Regulation of gap junction coupling in bovine ciliary epithelium

Abstract

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.47 ± 0.09 ( n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased Rf by ∼60% to 0.20 ± 0.07 ( n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing Rf from 0.41 ± 0.05 ( n = 15) to 0.17 ± 0.05 ( n = 20) after 10 min. Junctional currents were lowered by ∼50% ( n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP ( n = 6); thereafter, heptanol abolished the currents ( n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction ( n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

Mechanism of Aqueous Humor Secretion, Its Regulation and Relevance to Glaucoma

“…Gap junctions both within each of PE and NPE and between PE and NPE layers allow free exchange of metabolites and ions and provide direct electrical coupling between these cells (Raviola and Raviola, 1978). In accordance, gap junction dye coupling between the two cell layers has been demonstrated using intra injection of the fluorescent dye, Lucifer yellow (Oh et al, 1994;Wang et al, 2010). In addition, the fact that there is no difference in membrane potential (Carre et al, 1992;Wiederholt and Zadunaisky, 1986) and intracellular ion contents (Bowler et al, 1996) between PE and NPE cells indicates that the gap junctions allow direct intercellular communication that facilitates the two cell layers functioning as a syncytium.…”

“…In electron micrographs of thin sections, they appear as regions of intercellular contact where apposed plasma membranes of adjacent cells are separated by a gap junction of 2 -3nm (Saez et al, 1993). Gap junctions mediate both electrical and metabolic coupling between cells (Gilula et al, 1972;Wang et al, 2010).…”

“…There are still unanswered questions about the functions of each specific cell layers and their interactions. The presence of a large number of gap junctions between the PE and NPE suggests a coupling of these two layers in transepithelial transport (Coca-Prados et al, 1992;Helbig et al, 1989b;Wang et al, 2010). One of the characteristics of the CE is the asymmetric distribution of ion transporters on the membranes of PE and NPE cells, which is essential for mediating the vectorial ion and solute transport.…”

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium

“…Similar to our previous studies, 7,22 porcine CE cells were isolated from the freshly dissected porcine eyes by incubating with 0.25% trypsin (Gibco, Grand Island, NY, USA) for 30 minutes at 378C. The isolated cells were incubated in highglucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 1% penicillin-streptomycin (Gibco) and 10% fetal 0 end of the forward strands were labeled with FAM to assist the identification of transfected cells.…”

“…6 The inhibitory effect is possibly mediated by the uncoupling of gap junctions (i.e., decrease in junctional currents and gap junction permeability) between PE and NPE cells. 7 Generally, gap junction channels aggregate on the plasma membrane where they facilitate intercellular communication by exchanging solutes, metabolites, and signaling messengers between adjacent cells. 8 At present, over 20 gap junction connexin (Cx) isoforms have been identified.…”