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Ka Lok Li, Sze Wan Shan, Angela King Wah Cheng, Mortimer M. Civan, Chi-ho To, Chi-wai Do; Gap Junctions – their characterization and basis in porcine ciliary epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6405.
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© ARVO (1962-2015); The Authors (2016-present)
Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) cilary epithelial cells, and are therefore critical in the secretion of the aqueous humor by the ciliary epithelium. However, agreement has been incomplete concerning the connexin (Cx) composition of the gap junctions. We have now determined (1) the gene expression of Cxs in the porcine ciliary epithelium, a favorable model for the human; and (2) the contribution of the highest expressed Cx (Cx43) to secretion by this preparation.
Freshly-harvested porcine ciliary epithelial cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting (WB), respectively. The relative gene expressions of various connexins (Cx) were determined by quantitative PCR (qPCR). The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer.
Using RT-PCR and WB, Cx43, Cx45, Cx50 and Cx60, but not Cx26 and Cx40, were expressed in porcine ciliary epithelium. Cx43 was the most abundant isoform, over 200-fold higher than other Cxs. Knockdown of Cx43 by siRNA reduced mRNA and protein expressions by 59±3% (n=4, p<0.001) and 71±13% (n=3, p<0.05), respectively. The Cx43 knockdown significantly reduced dye transfer from PE to NPE cells by ~30% (n=4).
Similar to other species, Cx43 was found to be the major component of gap junctions in porcine ciliary epithelium. Knockdown of Cx43 reduced gap junctional permeability, supporting the functional significance of Cx43 in mediating fluid movement across porcine ciliary epithelium.
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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