Stress-Induced Alterations in Lens Epithelial Cells: Possible Regulatory Role of Gap Junctions

Abstract

Abstract: : Purpose: Earlier studies have shown that oxidative stress alters gap junction activity and communication in cells. Gap junction activity is regulated by a number of factors including drugs, growth factors, and protein kinases. The purpose of this study is to determine how PKC γ regulates the effect of H₂O₂ stress on lens epithelial cell gap junctions. Methods: DAG, Calphostin C, TPA, PKC γ and α pseudosubstrate inhibitors, LEDGF, EGF, IGF-1 and H₂O₂ were incubated with whole Sprague-Dawley rat lenses after which dye transfer was determined using lucifer yellow and rhodamine-dextran. The resulting gap junction activity was measured by confocal microscopy. Rabbit lens epithelial N/N1003A cells were grown in tissue culture until 90% confluent. The effect of H₂O₂ on cell viability was determined by trypan blue dye exclusion. Apoptosis was determined using flow cytometry. Cell culture gap junction activity was determined using the scrape load/dye transfer technique. Results: In lens epithelial cells 120µM H2O2 increased gap junction activity at 15 minutes without cell death. Gap junction activity was normalized by 10 ng/ml LEDGF but IGF-1 had no effect. Conclusions: H₂O₂ stress induced changes in gap junction activity. This could be controlled by stress-related growth factors such as LEDGF. Data suggest a role for gap junctions in lens epithelial cell stress responses.

Keywords: stress response • gap junctions/coupling • cell death/apoptosis