Molecular steps involved in light-induced oxidative damage to retinal rods.
OBJECTIVE
To define the molecular mechanism underlying light-induced oxidative damage to retinal photoreceptors.


METHODS
Oxidative stress was induced in isolated rod photoreceptors by bright 470- to 490-nm light and monitored by measuring the conversion of dihydrorhodamine 123 to rhodamine, with fluorescence microscopy. The effect of the wavelength on oxidant generation was investigated by applying prebleaching stimuli of either 485- or 520-nm light before the bright 470- to 490-nm light. The role of internal messengers in photooxidative stress and membrane damage by bright 470- to 490-nm light was investigated by patch-clamp recording.


RESULTS
Constant illumination with bright 470- to 490-nm light caused a rapid increase in generation of oxidants, which peaked after approximately 60 seconds, and a decrease in membrane resistance, eventually producing irreversible membrane damage. The time course and extent of oxidant generation were not affected by the absence of intracellular guanosine triphosphate (GTP) or adenosine triphosphate (ATP), suggesting that oxidative stress and membrane damage induced by 470- to 490-nm light do not require coupling to a GTP-binding protein. Prebleaching exposure to 520-nm light suppressed oxidative stress and membrane damage by subsequent application of bright 470- to 490-nm light, and the extent of suppression increased with prebleaching duration.


CONCLUSIONS
Oxidative stress and damage induced in rods in response to 470- to 490-nm light require rhodopsin activation, but not visual transduction steps downstream of active rhodopsin. Prebleaching with 485- or 520-nm light has a different effect on the level of a transient rhodopsin intermediate required for lipid peroxidation by 470- to 490-nm light.
Demontis
Gian Carlo
GC
Department of Psychiatry, Neurobiology, Pharmacology, and Biotechnology, University of Pisa, Pisa, Italy.
Longoni
Biancamaria
B
Marchiafava
Pier Lorenzo
PL
D000818:Animals; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D008027:Light; D018613:Microscopy, Confocal; D018384:Oxidative Stress / Q000528:radiation effects*; D018408:Patch-Clamp Techniques; D011833:Radiation Injuries, Experimental / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D011894:Rana pipiens; D012162:Retinal Degeneration / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D012235:Rhodamines / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014785:Vision, Ocular / Q000502:physiology
D012235:Rhodamines; D017392:Thiobarbituric Acid Reactive Substances; C058319:dihydrorhodamine 123; D012243:Rhodopsin; D019204:GTP-Binding Proteins