No 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.

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique "pulsing" nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na(+)-K(+)-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.