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Expression of nonphagocytic NADPH oxidase system in the ocular lens.
Mol Vis. 2004 Feb 19; 10:112-21.MV

Abstract

PURPOSE

The primary goal of this study was to characterize the Rac GTPase associated, NADPH oxidase-mediated Reactive Oxygen Species (ROS)-generating system in the lens tissue.

METHODS

NADPH oxidase activity in lens tissue was determined by quantifying superoxide-induced lucigenin photoemission. Immunological and PCR/RT-PCR techniques were utilized to determine expression of different components of the NADPH oxidase system in lens tissue. Growth factor stimulated ROS production was determined quantitatively in human lens epithelial cells using dichlorofluorescein diacetate.

RESULTS

Lens homogenates from different species showed generation of superoxide in a lucigenin-enhanced chemiluminescence assay in the presence of NADPH. This activity was found to be lens protein concentration dependent, heat sensitive, and inhibitable by superoxide dismutase and the flavoprotein inhibitor, diphenyleneiodonium (DPI). The distribution of superoxide generating activity in lens was confined predominantly to the lens epithelium, with very low levels in cortex and none in the nucleus. Immunological assays have demonstrated the presence of p67phox and p47phox in lens tissue, while PCR and RT-PCR reactions amplified DNA products corresponding to the p67phox, p40phox, p22phox, gp91phox, and Rac1 components of the NADPH oxidase complex from human and mouse lens cDNA libraries. Serum starved human lens epithelial cells stimulated with different growth factors including EGF, b-FGF, PDGF, TGF-beta, and LPA demonstrated increased production of ROS, a response which was blocked by inhibitors of NADPH oxidase, such as DPI and the antioxidant-N-acetyl cysteine (NAC). RT-PCR analysis of human lens RNA confirmed readily detectable levels of expression of low molecular weight protein tyrosine phosphatase (LMW-PTP), which is a well-characterized target of redox signaling pathway(s).

CONCLUSIONS

These data demonstrate the presence of a functional nonphagocytic NADPH oxidase system in lens that is predominantly localized to the lens epithelium. Several growth factors appear to stimulate the activity of lens NADPH oxidase, resulting in increased production of ROS in lens epithelial cells, indicating that redox signaling may have an important role in growth factor effects on lens growth and development.

Authors+Show Affiliations

Department of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA. rao00011@mc.duke.eduNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14978478

Citation

Rao, Ponugoti Vasantha, et al. "Expression of Nonphagocytic NADPH Oxidase System in the Ocular Lens." Molecular Vision, vol. 10, 2004, pp. 112-21.
Rao PV, Maddala R, John F, et al. Expression of nonphagocytic NADPH oxidase system in the ocular lens. Mol Vis. 2004;10:112-21.
Rao, P. V., Maddala, R., John, F., & Zigler, J. S. (2004). Expression of nonphagocytic NADPH oxidase system in the ocular lens. Molecular Vision, 10, 112-21.
Rao PV, et al. Expression of Nonphagocytic NADPH Oxidase System in the Ocular Lens. Mol Vis. 2004 Feb 19;10:112-21. PubMed PMID: 14978478.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression of nonphagocytic NADPH oxidase system in the ocular lens. AU - Rao,Ponugoti Vasantha, AU - Maddala,Rupalatha, AU - John,Faith, AU - Zigler,J Samuel,Jr Y1 - 2004/02/19/ PY - 2004/2/24/pubmed PY - 2004/3/6/medline PY - 2004/2/24/entrez SP - 112 EP - 21 JF - Molecular vision JO - Mol Vis VL - 10 N2 - PURPOSE: The primary goal of this study was to characterize the Rac GTPase associated, NADPH oxidase-mediated Reactive Oxygen Species (ROS)-generating system in the lens tissue. METHODS: NADPH oxidase activity in lens tissue was determined by quantifying superoxide-induced lucigenin photoemission. Immunological and PCR/RT-PCR techniques were utilized to determine expression of different components of the NADPH oxidase system in lens tissue. Growth factor stimulated ROS production was determined quantitatively in human lens epithelial cells using dichlorofluorescein diacetate. RESULTS: Lens homogenates from different species showed generation of superoxide in a lucigenin-enhanced chemiluminescence assay in the presence of NADPH. This activity was found to be lens protein concentration dependent, heat sensitive, and inhibitable by superoxide dismutase and the flavoprotein inhibitor, diphenyleneiodonium (DPI). The distribution of superoxide generating activity in lens was confined predominantly to the lens epithelium, with very low levels in cortex and none in the nucleus. Immunological assays have demonstrated the presence of p67phox and p47phox in lens tissue, while PCR and RT-PCR reactions amplified DNA products corresponding to the p67phox, p40phox, p22phox, gp91phox, and Rac1 components of the NADPH oxidase complex from human and mouse lens cDNA libraries. Serum starved human lens epithelial cells stimulated with different growth factors including EGF, b-FGF, PDGF, TGF-beta, and LPA demonstrated increased production of ROS, a response which was blocked by inhibitors of NADPH oxidase, such as DPI and the antioxidant-N-acetyl cysteine (NAC). RT-PCR analysis of human lens RNA confirmed readily detectable levels of expression of low molecular weight protein tyrosine phosphatase (LMW-PTP), which is a well-characterized target of redox signaling pathway(s). CONCLUSIONS: These data demonstrate the presence of a functional nonphagocytic NADPH oxidase system in lens that is predominantly localized to the lens epithelium. Several growth factors appear to stimulate the activity of lens NADPH oxidase, resulting in increased production of ROS in lens epithelial cells, indicating that redox signaling may have an important role in growth factor effects on lens growth and development. SN - 1090-0535 UR - https://www.unboundmedicine.com/medline/citation/14978478/Expression_of_nonphagocytic_NADPH_oxidase_system_in_the_ocular_lens_ L2 - http://www.molvis.org/molvis/v10/a15/ DB - PRIME DP - Unbound Medicine ER -