M Inomata1, M Hayashi, S Shumiya, S Kawashima, Y Ito. 1. Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Itabashi-ku, Japan. minomata@center.tmig.or.jp
Abstract
PURPOSE: Our previous results showed that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly suppresses lens opacification in Shumiya cataract rat (SCR). Therefore, we examine whether iNOS is upregulated and involved in cataract formation in SCR. METHODS: The expressions of iNOS mRNA and iNOS protein in SCR lenses were examined by RT-PCR and Western blotting, respectively. Calpain-mediated alphaB-crystallin proteolysis was analyzed by Western blotting using antibody specific to the calpain-generated fragment of alphaB-crystallin. Lens opacification was analyzed using computerized image analysis software connected to the Anterior Eye Segment Analysis System (EAS-1000, Nidek). Calcium contents in lenses were measured by atomic absorption spectrophotometry. RESULTS: High levels of iNOS mRNA and iNOS protein are expressed in cataractous lenses compared with normal lenses. The increases in their expression are markedly suppressed by the oral administration of AG, which acts to prevent lens opacification. The induction of iNOS protein is observed before the elevation in calcium content and the acceleration of calpain-mediated proteolysis, both of which are closely related to the development of lens opacification. CONCLUSIONS: These findings strongly suggest that iNOS is involved in cataract formation in SCR. The induction of iNOS occurs prior to the elevation of calcium content and its induction is inhibited by AG-treatment. Considering our previous result that the elevation of calcium content is also prevented by AG-treatment, it is conceivable that upregulation of iNOS causes calcium influx into lens cells and the subsequent activation of calpain.
PURPOSE: Our previous results showed that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly suppresses lens opacification in Shumiya cataractrat (SCR). Therefore, we examine whether iNOS is upregulated and involved in cataract formation in SCR. METHODS: The expressions of iNOS mRNA and iNOS protein in SCR lenses were examined by RT-PCR and Western blotting, respectively. Calpain-mediated alphaB-crystallin proteolysis was analyzed by Western blotting using antibody specific to the calpain-generated fragment of alphaB-crystallin. Lens opacification was analyzed using computerized image analysis software connected to the Anterior Eye Segment Analysis System (EAS-1000, Nidek). Calcium contents in lenses were measured by atomic absorption spectrophotometry. RESULTS: High levels of iNOS mRNA and iNOS protein are expressed in cataractous lenses compared with normal lenses. The increases in their expression are markedly suppressed by the oral administration of AG, which acts to prevent lens opacification. The induction of iNOS protein is observed before the elevation in calcium content and the acceleration of calpain-mediated proteolysis, both of which are closely related to the development of lens opacification. CONCLUSIONS: These findings strongly suggest that iNOS is involved in cataract formation in SCR. The induction of iNOS occurs prior to the elevation of calcium content and its induction is inhibited by AG-treatment. Considering our previous result that the elevation of calcium content is also prevented by AG-treatment, it is conceivable that upregulation of iNOS causes calcium influx into lens cells and the subsequent activation of calpain.
Authors: Tianlin Xiao; Mohammad Shoeb; M Saeed Siddiqui; Min Zhang; Kota V Ramana; Satish K Srivastava; Vasilis Vasiliou; Naseem H Ansari Journal: J Toxicol Environ Health A Date: 2009