Role of calcium-dependent protease(s) in globulization of isolated rat lens cortical fiber cells

L. F. Wang, B. N. Christensen, A. Bhatnagar, Satish Srivastava

Research output: Contribution to journalArticle

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Abstract

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 × 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 × 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 × 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).

Original languageEnglish (US)
Pages (from-to)194-199
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume42
Issue number1
StatePublished - 2001

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Lenses
Peptide Hydrolases
Calcium
leucylmethionine
Calpain
Cysteine Proteinase Inhibitors
Ethane
Edetic Acid
Acetic Acid
Proteolysis
Sucrose
Buffers
Esters
Salts
Fluorescence
Temperature
Ringer's solution

ASJC Scopus subject areas

  • Ophthalmology

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Role of calcium-dependent protease(s) in globulization of isolated rat lens cortical fiber cells. / Wang, L. F.; Christensen, B. N.; Bhatnagar, A.; Srivastava, Satish.

In: Investigative Ophthalmology and Visual Science, Vol. 42, No. 1, 2001, p. 194-199.

Research output: Contribution to journalArticle

Wang, LF, Christensen, BN, Bhatnagar, A & Srivastava, S 2001, 'Role of calcium-dependent protease(s) in globulization of isolated rat lens cortical fiber cells', Investigative Ophthalmology and Visual Science, vol. 42, no. 1, pp. 194-199.
Wang, L. F. ; Christensen, B. N. ; Bhatnagar, A. ; Srivastava, Satish. / Role of calcium-dependent protease(s) in globulization of isolated rat lens cortical fiber cells. In: Investigative Ophthalmology and Visual Science. 2001 ; Vol. 42, No. 1. pp. 194-199.
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abstract = "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 × 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 × 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 × 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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AU - Wang, L. F.

AU - Christensen, B. N.

AU - Bhatnagar, A.

AU - Srivastava, Satish

PY - 2001

Y1 - 2001

N2 - 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 × 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 × 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 × 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).

AB - 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 × 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 × 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 × 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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