Commensal bacteria that live in the colon express urease and use host-derived urea as a nitrogen source (Fuller & Reeds, 1998). We have previously reported the possible role in this host-microbial relationship of facilitative UT-A urea transporters in the mouse gastrointestinal tract, using an antibody raised against rat UT-A1 (Stewart et al. 2001). The aim of this study was to characterise two novel antibodies specifically raised against mouse UT-A proteins, and use them to further investigate the function of UT-A transporters in the mouse colon. Tissue was obtained from humanely killed male adult MF1 mice. Northern blot analysis revealed that four UT-A transcripts were present in mouse colon. These transcripts were different from the known renal mouse UT-A (mUT-A) isoforms, but had similar molecular weights to those in mouse testes. Two antisera, ML446 and ML194, targeted to the N- and C-termini of mUT-A1, respectively, were raised in rabbits. ML446 detected proteins at 34 and 48 kDa in the colon, as well as mUT-A1 (89 and 119 kDa) and mUT-A3 (48-53 kDa) in mouse kidney medulla. ML194 detected proteins at 48, 75 and 100 kDa in both testes and colon, in addition to mUT-A1 and mUT-A2 (43-55 kDa) in kidney medulla. Immunolocalisation in mouse colon using ML446 showed the presence of UT-A proteins in the cytoplasm of cells in the lower third of all colonic crypts. In contrast, ML194 specifically stained the plasma membranes of cells located in the lower portion of colonic crypts, in the proliferative and stem regions that extended to the beginning of the goblet cells. Refractive light flux experiments using colonic plasma membrane vesicles revealed a significant urea flux (n = 7, P < 0.01, ANOVA). This urea flux was completely inhibited by 500 µM phloretin (n = 5, P < 0.01, ANOVA), a known inhibitor of facilitative urea transporters.

Our results show that functional UT-A transporters are expressed in the plasma membranes of mouse colonic crypts and are thus ideally situated to transport urea into the colon.

This work was funded by the BBSRC and The Royal Society.

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