Induction of neuronal apoptosis by thiol oxidation: Putative role of intracellular zinc release

The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can indu ce Zn2+ release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neuro ns, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonylaspartate-fluoromethylketone. N,N,N',N'-Tetrakis( 2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelat ors also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK801, by the intra cellular calcium release antagonist dantrolene, or by high concentrations o f ryanodine. DTDP generated increases in fluorescence signals in cultured n eurons loaded with the zinc-selective dye Newport Green. The fluorescence s ignals following DTDP treatment also increased in fura-2- and magfura-2-loa ded neurons. These responses were completely reversed by TPEN, consistent w ith a DTDP-mediated increase in intracellular free Zn2+ concentrations. Our studies suggest that under conditions of oxidative stress, Zn2+ released f rom intracellular stores may contribute to the initiation of neuronal apopt osis.