Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium

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.

Thrombospondin‐1 mediates Rho‐kinase inhibitor‐induced increase in outflow‐facility

“…Similar to our previous study (Li et al, 2018 ), total RNA was extracted by the Qiagen RNeasy Micro Kit (Qiagen), quantified, and reverse‐transcribed to complementary DNA (cDNA) by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Polymerase chain reaction (PCR) was performed using a HotStarTaq Plus Master Mix Kit (Qiagen) to assess the messenger RNA (mRNA) expression of TSP1.…”

“…Similar to our previous study (Li et al, 2018), total RNA was extracted by the Qiagen RNeasy Micro Kit (Qiagen), quantified, and reverse-transcribed to complementary DNA (cDNA) by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).…”

Regulation of Aqueous Humor Secretion by Melatonin in Porcine Ciliary Epithelium

“…In contrast, Cl − secretion has been shown to be the major driving force for AH secretion in animal species, including pigs, oxen, and humans [ 38 , 45 , 46 , 49 ]. It has been suggested that pig eye serves as a good animal model for studying human AH dynamics [ 5 , 29 ], partly because of their similarities to humans regarding anatomical structures, AH’s electrolyte composition, and responses to drugs [ 50 , 51 , 52 , 53 ]. Our current results were also in good agreement with a previous study, which demonstrated that melatonin stimulated Isc and Cl − secretion in human colonic T84 cells [ 54 ].…”

“…As reported in our previous studies, LY dye diffusion was determined across freshly isolated porcine PE-NPE cell couplets [ 5 , 77 ]. For each PE-NPE cell couplet, a tight seal was formed between the micropipette and the plasma membrane of the PE cell (chosen as the donor cell).…”

“…Active Cl − transport across the ciliary epithelium has been shown to be the major driving force for AH secretion [ 1 , 2 ]. The intercellular gap junctions at the apical surfaces of PE and NPE cells link these two cell layers to work in a coordinated manner as a syncytium [ 3 , 4 , 5 ]. In addition, swelling-activated Cl − channels located at the basolateral membrane of NPE cells are thought to likely play important roles in regulating AH secretion rates via Cl − release [ 3 , 6 , 7 ].…”