Abstract
Both zinc (Zn(2+)) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn(2+) and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn(2+) rises that were divided into four phases in the course of 30 min of OGD. The first Zn(2+) rise was a transient, which was followed by a latent phase during which Zn(2+) levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn(2+) rise, which reached a sustained plateau called Zn(2+) overload. Zn(2+) rises were not observed when Zn(2+) was removed by TPEN (a Zn(2+) chelator) or thapsigargin (depleting Zn(2+) from intracellular stores) treatment, indicating that Zn(2+) was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn(2+) rise, indicating that the mitochondrial Zn(2+) accumulation contributes to Zn(2+) overload. We also detected two OGD-induced ROS rises. Two Zn(2+) rises preceded two ROS rises. Removal of Zn(2+) reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn(2+) contributes to mitochondrial ROS generation. There was a Zn(2+)-induced increase in the functional component of NADPH oxidase, p47(phox), thus suggesting that NADPH oxidase may mediate Zn(2+)-induced ROS accumulation. We suggest a new mechanism of cross talk between Zn(2+) and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn(2+) and ROS accumulations during the course of ischemic stress.