Nitric oxide-induced accumulation of lipofuscin-like materials is caused by inhibition of cathepsin S.
To determine whether nitric oxide (NO) is involved in accumulation of lipofuscin-like material (LFM) in retinal pigment epithelial (RPE) cells and if this formation is related to NO-mediated modification of cathepsin S (cat S). RPE cell cultures were fed once every day with porcine photoreceptor outer segments (POS) in the presence of NO-donor [S-nitroso-N-acetylpenicillamine (SNAP) or NOC18] for 2 weeks. LFM autofluorescence within the cells was measured by fluorophotometric flow cytometry (FACS). The activity of purified cat S was measured in the presence of NO-donor with or without dithiothreitol (DTT). The following results were observed. SNAP and NOC18 caused LFM accumulation in RPE cells in a dose-dependent manner, and this accumulation was reversed by the addition of NO-scavengers (hydroxycobalamin, carboxy-PTIO). Purified cat S activities were inhibited by NO-donors without DTT, but in the presence of DTT, NO-donors exhibited no inhibitory effect on its activity. Phagocytic challenge of RPE cells increased cat S activity, which was reduced by the addition of NO donors. These results indicated that cat S activity was inhibited by NO-donors and resulted in LFM accumulation in RPE cells. We conclude that NO-mediated inhibition of cat S was caused through protein modification of cat S and resulted in LFM accumulation.
Sugano
Eriko
E
Division of Biofunctional Science, Tohoku University Biomedical Engineering Research Organization, Sendai, Japan.
Tomita
Hiroshi
H
Ishiguro
Sei-ichi
S
Isago
Hitomi
H
Tamai
Makoto
M
D000818:Animals; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology*; D066298:In Vitro Techniques; D008062:Lipofuscin / Q000378:metabolism*; D009569:Nitric Oxide / Q000494:pharmacology*; D010587:Phagocytosis / Q000187:drug effects*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D013552:Swine
D016166:Free Radical Scavengers; D008062:Lipofuscin; D009569:Nitric Oxide; D002403:Cathepsins; C028217:cathepsin S; D004229:Dithiothreitol