Transepithelial cation absorption was measured as the short-circuit current (I_sc) across the epithelium from the apical side towards the basolateral side under conditions where the only major permeant ions on the apical side were either Na⁺ or K⁺ (Figure 1). In Na⁺-rich solution on the apical side, amiloride (100 μM) significantly inhibited the I_sc by 84% (Table 1) when the cells were grown on permeable supports. I_sc in K⁺-rich solution in the absence of amiloride was significantly smaller than in Na⁺-rich solution (Figure 1B). In K⁺-rich solution on the apical side, amiloride had no significant effect on the I_sc (Figure 1B, Table 1). A representative experiment is shown in Figure 1A and a summary of similar experiments is shown in Figure 1B. In K⁺-rich solution, I_sc was under 1 μA/cm² in 11 experiments, but in one experiment was 3.16 μA/cm². The single sample with the large current was likely due to a culture support with fewer perforations (lower permeability) than usual.

The currents were measured under conditions where the only major permeant ions were either Na⁺ or K⁺; Cl^- was replaced by gluconate. (Figures 2, 3A, 4). In Na⁺-rich bath solution, amiloride (100 μM) significantly inhibited the inward whole-cell current (carried mostly by bath Na⁺ at -120 mV) by 59.6% (Table 1). A representative experiment is shown in Figure 2 and 2a summary of similar experiments is shown in Figure 3A and 4. The reversal voltage in the absence of amiloride reflects the asymmetrical [Na⁺] and the leftward shift of the reversal voltage in the presence of amiloride is consistent with the block of a Na⁺-permeable channel. These whole-cell data are consistent with the observations of Na⁺-selective I_sc (above). In K⁺-rich bath solutions, the current at -120 mV was significantly smaller than in Na⁺ (Figure 4). Amiloride (100 μM) had no significant effect on the reversal voltage nor on the inward whole-cell current (carried mostly by bath K⁺ at -120 mV) (Table 1). A representative experiment is shown in Figure 2 and a summary of similar experiments is shown in Figure 3A and 4.

When primary SCCD cells were grown on impermeable supports (glass coverslips) and were bathed in Na⁺-rich solution, amiloride (100 μM) had no significant effect on the inward whole-cell current (carried mostly by bath Na⁺ at -120 mV; Figure 3B, Table 1). In K⁺-rich bath solution, amiloride (100 μM) led to a small inhibition of the inward whole-cell current (carried mostly by bath K⁺ at -120 mV) by 8.1% (Table 1). There was no significant difference in the amiloride-sensitive inward current between the Na⁺- rich bath at -120 mV (I_-120 = -9.7 ± 4.6 pA/pF, n = 6) and K⁺-rich bath solutions (I_-120 = -5.6 ± 1.2 pA/pF, n = 7) (Figure 3B). It was observed that culture on impermeable supports led to no significant change in Na⁺ -current, but to a marked increase in the K-current (factor of 18) (Figure 3, Table 1). These results are consistent with the expression of amiloride-insensitive, nonselective cation channels (see Discussion).

SCCD were dissected from neonatal Wistar rats (3-5 days), as described previously 8 12 and followed protocols in compliance with internationally recognized guidelines with approval by the Kansas State University Institutional Animal Care and Use Committee. Epithelial cells from SCCD, exclusive of common crus, were dispersed and seeded on Transwell permeable supports (6.5 mm diameter, Costar 3470, Corning, NY) or on glass cover slips (3 mm × 5 mm) and cultured with DMEM/F-12 medium (Invitrogen 12500-062, Carlsbad, CA) supplemented with 5% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich #P-7539). The cells were used within 6 to 12 days after seeding and confluence of the primary cultures on permeable supports (4-6 days after seeding) was verified by measurement of transepithelial resistance. Cultures were treated with dexamethasone (100 nM) for 24 h before recordings of I_sc or whole cell currents.

I_sc was measured as described previously 8 from confluent monolayers of SCCD in an Ussing chamber (AH 66-0001, Harvard Apparatus, Holliston, MA) modified to accept the Transwells, maintained at 37°C and connected to a voltage/current clamp amplifier (model VCC MC8, Physiologic Instruments, San Diego, CA) via Ag/AgCl electrodes and HEPES-buffered bath solution bridges with 3% agar. I_sc was directly measured under voltage clamp (short circuit conditions) and both apical and basolateral side solutions were stirred by bubbling air.

Cover slips or filter inserts with an SCCD epithelial monolayer were mounted in a chamber (150 μl) on an inverted microscope and perfused with bath solution at a rate of 150 μl/s. The patch pipettes were pulled in two steps from borosilicate glass capillaries (WPI, 1B150F-4) using a vertical puller (PP-83, Narishige) and had a resistance of 8-12 MΩ. After achieving a high-resistance seal (> 1.5 GΩ), the whole cell patch configuration was achieved by rupturing the cell membrane with suction; we were only successful about 1 time out of ~80-100 trials due to the thinness of the cells and the rigidity of the support. Electrodes were connected to an Axopatch 200A amplifier (Axon Instruments/Molecular Devices Corp., Sunnyvale, CA) at a low-pass filter bandwidth of 1 kHz (four-pole Bessel). Recordings were made at 36-37°C. Membrane currents were recorded with a Digidata 1322A (Axon Instruments) interface and pCLAMP 9 software (Axon Instruments) at a sampling rate of 10 kHz. The same software was used for the analyses. Stability of the patch was ascertained by monitoring the cell capacitance (C_cell) and resistance (R_cell) at the beginning and the end of recordings; C_cell changed less than 1 pF during experiments. The initial C_cell for cells on permeable supports was 20.9 ± 3.6 pF (n = 14 and on impermeable supports was 15.5 ± 2.0 pF (n = 13). The holding potential was 0 mV and the voltage command protocol consisted of 10 voltage steps with 20 mV spacing from -120 mV to +60 mV, 200 ms per step. Data are presented with command voltages corrected for liquid junction potentials (16.1 mV in Na⁺-rich bath and 20.4 mV in K⁺-rich bath, calculated with Axon Instruments implementation of the JPCalcW program by Peter H. Barry and additional ion mobility values, http://web.med.unsw.edu.au/phbsoft/mobility_listings.htm). Currents were normalized by cell capacitance as a measure of cell membrane surface area.

The impermeant anions gluconate^- and methanesulfonate^- were used to exclude any possible contributions from anions to the measured currents. The bath solutions were 1)

Na⁺-rich

K⁺-rich

The pipette electrodes were filled with the following two solutions in each electrode; tip solutions dialyzed the cells and the back fill solutions made contact with the Ag/AgCl electrode. For use with the Na⁺-rich bath, ~10 mm of the pipette tip was filled with (mM): 135 NMDG-methanesulfonate, 15 Na-gluconate, 5 Mg-ATP, 1 EGTA and 10 HEPES, pH 7.4 and the backfilling solution was identical except that 15 Na-gluconate was replaced by 15 NaCl and the water-soluble dye fast green added for visualization. For use with the K⁺-rich bath, the pipette tip solution contained (mM): 135 NMDG-methanesulfonate, 15 K-gluconate, 5 Mg-ATP, 1 EGTA and 10 HEPES and the backfilling solution was identical except that 15 K-gluconate was replaced by 15 KCl.

The composition of the basolateral side solution was (mM) 145 NaCl, 5 KCl, 1.2 MgCl2, 1.2 CaCl2, 10 HEPES and 5 Glucose, pH 7.4 adjusted with NaOH. The composition of the apical side solution was (mM) 150 NaCl, 0.25 MgCl2, 0.25 CaCl2, 10 HEPES, pH 7.4 adjusted with NaOH; in the substitution experiments, 150 mM NaCl was replaced by 150 mM KCl, pH 7.4 adjusted with KOH.