Chapter 10 - Redox Clamp Model for Study of Extracellular Thiols and Disulfides in Redox Signaling
Introduction
Recognition of the highly regulated nature of extracellular thiol/disulfide couples, measured as GSH/GSSG and cysteine/cystine (Cys/CySS) redox potentials (Jones et al., 2000), prompted studies to determine whether variation in extracellular redox state changed with cell differentiation (Nkabyo et al., 2002) or affected cell growth and proliferation (Jonas et al., 2002, Jonas et al., 2003). The latter studies revealed that human cells in culture regulate the Cys/CySS redox potential (EhCySS) of the culture medium to the same value as found in plasma of young health adults (Fig. 10.1). Subsequent human research has revealed that EhCySS values are more oxidized in association with disease risk factors and specific diseases (Jones and Liang, 2009). In the present chapter, we describe the redox clamp approach which is useful to study mechanisms whereby variation in extracellular Eh value contributes to disease.
Section snippets
Key Concepts for Use
The GSH/GSSG and Cys/CySS couples represent distinct redox signaling nodes (Jones et al., 2004) which have not been fully delineated. The initial observations showed that the GSH/GSSG redox potential (EhGSSG) in human plasma is maintained at a more reducing steady-state value (approximately − 140 mV) compared to the EhCySS value (approximately − 80 mV). Importantly, the Cys/CySS pool size (> 90 μM in Cys equivalents) in human plasma is at least 10-fold greater than the GSH/GSSG pool (< 9 μM in GSH
Principles for Experimental Design
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Desired Ehvalues are obtained by addition of selected concentrations of thiol and disulfide.
A redox clamp is obtained by using a relatively large pool of Cys plus CySS in which the concentrations of Cys and CySS are set to values which give desired Eh values according to the Nernst equation:In this equation, Eo is the standard half-cell potential (− 250 mV for pH 7.4) for the Cys/CySS couple relative to a standard hydrogen electrode, R is the gas constant, T is
Summary of Available Redox Clamp Studies
Hwang and Sinskey (1991) provided an important contribution to the understanding of redox potential and mammalian cell growth in a study designed to optimize in vitro conditions for production of biologic products using cultured mammalian cells. They noted that three parameters were critical to rapidly obtain maximum cell density, i.e., pO2, pH, and redox potential. They found that with pO2 and pH controlled, the redox potential (measured with a potentiometric electrode) could be maintained by
Perspectives and Conclusion
The redox clamp approach as described provides a straightforward and convenient means to test for redox signaling processes which are dependent upon extracellular EhCySS or EhGSSG. The cumulative evidence indicates that multiple extracellular and cell surface proteins are responsive to thiols and disulfides in the plasma and other extracellular fluids. Although information on Eh values for thiol/disulfide couples in biologic fluids is limited, the available data has been recently reviewed (Go
Acknowledgments
This work was supported by NIH grants ES011195 and ES009047.
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2019, Current Opinion in ToxicologyCitation Excerpt :ENMs have the potential to affect these thiols through direct interaction, but also by changing the extracellular oxidation state. An oxidizing extracellular state leads to a more oxidized state of extracellular thiols thereby resulting in activation of NF-kB as well as increased mitochondrial ROS production [36,37]. Interestingly, all of these events could occur without uptake of the ENM into the cell, which has yet to be explored.
Selenium supplementation prevents metabolic and transcriptomic responses to cadmium in mouse lung
2018, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Protein concentration was measured in 5 μL of tissue homogenates using Bio-Rad DC kit. Indicators of oxidative stress, glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), cystine (CySS) and calculated Nernst potentials (Eh) for the GSH/GSSG and Cys/CySS couples, were measured [43,44] but are not included because no significant effects were observed. Total RNA was isolated from mouse lung (n = 5/group) with the mirVana RNA isolation kit (Life Technologies, Grand Island, NY) and stabilized in RNAlater (QIAGEN, Valencia, CA).
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2014, Advances in GeneticsThe thiol pool in human plasma: The central contribution of albumin to redox processes
2013, Free Radical Biology and MedicineCitation Excerpt :The extracellular concentrations of different thiols and oxidized derivatives are tightly regulated, and its alterations are associated with cellular responses. Cells in culture respond to increased ratios of cysteine disulfide to cysteine in the extracellular medium through downstream events that impact on proliferation, apoptosis, and proinflammatory signaling (for review see [29]). In line with this concept, the in vivo concentrations of plasma low molecular weight thiols and disulfides have been found altered in association with conditions such as aging, smoking, obesity, and alcoholism among others [30].
Application of a metal-insulator-semiconductor (MIS) sensor for extracellular redox potential detection
2013, Sensors and Actuators, B: ChemicalCitation Excerpt :The value can be calculated as Eh = −250 + 29.5log([CySS]/[Cys]2) [3,16,18–20] at pH 7.4, where ‘−250′ (in mV) represents the standard half-cell potential relative to a standard hydrogen electrode when a platinum electrode is used as the indicating electrode, the sensitivity is treated as a constant (29.5) at room temperature (25 °C) for a two-electron transfer reaction, the concentration values (in molar) is obtained through high-performance liquid chromatography (HPLC), and the Eh value is expressed in millivolts. Besides, the extracellular thiol/disulfide redox environments can be clamped through addition of thiols and disulfides to obtain required redox state [6,8,18]. Potentiometric measurements of redox potential were often carried out by directly detecting the half-cell potential of an indicating electrode (redox electrode) relative to a reference electrode.
Cysteine/cystine redox signaling in cardiovascular disease
2011, Free Radical Biology and MedicineCitation Excerpt :In this section, we focus on recent advances that show that oxidized extracellular EhCySS stimulates inflammatory redox signaling associated with mechanisms in the pathogenesis of vascular disease (Fig. 1). Downstream signaling due to changes in extracellular EhCySS has been studied using a redox clamp in which Cys and CySS concentrations are used to obtain a series of controlled EhCySS [35]. The extracellular/plasma Eh is controlled mainly by Cys/CySS and GSH/GSSG, whereas redox states of other subcellular compartments (cytoplasm, mitochondria, nucleus, endoplasmic reticulum) are maintained by different thiol/disulfide couples (see Fig. 2).