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A.C. Long, C.M. H. Colitz, J.A. Bomser; Regulation Of Gap Junction Communication By Protein Kinase C In Primary Canine Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4090.
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Gap junction communication (GJC) is important in maintaining lens epithelial cell homeostasis and reductions in GJC may be associated with the development of senile cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (Cx43) proteins that compose gap junction channels. The purpose of this study is to examine the role of PKC activation in modulating GJC in a primary canine lens epithelial cell (LEC) line.
TPA (12–O–tetradecanoyl–phorbol–acetate), a potent PKC activator and inhibitor of GJC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0–1000ng/ml) for 30 minutes and GJC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of Cx43 protein. Inhibition of general and Ca++–dependent PKC activity was achieved by pretreatment of cells with calphostin C and Gö6976, respectively.
Treatment with TPA (1–1000ng/ml) significantly decreased GJC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJC by 80% as compared to controls and increased Cx43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with Gö6976, but not calphostin C, blocked TPA–induced reductions in GJC by approximately 60%.
Treatment with TPA significantly reduces GJC in canine LEC. These effects are mediated, in part, by activation of Ca++–dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJC and cataractogenesis. Understanding the molecular mechanisms of GJC in the lens may lead to therapeutic strategies to maintain GJC and slow the progression of senile cataract.
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