Oxidative stress, lens gap junctions, and cataracts

Abstract

The eye lens is constantly subjected to oxidative stress from radiation and other sources. The lens has several mechanisms to protect its components from oxidative stress and to maintain its redox state, including enzymatic pathways and high concentrations of ascorbate and reduced glutathione. With aging, accumulation of oxidized lens components and decreased efficiency of repair mechanisms can contribute to the development of lens opacities or cataracts. Maintenance of transparency and homeostasis of the avascular lens depend on an extensive network of gap junctions. Communication through gap junction channels allows intercellular passage of molecules (up to 1 kDa) including antioxidants. Lens gap junctions and their constituent proteins, connexins (Cx43, Cx46, and Cx50), are also subject to the effects of oxidative stress. These observations suggest that oxidative stress-induced damage to connexins (and consequent altered intercellular communication) may contribute to cataract formation.

FIG. 1.

Diagram of the lens showing the distribution of connexin isoforms. Cells from the anterior epithelial layer express Cx43 and Cx50, cells from the equatorial region express Cx43, Cx46, and Cx50, and fiber cells (including those of the nucleus) contain Cx46 and Cx50.

FIG. 2.

Pathways for generation of ROS in the lens. Normal electron transport and activity of cytochrome P450 (P450) can generate ·O₂⁻· The NADPH oxidase complex, which assembles with participation of Rac GTPases and is associated with activation of plasma membrane receptors (R) by extracellular signals (e.g., growth factors), can also generate ·O₂⁻· Growth factor receptors may also be activated by UV radiation-generated reactive oxygen species (ROS). H₂O₂ within the cell can be produced by the activity of superoxide dismutase (SOD) on ·O₂⁻ or it can originate by diffusion through the plasma membrane from the aqueous humor. H₂O₂ can also be generated by ascorbate and molecular oxygen in the presence of ferric ions. Reaction of H₂O₂ with metal ions (M+) can generate other ROS including ·OH or ·O₂⁻ through the Fenton reaction. H₂O₂ can be decomposed enzymatically by GSH-dependent peroxidases (GSHPx) or catalase, and nonenzymatically by reduced glutathione (GSH).

FIG. 3.

Redox pathways involved in oxidative stress in the eye lens. AFR·, ascorbate free radical; AFRR, NADH-dependent ascorbate free radical reductase; DHA, dehydroascorbate; GR, GSSG reductase; GSSG, oxidized glutathione; GSH, reduced glutathione; PDI, protein disulfide isomerase; PSH, sulfhydryl-containing protein; PSSG, glutathionated protein; PSSP, oxidized disulfide bond-containing protein; ROS, reactive oxygen species; TrxR, thioredoxin reductase; Trxox, oxidized thioredoxin; Trxred, reduced thioredoxin; TTase, thioltransferase.

FIG. 4.

Regional distribution of components of the redox system within the lens. This graphical representation summarizes data from many studies regarding the relative levels/activities (shown in arbitrary units) of the different molecules/enzymes in the different regions of the lens. The levels/activities of most components of the redox system are higher in the epithelium. AFRR, NADH-dependent ascorbate free radical reductase; GR, GSSG reductase; GSH, reduced glutathione; GSHPx-1, glutathione-dependent peroxidase-1; MsrA, methionine sulfoxide reductase A; PRDX3, peroxiredoxin 3; PRDX6, peroxiredoxin 6; PSSC, protein-bound cysteine; PSSG, glutathionated protein; SOD, super-oxide dismutase; TrxR-1, thioredoxin reductase 1; TTase, thioltransferase.

FIG. 5.

Effects of TPA on Cx56 and PKCγ in cultured chicken embryonic lens cells. (A) Immunoblot of Cx56 from homogenates of lentoid-containing cultures that were left untreated (Control) or that were treated with 200 nM TPA for 3 or 24 h. The migration positions of the molecular mass standards are indicated. (B, C). Photomicrographs show the distribution of anti-PKCγ immunoreactivity in lentoid cells left untreated (B) or treated with 200 nM TPA for 30 min (C). TPA treatment activated PKCγ (as demonstrated by its translocation to the plasma membrane) and produced a change in the immunoblot pattern of phosphorylated Cx56 forms. Bar, 12 μm in B and 15 μm in C.

FIG. 6.

Effects of oxidative stress on Cx56 in cultured embryonic chicken lens cells. (A) Immunoblot of Cx56 from homogenates of lentoid-containing cultures that were left untreated (Control) or that were treated with 100 or 500 μM H₂O₂ for 3 h. (B) Immunoblot of Cx56 from homogenates of lentoid-containing cultures that were left untreated (Control) or that were treated with 500 μM H₂O₂ for 30 min or 3 h. The migration positions of the molecular mass standards are indicated. Only subtle changes in the immunoblot pattern of Cx56 bands were detected after treatment with 100 μM H₂O₂ for 3 h. A prominent degradation product of Cx56 was generated after treatment with 500 μM H₂O₂ for 3 h.