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Electrophysiological characterization of rat type II pneumocytes in situ by whole‐cell patch‐clamp
Author(s) -
Shlyonsky Vadim,
Goolaerts Arnaud,
Mies Frédérique,
Naeije Robert
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a549-b
Subject(s) - amiloride , tetrodotoxin , patch clamp , chemistry , biophysics , electrophysiology , lamellar granule , pulmonary surfactant , channel blocker , microbiology and biotechnology , sodium , biology , medicine , biochemistry , calcium , receptor , organic chemistry
We present a novel functional in vitro model of rat lung slices 200 μm thick, void of supporting agarose and attached to polycarbonate filters. Cells in slices were identified using specific dyes for live, dead, and surfactant‐producing cells. Slices cultured on paper filters saturated with culture medium retained a majority of living cells for up to 72 hours following sectioning. Surfactant producing cells (type II pneumocytes) continued to proliferate for up to 48 hours in culture, after which they lost their cubic appearance and the majority of lamellar bodies and appeared squamous. Dexamethasone significantly attenuated the disappearance of surfactant producing cells and the loss of lamellar bodies. When patched in whole‐cell configuration, type II pneumocytes in situ had a mean capacitance of 11.6±1.2 pF (range 5–26 pF) and a resting membrane voltage of −4.3±2.8 mV (range −12 to +8 mV). Tetraethylammonium significantly inhibited outward currents, indicating the presence of K‐channels, while replacement of Na + in the bath with NMDG + significantly decreased inward currents. These inward currents were not sensitive to amiloride and proteolytic treatment of cells with trypsin (5 μg/ml) did not activate amiloride‐sensitive currents. These results indicate that ENaC channels are not present in type II pneumocytes from freshly prepared rat lung slices. Exposure of slices to 1 μM dexamethasone for 48 hours induced an increase in Na‐entry currents, which became partially inhibitable by amiloride. Acute exposure of type II pneumocytes in situ to 100 μM cpt‐cAMP resulted in hyperpolarization of these cells by ~ 15–20 mV and activated Cl‐currents inhibitable by NPPB, a blocker of CFTR channels. This work was funded by grants from ULB, FNRS and Fonds E.Defay. VS was a recipient of ERS research fellowship (No 306)