Odgaard Elvin, Jakobsen Jakob K, Frische Sebastian, Praetorius Jeppe, Nielsen Søren, Aalkjaer Christian, Leipziger Jens
Institute of Physiology, Univeristy of Aarhus, Denmark.
J Physiol. 2004 Feb 15;555(Pt 1):205-18. doi: 10.1113/jphysiol.2003.046474. Epub 2003 Dec 12.
The electroneutral Na(+)-dependent HCO3- transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up-regulated during NH4(+)-induced metabolic acidosis. Here we used in vitro perfusion and BCECF video-imaging of mTAL tubules to investigate functional localization and regulation of Na(+)-dependent HCO3- influx during NH4(+)-induced metabolic acidosis. Tubule acidification was induced by removing luminal Na+ (DeltapHi: 0.88 +/- 0.11 pH units, n = 10). Subsequently the basolateral perfusion solution was changed to CO2/HCO3- buffer with and without Na+. Basolateral Na(+)-H+ exchange function was inhibited with amiloride. Na(+)-dependent HCO3- influx was determined by calculating initial base flux of Na(+)-mediated re-alkalinization. In untreated animals base flux was 8.4 +/- 0.9 pmol min(-1) mm(-1). A 2.4-fold increase of base flux to 21.8 +/- 3.2 pmol min(-1) mm(-1) was measured in NH4(+)-treated animals (11 days, n = 11). Na(+)-dependent re-alkalinization was significantly larger when compared to control animals (0.38 +/- 0.03 versus 0.22 +/- 0.02 pH units, n = 10). In addition, Na(+)-dependent HCO3- influx was of similar magnitude in chloride-free medium and also up-regulated after NH4+ loading. Na(+)-dependent HCO3- influx was not inhibited by 400 microm DIDS. A strong up-regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT-PCR analysis revealed no evidence for the Na(+)-dependent HCO3- transporter NBC4 or the two Na(+)-dependent CI-/HCO3- exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS-insensitive NBCn1. Function and protein are strongly up-regulated during NH4(+)-induced metabolic acidosis. We suggest that NBCn1-mediated basolateral HCO3- influx is important for basolateral NH3 exit and thus NH4+ excretion by means of setting pHi to a more alkaline value.
电中性的Na(+)-依赖型HCO3-转运体NBCn1在大鼠髓袢升支粗段细胞（mTAL）的基底外侧膜中大量表达，并且在NH4(+)-诱导的代谢性酸中毒期间上调。在此，我们利用mTAL小管的体外灌注和BCECF视频成像技术，研究了NH4(+)-诱导的代谢性酸中毒期间Na(+)-依赖型HCO3-内流的功能定位和调节。通过去除管腔中的Na+诱导小管酸化（ΔpH i：0.88±0.11 pH单位，n = 10）。随后，将基底外侧灌注液更换为含或不含Na+的CO2/HCO3-缓冲液。用氨氯吡咪抑制基底外侧Na(+)-H+交换功能。通过计算Na(+)-介导的再碱化的初始碱通量来确定Na(+)-依赖型HCO3-内流。在未处理的动物中，碱通量为8.4±0.9 pmol min(-1) mm(-1)。在NH4(+)-处理的动物（11天，n = 11）中测得碱通量增加了2.4倍，达到21.8±3.2 pmol min(-1) mm(-1)。与对照动物相比，Na(+)-依赖型再碱化显著更大（0.38±0.03对0.22±0.02 pH单位，n = 10）。此外，在无氯培养基中，Na(+)-依赖型HCO3-内流的幅度相似，并且在NH4+负荷后也上调。400 μM DIDS不抑制Na(+)-依赖型HCO3-内流。免疫标记实验证实了NBCn1染色的强烈上调。RT-PCR分析未发现Na(+)-依赖型HCO3-转运体NBC4或两种Na(+)-依赖型Cl-/HCO3-交换体NCBE和NDCBE的证据。这些数据强烈表明，大鼠mTAL小管在功能上表达基底外侧对DIDS不敏感的NBCn1。在NH4(+)-诱导的代谢性酸中毒期间，功能和蛋白强烈上调。我们认为，NBCn1介导的基底外侧HCO3-内流对于基底外侧NH3的排出很重要，因此通过将pH i设置为更碱性的值来促进NH4+的排泄。
