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S.S. Lobell, D. Lin, L. Willard, E. Gustafson, M. Barnett, D. Madgwick, D.J. Takemoto; PKC Knockout Mouse Lenses Are More Susceptible to Oxidative Damage . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4091.
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© ARVO (1962-2015); The Authors (2016-present)
Protein kinase C γ (PKCγ) plays critcal roles in control of gap junctional communication and oxidative stress responses in the lens epithelial cells. In this report we wished to determine if the deletion of endogenous PKC γ causes lenses to be more sensitive to oxidative stress.
Animals were 6 weeks old unless otherwise indicated. Both the control (b6129pf21j100903) mice and PKC γ knockout (B6;129p–Prkcctm1St1) mice were from Jackson Laboratory (Bar Harbor, MA). For light microscopy, all eyes were removed immediately after death and treated with hydrogen peroxide for 1 hour. The concentration for 6 week old mice was 50 µM, and the concentration for the 3 day old mice was 10 µM. Lenses were then fixed in a solution of 2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M cacodylate. Lenses were post–fixed with osmium tetroxide, dehydrated with increasing concentrations of ethanol, and embedded in epon (LX112). Sections (one µm thick) were stained with toludine blue. PKC γ enzyme activity was measured by use of the PepTag PKC assay kit. Cx 50 phosphorylation was measured by western blot with anti–phosphoserine antibodies. Whole lens gap junction activity was determined by dye transfer assay. Lucifer yellow and rhodamine dextran were microinjected and dye transfer was measured by confocal microscopy.
In control mouse lenses, endogenous PKCγ enzyme was activated by application of phorbol ester (TPA 200nM) or H2O2 (100µM). However, no PKCγ enzyme activity was detected in PKCγ knockout lenses. Cx50 phosphorylation experiments showed that activation of PKCγ by TPA or H2O2 caused Cx50 phosphorylation on serines in control mice but not in knockout mice. Activation of PKCγ by H2O2 caused a decrease in dye transfer in control lenses, but no decrease in dye transfer was observed in PKCγ knockout lenses. Structural studies by light microscopy demontrated that H2O2 challenge resulted in extensive structural damage in knockout lenses, while the control lenses showed much less damage. Data indicates that open gap junctions causes lens to be more sensitive to oxidative stress in PKCγ knockout mice.
PKC γ phosphorylates connexin proteins in response to oxidative stress which, in turn, causes inhibition of gap junctions in the lens. PKC γ is required for protection of lens from oxidative damage. A failure to inhibit gap junctions in response to oxidative stress may contribute to the oxidative damage of lenses in PKCγ knockout mice.
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