CONTRIBUTION OF OSMOTIC CHANGES TO DISINTEGRATIVE GLOBULIZATION OF SINGLE CORTICAL FIBERS ISOLATED FROM RAT LENS

In this study the contribution of osmotic changes to disintegrative gl obulization of lens cortical fibers was examined, Single fiber cells w ere isolated by trypsinization of adult rat lens cortex, and morpholog ical changes elicited by exposure to different external solutions were monitored optically, The survival of the fiber-shaped cells was analy sed in accordance with the Weibull distribution, Changes in [Ca2+](i) were measured using the fluorescent calcium-sensitive dye-Fluo-3. Expo sure 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 incr ease in [Ca2+](i) was delayed by removing Cl- ions from the external m edia, Globulization was also delayed by adding 20% bovine serum albumi n (T-g = 107 +/- 3 min) or chloride channel inhibitors 5, nitro-2-(3-p henylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, an d tamoxifen. When the fiber cells were suspended in isotonic (280 mM s ucrose) HEPES-sucrose (HS) or HEPES-EDTA-sucrose (HES) solution, no gl obulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mM) HES solution led to disintegration of f iber cells in 75 +/- 7 min. Disintegration of the fiber induced by hyp otonic 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 n o effect on T-g in hypotonic HES, Addition of 2 mM Ca2+ to HES solutio n accelerated globulization, and T-g was 57 +/- 4, 69 +/- 5 and 102 +/ - 6 min for hypo-, iso- and hyper-tonic solutions, respectively. Trans ient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results su ggest that in ionic media, part of the calcium influx in isolated fibe r cells is mediated by the influx of chloride ions. In the absence of other ions, the fiber cells still accumulate calcium. although this ca lcium influx was independent of medium tonicity. Globulization-induced by hypotonic sucrose solution appears to be mediated by the activatio n of intracellular proteases and by cell swelling-induced release of c alcium from internal stores. Such swelling-mediated disintegrative glo bulization of fiber cells may be of significance in understanding the cellular basis of diabetic cataracts. (C) 1997 Academic Press Limited.