Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts

Abstract

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.

Molecular and functional characterization of recombinant human γ‐glutamyltransferase

“…Firstly, V79 cells are known to share GSH through intercellular contacts, enhancing GSH content heterogeneity between cells at low cell density [44]. Second, Atzori et al [45] observed that trypsin treatment of human bronchial fibroblasts transiently affected GSH content. Lastly, a detailed study on GGT expression as a function of cell density led to a lower GGT specific activity after 24-h growth.…”

Changes in glutathione homeostasis during liver regeneration in the rat

“…[1][2][3][4][5][6][7][8] Many studies involving lymphocytes and fibroblasts showed that an increased GSH level was associated with an early proliferative response and was essential for the cell to enter the S phase. 2,4,7,8 We showed previously that plating primary cultures of rat hepatocytes under low density, which stimulates hepatocytes to shift from the G 0 to the G 1 phase of the cell cycle, resulted in increased levels of GSH and cysteine, and increased activity of ␥-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, as early as 4 hours after plating.…”

cAMP-induced Mitochondrial Compartment Biogenesis

“…thiol residue status) are most likely the main regulatory processes besides phosphorylation (36). Evidence is now cumulating that cell fate and proliferation depend on its redox status (43)(44)(45)(46). Our study pinpoints a crucial intermediate in the process of redox control of mitochondrial biogenesis and cell growth that is the glutathione redox state.…”