Hanna Mäenpää

Expression of glutamate transporter subtypes in cultured retinal pigment epithelial and retinoblastoma cells

Purpose. Glutamate is the major excitatory neurotransmitter in the retina and glutamate uptake is essential for normal glutamate signalling. Retinal diseases may induce neurochemical changes which affect retinal cells including retinal pigment epithelium (RPE). The aim of the study was to investigate the expression of glutamate transporter subtypes in RPE and retinoblastoma cells and to clarify the effect of proliferation modulators on the levels of the expressed transporter in the RPE cell line. Methods. Cultured pig RPE cells and two human RPE cell lines, D407 and ARPE-19, as well as the human retinoblastoma cell line Y79 were used. Glutamate transporter expression was evaluated with Western blot analysis and immunocytochemistry. Results. The study revealed unexpected expression of neuronal glutamate transporter/chloride channel EAAT4 in these three cell lines, but not in cultured pig RPE cells, whereas another glutamate carrier, EAAC1, was present in all cell types utilized. Other transporter subtypes, GLT1, GLAST and EAAT5 were not found. Neither tamoxifen, known to inhibit both proliferation and glutamate uptake in RPE cells, nor retinoic acid nor insulin, also known to affect cell proliferation rates, were capable of changing the total levels of EAAT4 in APRE-19 cells. Conclusions. Neuronal glutamate transporter EAAC1 is expressed in RPE cells. The robust expression of EAAT4 in cell lines may reflect a role of EAAT4 in cell proliferation and migration. Unaltered steady-state expression of this carrier and chloride-channel protein hints at posttranslational mechanisms of regulation of EAAT4.

Toxicity of selected cationic drugs in retinoblastomal cultures and in cocultures of retinoblastomal and retinal pigment epithelial cell lines.

Tamoxifen and toremifene are antiestrogenic drugs successfully used in the therapy of breast cancer. Rheumatoid arthritis and malaria have been treated with chloroquine for decades. Unfortunately, tamoxifen and chloroquine are reported to induce retinal changes as a side effect. We now studied the effects of tamoxifen, toremifene, and chloroquine on the viability of the human retinoblastomal cell line Y79, using the WST-1 test or measurement of the cellular ATP content. The studies were made on Y79 cell cultures and on cocultures of Y79 cells and retinal pigment epithelial cell line ARPE-19. The cocultures were used to clarify the effect of retinal pigment epithelium on toxicity to Y79 cells. In the coculture, the drugs were applied to ARPE-19 cells growing in the culture inserts on top of Y79 cells and the viability of ARPE-19 and Y79 cells was assessed separately. Tamoxifen, toremifene, and chloroquine reduced dose-dependently the viability of Y79 cells after 24-h exposure. The ARPE-19 cells proved to be protective after chloroquine exposure in the coculture. The results shed light on the toxicity of tamoxifen and chloroquine in Y79 cells in vitro. With the coculture we were able to simulate the in vivo route of chloroquine to the retina via the retinal pigment epithelium.

Metabolism-Induced Toxicity of Selegiline and Carbamazepine Studied with an In Vitro Method

Carbamazepine and selegiline, although neuroprotective themselves, are presumed to have toxic metabolites. The aim of this study was to investigate the possible metabolism-induced toxicity of selegiline and carbamazepine with a novel in vitro method: The drugs were incubated with target cells (neuroblastoma SH-SY5Y) with or without a pre- incubation with mouse or human hepatocytes. The viability of SH-SY5Y cells was then measured by using total cellular ATP as an indicator of the cell viability. For the pre-incubation with hepatocytes two different methods were used: Hepa- tocytes were grown either in multiwell plates (Model 1) or in filter inserts (Model 2). Selegiline itself increased SH-SY5Y viability, but the pre-incubation with both mouse and human hepatocytes made se- legiline slightly toxic to SH-SY5Y cells. The biotransformation of carbamazepine seemed to be more complex and showed variation in different hepatocyte models. In general, human hepatocytes increased carbamazepine toxicity to SH- SY5Y cells, whereas mouse hepatocytes had no such effect. The methodology used (especially Model 1) could form a ba- sis in developing a test system for a qualitative detection of metabolism-induced (neuro)toxicity in the early phase of drug discovery. In this respect, the present study might be promising for further evaluation by means of a larger number of in- dependent experiments and different types of compounds.