Purpose. To study the mechanism of regulation of GSH in HLE-B3 cells expressing αA-crystallin (αA) and in αA knockout mouse lenses. Methods. GSH levels and maximal rates of GSH synthesis were measured in immortalized, αA-transfected HLE-B3 cells containing varying amounts of αA. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, γ-glutamylcysteine synthetase (GCS), were also determined in αA- and mock-transfected cells by Northern blot analysis and Western blot analysis of heavy (GCS-HS) and light (GCS-LS) subunits. The effect of absence of αA and αB on lens GSH concentrations was evaluated in whole lenses of αA knockout and αB knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous αA knockout lenses. Results. GSH levels were significantly higher in HLE-B3 cells expressing αA- compared with mock-transfected cells and were correlated positively with αA content. Mean rate of GSH synthesis was also higher in αA-expressing cells than in mock controls (0.84 vs. 0.61 nmol · min-1 per mg protein, respectively). GCS-HS mRNA and GCS-LS mRNA were approximately twofold higher in αA-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In αA(-/-) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to ∼50% of controls. On the other hand, GCS-HS and GCS-LS proteins showed a significant decrease before cataract formation as early as 15 days after birth. GSH level in cataract-free αB(-/-) lenses was similar to that of wild type for up to 14 months. Conclusions. Expression of αA caused an increase in cellular GSH, in part, because of an increase in mRNA and protein of both GCS subunits. GSH levels decreased with increasing age in cataractous αA(-/-) lenses but not in the noncataractous αB(-/-) lenses. It is suggested that neonatal precataractous lenses (with normal GSH and decreased GCS) may maintain their GSH level by other compensatory mechanisms such as increased GSH transport.
|Number of pages||8|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - 2001|