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 P-CO2. Immunoperoxidase labeling localized NHE3 on microvilli and vesicle membranes. Under the confocal microscope, a fluorescent band along apical membranes at P-CO2 70 mm Hg became a subapical haze at P-CO2 21 mm Hg. This pattern was not affected by carbonic anhydrase inhibition or when pH or [HCO3-] was changed, but P-CO2 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.