de Jong Joke C, Willems Peter H G M, van den Heuvel Lambertus P W J, Knoers Nine V A M, Bindels René J M
Department of Physiology, University Medical Centre Nijmegen, Nijmegen, The Netherlands.
J Am Soc Nephrol. 2003 Oct;14(10):2428-35. doi: 10.1097/01.asn.0000089832.52063.f5.
The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na(+) and Cl(-). To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na(+) uptake and transepithelial transport activity. Na(+) uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phospho-inositide 3-kinases or protein kinase C. Aldosterone did not alter Na(+) uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.
噻嗪类敏感的Na(+)-Cl(-)共转运蛋白（NCC）表达于远曲小管上皮细胞的顶端膜，负责重吸收5%至10%的滤过Na(+)和Cl(-)。迄今为止，由于缺乏表达这种共转运蛋白的合适细胞系统，关于NCC定位运输和离子转运活性的结构及调控要求的功能研究受到阻碍。本文报道了人NCC（hNCC）在源自肾脏的极化哺乳动物细胞——即高电阻的Madin-Darby犬肾（MDCK）细胞中的功能表达。蛋白质印迹检测显示，这些细胞主要表达复杂糖基化（约140 kD）形式的hNCC。hNCC主要存在于细胞的顶端部分。噻嗪类敏感的Na(+)摄取增加和跨上皮运输活性证明了hNCC的功能。用福司可林短期（15分钟）处理后，Na(+)摄取显著增加，而环磷酸鸟苷、渥曼青霉素、佛波酯12-肉豆蔻酸酯13-乙酸酯和星形孢菌素均无作用。这表明hNCC活性是通过环磷酸腺苷调节的，而不是通过环磷酸鸟苷、磷酸肌醇3激酶或蛋白激酶C。醛固酮在短期内（15分钟）不会改变Na(+)摄取，但在长期（16小时）会显著增加运输活性。醛固酮的后一种作用是由于其对驱动hNCC表达的巨细胞病毒启动子/增强子的影响。hNCC-MDCK细胞是研究hNCC顶端运输和离子转运活性调节的良好模型。
