Contribution of osmotic changes to disintegrative globulization of single cortical fibers isolated from rat lens

Li Fei Wang, Prajay Dhir, Aruni Bhatnagar, Satish Srivastava

Research output: Contribution to journalArticle

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Abstract

In this study the contribution of osmotic changes to disintegrative globulization of lens cortical fibers was examined. Single fiber cells were isolated by trypsinization of adult rat lens cortex, and morphological changes elicited by exposure to different external solutions were monitored optically. The survival of the fiber-shaped cells was analysed in accordance with the Weibull distribution. Changes in [Ca(2)+](i) were measured using the fluorescent calcium-sensitive dye-Fluo-3. Exposure of isolated fiber cells to Ringer's solution (containing 2 mM Ca2+) led to an exponential increase in [Ca2+](i) with a time constant of 10.2 ± 0.8 min, and caused disintegrative globulization in 25 ± 4 min (= T(g)). The process of globulization as well as the rate of increase in [Ca2+](i) was delayed by removing Cl- ions from the external media. Globulization was also delayed by adding 20% bovine serum albumin (T(g) = 107 ± 3 min) or chloride channel inhibitors 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, and tamoxifen. When the fiber cells were suspended in isotonic (280 mM sucrose) HEPES-sucrose (HS) or HEPES-EDTA- sucrose (HES) solution, no globulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mM) HES solution led to disintegration of fiber cells in 75 ± 7 min. Disintegration of the fiber induced by hypotonic HES solution could be delayed by either 0.05 mm leupeptin (T(g) = 97 ± 6 min) or by pre-loading the fibers with BAPTA (T(g) = 100 ± 4 min). Inhibition of membrane calcium transport by 0.5 mm La3+ had no effect on T(g) in hypotonic HES. Addition of 2 mM Ca2+ to HES solution accelerated globulization, and T(g) was 57 ± 4. 69 ± 5 and 102 ± 6 min for hypo-, iso- and hyper- tonic solutions, respectively. Transient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results suggest that in ionic media, part of the calcium influx in isolated fiber cells is mediated by the influx of chloride ions. In the absence of other ions, the fiber cells still accumulate calcium, although this calcium influx was independent of medium tonicity. Globulization-induced by hypotonic sucrose solution appears to be mediated by the activation of intracellular proteases and by cell swelling-induced release of calcium from internal stores. Such swelling- mediated disintegrative globulization of fiber cells may be of significance in understanding the cellular basis of diabetic cataracts.

Original languageEnglish (US)
Pages (from-to)267-275
Number of pages9
JournalExperimental Eye Research
Volume65
Issue number2
DOIs
StatePublished - Aug 1997

Fingerprint

HEPES
Lenses
Sucrose
Edetic Acid
Calcium
Ions
Niflumic Acid
Hypotonic Solutions
Benzoates
Tamoxifen
Bovine Serum Albumin
Cataract
Chlorides
Peptide Hydrolases
Coloring Agents
Observation
Membranes

Keywords

  • Cataractogenesis
  • Chloride channels
  • Donnan swelling
  • Globulization
  • Intracellular calcium
  • Lens fiber cells

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Contribution of osmotic changes to disintegrative globulization of single cortical fibers isolated from rat lens. / Wang, Li Fei; Dhir, Prajay; Bhatnagar, Aruni; Srivastava, Satish.

In: Experimental Eye Research, Vol. 65, No. 2, 08.1997, p. 267-275.

Research output: Contribution to journalArticle

Wang, Li Fei ; Dhir, Prajay ; Bhatnagar, Aruni ; Srivastava, Satish. / Contribution of osmotic changes to disintegrative globulization of single cortical fibers isolated from rat lens. In: Experimental Eye Research. 1997 ; Vol. 65, No. 2. pp. 267-275.
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N2 - In this study the contribution of osmotic changes to disintegrative globulization of lens cortical fibers was examined. Single fiber cells were isolated by trypsinization of adult rat lens cortex, and morphological changes elicited by exposure to different external solutions were monitored optically. The survival of the fiber-shaped cells was analysed in accordance with the Weibull distribution. Changes in [Ca(2)+](i) were measured using the fluorescent calcium-sensitive dye-Fluo-3. Exposure of isolated fiber cells to Ringer's solution (containing 2 mM Ca2+) led to an exponential increase in [Ca2+](i) with a time constant of 10.2 ± 0.8 min, and caused disintegrative globulization in 25 ± 4 min (= T(g)). The process of globulization as well as the rate of increase in [Ca2+](i) was delayed by removing Cl- ions from the external media. Globulization was also delayed by adding 20% bovine serum albumin (T(g) = 107 ± 3 min) or chloride channel inhibitors 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, and tamoxifen. When the fiber cells were suspended in isotonic (280 mM sucrose) HEPES-sucrose (HS) or HEPES-EDTA- sucrose (HES) solution, no globulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mM) HES solution led to disintegration of fiber cells in 75 ± 7 min. Disintegration of the fiber induced by hypotonic HES solution could be delayed by either 0.05 mm leupeptin (T(g) = 97 ± 6 min) or by pre-loading the fibers with BAPTA (T(g) = 100 ± 4 min). Inhibition of membrane calcium transport by 0.5 mm La3+ had no effect on T(g) in hypotonic HES. Addition of 2 mM Ca2+ to HES solution accelerated globulization, and T(g) was 57 ± 4. 69 ± 5 and 102 ± 6 min for hypo-, iso- and hyper- tonic solutions, respectively. Transient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results suggest that in ionic media, part of the calcium influx in isolated fiber cells is mediated by the influx of chloride ions. In the absence of other ions, the fiber cells still accumulate calcium, although this calcium influx was independent of medium tonicity. Globulization-induced by hypotonic sucrose solution appears to be mediated by the activation of intracellular proteases and by cell swelling-induced release of calcium from internal stores. Such swelling- mediated disintegrative globulization of fiber cells may be of significance in understanding the cellular basis of diabetic cataracts.

AB - In this study the contribution of osmotic changes to disintegrative globulization of lens cortical fibers was examined. Single fiber cells were isolated by trypsinization of adult rat lens cortex, and morphological changes elicited by exposure to different external solutions were monitored optically. The survival of the fiber-shaped cells was analysed in accordance with the Weibull distribution. Changes in [Ca(2)+](i) were measured using the fluorescent calcium-sensitive dye-Fluo-3. Exposure of isolated fiber cells to Ringer's solution (containing 2 mM Ca2+) led to an exponential increase in [Ca2+](i) with a time constant of 10.2 ± 0.8 min, and caused disintegrative globulization in 25 ± 4 min (= T(g)). The process of globulization as well as the rate of increase in [Ca2+](i) was delayed by removing Cl- ions from the external media. Globulization was also delayed by adding 20% bovine serum albumin (T(g) = 107 ± 3 min) or chloride channel inhibitors 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, and tamoxifen. When the fiber cells were suspended in isotonic (280 mM sucrose) HEPES-sucrose (HS) or HEPES-EDTA- sucrose (HES) solution, no globulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mM) HES solution led to disintegration of fiber cells in 75 ± 7 min. Disintegration of the fiber induced by hypotonic HES solution could be delayed by either 0.05 mm leupeptin (T(g) = 97 ± 6 min) or by pre-loading the fibers with BAPTA (T(g) = 100 ± 4 min). Inhibition of membrane calcium transport by 0.5 mm La3+ had no effect on T(g) in hypotonic HES. Addition of 2 mM Ca2+ to HES solution accelerated globulization, and T(g) was 57 ± 4. 69 ± 5 and 102 ± 6 min for hypo-, iso- and hyper- tonic solutions, respectively. Transient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results suggest that in ionic media, part of the calcium influx in isolated fiber cells is mediated by the influx of chloride ions. In the absence of other ions, the fiber cells still accumulate calcium, although this calcium influx was independent of medium tonicity. Globulization-induced by hypotonic sucrose solution appears to be mediated by the activation of intracellular proteases and by cell swelling-induced release of calcium from internal stores. Such swelling- mediated disintegrative globulization of fiber cells may be of significance in understanding the cellular basis of diabetic cataracts.

KW - Cataractogenesis

KW - Chloride channels

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KW - Intracellular calcium

KW - Lens fiber cells

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