Calcium homeostasis of isolated single cortical fibers of rat lens

Satish Srivastava, Li Fei Wang, Naseem Ansari, Aruni Bhatnagar

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Abstract

Purpose. To investigate the calcium homeostasis in single fiber cells isolated from rat ocular lens cortex and to quantify the changes in the concentration of free intracellular calcium [Ca2+](i) during the process of disintegrative globulization. Methods. Individual fiber cells from the cortex of the adult rat lens were isolated by treatment with trypsin in ion-free buffered sucrose. The isolated fiber cells were loaded with the acetoxymethyl esters of Fluo-3 or Calcium Green-2, or with Fluo-3 and Fura Red, and changes in [Ca2+](i) of single cortical fibers were measured using a microfluorometer. The time course of increase of [Ca2+](i) in fiber cells exposed to Ringer's solution was measured, and die effects on the increase of [Ca2+](i) of calcium channel blocker, verapamil, Na-Ca exchange inhibitors Ni2+ and Zn2+, and protease inhibitor, leupeptin, Na+-free and K+-free media and Ca2+-containing isotonic sucrose solution, were investigated. Results. In Hepes sucrose solution (containing ~1,5 μM Ca2+), the isolated fiber cells maintained stable values of [Ca2+](i) at 99.6 ± 10 nM (n = 32). Exposure of the isolated fibers to Ringer's solution (containing 2 mM Ca2+) led to a monoexponential increase of [Ca2+](i) at a rate of 0.12 min-1. This increase in [Ca2+](i) was accompanied by disintegration of the isolated fibers into discrete but resealed globules. Changes in [Ca2+](i), monitored by using a two-dye ratiometric method using Fura Red and fluo-3, showed a progressive increase in [Ca2+](i) in fibers exposed to Ringer's solution, preceding globulization. The [Ca2+](i) in the globules in Ringer's solution, determined using Calcium Green-2, was 3.6 ± 0.7 μM (n =23). Compared with that in fibers in Ringer's solution, the rate of increase of [Ca2+](i) in fibers was much slower in the presence of 50 μM verapamil (0.047 min-1), in Na+-free (0.086 min-1) and in K+-free (0.062 min- 1) Ringer's solution, or when the fibers were suspended in Hepes-sucrose solution, containing 2 mM Ca2+ (0.046 min-1). After 30 minutes, the [Ca2+](i) of fiber cells exposed to Ringer's solution, containing 2 mM Ni2+ (574.7 ± 29 nM; n = 7) or Zn2+ (402.6 ± 77 nM; n = 7) was significantly lower (P < 0.001) compared with that in fiber cells exposed to Ringer's solution alone (1995 ± 461 nM, n = 10). In Ringer's solution, leupeptin delayed globulization without significantly affecting the increase in [Ca2+](i). The [Ca2+](i) of fiber cells isolated from outer and inner cortex and suspended in Hepes-sucrose was comparable; however, after 15 minutes of exposure to Ringer's solution, [Ca2+](i) in fibers from the outer cortex was approximately three times higher than [Ca2+](i) in those from the inner cortex. Conclusions. Exposure to high (millimolar) concentrations of calcium in the external medium leads to an increase in [Ca2+](i) of isolated individual fiber cells, which precedes disintegrative globulization. The protective effects of Na+-free and K+-free solutions on globulization appear to be due to a lower rate of increase of [Ca2+](i). Part of the calcium influx may be mediated by L-type calcium channels and by Na-Ca exchange, operating in reverse. Proteolytic inhibitors do not affect the increase in [Ca2+](i) but delay globulization by inhibiting calcium- mediated proteolysis. The isolated fiber cells and the disintegrated globules maintain a 100- to 300-fold gradient of calcium across their plasma membranes.

Original languageEnglish (US)
Pages (from-to)2300-2312
Number of pages13
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number11
StatePublished - Oct 1997

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Keywords

  • Calcium homeostasis
  • Fiber cells
  • Globulization
  • Lens
  • Proteolysis

ASJC Scopus subject areas

  • Ophthalmology

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