S Chen1, M Sears. 1. Department of Ophthalmology and Visual Science, Yale School of Medicine, New Haven, CT, USA.
Abstract
PURPOSE: Chloride efflux plays an important role in aqueous humor production. Chloride currents have been described in bovine non-pigmented ciliary epithelium (NPE), in transformed cultured human NPE, and in bovine volume-activated chloride channels described in the latter. It is the basolateral membranes of the NPE of ocular ciliary processes that comprise the exit pathway for the process of aqueous secretion performed by the double syncytial layer of ciliary epithelium, however. Therefore, we studied both cell-attached, and, excised, inside-out patches from basolateral membranes of the NPE. METHODS: Cell-attached and cell-free excised patches were formed from the basolateral membranes of NPE and single channel currents recorded with a Dagan 3900A patch clamp amplifier. RESULTS: A low conductance channel of 14 pS was observed and recorded in 30% of cell attached patches and in 35% of excised inside out patches under symmetrical conditions (160 mM chloride). This channel displayed a nearly linear current-voltage relationship, with an open probability that was not voltage-dependent. The channel was chloride-selective: N-methyl-D-glucamine (NMDG) used as cation did not alter the current profile nor the reversal protein. Further, with inside-out patches, the reversal potential was close to zero (0.3 +/- 0.5 mV (10) in symmetrical (160 mV) chloride, but shifted to -32.3 +/- 0.5 mV (5) when the concentration of chloride in the bathing solution was reduced to 40 mM while the recording pipette was held at 160 mM. This value approaches the theoretical equilibrium potential of chloride for these conditions. The channel anion permeability sequence was: I- > NO3- > or = Br- > Cl- > gluconate- approximately equal to aspartate-. Three different chloride-channel blockers inhibited the channel activity. CONCLUSION: A low conductance channel, selective for chloride, and, modulated by beta adrenergic and VIP stimulation, based on it sensitivity to exogenously added cAMP, ATP and the catalytic subunit of PKA, is present in the exit basolateral membranes of rabbit NPE and contributes to the process of aqueous humor formation.
PURPOSE:Chloride efflux plays an important role in aqueous humor production. Chloride currents have been described in bovine non-pigmented ciliary epithelium (NPE), in transformed cultured human NPE, and in bovine volume-activated chloride channels described in the latter. It is the basolateral membranes of the NPE of ocular ciliary processes that comprise the exit pathway for the process of aqueous secretion performed by the double syncytial layer of ciliary epithelium, however. Therefore, we studied both cell-attached, and, excised, inside-out patches from basolateral membranes of the NPE. METHODS: Cell-attached and cell-free excised patches were formed from the basolateral membranes of NPE and single channel currents recorded with a Dagan 3900A patch clamp amplifier. RESULTS: A low conductance channel of 14 pS was observed and recorded in 30% of cell attached patches and in 35% of excised inside out patches under symmetrical conditions (160 mM chloride). This channel displayed a nearly linear current-voltage relationship, with an open probability that was not voltage-dependent. The channel was chloride-selective: N-methyl-D-glucamine (NMDG) used as cation did not alter the current profile nor the reversal protein. Further, with inside-out patches, the reversal potential was close to zero (0.3 +/- 0.5 mV (10) in symmetrical (160 mV) chloride, but shifted to -32.3 +/- 0.5 mV (5) when the concentration of chloride in the bathing solution was reduced to 40 mM while the recording pipette was held at 160 mM. This value approaches the theoretical equilibrium potential of chloride for these conditions. The channel anion permeability sequence was: I- > NO3- > or = Br- > Cl- > gluconate- approximately equal to aspartate-. Three different chloride-channel blockers inhibited the channel activity. CONCLUSION: A low conductance channel, selective for chloride, and, modulated by beta adrenergic and VIP stimulation, based on it sensitivity to exogenously added cAMP, ATP and the catalytic subunit of PKA, is present in the exit basolateral membranes of rabbit NPE and contributes to the process of aqueous humor formation.