Redox clamp model for study of extracellular thiols and disulfides in redox signaling

Abstract

Extracellular thiol/disulfide redox environments are highly regulated in healthy individuals and become oxidized in disease. This oxidation affects the function of cell surface receptors, ion channels, and structural proteins. Downstream signaling due to changes in extracellular redox potential can be studied using a redox clamp in which thiol and disulfide concentrations are varied to obtain a series of controlled redox potentials. Previous applications of this approach show that cell proliferation, apoptosis, and proinflammatory signaling respond to extracellular redox potential. Furthermore, gene expression and proteomic studies reveal the global nature of redox effects, and different cell types, for example, endothelial cells, fibroblasts, monocytes, and epithelial cells, show cell-specific redox responses. Application of the redox clamp to studies of different signaling pathways could enhance the understanding of redox transitions in many aspects of normal physiology and disease.

Figure 10.1

Changes in cysteine/cystine redox potential in culture medium of HT29 cells. Cells were grown to 90% confluence, culture media was removed, and cells were washed once with PBS. Cys and CySS-free DMEM with 10% neonatal calf serum was added with Cys and CySS concentrations calculated as in Table 10.1 to give either −150 or 0 mV. Samples were collected over a 24-h time course and Cys and CySS concentrations were measured by HPLC. E_h was calculated using the Nernst equation.

Figure 10.2

Human plasma redox states of thiol/disulfide couples, GSH/GSSG, and Cys/CySS, and control of extracellular E_hCySS by cultured cells. (A) Histogram showing frequency of E_hCySS and E_hGSSG in human plasma. Data are fasting values for young, healthy, and physically fit individuals. More reduced, normal and more oxidized ranges of plasma redox potential are indicated by the labels at the top. (B) Changes in extracellular E_hCySS with time were measured in cultured bovine aortic endothelial cells (Go and Jones, 2005), human retinal pigment epithelial cells ( Jiang et al., 2005), and human colonic cancer cells, HT-29 (Anderson et al., 2007). Cells were incubated with different initial E_hCySS (−159, −130, −77, −35, −23, −8 mV) and concentrations of Cys and CySS were quantified as function of time (0, 1, 2, 4, 10 h). E_hCySS values calculated from the Nernst equation are shown as mean ± S.E.M., n = 3.