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C. W. Do, Z. Wang, V. Valiunas, A. F. Clark, M. B. Wax, J. E. Chatterton, M. M. Civan; Regulation of Gap-Junction Coupling in Bovine Ciliary Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2103.
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© ARVO (1962-2015); The Authors (2016-present)
Aqueous humor is formed by fluid transfer from ciliary stroma sequentially across pigmented ciliary epithelial (PE) cells, gap junctions and nonpigmented (NPE) cells. Gap junctions linking PE and NPE cells arise from Cx43 and Cx40 connexons, but their relative functional importance and regulation are unknown. We tested whether siRNA directed against Cx43 affects PE-NPE communication and whether cAMP regulates this communication.
Bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, real-time PCR and Western blot.
Transfection with 30 nM Cx43 siRNA inhibited Cx43 mRNA expression by 57 ±4% after 24 hrs and by 52 ±6% after 48 hrs, in comparison to a mock-transfected control. After 48 hrs, siRNA reduced both Cx43 protein expression and LY dye 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 a non-targeting control siRNA. The Cx43 siRNA decreased Rf to 0.20 ±0.07 (N=13), an inhibition of ~60% (P<0.02). cAMP (500 µM) also reduced LY dye transfer after 10 min from 0.41 ±0.05 (N=15) in control couplets to 0.17 ±0.05 (N=20), an inhibition of ~60%. Junctional currents were also reduced by 53 ±7% (N=6) after perfusion for 10 min with 500 µM cAMP (N=6); thereafter, heptanol reduced currents by an additional 25% (N=6). Preincubation with the PKA inhibitor H89 (2 µM) for 15 min entirely prevented cAMP-triggered inhibition (N=6).
(1) siRNA knockdown of Cx43 can reduce communication between PE and NPE cells by more than 50%, documenting that Cx43 is a major functional component of PE-NPE gap-junctions. (2) Although cAMP can either up- and down-regulate Cx43 and Cx40 gap junctions in other cells, cAMP acts through PKA to inhibit PE-NPE gap-junctional communication.
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