Novel bUT-B2 urea transporter isoform is constitutively activated

Abstract

Our previous studies have detailed a novel facilitative UT-B urea transporter isoform, bUT-B2. Despite the existence of mouse and human orthologs, the functional characteristics of UT-B2 remain undefined. In this report, we produced a stable MDCK cell line that expressed bUT-B2 protein and investigated the transepithelial urea flux across cultured cell monolayers. We observed a large basal urea flux that was significantly reduced by known inhibitors of facilitative urea transporters; 1,3 dimethylurea (P < 0.001, n = 17), thionicotinamide (P < 0.05, n = 11), and phloretin (P < 0.05, n = 9). Pre-exposure for 1 h to the antidiuretic hormone vasopressin had no effect on bUT-B2-mediated urea transport (NS, n = 3). Acute vasopressin exposure for up to 30 min also failed to elicit any transient response (NS, n = 9). Further investigation confirmed that bUT-B2 function was not affected by alteration of intracellular cAMP (NS, n = 4), intracellular calcium (NS, n = 3), or protein kinase activity (NS, n = 4). Finally, immunoblot data suggested a possible role for glycosylation in regulating bUT-B2 function. In conclusion, this study showed that bUT-B2-mediated transepithelial urea transport was constitutively activated and unaffected by known regulators of renal UT-A urea transporters.

UT-B Urea Transporter Localization in the Bovine Gastrointestinal Tract

“…This result is similar to a previous study for the MCT1 short-chain fatty acid transporter, whose abundance did not differ between dorsal and ventral sacs in developing calves (Flaga et al 2015). Since we have previously reported that UT-B2 transporters are constitutively activated (Tickle et al 2009), this strongly suggests that overall there will be no difference in UT-B mediated trans-epithelial transport in different ruminal sacs at the cellular level. However, in contrast to this, physical measurements showed that there were significant differences in the size of papillae from the three ruminal sacs, with those from the dorsal sac being considerably smaller (Table 1).…”

“…d In contrast, preincubation of hUTBc19 with the same concentration of a nonspecific peptide (1 lg/ml) had minimal effect on the strong stratum basale staining. Scale bar represents 100 lm protein was deglycosylated to a core protein of *40 kDa, which is similar to the *36 kDa protein previously reported for bovine UT-B2 using a different antibody (Simmons et al 2009;Tickle et al 2009). The weak, unglycosylated signals obtained in the cytoplasm-enriched protein sample are most likely non-specific signals, similar to those previously observed with the hUTBc19 antibody (Walpole et al 2014).…”

“…The predominantly basolateral localization found for UT-B in the bovine abomasum, colon and rectum is similar to that reported for UT-B1 transporters in the human colon (Collins et al 2010). At present, in terms of mammalian gastrointestinal UT-B transporters, it appears that while ruminal UT-B2 transporters are targeted to both apical and basolateral membranes (Stewart et al 2005;Simmons et al 2009;Tickle et al 2009), UT-B1 (or ''UT-B1-like'') transporters in all other tissues are only present at the basolateral membrane. Further studies are now urgently Fig.…”

Triazolothienopyrimidine Inhibitors of Urea Transporter UT-B Reduce Urine Concentration

“…UT-A1 and UT-A3 are cAMP-regulated urea transporters expressed in kidney tubule epithelia. [23][24][25] MDCK cells were cultured on collagen-coated porous filters for measurement of urea accumulation in the apical solution (basolateral to apical transport) after addition of urea to the basolateral solution. The top panel in Figure 5A show increased urea permeability in each of the transfected cell lines.…”

Differential protein abundance and function of UT-B urea transporters in human colon

“…In other mammals, particularly ruminants, there is gathering evidence that these transporters allow the passage of blood urea into the lumen of the gastrointestinal tract (20,22,31,33). They therefore play a vital role in the urea nitrogen salvaging (UNS) process in which urea supplied by the host is broken down by bacterial urease to release nitrogen for bacterial growth.…”