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1 From the Departments of Human Biological Chemistry and Genetics and 2 Physiology and Biophysics, University of Texas Medical Branch, Galveston; and the 3 Division of Cardiology, Department of Medicine, University of Louisville, Kentucky.
PURPOSE. To investigate the role of calcium-activated proteases in calcium-dependent disintegrative globulization of isolated rat lens cortex fiber cells.
METHODS. Rat lens fiber cells were isolated and plated on coverslips at the bottom of a temperature-controlled chamber. The fiber cells were incubated with 10 µM protease substrate, (t-butoxycarbonyl-leu-met-7-amino-4-chloromethylcoumarin:BOC-Leu-Met-CMAC) and the proteolytic activity in the fiber cells was determined by observing the increase in fluorescence, using an excitation wavelength of 360 nm, and measuring emission at 410 nm. Free intracellular calcium was measured using the cell-permeable calcium indicator Fluo-3-AM, and the globulization time (Tg) was determined using image analysis.
RESULTS. Tg of fiber cells superfused with Ringer’s
solution containing 2 x 10-3 M,
10-6 M, and 10-8 M
[Ca2+]o were: 24.7 ± 1.3, 53.0 ±
2.8, and more than 120 minutes, respectively. A significant increase in
Tg (
95 minutes) was observed when the
fibers were preincubated with acetoxymethyl ester of 1,2-bis
(2-amino-phenoxy) ethane N, N,
N, N-tetra-acetic acid (BAPTA-AM) to
buffer changes in [Ca2+]i, or the protease
substrate to competitively inhibit degradation of cellular proteins. In
the presence of Ringer’s solution containing 2 x
10-3 M [Ca2+]o and
0.5 mM of the cysteine protease inhibitor, leupeptin,
Tg increased to 100 minutes, without
affecting [Ca2+]i. The proteolytic activity
of fiber cells in Ringer’s solution containing
10-6 M and 2 x
10-3 M [Ca2+]o
increased by approximately 7- and 12-fold, respectively, compared with
sucrose-EDTA solution or Ringer’s solution containing
10-8 M [Ca2+]o. This
increase in proteolytic activity was inhibited by leupeptin.
CONCLUSIONS. Elevation of calcium in the medium results in a proportionate increase in [Ca2+]i and the proteolytic activity in isolated lens fiber cells. The increase in the proteolytic activity is accompanied by an increase in the rate of globulization of the fiber cells. Inhibition of the proteolytic activity by leupeptin increases Tg without affecting the gain in [Ca2+]i. These results suggest that globulization of isolated fiber cells in physiological salt solutions is mediated by Ca2+-activated protease(s).
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