Calcium homeostasis of isolated single cortical fibers of rat lens

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.