Glutathione Depletion Is an Early and Calcium Elevation Is a Late Event of Thymocyte Apoptosis

According to current understanding, several metabolic alterations form part of the common phase of the apoptosis process. Such alterations include a disruption of the mitochondrial transmembrane potential (Δψ_m), a depletion of nonoxidized glutathione (GSH) levels, an increase in the production of reactive oxygen species (ROS), and an elevation in cytosolic free Ca²⁺ levels. Using a cytofluorometric approach, we have determined each of these parameters at the single cell level in thymocytes or T cell hybridoma cells undergoing apoptosis. Regardless of the apoptosis induction protocol (glucocorticoids, DNA damage, Fas cross-linking, or CD3ε cross-linking), cells manifest a near-to-simultaneous Δψ_m dissipation and GSH depletion early during the apoptotic process. None of the protocols for apoptosis inhibition (antioxidants, Δψ_m, stabilization, Bcl-2 hyperexpression, or inhibition of IL-1-converting enzyme) allowed for the dissociation of Δψ_m disruption and GSH depletion, indicating that both parameters are closely associated with each other. At a later stage of the apoptotic process, cells manifest a near-simultaneous increase in ROS production and intracellular Ca²⁺ levels. Whereas the thapsigargin- or ionophore-induced elevation of calcium levels has no immediate consequence on Δψ_m, cellular redox potentials, or ROS production, pro-oxidants and menadione, an inducer of mitochondrial superoxide anion generation, cause a rapid (15 min) Ca²⁺ elevation. Together, these data suggest a two-step model of the common phase of apoptosis. After an initial Δψ_m dissipation linked to GSH depletion (step 1), cells hyperproduce ROS with an associated disruption of Ca²⁺ homeostasis (step 2).