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Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses.
Invest Ophthalmol Vis Sci. 2001 Feb; 42(2):409-16.IO

Abstract

PURPOSE

To study the mechanism of regulation of GSH in HLE-B3 cells expressing alphaA-crystallin (alphaA) and in alphaA knockout mouse lenses.

METHODS

GSH levels and maximal rates of GSH synthesis were measured in immortalized, alphaA-transfected HLE-B3 cells containing varying amounts of alphaA. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase (GCS), were also determined in alphaA- 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 alphaA and alphaB on lens GSH concentrations was evaluated in whole lenses of alphaA knockout and alphaB knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous alphaA knockout lenses.

RESULTS

GSH levels were significantly higher in HLE-B3 cells expressing alphaA- compared with mock-transfected cells and were correlated positively with alphaA content. Mean rate of GSH synthesis was also higher in alphaA-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 alphaA-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In alphaA(-/-) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to approximately 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 alphaB(-/-) lenses was similar to that of wild type for up to 14 months.

CONCLUSIONS

Expression of alphaA 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 alphaA(-/-) lenses but not in the noncataractous alphaB(-/-) 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.

Authors+Show Affiliations

Division of Gastrointestinal and Liver Diseases, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA. kannan@hsc.usc.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11157875

Citation

Kannan, R, et al. "Regulation of GSH in alphaA-expressing Human Lens Epithelial Cell Lines and in alphaA Knockout Mouse Lenses." Investigative Ophthalmology & Visual Science, vol. 42, no. 2, 2001, pp. 409-16.
Kannan R, Ouyang B, Wawrousek E, et al. Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses. Invest Ophthalmol Vis Sci. 2001;42(2):409-16.
Kannan, R., Ouyang, B., Wawrousek, E., Kaplowitz, N., & Andley, U. P. (2001). Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses. Investigative Ophthalmology & Visual Science, 42(2), 409-16.
Kannan R, et al. Regulation of GSH in alphaA-expressing Human Lens Epithelial Cell Lines and in alphaA Knockout Mouse Lenses. Invest Ophthalmol Vis Sci. 2001;42(2):409-16. PubMed PMID: 11157875.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses. AU - Kannan,R, AU - Ouyang,B, AU - Wawrousek,E, AU - Kaplowitz,N, AU - Andley,U P, PY - 2001/2/7/pubmed PY - 2001/3/10/medline PY - 2001/2/7/entrez SP - 409 EP - 16 JF - Investigative ophthalmology & visual science JO - Invest. Ophthalmol. Vis. Sci. VL - 42 IS - 2 N2 - PURPOSE: To study the mechanism of regulation of GSH in HLE-B3 cells expressing alphaA-crystallin (alphaA) and in alphaA knockout mouse lenses. METHODS: GSH levels and maximal rates of GSH synthesis were measured in immortalized, alphaA-transfected HLE-B3 cells containing varying amounts of alphaA. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase (GCS), were also determined in alphaA- 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 alphaA and alphaB on lens GSH concentrations was evaluated in whole lenses of alphaA knockout and alphaB knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous alphaA knockout lenses. RESULTS: GSH levels were significantly higher in HLE-B3 cells expressing alphaA- compared with mock-transfected cells and were correlated positively with alphaA content. Mean rate of GSH synthesis was also higher in alphaA-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 alphaA-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In alphaA(-/-) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to approximately 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 alphaB(-/-) lenses was similar to that of wild type for up to 14 months. CONCLUSIONS: Expression of alphaA 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 alphaA(-/-) lenses but not in the noncataractous alphaB(-/-) 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. SN - 0146-0404 UR - https://www.unboundmedicine.com/medline/citation/11157875/Regulation_of_GSH_in_alphaA_expressing_human_lens_epithelial_cell_lines_and_in_alphaA_knockout_mouse_lenses_ L2 - https://iovs.arvojournals.org/article.aspx?volume=42&issue=2&page=409 DB - PRIME DP - Unbound Medicine ER -