Blue light-induced generation of reactive oxygen species in photoreceptor ellipsoids requires mitochondrial electron transport.
OBJECTIVE
To investigate whether photoreceptor ellipsoids generate reactive oxygen species (rOx) after blue light illumination.


METHODS
Cultured salamander photoreceptors were exposed to blue light (480 +/- 10 nm; 10 mW/cm(2)). The light-induced catalytic redox activity in the culture was monitored with the use of 3,3'-diaminobenzidine (DAB). Tetramethylrhodamine ethyl ester (TMRE) and 2',7'-dichlorodihydro-fluorescein acetate (DHF-DA) were used as probes to measure the mitochondrial membrane potential and intracellular rOx, respectively.


RESULTS
A significant deposit of DAB polymers was found in the culture after exposure to blue light. Basal levels of rOx were observed in photoreceptor ellipsoids when cells were stained with DHF-DA. This staining colocalized with TMRE. After exposure to blue light, a sharp increase of rOx immediately occurred in the ellipsoids of most photoreceptors. When the light intensity was reduced, the response kinetics of rOx generation were slowed down; however, comparable amounts of rOx were generated after a standard time of exposure to light. The production of rOx in photoreceptors was markedly decreased when an antioxidant mixture was included in the medium during exposure to light. Rotenone or antimycin A, the respiratory electron transport blockers at complex I and III, respectively, significantly suppressed the light-evoked generation of rOx.


CONCLUSIONS
A robust amount of rOx is produced in the ellipsoid when photoreceptors are exposed to blue light. This light-induced effect is antioxidant sensitive and strongly coupled to mitochondrial electron transport. The cumulative effect of light on rOx generation over time may implicate a role for mitochondria in light-induced oxidative damage of photoreceptors.
Yang
Jun-Hai
JH
Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 77030-2702, USA. jhyang@bmc.tmc.edu
Basinger
Scott F
SF
Gross
Ronald L
RL
Wu
Samuel M
SM
D015100:3,3'-Diaminobenzidine / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D005452:Fluoresceins; D008027:Light; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D009924:Organ Culture Techniques; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism* / Q000528:radiation effects*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012235:Rhodamines; D012402:Rotenone / Q000494:pharmacology; D014562:Caudata
D005452:Fluoresceins; D017382:Reactive Oxygen Species; D012235:Rhodamines; C401833:tetramethylrhodamine methyl ester; D012402:Rotenone; C065013:2',7'-dichlorodihydrofluorescein; D015100:3,3'-Diaminobenzidine; D000968:Antimycin A