Na⁺/HCO₃^- cotransporter (NBC) is located in the basolateral membrane of the gastrointestinal epithelium, where it serves the import of HCO₃^- during secretagogue-stimulated anion secretion. While the role of electroneutral NBC (NBCn1) in the intestine is still unclear, the relevance of electrogenic NBC (NBCe1-B) is supported by a HCO₃^- secretory defect in the colon of NBCe1 deficient mice.

After having demonstrated secretagogue activation of NBC in murine colonic crypts, we now asked whether vesicle traffic and exocytosis as increasingly recognized mechanisms for transporter regulation are involved in this process. To this end, crypts were isolated from the colon of C57BL/6 mice, NBCn1 and NBCe1 subtype expression was investigated with RT-PCR and QRT-PCR, NBCe1-B membrane expression was studied by immunohistochemistry and confocal microscopy, and NBCe1-B membrane abundance was quantified using cell surface protein biotinylation and Western blot. PCR revealed NBCe1-B expression in colonic crypts and, to a lesser extent, in the kidney, while NBCe1-A was exclusively found in the kidney. Furthermore, NBCe1-B was expressed at significantly higher levels than NBCn1 in colonic crypts as determined by QRT-PCR. In isolated colonic crypts pre-treated with forskolin (10^-5 M) and carbachol (10^-4 M) for 10 minutes, respectively, the NBCe1-B signal showed a markedly stronger merge with the E-cadherin signal which was used as a membrane marker, as compared with the untreated control. In the biotinylation experiments, a time-dependent increase in biotinylated NBCe1-B was observed, which occured slower with a peak of +54.8% after 20 minutes with forskolin, and more rapidly with a peak of +59.8% after 10 minutes with carbachol. Pre-incubation with the actin polymerization inhibitor cytochalasin D did not change the observed increase, while the microtubule polymerization inhibitor colchicine alone enhanced NBCe1-B membrane expression, possibly via inhibition of vesicle endocytosis, without an additional increase after carbachol or forskolin.

Taken together, our results demonstrate a secretagogue-induced increase of NBCe1-B membrane expression, which parallels the previously reported NBC activity increase in murine colonic crypts. Vesicle traffic and exocytosis might thus represent a novel mechanism of secretagogue-dependent activation of intestinal NBC.