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 i nclude a disruption of the mitochondrial transmembrane potential (Delt a Psi(m)), depletion of nonoxidized glutathione (GSH) levels, an incre ase in the production of reactive oxygen species (ROS), and an elevati on in cytosolic free Ca2+ levels. Using a cytofluorometric approach, w e 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 CD3 epsilon cross-linking), cells manifest a near-t o-simultaneous Delta Psi(m) dissipation and GSH depletion early during the apoptotic process, None of the protocols for apoptosis inhibition (antioxidants, Delta Psi(m), stabilization, Bcl-2 hyperexpression, or inhibition of IL-1-converting enzyme) allowed for the dissociation of Delta Psi(m) disruption and GSH depletion, indicating that both param eters 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 conseq uence on Delta Psi(m), cellular redox potentials, or ROS production, p ro-oxidants and menadione, an inducer of mitochondrial superoxide anio n generation, cause a rapid (15 min) Ca2+ elevation. Together, these d ata suggest a two-step model of the common phase of apoptosis. After a n initial Delta Psi(m) dissipation linked to GSH depletion (step 1), c ells hyperproduce ROS with an associated disruption of Ca2+ homeostasi s (step 2).