Relationship to S-thiolation of cellular proteins

Stimulation of the respiratory burst ill phagocytes induces the formation of mixed disulfides between sulfhydryl groups of proteins and low-molecular-weight thiols, We hypothesized that this process (S-thiolation) might be involved hi turning off the respiratory burst, However, induction of S-thiolation by pretreatment of neutrophils with diamide, a direct thiol oxidizing agent, actually primed the cells for a two- to fivefold increase in total release and fourfold increase ill rate of release of O-2- on stimulation by f-Met-Leu-Phe. Generation of intracellular oxidants (hydroethidine fluorescence) was increased ninefold, Priming and S-thiolation were apparent at 1 min of incubation and peaked at 5-10 min, Diamide pretreatment also reduced the lag time between addition of phorbol diester and release of O-2- by a mean of 23 s (41%). Dithioerythritol, a sulfhydryl-reducing agent, abolished both the S-thiolation and printing mediated by diamide. H2O2 also induced priming and S-thiolation; and these were eliminated by dithioerythritol, In contrast to the effect of endotoxin, diamide priming did not affect Ca2+ homeostasis of the neutrophils. Diamide did not significantly alter NADPH oxidase activity in a cell-free system. These findings suggest that sulfhydryl groups on one or more proteins play an important role in modulating the respiratory burst.