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J.–O. Karlsson, A. Petersen, M. Zetterberg, H. Zetterberg, J. Sjostrand; Inhibition of Glycogen Synthase Kinase (GSK–3) Protects Against Oxidative Stress and Attenuates Apoptosis in Human Lens Epithelial Cells and the Mouse Lens in Organ Culure . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3867.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: GSK–3 may regulate Wnt signaling, gene expression, the cell cycle, cell differentiation and apoptosis. Inhibition of GSK–3 can be obtained via the structurally unrelated substances lithum or Kenpaullone. The lens and the lens epithelial cells are excellent models to study the role of this enzyme. Methods: Primary cultures of human lens epithelial cells (HLEC) or the mouse lens in organ culture were exposed to the GSK–3 inhibitors lithium (2 mM) or Kenpaullone (2 µM) for times upp to 24h.The cells were, before or after treatment, placed in medium containing fluorogenic indicators of oxidative damage. DCFH–DA was used to assay peroxides, mitochondrial function was evaluated with Rhodamine 123 and JC–1, monochlorobimane was used to assay intracellular glutathione (GSH) levels, Proteolytic activities were assayed, on line, with cell–permeable fluorogenic substrates.Proteasome and calpain activities were assayed with LLVY–AMC, Cathepsin B with RR–AMC or FR–AMC. Metalloproteases were assayed with AAF–AMC. Caspase–3, 8 and 9 were assayed in cell extracts with DEVD–, IETD– or LEHD–AMC, respectively. Results: The mitochondrial membrane potential and the level of GSH increased by 10% after treatment of HLEC with Li or Kenpaullone for 24h. No change was observed for peroxide production. The basal (low) level of caspase–3 activity was decreased by at least 20%. No significant effects were found concerning caspase–8 or 9 activities. No effect was observed on the activity of calpain, the proteasome, metalloproteases and cathepsin D/E activity.The whole mouse lens in organ culture showed essentially the same elevated mitochondrial potential. The GSH increase was even more evident in the whole lens preparation. Conclusions: Inhibition of GSK–3 may protect against oxidative stress (and cataract) via prevention of MPT induction and attenuate apoptosis in HLEC.
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