Biochemical characterization of cell specific enzymes in light-exposed rat retinas: oxidative loss of all-trans retinol dehydrogenase activity.
OBJECTIVE
To determine the effect of acute, intense, visible light on the activities of all-trans retinol dehydrogenase (t-RDH) and glutamine synthetase (GS), two oxidatively sensitive enzymes located in the photoreceptors and Müller cells, respectively.


METHODS
Male albino rats, previously maintained in a weak cyclic light- or dark-rearing environment, were exposed to intense light (490-580 nm) for as long as 24 hrs. One-half of the experimental animals were pre-treated with the antioxidant 1,3-dimethylthiourea (DMTU), at 500 mg/kg, i.p., 24 hrs before and just before light exposure. Upon sacrifice, retinas were excised for the determination of t-RDH and GS activity, or for the preparation of rod outer segments (ROS). Other light-exposed rats were maintained in darkness for 2 weeks before sacrifice, for rhodopsin determinations. Retinal homogenates were also treated in vitro under oxidizing conditions to compare enzymatic inactivation with the in vivo effects of light exposure.


RESULTS
In cyclic light-reared rats 24 hr light exposures resulted in a significant loss of t-RDH activity in retinal homogenates and in isolated ROS. In both the retina and ROS, pretreatment of the animals with DMTU completely prevented the loss of t-RDH activity. As measured by rhodopsin levels 2 weeks after light exposure, DMTU-treated rats exhibited no loss of photoreceptor cells, whereas those not given the antioxidant lost over 50% of their photoreceptors. Retinal GS activity was unchanged by 24 hr intense light exposures. In dark-reared rats 4 hr light exposures did not alter retinal t-RDH or GS activity, despite the loss or approximately 70% of the rhodopsin content or the eye, measured 2 weeks later. When 4 hr light-exposed rats were held in darkness for an additional 20 hrs, a significant loss of retinal t-RDH occurred, but no change in GS activity was measured. In these rats DMTU treatment also prevented the loss of t-RDH activity. In contrast to the lack of an in vivo light effect on retinal GS, oxidation in vitro completely inactivated the enzyme after only 1 hr.


CONCLUSIONS
The light-induced loss of t-RDH in both cyclic light- and dark-reared rats is an oxidative and time dependent process that is not strictly photochemical in nature. The loss of rhodopsin and t-RDH activity, but not GS activity, following intense light exposure are manifestations of light's effect on photoreceptor cells without a comparable effect in the adjacent retinal Müller cells. Additional work will be needed to understand the differences in light damage susceptibility between retinal photoreceptors and glial cells and between cyclic light- and dark-reared rats.
Darrow
R A
RA
Petticrew Research Laboratory, Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA.
Darrow
R M
RM
Organisciak
D T
DT
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D000072467:Cytochrome P450 Family 2; D003623:Dark Adaptation / Q000502:physiology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D008027:Light; D008297:Male; D010084:Oxidation-Reduction; D017440:Photoperiod; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects*; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000528:radiation effects*; D013237:Stereoisomerism; D013347:Subcellular Fractions / Q000201:enzymology
D000975:Antioxidants; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2; D005974:Glutamate-Ammonia Ligase