Figure 3. Ex vivo aldosterone induces ENaC activity and bumetanide-sensitive K⁺ secretion and increases NKCC1 and BKα mRNA abundance in normal rat distal colon.

I_SC (A) and transepithelial resistance (G_TE; B) were continuously monitored in normal rat distal colonic mucosae mounted under voltage clamp conditions in the presence and absence of 10 nM aldosterone, or 10 nM aldosterone plus 10 μM spironolactone. Following an 8-hour incubation period, I_SC and G_TE were progressively increased (evidence for induced ENaC activity) in aldosterone-treated tissues (closed circles), while no change was observed in control (open circles) or aldosterone plus spironolactone-treated tissues (grey triangles). I_SC and G_TE data over the 8-hour incubation period are not shown here, so as to prevent the depicted data from being distorted by the lengthy time scale. I_SC recordings depicted here were begun at 8 hours post-treatment. All tissues were treated with mucosal amiloride (AMIL; 100 μM), followed by serosal bumetanide (BUMET; 200 μM) where indicated. (C-D) Group data showing the bumetanide-sensitive change in Isc (ΔI_SC; C) and G_TE (ΔG_TE; D) in control, aldosterone-, and aldosterone plus spironolactone-treated tissues. Average I_SC and G_TE values over the course of 1 minute immediately prior to bumetanide addition, and 1 minute at the end of the recording period were calculated for each tissue. ΔI_SC and ΔG_TE were calculated by subtracting the basal value from the value at the end of the recording period, after bumetanide application. (E) NKCC1 and BKα mRNA transcript abundance measured by qRT-PCR analysis using RNA extracted from control, aldosterone- and aldosterone plus spironolactone-treated mucosae used in the above experiments. Lines and error bars represent means ± SEM. * p<0.05 and ** p<0.01, as determined by one-way ANOVA with Tukey’s post-hoc.