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C. W. Do, Z. Wang, V. Valiunas, M. M. Civan; Monitoring of Gap-Junctional Coupling in Bovine Ciliary Epithelium by Dual Whole-Cell Patch-Clamping and Lucifer Yellow Fluorescence Microscopy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2813.
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The aqueous humor is secreted by the bilayered ciliary epithelium. Fluid is taken up by the pigmented ciliary epithelial (PE) cells from the stroma, transferred through homomeric, homotypic Cx43 and Cx40 gap junctions to the nonpigmented ciliary epithelial (NPE) cells, and released into the aqueous humor. Interrupting gap junctions with heptanol inhibits aqueous humor secretion in vitro. Since the PE-NPE cell couplet is the fundamental unit for secretion, this study aims to determine the coupling characteristics using both dual patch clamping and fluorescence microscopy.
Freshly-harvested couplets from bovine ciliary epithelium were studied. Gap-junctional communication was assessed by both dual-cell patch clamping and Lucifer Yellow (LY) dye transfer.
Dual patch clamping of PE-NPE cell couplets revealed a wide range of junctional conductances, with a mean ± SEM of 6.1 ± 3.1 nS (n=6). Perfusion with 4.3 mM heptanol decreased the total junctional conductance to 0.004 ± 0.013 nS (n=6). Total junctional conductance reflects contributions from Cx43 and Cx40. However, LY is known to be five times more permeable through Cx43 than through Cx40, so that dye transfer measurements largely reflect passage through Cx43. We found that LY is transferred through the untreated gap junctions < 5 min. After applying LY for 10 min, the ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.63 ± 0.06 (n=17) and 0.66 ± 0.10 (n=12) 24 and 48 hrs after harvesting the couplets, respectively. Heptanol inhibited LY transfer (n=6, P=0.001).
We have demonstrated for the first time the heptanol-inhibitable PE-NPE cell gap-junctional coupling by dual whole-cell patch-clamping to assess permeation through Cx43 and Cx40 connexons. We have also measured heptanol-inhibitable LY transfer, which largely reflects permeation through Cx43 gap junctions.
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