Nitric oxide regulates glutathiolation of proteins in the lens of normoglycemic and hyperglycemic rats

Abstract

Abstract: : Purpose: Glutathiolation of various lens proteins including aldose reductase (AR), various antioxidative enzymes and glyceraldehyde 3–phosphate dehydrogenase etc play a significant role in cataractogenesis. Since the lens has extremely low levels of GSSG (5–10 µM), alternative glutathiolating agents would be required. We have identified that GSNO can glutathiolate proteins in low µM range. Therefore, the purpose of the present study is to investigate how NO levels, increased by injecting iNOS substrate, L–arginine or by nitroglycerin (NG)–patch could glutathiolate lens proteins, specifically AR. Methods: Normal and streptozotocin–induced diabetic rats were injected with iNOS substrate, L–arginine or iNOS inhibitor, L–NAME for 10 days. In some cases NG– patches that slowly releases NO were applied. The eye balls were dissected out and lenses were removed, homogenized and Western blot analysis was carried out to identify the formation of protein mixed disulfides by using specific anti–PSSG antibodies. Mixed disulfide of AR (AR–SSG) was also determined by performing immunoprecipitation studies. The in vivo results were confirmed by performing in vitro studies using cultured human lens epithelial cells and ex vivo studies using lenses from normal and diabetic rats. Results: L–arginine and NG patch significantly induced mixed disulfide formation of various proteins, molecular mass ranging from 12–150 kDa including AR, whereas L–NAME significantly inhibited mixed disulfide formation in normal and diabetic rat lenses. Treatment of ex vivo lens with NO–donors, SNAP and GSNO resulted in the inactivation of AR activity and sorbitol formation in normal and diabetic rats. Further, incubation of HLEC with SNAP or GSNO resulted in the glutathiolation and inactivation of AR. Conclusions: The results suggest that under normoglycemic conditions, AR activity and sorbitol formation are partially repressed in lens by concurrent generation of NO, and that during hyperglycemia, the NO synthesis decreases and the polyol pathway activity increases. Thus, restoring NO availability in diabetic animals should inhibit AR and prevent sorbitol accumulation. This may represent a potentially useful strategy for preventing or delaying the development of diabetic cataractogenesis and other secondary diabetic complications.

Keywords: diabetes • nitric oxide • protein modifications–post translational