Superficial membrane -SH groups inaccessible by intracellular GSH

The importance of membrane -SH groups in the epithelium and posterior fiber cells of rabbit lens was demonstrated by employing a non-penetrating sulfhydryl reagent parachloromercuribenzoate sulfonic acid (PCMBS). Both fiber cell and epithelial membrane preparations contain substantial amounts of -SH, 31 nmoles/mg membrane protein. PCMBS-treatment of anterior and posterior surfaces of the lens leads to dramatic increases in the calcium influx across both anterior and posterior surfaces, indicating that the importance of membrane -SH groups is not limited to the epithelium. When the entire lens is bathed in PCMBS (0.1 mM) for short duration and transferred to normal medium, calcium continues to increase from 0.4 mM to nearly 1 mM over a 20 hr period. At this point in time, GSH levels are normal, indicating that intracellular GSH does not gain access to PCMBS-binding sites. In contrast, external GSH or cysteine, at lower levels (5 mM) quickly reverses PCMBS binding with membrane -SH groups and leads to near normal levels of lens calcium during subsequent culture. This in addition to the fact that PCMBS is not found in the cell interior where GSH levels are undiminished, suggests that the critical -SH groups involved in control of barrier properties are externally located where little protection from intracellular GSH is afforded. These data indicate that aqueous humor GSH may play a critical role in maintaining reduced -SH groups controlling membrane permeability located on the surface of membranes.