Acid-base effects on intestinal Cl- absorption and vesicular trafficking.

In rat ileum and colon, apical membrane Cl(-)/HCO(3)(-) exchange and net Cl(-) absorption are stimulated by increases in Pco(2) or [HCO(3)(-)]. Because changes in Pco(2) stimulate colonic Na(+) absorption, in part, by modulating vesicular trafficking of the Na(+)/H(+) exchanger type 3 isoform to and from the apical membrane, we examined whether changes in Pco(2) affect net Cl(-) absorption by modulating vesicular trafficking of the Cl(-)/HCO(3)(-) exchanger anion exchanger (AE)1. Cl(-) transport across rat distal ileum and colon was measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at Pco(2) 70 mmHg than at Pco(2) 21 mmHg but was not affected by pH changes in the absence of CO(2). AE1 was internalized when Pco(2) was reduced and exocytosed when Pco(2) was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl(-) absorption caused by an increase in Pco(2). Increases in Pco(2) stimulated ileal Cl(-) absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by Pco(2). We conclude that CO(2) modulation of colonic, but not ileal, Cl(-) absorption involves effects on vesicular trafficking of AE1.

Effect of secretagogues and pH on intestinal transport in guanylin-deficient mice.

The small and large intestine secrete guanylin, a peptide homologous to heat stable enterotoxin (STa) elaborated by enterotoxigenic Escherichia coli. Guanylin's role in intestinal electrolyte transport was investigated in guanylin-deficient knockout mice and heterozygous littermate controls. Segments of mid-jejunum, distal ileum, and proximal and distal colon were studied in Ussing chambers in HCO3- Ringer under short circuit conditions. We found that (1) under basal conditions, all segments in control and knockout mice absorb Na+, and the knockout mouse proximal colon secretes Cl-; (2) all segments except the jejunum of knockout mice respond by increasing absorption in response to reductions in pH from 7.6 to 7.1; (3) all segments exhibit decreased absorption in response to 1 mM cAMP; (4) the jejunum and ileum of knockout and control mice, and the proximal colon of control mice (but not knockout mice) respond to the mucosal addition of 50 nM STa with decreases in absorption; and (5) mucosal guanylin caused similar decreases in proximal colon absorption in control and guanylin-deficient mice. These findings suggest that guanylin deficiency causes basal Cl- secretion and reduced responsiveness to STa in mouse proximal colon. The effectiveness of guanylin in this segment suggests a difference in the intestinal secretory actions of STa and guanylin.

Non-catalytic role of carbonic anhydrase in rat intestinal absorption.

Carbonic anhydrase (CA) inhibition reduces NaCl absorption in rat distal ileum, a pH-sensitive, low CA activity tissue, and in distal colon, a CO(2)-sensitive, high CA activity tissue. We hypothesized that CA plays a non-catalytic role in NaCl absorption in these segments. Unidirectional fluxes of Na(+) and Cl(-), and total HCO(3)(-) generation (estimated as the sum of radiolabeled HCO(3)(-) and CO(2) produced from glucose) were measured in Ussing chambers in nominally CO(2), HCO(3)(-)-free HEPES Ringer. Measurements were made in the presence and absence of 0.1 mM methazolamide, a membrane-permeant CA inhibitor. Ringer pH reduction from 7.6 to 7.1 stimulated ileal but not colonic Na(+) and Cl(-) absorption. In the ileum, methazolamide reduced J(ms)(Na) and J(ms)(Cl) and caused net Cl(-) secretion at pH 7.6, and prevented the stimulatory effect of lowering pH. In the colon, methazolamide reduced Na(+) and Cl(-) absorption at pH 7.6. Total HCO(3)(-) generation was minimal in HEPES at pH 7.6 and 7.1 in both segments, was minimally affected by methazolamide, and did not account for the changes in Cl(-) absorption caused by pH or methazolamide. We conclude that CA plays a role in ileal and colonic NaCl absorption independent of its catalytic function.

Acid-base effects on intestinal Na(+) absorption and vesicular trafficking.

We examined for vesicular trafficking of the Na(+)/H(+) exchanger (NHE) in pH-stimulated ileal and CO(2)-stimulated colonic Na(+) absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at x27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na(+) absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na(+) absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a PCO(2) value of 70 than 21 mmHg, was internalized when PCO(2) was reduced, and was exocytosed when PCO(2) was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na(+) absorption but is important in CO(2)-stimulated colonic Na(+) absorption.

Carbon dioxide affects rat colonic Na+ absorption by modulating vesicular traffic.

BACKGROUND & AIMS: We examined whether CO2 affects colonic Na+ absorption by endosome recycling of the Na+/H+ exchanger NHE3. METHODS: Rat distal colon segments exposed to various acid-base conditions were examined by transmission electron microscopy at 27,500x magnification and subapical vesicles quantified. Immunocytochemistry was used to identify vesicular NHE3. Endocytosis was tested for by observing internalization of apical membrane labeled with fluorescein isothiocyanate-phytohemagglutinin and Cy-3-NHE3 antibody using confocal microscopy. The effects of mucosal 5-(N,N-dimethyl)-amiloride (DMA), which inhibits NHE2 and/or NHE3, and wortmannin, which inhibits phosphatidylinositol 3-kinase, on CO2-stimulated Na+ absorption were measured in the Ussing chamber. RESULTS: The number of (coated and uncoated) subapical vesicles in epithelial cells was specifically and inversely related to net colonic Na+ absorption and PCO2. Immunoperoxidase labeling localized NHE3 on microvilli and vesicle membranes. Under the confocal microscope, a fluorescent band along apical membranes at PCO2 70 mm Hg became a subapical haze at PCO2 21 mm Hg. This pattern was not affected by carbonic anhydrase inhibition or when pH or [HCO3-] was changed, but PCO2 was held constant. DMA inhibition indicated that NHE3 mediates CO2-stimulated Na+ absorption. Wortmannin inhibited CO2-stimulated vesicle movement (exocytosis) and Na+ absorption. CONCLUSIONS: CO2 affects Na+ absorption in rat distal colon epithelium in part by modulating the movement of NHE3-containing vesicles to and from the apical membrane.