Connexin Hemichannels Protect Lens Fiber Cells Against Oxidative Stress

Abstract

Purpose : 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. However, the biological importance of these lens connexin hemichannels in lens cells remains largely unknown.

Methods : Lens primary cell cultures were prepared using embryonic day-11 fertilized chicken eggs and were cultured up to 15 days with the gradual formation of lentoid body structures, an indicator of lens cell differentiation. High titer recombinant retroviruses containing vehicle, chick Cx50 or Cx46, were prepared and were used to infect chick embryonic fibroblast (CEF) cells and lens primary cell culture. Both CEF cells and primary lens cell culture were treated with H₂O₂ at 0.3-0.5 mM for 20 min to 2 hrs. Hemichannel activity was studied using dye uptake assay with ethidium bromide (EtBr) dye and quantified using NIH Image J software. Cell viability was assayed with annexin V and propidium iodide (PI) labeling, and numbers of the positive cells were quantified with fluorescence microcopy.

Results : Cx50 hemichannels were open in response to the treatment with H₂O₂. The dye uptake assay showed that chick embryonic fibroblast (CEF) cells or lentoids bodies in lens primary culture were highly sensitive to H₂O₂, and hemichannels 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, but not gap junctions. H₂O₂ treatment also opened hemichannels in cultured lens epithelial cells and this opening was inhibited by Cx43 blocking antibody, but not by Cx50 dominant negative mutants, suggesting that epithelial hemichannels are likely formed by Cx43, not Cx50. The treatment of H₂O₂ increased the numbers of cells under apoptosis and this increase was augmented in cells expressing those two dominant negative mutants that disrupted Cx50 hemichannels, while Cx50E48K that only impaired gap junctions did not have such effect.

Conclusions : These results showed that oxidative stress activates connexin hemichannels in the lens cells and functional connexin hemichannels, not gap junctions are likely to play a cell protective role against oxidative damage in lens.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.