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Deepak Chandra, Kota V. Ramana, Lifei Wang, Burgess N. Christensen, Aruni Bhatnagar, Satish K. Srivastava; Inhibition of Fiber Cell Globulization and Hyperglycemia-Induced Lens Opacification by Aminopeptidase Inhibitor Bestatin. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2285-2292.
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purpose. To examine the role of calcium-dependent and -independent proteolytic activity in the globulization of isolated fiber cells and glucose-induced lens opacification.
methods. Fiber cells from rat lens cortex were isolated, and the [Ca2+]i and protease activity in the isolated fibers were determined by using a calcium binding dye and the protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (BOC-Leu-Met-CMAC). The activity of calpain in the lens cortex homogenate was determined with fluorescein-casein in the presence of Ca2+ and that of fiber cell globulizing aminopeptidase (FCGAP) with BOC-Leu-Met-CMAC and reduced glutathione (GSH) in the absence of Ca2+. The lens proteases-calpain and the novel aminopeptidase FCGAP were partially purified by diethylaminoethyl (DEAE) gel column chromatography. Single fiber cells were isolated from rat lens, plated on coverslips, and placed in a temperature-controlled chamber. Their globulization time was determined by the appearance of light-scattering globules in the absence and the presence of protease inhibitors including the aminopeptidase inhibitor bestatin. To investigate the effect of the protease inhibitors E-64 and bestatin on the prevention of hyperglycemic cataract, the rat lenses were cultured in medium 199 in the presence of 5.5 and 50 mM glucose and in the absence and the presence of protease inhibitors. Changes in light transmission by the lenses were determined by digital image analysis.
results. Normal levels of lens fiber cell [Ca2+]i, determined by using a cell-permeable dye were approximately 100 nM, and the protease activity determined with BOC-Leu-Met-CMAC was maximum at [Ca2+]i of approximately 500 nM. A large fraction of the FCGAP that cleaves BOC-Leu-Met-CMAC was separated from calpain, which cleaves fluorescein-casein, by diethylaminoethyl (DEAE) gel column chromatography. The FCGAP did not bind to the column, whereas calpain bound to the column and was eluted by approximately 180 mM NaCl. Unlike calpain, the FCGAP did not require calcium for activation and did not cleave fluorescein-casein. However, the Ca2+-dependent calpain activated FCGAP, indicating that the latter may exist in pro-protease form. The FCGAP was selectively inhibited by the specific aminopeptidase inhibitor bestatin, indicating that FCGAP could be an aminopeptidase. However, the FCGAP was found to be immunologically distinct from leucine aminopeptidase and calpain. Perfusion of the isolated rat lens fiber cells with Ringer’s solution led to their globulization in 30 ± 3 minutes. Addition of 0.5 mM of the protease inhibitors E-64 and leupeptin increased the globulization time to 60 and 100 minutes, respectively, whereas no globulization of the fiber cells was observed for 4 hours in the presence of 0.05 mM bestatin. In rat lens cultured in medium containing 50 mM glucose, both E-64 and bestatin (0.05 mM each) significantly reduced the extent of opacification, indicating that an aminopeptidase, downstream to a Ca2+-dependent protease, may be involved in mediating cataractogenic changes.
conclusions. In addition to calpain, a Ca2+-independent novel protease, FCGAP, a novel aminopeptidase, represents a significant fraction of the total proteolytic activity in the lens. Inhibition of FCGAP by bestatin attenuates Ca2+-induced globulization of the isolated fiber cells in vitro and hyperglycemia-induced opacification of cultured rat lens.
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