Figure 1.
ISC and CT recordings with and without hormonal supplementation.
(A) Representative ISC (black trace) and CT (grey trace) recording from mpkCCD cells mounted in modified Ussing chambers and stimulated with (10 µM) forskolin. Addition of 10 µM amiloride at the end of the trace demonstrated the majority of the recorded ISC was Na+ transport via ENaC. (B) A similar trace from mpkCCD cells cultured in the absence of dexamethasone supplementation. (C) Summarized data for stimulated amiloride-sensitive current (INa) and CT response to forskolin stimulation (n = 14) in cells with (+) and without (-) full supplementation.
Figure 2.
Aldosterone restores ISC and CT responses to cAMP stimulation.
(A) Cells in unsupplemented media were incubated with 100 nM aldosterone for increasing time and stimulated with forskolin. The basal (-cAMP) and forskolin stimulated (+cAMP) currents from cells with no aldosterone (unstimulated), 6, 12 and 24 hr aldosterone stimulation were compared to cells which received fully supplemented medium (supplemented). The average ISC response to cAMP stimulation is plotted as a bar graph on the right (n = 16). (B) The change in capacitance (ΔCT) versus change in amiloride-sensitive ISC (ΔINa) in response to forskolin stimulation is summarized for cells without supplementation and 24 hr stimulation with aldosterone. Both ISC and CT responses were significantly greater in the aldosterone treated cells compared to unsupplemented controls (n = 16, p<0.01). (C) The change in capacitance versus total stimulated ENaC ISC. The INa and ΔCT of the fully supplemented cells was not significantly different from cells treated with aldosterone for either 12 hr or 24 hrs, but these 3 groups were significantly greater than cells that received no supplementation (p<0.01). (D) Data for all recordings were pooled (regardless of supplemented state) and categorized into groups that responded to cAMP with 0–5, 5–10 and >10 µA/cm2ΔISC. The average ΔCT was then plotted for each group. A significantly larger capacitance response was observed in cells with ΔISC greater than 10 µA/cm2 compared to those with less than 5 µA/cm2.
Figure 3.
ENaC siRNA reduces ISC and cAMP CT response.
(A) Western blots of whole cell lysate from βENaC knockdown demonstrate reduction in expression of ENaC compared to control siRNA transfected mpkCCD cells. The actin-corrected percent reduction in expression (n = 3) are summarized to the right of each representative blot. (B) Representative traces for ISC (top traces) and CT recordings (bottom) for control (black traces) and ENaC knockdown (grey traces) cells stimulated with forskolin (10 µM). (C) Summarized data for ISC versus CT responses to forskolin stimulation for control (n = 45) and βENaC (n = 55) knockdown cells similar to those presented in (B).
Figure 4.
Inhibiting ENaC activity does not alter trafficking.
(A) Representative ISC (black trace) and CT (grey trace) traces from mpkCCD cells pretreated with 50 µM FCI for 24 hrs before recordings and stimulated with forskolin (10 µM). (B) Single ISC recording from FCI inhibited cells (as in A) where 1 µM trypsin was added to the apical bath (light blue bar) to activate uncleaved channels. (C) Summarized ISC versus ΔCT plots for control unsupplemented, aldosterone treated and aldo+FCI treated cells (n>16 per point). The ΔCT for uncleaved channels was significantly greater (p<0.01) than the unsupplemented controls, but not significantly different from aldosterone stimulated cells.
Figure 5.
. (A) Representative ISC trace from FTR epithelial cells transfected with α,β,γENaC (black trace) or control plasmid (grey trace), mounted in Ussing chambers and stimulated with 10 µM forskolin (grey bar). The expression of ENaC was confirmed by the addition of 10 µM amiloride (black bar) at the end of the recording. (B) Data from a number of similar experiments (n = 34) are summarized. Basal (-cAMP) and forskolin-stimulated (+cAMP) INa are presented on the left and the change in ISC with foskolin stimulation (ΔISC) on the right (bar graph). (C) Representative capacitance trace from the same samples provided in (A). (D) Summarized data for capacitance changes under basal and forskolin-stimulated conditions as in (B). (* - indicates significantly different from untransfected p<0.05).
Figure 6.
Confocal fluorescent images of FM1-43X endocytosis in mpkCCD cells.
(A) Representative maximum projection images from confocal stacks obtained at 0 min and 10 min in basal (no forskolin) and forskolin stimulated (10 µM) cells previously cultured in fully supplemented media. (B) Representative images as in (A) from cells cultured without dexamethasone supplementation. (Bars represent 10 µm). (C) The number of vesicles internalized per cell is presented as a percentage of the fully supplemented counts at time 0 min (N = 3, n>200). Counts from cells cultured without dexamethasone supplementation (Unsupplemented) were significantly lower than cells in full supplementation for all conditions (p<0.05). For fully supplemented cells, there was a significantly greater number of vesicles internalized in the presence of forskolin than from cells without forskolin stimulation after 10 minutes (*, p<0.05). The percent of internalized vesicles in unsupplemented cells stimulated with forskolin was smaller than the equivalent vesicle number at 10 min in cells with full supplementation (‡, p<0.05).