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L. D. Haarsma, S. L. Bardolph, J. L. Ubels; Recording of K+ Currents From Lacrimal Gland Duct Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4175.
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Lacrimal gland duct cells are believed to secrete the relatively high [K+] in tears. Channels and transporters for K+ and Cl- in an arrangement consistent with secretion of these ions have been identified in lacrimal ducts by immunostaining (Ubels et al. IOVS 47:1876, 2006), but their functional properties have not been measured. Electrophysiologic studies have characterized ion channels in lacrimal acinar cells, but due to relative paucity and inaccessibility such studies have not been done on duct cells. The goal of this project was to develop a method for isolation of viable, single duct cells and demonstrate their utility for patch-clamp recording.
Exorbital lacrimal glands from Sprague-Dawley rats (male, 6-8 weeks) were injected with DMEM, 1 mg/ml BSA, 100 U/ml collagenase. Gland slices were incubated in the same collagenase solution for 45 min at 37oC. Under a dissecting microscope the glands were microdissected in cold DMEM, 3% BSA. Ducts were removed from the glands and kept on ice in the same solution. Ducts were then incubated in DMEM, 150 U/mL elastase, 300 U/mL collagenase for 50 min, triturated and incubated for an additional 30 min. Duct cells were plated on a cover slip coated with BD Cell-Tak (3.5 µg/cm2) in recording medium containing (in mM) 160 Na aspartate, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES Na+ salt at pH 7.4 and placed in an incubator for 60 min before recordings were started. Dissociated duct cells were distinguished from acinar cells in vitro by their smaller size and lack of granularity. Whole-cell K+ currents were recorded from duct cells as a function of voltage (-80 to +100 mV) using the perforated-patch technique (amphotericin-B) and pipettes with resistances 4-7 MΩ. EGTA and CaCl2 in the pipette solution were adjusted to give 1 uM free Ca++.
Most cells (n=7 of 11) showed K+ currents that activated at command voltages near 0 mV and reached amplitudes near 1 nA at +100 mV. Currents reached peak amplitude less than 20 ms after depolarization and did not inactivate. Currents were blocked reversibly by TEA (25 mM). Cells recorded with the ruptured-patch method showed no whole-cell K+ currents (n=4).
A method has been established for isolation and dissociation of adequate numbers of lacrimal duct cells for electrophysiologic studies. Recording from these cells is challenging due to their small size and the need to use high resistance recording pipettes; however, the data are consistent with the presence of voltage-activated K+ channels. In future work appropriate recording conditions and blocking agents will be used to identify specific K+ channels types, as well as the CFTR Cl- channel.
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