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 (delta psi(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 CD3epsilon cross-linking), cells manifest a near-to-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 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 delta psi(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 delta psi(m) dissipation linked to GSH depletion (step 1), cells hyperproduce ROS with an associated disruption of Ca2+ homeostasis (step 2).
Macho
A
A
National Center for Scientific Research, Unit 420, Villejuif, France.
Hirsch
T
T
Marzo
I
I
Marchetti
P
P
Dallaporta
B
B
Susin
S A
SA
Zamzami
N
N
Kroemer
G
G
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D007425:Intracellular Membranes / Q000502:physiology; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000502:physiology*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology*; D013997:Time Factors
D017382:Reactive Oxygen Species; D005978:Glutathione; D002118:Calcium