Abstract
Purpose::
We hypothesize that Retinol dehydrogenases (RDHs) 11 and 12 protect photoreceptors from toxic aldehydes. Both enzymes are located in photoreceptor inner segments and can reduce aldehydes to alcohols, using NADPH as a coenzyme. Their substrate specificity is directed towards retinaldehydes and the short-chain aldehydes produced during lipid peroxidation. These molecules are particularly dangerous because they can induce oxidative stress in cells.
Methods::
We first investigated by quantitative RT-PCR (Q-PCR) and immunoblot if the expression of endogenous RDH enzymes (RDH11, RDH12, RDH13, RDH14, and retSDR1) was modified during light-induced oxidative stress, in the photoreceptor cell line 661W. We then measured the total RDH activity in vitro using membrane fractions prepared from light-exposed 661W cells and all-trans retinal as substrate.We then transfected 661W cells with RDH11, RDH12, and RDH8 and investigated whether or not these enzymes could protect against light-induced or 4-hydroxy-2-nonenal (4-HNE)-induced oxidative stress, by measuring cell survival during treatments.
Results::
We found that the reducing activity towards all-trans-retinal (total RDH activity) in microsomal fractions of 661W cells was significantly decreased after 4 hours of light exposure at 25,000 lux. However, Q-PCR and immunoblot analysis showed that expression of endogenous RDHs was either unchanged or induced at 4 hours of light exposure. When transfected in 661W cells, RDH8, RDH11 and RDH12 protected the cells against light-induced apoptosis with RDH8 displaying the highest protection. We also found that only RDH11 and RDH12 were protective against 4-HNE-induced cell death with RDH11 displaying the highest protection.
Conclusions::
RDHs could protect against oxidative stress by reducing toxic aldehydes to less toxic alcohols. However, their activity is unpaired during light exposure, which probably precipitates the process of cell death. This decrease of activity could not be explained by a decrease of expression, and we hypothesize that a post-transcriptional modification is taking place during light exposure and is responsible for reduced catalytic activity.
Keywords: retinoids/retinoid binding proteins • protective mechanisms • photoreceptors