Probucol, an
antioxidant and
anti-inflammatory agent counteracting
atherosclerosis and restenosis, is partially effective by influencing suicidal cell death or apoptosis. In analogy to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling with
phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase in cytosolic Ca(2+) activity, for example, after energy depletion or oxidative stress. The present study explored whether
probucol influences eryptosis.
Phosphatidylserine exposure was estimated from
annexin-V-binding, cell volume from forward scatter (FSC), and cytosolic Ca(2+) concentration from
fluo-3 fluorescence in flow cytometry. As a result, energy depletion (48-hour
glucose removal) increased
annexin-V-binding, decreased FSC, and increased
fluo-3 fluorescence.
Probucol (≤30 μM) did not significantly modify
annexin-V-binding, FSC, or
fluo-3 fluorescence in the presence of
glucose but (at ≥5 μM) blunted the effect of
glucose depletion on
annexin-V-binding.
Probucol (≥20 μM) only slightly blunted the effects of
glucose depletion on FSC and
fluo-3 fluorescence. Ca(2+)
ionophore ionomycin (1 μM) and oxidative stress (30-minute exposure to 0.3 mM of
tert-butylhydroperoxide) increased
annexin-V-binding, effects again blunted by 30 μM of
probucol. In conclusion,
probucol blunts cell membrane scrambling after energy depletion and oxidative stress, effects primarily because of interference with the scrambling effects of increased cytosolic Ca(2+) concentration.