PubMed · 7979112
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R Siebers. 1994. Laboratory blunders revisited.. https://pubmed.ncbi.nlm.nih.gov/7979112/
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Stimulation of the respiratory burst in mouse macrophages or human neutrophils results in the formation of disulfide bonds between low m.w. thiols and sulfhydryl groups on specific cytosolic proteins (S-thiolation). S-thiolation is reversible in certain chemical systems. The aim of the present study was to analyze the dynamic nature of this process in human monocytes under physiologic conditions. We report here that the extent of S-thiolation and the rate of respiratory burst stimulated by opsonized zymosan or phorbol diester increased for 10 to 20 min and then declined (dethiolation) in close association. Individual proteins underwent S-thiolation and dethiolation at different rates. H2O--appeared particularly effective in mediating S-thiolation, based on inhibition of S-thiolation by added catalase and accentuation by azide, which inhibits cellular catalase. S-thiolation did not occur in stimulated monocytes from patients with chronic granulomatous disease. The addition of H2O2 to monocytes or lymphocytes induced rapid S-thiolation (1 to 3 min); a subsequent dethiolation returned most of the proteins to baseline by 15 to 30 min. At 0 degrees C and after addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea, there was effective S-thiolation on exposure to H2O2, but dethiolation was inhibited, suggesting a possible role for glutathione (GSH)/thioredoxin reductase systems in this process. GSH was determined to be the most abundant low m.w. thiol bound to S-thiolated proteins, but gamma-glutamylcysteine and cysteine were also bound. The time of maximal reduction in cytosolic GSH during the respiratory burst (10 min) coincided with the time at which protein-bound GSH was highest. S-thiolation-dethiolation represents a reversible post-translational modification that could protect cellular proteins from irreversible oxidative damage.
This study was undertaken to identify the signaling events involved in activation of neutrophil superoxide anion (O2-) production by eosinophil granule major basic protein (MBP). MBP did not produce an immediate increase in the cytosolic free calcium concentration ([Ca2+]i), characteristic of phospholipase C activation, but did cause a gradual increase in [Ca2+]i in cytochalasin B-treated cells. Preincubation with 0.01 to 3 micrograms/mL pertussis toxin did not inhibit MBP-stimulated O2- production, and MBP did not stimulate an increase in diradylglycerol levels. MBP did stimulate a low level of phospholipase D activity, as measured by a time-dependent increase in phosphatidic acid and, in the presence of 0.5% ethanol, phosphatidylethanol. Inhibition of MBP-stimulated O2- production by genistein and Western blot analysis using an antiphosphotyrosine antibody showed tyrosine kinase activation by MBP. Calmodulin antagonists (calmidazolium and W-7) caused up to 80% inhibition of MBP-stimulated O2- production. In agreement with the pharmacologic sensitivity, MBP did not stimulate any 51Cr release. These data indicate that tyrosine kinase and calmodulin-dependent steps are involved in the noncytotoxic stimulation of neutrophil O2- production by MBP.
In vivo generated nitric oxide, NO, circulates in plasma mainly as an S-nitroso adduct of serum albumin. Compared to free NO, this NO-adduct is relatively long-lived. It exerts EDRF-like effects of vasodilation and platelet inhibition. Free NO is directly inactivated ('quenched') by advanced glycosylation end products (AGEs), glucose-derived protein moieties that form nonenzymatically and accumulate primarily on long-lived tissue proteins. They have been implicated in many of the long-term complications of diabetes mellitus. We found that the antiproliferative effects of thiol-stabilized NO (SNO-BSA) on Con A-stimulated lymphocytes from peripheral blood were even stronger than those of the NO-generating drug SNAP. The antimitogenic activity of SNO-BSA, however, was not significantly enhanced by the low molecular weight NO-carrier glutathione. NO liberated from SNO-BSA in molar excess was almost completely quenched by AGE-BSA. NO-dependent activating effects such as enhanced rate of glucose uptake or generation of cGMP in resting peripheral mononuclear cells (PBMC) and the antiproliferative activity of the NO-carrier BSA on Con A-stimulated cells were thereby abolished. In contrast, advanced glycosylation impaired the ability of BSA to function as NO-carrier, as evidenced by the lack of antiproliferative activity of NO-AGE-BSA and its inability to activate glucose transport or cGMP generation.