Effects of Antiestrogens on Retinal Cells in Vitro -Glutamate transporter as a novel target?

Glutamate is the main excitatory neurotransmitter in the retina and brain, but it may also exert excitotoxic harmful effects on cells, a phenomenon related to retinal diseases such as glaucoma and diabetic retinopathy. An excess of extracellular glutamate may activate an excitotoxic reaction cascade via its synaptic receptors. Efficient glutamate transport is crucial in keeping extracellular glutamate below toxic levels in the retina. Müller glial cells and the retinal pigment epithelium (RPE) are believed to regulate the glutamate concentration in the subretinal space, but little is known of the specific features of the glutamate transporter expressed in the RPE. The purpose of the present study was to investigate the functional characteristics and the expression of glutamate transporter in RPE cells in vitro. The main hypothesis was that selected drugs (tamoxifen and chloroquine) affect the function of the glutamate transporter in the RPE. Tamoxifen and chloroquine have caused retinal defects as an adverse effect of the treatment of breast cancer and rheumatoid arthritis, respectively. In the present study, the effects of these drugs on glutamate uptake in RPE cells and the cytotoxic effects on retinoblastoma Y79 cells were evaluated in vitro. Pig RPE cultures, two human RPE cell lines ARPE-19 and D407 and a human retinoblastoma cell line Y79 were used in the experiments. As a more complex in vitro model, a retinal coculture of ARPE-19 and Y79 cell lines was also established. Glutamate transport was characterized by glutamate uptake assays and the expression of glutamate transporter subtypes detected by immunoblotting and immunocytochemistry. Changes in cell viability were estimated by measuring mitochondrial enzyme activity and cellular ATP levels. The RPE cells expressed active, Na-dependent high-affinity glutamate transporters. Their activity was affected by tamoxifen and its structural analogue toremifene, whereas chloroquine was ineffective. In fact, tamoxifen and toremifene competitively inhibited glutamate transport in both the pig RPE cultures and the RPE cell line, suggesting that this function might constitute a new cellular target for these drugs. The glutamate transporter subtypes expressed in the retinal cells were EAAC1 and EAAT4, which were now detected in the RPE cells and in the retinoblastoma cells for the first time. The results corroborate atypical, strong expression of EAAT4 in the cell lines. The drugs effected a dose-dependent reduction in the viability of Y79 cells, this being discernible with antiestrogens at concentrations attainable in tissues at clinically relevant drug doses. Evaluation of cell viability in the Y79 cell line implies that robust cytotoxicity can be studied with cell lines, while retinal co-cultures and more specific cellular mechanisms such as glutamate uptake may be used for more detailed evaluations.