Functional Interplay of Connexin Hemichannels in Glutathione Export from Lens Epithelial Cells and in Fiber Cell protection against Oxidative Stress

Abstract

Purpose : As an avascular organ, lens depends on a unique network of transport system for its survival and metabolic homeostasis, and connexin channels are functionally involved. The purpose of this study is to test hypothesis that mechanical-activated connexin (Cx) hemichannels (HCs) mediate glutathione (GSH) transport from lens epithelial cells to lens fibers and protect fiber cells against oxidative insults.

Methods : Primary embryonic chick lens epithelial culture and differentiated chick lens cultures (containing lentoid bodies) were used to mimic lens epithelial cells and lens fibers, respectively. Fluid flow shear stress (FFSS, 1 dyne/cm²) were used as a mechanical stimulation on primary embryonic chick lens cell culture. Conditioned medium was collected from the culture subjected to fluid flow (FFCM) or non-fluid flow control (NFCM). These CM were used to treat primary lens epithelial cells and differentiated lens culture with lentoids for 2 hrs. HCs opening was assessed by dye (ethidium bromide (EtBr)/FITC-dextran) uptake and GSH uptake by Thiol Tracker indicator and fluorescence intensity. Cell survival after H₂O₂ treatment was evaluated by nnexin V/propidine iodide (PI) staining. Approaches that specifically inhibit hemichannel function include:1) HCs specific antibody (Cx43(E2)); 2) exogenously expressing Cx50 H156N, a Cx50 dominant negative mutant impairing hemichannel function through RCAS(A) retroviral infection.

Results : Dye uptake was increased in primary chick lens epithelial cells in response to FFSS and this increase was reduced with Cx43(E2) antibody treatment. GSH levels were increased in FFCM compared with NFCM as a result of HC opening in response to FFSS. Inhibition of Cx50 HCs by HC-impaired Cx50 H156N mutant via RCAS(A) retroviral infection in differentiated lens epithelial cell culture increased apoptotic lentoid cells indicated by Annexin V/PI positive signals under H₂O₂. In addition, increased GSH level was observed after FFCM compared to NFCM treatment under H₂O₂.

Conclusions : The results suggest that Cx43 HCs in lens epithelial cells are induced open by FFSS that leads to GSH release. The released GSH is up taken by Cx50 HCs on lens fiber cells and protects lens fibers against oxidative stress.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.