Functional coupling in bovine ciliary epithelial cells is modulated by carbachol

Abstract

The functional coupling of the ciliary epithelium was studied in isolated pairs (couplets) of pigmented ciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE) cells using the whole cell patch clamp and the fluorescent dye lucifer yellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2–5 MΩ electrode, containing 1–2 mM lucifer yellow, in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells were coupled. The electrical coupling of the cells was also studied by calculating the capacitance (using the time-domain technique), assuming a “supercell” model for coupled cells. The mean capacitance of coupled pairs was 79.8 ± 4.3 (SE) pF ( n = 47) compared with single cell capacitances of 36.8 ± 3.4 pF ( n = 10) for PCE cells and 38.1 ± 3.1 pF ( n = 15) for NPCE cells. Octanol, carbachol (CCh), and raised extracellular Ca2+ concentration ([Ca2+]o) all caused uncoupling in pairs (couplets) of coupled NPCE and PCE cells. At room temperature (22–24°C), the capacitance of the couplets decreased from 70.5 ± 8.0 to 48.0 ± 5.2 pF ( n = 5) when exposed to octanol (1 mM), from 73.8 ± 9.2 to 43.2 ± 9.5 pF ( n = 4) when exposed to CCh (100 μM), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF ( n = 4) when exposed to 10 mM [Ca2+]o. The response to CCh was dose dependent; at higher temperatures of 34–37°C, 10 μM CCh caused a 38% reduction in capacitance, from 53.7 ± 9.7 to 33.5 ± 3.3 pF ( n = 7) with a half-time of 249 s, and 100 μM CCh caused a 49% reduction in capacitance, from 51.3 ± 5.6 to 26.0 ± 2.4 pF ( n = 7) with a half-time of 124 s. After pairs uncoupled and the uncoupling agent was washed out, the cell pairs often exhibited an increase in capacitance that we interpreted as “recoupling” or a reopening of the gap junctional communication pathway; the half-time for this process was 729 s after uncoupling with 100 μM CCh and 211 s after uncoupling with 10 μM CCh. This interpretation was confirmed optically by the spread of lucifer yellow into both cells of an uncoupled pair with a time course corresponding to the increase in electrical coupling. The controllable coupling of ciliary epithelial cells extends the idea of a functional syncytium involved in active transport. PCE cells take up solute and water from the blood, which then cross to NPCE cells via gap junctions and from there are secreted into the posterior chamber of the eye. Modulation of the coupling between NPCE and PCE cells may provide a mechanism to control secretion.