Abstract
Abstract: :
Purpose: To demonstrate the effects of H2O2 on activation of PKCγ, subsequent interactions with caveolin–1(Cav–1), connexin 46 (Cx46), or connexin 50 (Cx50) in lipid rafts, and its regulation on gap junctions in the lens. Methods:Six–week old rat whole lenses were used. Coimmunoprecipitation and Western blotting were employed to detect protein phosphorylation and their interactions. Lipid rafts from lens cells were analyzed by sucrose gradients and by consequent Western blotting. Dye transfer/confocal microscopy was employed to measure gap junction activity. PKCγ activity was measured using PepTag nonradioactive protein kinase C assay kit. Results:H2O2 stimulated activation and autophosphorylation of PKCγ, phosphorylation of Cx50 and Cx46 at threonines and serines in rat whole lenses. Cx50 and Cx46 were associated with Cav–1 in lipid rafts. H2O2 recruited PKCγ into Cav–1 containing lipid rafts, and stimulated the interactions of PKCγ with Cav–1, Cx46, and Cx50. Transient H2O2 stimulation induced redistribution of Cx46/Cx50 from light density fractions to higher density fractions on sucrose gradients. H2O2 decreased gap junction activity in the lens. Conclusions:Activation of PKCγ by H2O2 stimulated phosphorylation and redistribution of Cx46/Cx50 within lipid rafts which may result in inhibition of gap junctional communication. PKCγ may serve as an oxidative stress sensor in the lens.
Keywords: cataract • antioxidants • gap junctions/coupling