Effect of Inflammation on Colonic Anion Secretion and the Associated Ion Transporters in IL10 Knockout Mice
|dc.contributor.advisor||Butt, Adrian Grant|
|dc.identifier.citation||Fan, S. (2014). Effect of Inflammation on Colonic Anion Secretion and the Associated Ion Transporters in IL10 Knockout Mice (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4936||en|
|dc.description.abstract||Intestinal inflammation, such as that associated with inflammatory bowel disease (IBD), is reported to cause a marked reduction in intestinal fluid and electrolyte secretion. However, the mechanisms of the reduced secretion are contentious. In this study, the effects of inflammation on colonic anion secretion and the associated transporter proteins were investigated in an animal model of IBD, interleukin10 knockout (IL10-/-) mice infected with Helicobacter typhlonius. These mice develop significant colitis, which is most severe in the proximal colon. Uninfected wild type and IL10-/- mice, which in the Otago University Animal facility do not develop colitis, served as controls, and wild type mice infected with H. typhlonius were included to identify possible effects of infection that were independent of inflammation. To assess regional differences, the entire colon was divided into four segments of approximately equal length, proximal, proximal mid, distal mid, and distal colon. Electrogenic anion secretion was measured using the short circuit current (Isc) technique. Expression of transport proteins was determined with immunohistochemistry and western blotting. Finally, using crypts isolated from the proximal colon, the effect of inflammation on the activity of the electrogenic Na+/HCO3- cotransporter, NBCe1, was investigated by fluorescence microscopic measurement of intracellular pH (pHi) recovery from an acid load induced by ammonium prepulse. The pHi measurements were carried out in the cells within the upper third of the crypt and surface epithelium (surface cells), and the cells located in the lower third of the crypts (crypt cells). In intact colonic sheets (i.e., epithelium plus muscle layers), electrogenic anion secretion stimulated by forskolin (10 μM mucosal [m] and serosal [s]) was markedly reduced by inflammation throughout the colon. However, when the underlying muscle layer was removed, the forskolin response was comparable in inflamed and non-inflamed tissues. In the proximal mid and all distal colonic segments, the forskolin response was partially (50 – 60%) inhibited by bumetanide (100 μM s) but unaffected by 4,4'-diisothiocyanatostilbene-2,2ʹ-disulfonic acid (DIDS, 500 μM s). In contrast, in the proximal colon the forskolin response consisted of both bumetanide- and DIDS-sensitive components, which were of comparable magnitude. Bumetanide-sensitive Cl- secretion was dependent on basolateral Na+/K+/2Cl- cotransporter (NKCC1), whereas the DIDS-sensitive component was Cl- secretion driven by basolateral Cl-/HCO3- exchanger and intracellular generation of HCO3- by hydration of metabolic CO2. Neither the bumetanide- nor the DIDS-sensitive secretory component was significantly affected by inflammation in the proximal colon. Consistent with the Isc measurements, the expression of NKCC1 and Na+/K+-ATPase, two major transporters associated with Cl- secretion, was not altered by colonic inflammation in any region of the colon. In contrast, inflammation significantly reduced the expression of NBCe1 in the colon. In non-inflamed tissues, NBCe1 was expressed in all epithelial cells in the proximal mid colon, and predominantly expressed in the surface cells and the upper third of the crypts of the proximal colon. However, NBCe1 immunoreactivity was absent in the corresponding regions of the inflamed tissues and there was a marked decrease in the total amount of NBCe1 protein compared with that in the control animals. In the distal colon, although NBCe1 immunoreactivity was absent in all tissues, western blots detected NBCe1 protein in non-inflamed tissues, and this was significantly reduced in the inflamed ones. Consistent with the pattern of NBCe1 expression in the proximal colon, measurements of pHi recovery from acid loading indicated that there was no NBCe1 activity in the crypt cells. This was true under basal conditions and following stimulation with forskolin. In contrast, in the surface cells of the control animals, while NBCe1 was inactive under basal conditions its activity was stimulated by forskolin, and this was significantly blocked by DIDS. However forskolin did not stimulate NBCe1 activity in the surface cells of the inflamed tissues. This suggests inflammation reduces basolateral HCO3- uptake in response to forskolin in colonic surface epithelial cells, the predominant sites for electroneutral HCO3- secretion. Taken together, these data indicate that while inflammation has no effect on electrogenic anion secretion in the colon, it is likely to inhibit electroneutral HCO3- secretion. This may affect mucosal surface pH regulation and mucus formation, which will potentially disturb the bacterial growth and mucus barrier, exacerbating inflammation. Measurements of electroneutral HCO3- secretion and mucus properties in non-inflamed and inflamed colon would be required to clarify these potential consequences.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.subject||Colonic anion secretion|
|dc.subject||Epithelial ion transport|
|dc.title||Effect of Inflammation on Colonic Anion Secretion and the Associated Ion Transporters in IL10 Knockout Mice|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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