Cysteine starvation activates the redox-dependent mitochondrial permeability transition in retinal pigment epithelial cells

Purpose. Glutathione (GSH) plays a key role in protection against oxidative stress. L-Cysteine is thought to be rate-limiting for the synthesis of glutathione (GSH) and therefore may be a critical component in protection against oxidative stress. The purpose of this study was to investigate the role of L-cysteine in GSH metabolism and oxidative stress in human retinal pigment epithelial (hRPE) cells. Methods. To identify the role of cysteine in GSH metabolism in hRPE cells, a strategy of cysteine starvation was used to determine (1) GSH levels and oxidative stress by measuring reactive oxygen species (ROS) production, (2) mitochondrial membrane potential (Deltapsi(m)) and mitochondrial ultrastructure by using conventional electron microscopy (EM), and (3) indices of cell viability and apoptosis including analysis of cells containing hypodiploid amounts of DNA. Results. Cysteine starvation resulted in approximately a 95% decrease in GSH concentrations over 24 hours. The GSH Nernst redox potential (E-h) increased approximately 70 mV (E-h=-248+/-2.9 mV in control cells compared with E-h=-179+/-2.0 mV in cysteine-starved cells) indicating significant intracellular oxidation. Cysteine starvation increased the production of ROS by mitochondrial respiratory complex III (cytochrome bc(1)), determined using a pharmacological strategy that resulted in the loss of Deltapsi(m) and cell death. The loss of Deltapsi(m) and cell death was prevented with bongkrekic acid, an inhibitor of the adenine nucleotide translocator inhibitor, suggesting activation of the mitochondrial permeability transition (MPT). This conclusion was further supported by electron microscopic studies that showed significant mitochondrial swelling, a hallmark of MPT activation. Cell death was not prevented with either the broad-spectrum caspase inhibitor zVADfmk or the caspase 3-specific inhibitor DEVD-CHO, indicating that cytochrome bc(1)-mediated ROS production results in the MPT and necrosis. Conclusions. These results show that cysteine is a required component for normal GSH metabolism and protection against oxidative stress in hRPE cells.