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Title: Characterization and regulation of gap junctions in porcine ciliary epithelium
Authors: Li, SKL 
Shan, SW 
Li, HL 
Cheng, AKW 
Pan, F 
Yip, SP 
Civan, MM
To, CH 
Do, CW 
Issue Date: 2018
Source: Investigative ophthalmology and visual science, July 2018, v. 59, no. 8, p. 3461-3468
Abstract: PURPOSE. Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion.
Methods. Freshly-harvested porcine CE 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 Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer.
Results. Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%.
Conclusions. Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.
Keywords: Gap junctions
Connexin 43
Ciliary epithelium
Aqueous humor formation
Publisher: Association for Research in Vision and Ophthalmology
Journal: Investigative ophthalmology and visual science 
ISSN: 0146-0404
EISSN: 1552-5783
DOI: 10.1167/iovs.18-24682
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