Tickle P, Thistlethwaite A, Smith C P, Stewart G S
Faculty of Life Sciences, The University of Manchester, Manchester, UK.
Am J Physiol Regul Integr Comp Physiol. 2009 Aug;297(2):R323-9. doi: 10.1152/ajpregu.00199.2009. Epub 2009 May 27.
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.
我们之前的研究详细介绍了一种新型的易化性UT-B尿素转运体亚型，即bUT-B2。尽管存在小鼠和人类的直系同源物，但UT-B2的功能特性仍不明确。在本报告中，我们构建了一个稳定表达bUT-B2蛋白的MDCK细胞系，并研究了跨培养细胞单层的跨上皮尿素通量。我们观察到一个较大的基础尿素通量，已知的易化性尿素转运体抑制剂可使其显著降低；1,3-二甲基脲（P < 0.001，n = 17）、硫代烟酰胺（P < 0.05，n = 11）和根皮素（P < 0.05，n = 9）。预先暴露于抗利尿激素血管加压素1小时对bUT-B2介导的尿素转运没有影响（无显著性差异，n = 3）。急性暴露于血管加压素长达30分钟也未能引发任何瞬时反应（无显著性差异，n = 9）。进一步研究证实，bUT-B2的功能不受细胞内cAMP（无显著性差异，n = 4）、细胞内钙（无显著性差异，n = 3）或蛋白激酶活性（无显著性差异，n = 4）改变的影响。最后，免疫印迹数据表明糖基化在调节bUT-B2功能中可能起作用。总之，本研究表明bUT-B2介导的跨上皮尿素转运是组成性激活的，且不受肾脏UT-A尿素转运体已知调节因子的影响。
