I_sc responses to apical and basolateral bumetanide in early and late distal colon of rats.

<p>Measurements were made in normal Ringer solution. Note that while apical bumetanide (100 μM) had no effect on either colonic segment, basolateral bumetanide (100 μM) induced two opposite I_sc responses: inhibitory in early but stimulatory in late distal colon. ap, apical; bl, basolateral. ** P<0.01 <i>vs</i>. absence of bumetanide using paired <i>t</i> test. N = 10.</p

Differential expression of KCC1 protein in late distal colon of rats relative to early distal colon.

<p><b>Upper panel</b>, Western blots of KCC1; <b>Middle panel</b>, Ponceau S staining; <b>Lower panel</b>, The Western blots were quantified and normalized to ponceau S staining. The y-axis represents relative values (changes in fold) with respect to the values of early distal colon. * P<0.05 <i>vs</i>. early distal colon. N = 5.</p

Effects of Cl^- channel inhibitors on I_sc responses to basolateral bumetanide in early and late distal colon of rats.

<p>Measurements were made in normal Ringer solution. Note that pretreatment with NPPB (100 μM, apical) and glibenclamide (300 μM, apical) significantly attenuated I_sc responses to bumetanide (100 μM, basolateral) and forskolin (500 nM, basolateral), but not to amiloride (10 μM, apical) or carbachol (CCH, 100 μM, basolateral). **P<0.05 <i>vs</i>. (-) NPPB/Glibenclamide. N = 5.</p

Effects of dietary Cl^- depletion and repletion on I_sc responses to basolateral bumetanide in early and late distal colon.

<p>Note that dietary Cl^- depletion enhanced whereas dietary Cl^- repletion reduced the positive I_sc induced by basolateral bumetanide (100 μM) in late distal colon but had no effect on the induced negative I_sc in early distal colon. * P<0.05 and ** P<0.01 <i>vs</i>. normal Cl^- diet. N = 6–10.</p

I_sc^Amiloride responses to low and high salt diet treatments in rat distal colon.

<p>I_sc^Amiloride responses to low and high salt diet treatments in rat distal colon.</p

I_sc responses to basolateral bumetanide in the presence of amiloride and barium.

<p>I_sc was measured in normal Ringer solution. Note that amiloride (10 μM, apical) significantly decreased and barium (5 mM, apical) significantly increased I_sc in late distal colon. However, these treatments did not alter the subsequent I_sc responses to bumetanide (100 μM, basolateral) in either colonic segment. Subsequent addition of forskolin (500 nM, basolateral) caused I_sc stimulation in both segments (see text). * P<0.05 and ** P<0.01 <i>vs</i>. prior treatment by paired <i>t</i> test. N = 10.</p

Effects of amiloride and benzamil on ΔI_sc^Amiloride and ΔI_sc^Bumetanide in rat early and late distal colon^*.

<p>Effects of amiloride and benzamil on ΔI_sc^Amiloride and ΔI_sc^Bumetanide in rat early and late distal colon^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171045#t002fn001" target="_blank">*</a>}.</p

Differential expression of gene transcripts in rat distal colon.

<p>Differential expression of gene transcripts in rat distal colon.</p

Transepithelial conductance (G_T) responses to bumetanide (100 μM, basolateral) and forskolin (500 nM, basolateral) in early and late distal colon of rats.

<p>The experiments were performed in normal Ringer solution. ** P<0.01 <i>vs</i>. prior drug treatment. N = 10.</p

Effects of ion substitution on changes in I_sc responses to basolateral bumetanide (100 μM) in early <i>vs</i>. late distal colon.

<p>Note the two opposing ΔI_sc^Bumetanide: negative or inhibitory in early distal colon and positive or stimulatory in late distal colon_. While Na⁺ substitution caused no significant change in the I_sc response in either colonic segment, removal of Cl^- from media abolished the I_sc responses in both segments. ** P<0.01 <i>vs</i>. control. N = 8–12.</p