Involvement of Na+, K+-ATPase inhibitors in cataract formation

Fundamental to the maintenance of ionic concentration gradients and transparency of the lens is the activity of the Nd, KC-ATPase in the epithelial layer. Recently we have identified endogenous digitalis-like compounds (DLC), 19-norbufalin and its peptide derivatives, in human cataractous lenses (Lichtstein et al. fur. J. Biochem. 216: 261-268. 1993). These compounds inhibit the activity of the Na+, K+-ATPase and were suggested to be involved in cataract formation. The present experiments were designed to test this hypothesis by determining the ability of digitalis and DLC to induce changes in protein composition and leakage from rat lenses in organ-culture. DLC were determined in rat lenses using three independent assays: interaction with ouabain and bufalin antibodies and inhibition of [H-3-ouabain] binding to red blood cells. Rat lenses were incubated in modified TC-199 medium in 5% CO2 atmosphere at 37 degreesC for the time of the experiment. The onset of cataractogenesis was assessed by measuring protein leakage from lenses as well as by crystalline composition in the lens and media. DLC is present in rat lens with concentration 7-30 fold higher in the capsular-epithelial layer as compared to the lens fibers regions. Ouabain, bufalin, digoxin and DLC induced dose and time dependent leakage of protein from rat lenses. Lenses incubated with these compounds showed alterations in crystallin content consistent with changes that initiate opacity. These results, together with the demonstration of DLC in the normal lens, and their increased level in human cataractous lenses, strongly suggests their involvement in the molecular mechanisms responsible for cataract formation.