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

Abstract

Stimulation of the respiratory burst in 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 in 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 in rate of release of 02- 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 02- by a mean of 23 s (41%). Dithioerythritol, a sulfhydryl-reducing agent, abolished both the S-thiolation and priming mediated by diamide. H202 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.

Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth

“…Support for this conclusion came from experiments using the SOD linked to polyethylene glycol (PEG-SOD) [55] or high concentrations of extracellularly added SOD [56]. The increase in the HE-derived fluorescence observed after treatment of the cells with H 2 O 2 was attributed to intracellular stimulation of superoxide production by H 2 O 2 [53,54,57-59]. Based on these results, the HE-derived red fluorescence became widely used for the detection and quantitation of intracellularly generated superoxide [60-68].…”

Redox systems of the cell: Possible links and implications

“…This process was reversible, appeared dependent on hydrogen peroxide (37), and resulted in an increased cell uptake of glutathione (38). Although anticipated to be a mechanism that might shut off the respiratory burst, enhancement of oxidative stress during the burst caused a paradoxical priming, or further stimulation, of respiratory burst activity (39). Because decreased GAPDH activity would result, it is conceivable that increased glucose metabolism through the hexose monophosphate shunt and decreased glucose utilization through glycolysis could have a role in this effect, and, further, that such changes could impact inf lammator y cell sur vival (40 -43) and͞or mode of death (44).…”

Characterization of UPF peptides, members of the glutathione analogues library, on the basis of their effects on oxidative stress-related enzymes

“…Controversially, research has shown that binding of thiol groups does not always lead to an inhibition of NAD(P)H oxidase. Moriguchi et al [42] induced S-thiolation by pre-treating the neutrophils with a direct thiol oxidizing compound diamide and showed the enhanced release of superoxide anion by NAD(P)H oxidase after binding the thiol groups. Different effects of thiol binding could be explained by the exact location of the modification and previous redox status of the enzyme.…”