AbstractsBiology & Animal Science

Effect of Inflammation on Colonic Anion Secretion and the Associated Ion Transporters in IL10 Knockout Mice

by Shujun Fan

Institution: University of Otago
Year: 0
Keywords: Inflammation; Colonic anion secretion; Epithelial ion transport
Record ID: 1299859
Full text PDF: http://hdl.handle.net/10523/4936


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…