Pancreatic beta-cell death induced by oxidative stress plays an important role in the pathogenesis of
diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic beta-cell line NIT-1 cells exposed to H2O2 or
alloxan. Intracellular pH was measured by a pH-sensitive
dye, and cell damage by double staining with
Annexin-V and
propidium iodide using flow cytometry. H2O2 and
alloxan caused a rapid fall in intracellular pH and suppressed Na+/H+ exchanger activity in the NH4Cl prepulse method. H2O2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using
nigericin (unclamped cells vs clamped cells,
necrosis 43.8 +/- 5.8% vs 21.1 +/- 10.6%, P < 0.05; apoptosis 8.0 +/- 1.9% vs 44.5 +/- 5.0%, P < 0.01). pH-clamped cells showed enhanced
caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to
alloxan toxicity, but treatment with
alloxan and
nigericin strikingly enhanced the cytotoxicity.
Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H2O2 -treated beta cells, whereas no link between cell death and acidification was demonstrated in
alloxan toxicity.