Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis.

When cells undergo nuclear apoptosis (chromatin condensation, DNA fragmentation), they already manifest at least three alterations that can be quantified cytofluorometrically at the single-cell level: 1) a loss of mitochondrial transmembrane potential (delta psi m), 2) an increased production of superoxide anions, and 3) the aberrant exposure of phosphatidylserine (PS) residues on the outer plasma membrane leaflet. This latter alteration allows for the phagocytic recognition/elimination of apoptotic cells. In this work, we show that cells first undergo the delta psi m disruption and that PS exposure only affects cells that already have a low delta psi m. Pharmacologic modulation of apoptosis with inhibitors of macromolecule synthesis or proteases, as well as with drugs stabilizing the delta psi m, indicates that delta psi m disruption and PS exposure are coregulated. Interventions on apoptosis-regulatory genes (p53, bcl-2) confirm the coregulation of delta-psi-m disruption, PS exposure, and nuclear signs of apoptosis. In all conditions in which apoptosis is prevented, the delta psi m remains stable and PS cannot be detected on the cell surface. Reactive oxygen species do not contribute to PS exposure, based on two lines of evidence. First, among thymocytes undergoing apoptosis in response to dexamethasone, delta psi mlow cells first expose PS and then hyperproduce superoxide anion. Second, exogenous sources of reactive oxygen species or the superoxide anion-generating drug menadione fail to cause rapid PS exposure. Instead, direct interventions on mitochondria using inhibitors of the respiratory chain or the F1 ATP synthase cause PS exposure in cells subsequent to delta psi m disruption. This effect is also obtained in anucleate cells, indicating that the nucleus does not intervene in the sequence of events coupling mitochondrial dysfunction to PS exposure. Altogether, these data underline the functional impact of mitochondrial alterations on the apoptotic process.