Diamide induces reversible changes in morphology, cytoskeleton and cell-cell coupling in lens epithelial cells

The isolated frog lens epithelium can be maintained with its cell shape, cytoskeletal organization and membrane electrophysiological characteristics intact for more than 24 hr. Perifusion with the permeant oxidant diamide (1 mM) led to drastic, but reversible, changes in all the above parameters. After a 20 min exposure to diamide, the regular polygonal arrangement of the epithelial cells become increasingly disrupted as the cells reorganized and a 'rosette' pattern formed. The cells at the edges of the rosette pulled apart from one another while those in the centre maintained a relatively normal appearance. Blebs formed on the apical surface of all of the cells on prolonged exposure and the internal structure was also found to be severely disrupted. The cytoplasm became granular, vacuolated and the nucleus had a banded, non-homogeneous appearance. Phalloidin staining of F-actin microfilaments revealed that there was a general disruption of organization, with actin losing its association with the membrane. The microtubule array, organized around the centrosome, was also severely disrupted although microtubules were still discernible in most cells. During exposure to diamide the membrane potential depolarized and both electrical and dye coupling, which are normally extremely efficient in these cells, were disturbed. If the epithelium was exposed to 1 mM diamide for more than 45 min then all of the above changes were irreversible and cell death followed. If exposure was restricted to less than 30 min, then all of the above changes occurred and, in fact, progressed for over 1 hr; but if the epithelium was perifused for a further 20 hr in control medium, then most of the changes were reversible.