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Wen Shi, Manuel A Riquelme, Sumin Gu, Jean X Jiang; Lens Connexin Hemichannels are Responsive to Mechanical Stimulation and Oxidative Stress, and Protect Cell against Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Gap junctions formed by connexins play important roles in lens homeostasis and transparency. In addition to their roles in forming gap junctions, connexins form hemichannels in lens fibers. However, the physiological importance of these lens connexin hemichannels remains largely unknown.
Lens primary cell cultures were prepared using lens at embryonic day-11 and cultured up to 15 days with the gradual formation of lentoid bodies, an indicator of lens cell differentiation. High titer recombinant retroviruses containing vehicle, chick Cx50 or Cx46, were prepared and used to infect chick embryonic fibroblast (CEF) cells and lens primary cell culture. The cells were treated with H2O2 at 300 µM for 20 min to 2 hrs or were subject to mechanical stimulation through fluid flow shear stress (FFSS) at 8 dynes/cm2 for 15 min using a parallel flow chamber. Hemichannel activity was studied using dye uptake assay with ethidium bromide (EtBr) dye. Cell viability was assayed with annexin V and propidium iodide (PI) labeling.
Hemichannels formed by either Cx50 or Cx46 were open in response to the treatment with H2O2 or FFSS. The results of dye uptake assay showed that lentoids bodies in lens primary culture were highly sensitive to H2O2 and FFSS, and hemichannel activity was inhibited by a connexin channel blocker carbenoxolone. Interestingly, this activity was significantly inhibited by two dominant negative mutants of Cx50; Cx50P88S which inhibits both gap junctions and hemichannels and Cx50H156N which only inhibits hemichannels, not gap junctions, implying the role of hemichannels. H2O2 treatment also caused the opening of hemichannels in cultured epithelial cells, but these dominant negative mutants failed to inhibit these hemichannel activities, suggesting that epithelial hemichannels are unlikely formed by Cx50 in epithelial cells. The treatment of H2O2 increased the numbers of cells under apoptosis and this increase was augmented in cells expressing these two dominant negative mutants.
These results show that both oxidative stress and mechanical stimulation activate connexin hemichannels in the lens. Functional connexin hemichannels are likely to play a cell protective role against oxidative damage in the lens.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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