Peroxynitrite-induced apoptosis in photoreceptor cells.
OBJECTIVE
To study the mechanisms of peroxynitrite-induced photoreceptor cell damage, using retinal cultures and a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1).


METHODS
Retinal explants obtained from 20-day-old Lewis rat pups, were exposed to SIN-1 for varying lengths of time at varying concentrations. Apoptosis in the photoreceptor cells was detected using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a DNA fragmentation assay. Selected retinal samples were processed for an ultrastructural analysis to confirm apoptosis. The retinas exposed to SIN-1 were tested for the expression of caspase-3 by immunohistochemistry and a Western blot analysis. The retinas were also evaluated for the prevention of apoptosis in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk).


RESULTS
The retinal explants exposed to SIN-1 showed a significant increase in the presence of TUNEL-positive photoreceptor cells. Similarly lineal increases in TUNEL-positive cells were seen in the presence of increasing concentrations of SIN-1. DNA ladder formation was seen with the exposure of SIN-1. Ultrastructurally, SIN-1 exposed retinas revealed typical apoptotic changes in the photoreceptor cell nuclei. The retinas preincubated with urate for 6 hours and exposed to SIN-1 for 16 hours showed significantly fewer TUNEL-positive cells compared to the retinas exposed to SIN-1 alone (p < 0.05). Moreover, the retinas exposed to SIN-1 showed the expression of caspase-3. This expression, as well as the number of apoptotic photoreceptors, significantly decreased in the presence of Z-VAD-fmk.


CONCLUSIONS
These results show that peroxynitrite induces apoptosis in photoreceptor cells and that such retinal damage appears to be mediated by caspase-3. The apoptotic process can be minimized by peroxynitrite scavenger urate, as well as by the caspase inhibitor Z-VAD-fmk.
Ito
Shinji
S
Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Wu
Guey-Shuang
GS
Kimoto
Takashi
T
Hisatomi
Toshio
T
Ishibashi
Tatsuro
T
Rao
Narsing A
NA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology / Q000473:pathology* / Q000648:ultrastructure; D051381:Rats; D011917:Rats, Inbred Lew; D014527:Uric Acid / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D020030:Nitric Oxide Donors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D030421:Peroxynitrous Acid; D014527:Uric Acid; C002385:linsidomine; D004247:DNA; D008981:Molsidomine; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases