Hypertension and Vascular Research Div., Dept. of Internal Medicine, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA.
Am J Physiol Renal Physiol. 2010 Nov;299(5):F1193-202. doi: 10.1152/ajprenal.00307.2010. Epub 2010 Aug 18.
The Na-K-2Cl cotransporter (NKCC2) mediates NaCl absorption by the thick ascending limb of Henle's loop (THAL). Exocytosis and endocytosis regulates surface expression of most transporters. However, little is known about the mechanism of NKCC2 trafficking in the absence of stimulating hormones and whether this mechanism contributes to regulation of steady-state surface expression of apical NKCC2 in the THAL. We tested whether NKCC2 undergoes constitutive endocytosis that regulates steady-state surface NKCC2 and NaCl reabsorption in THALs. We measured steady-state surface NKCC2 levels and the rate of NKCC2 endocytosis by surface biotinylation and Western blot and confocal microscopy of isolated perfused rat THALs. We observed constitutive NKCC2 endocytosis over 30 min that averaged 21.5 ± 2.7% of the surface pool. We then tested whether methyl-β-cyclodextrin (MβCD), a compound that inhibits endocytosis by chelating membrane cholesterol, blocked NKCC2 endocytic retrieval. We found that 30-min treatment with MβCD (5 mM) blocked NKCC2 endocytosis by 81% (P < 0.01). Blockade of endocytosis by MβCD induced accumulation of NKCC2 at the apical membrane as demonstrated by a 60 ± 16% (P < 0.05) increase in steady-state surface expression and enhanced apical surface NKCC2 immunostaining in isolated, perfused THALs. Acute treatment with MβCD did not change the total pool of NKCC2. MβCD did not affect NKCC2 trafficking when it was complexed with cholesterol before treatment. Inhibition endocytosis with MβCD enhanced NKCC2-dependent NaCl entry by 57 ± 16% (P < 0.05). Finally, we observed that a fraction of retrieved NKCC2 recycles back to the plasma membrane (36 ± 7%) over 30 min. We concluded that constitutive NKCC2 trafficking maintains steady-state surface NKCC2 and regulates NaCl reabsorption in THALs. These are the first data showing an increase in apical membrane NKCC2 in THALs by altering the rates of constitutive NKCC2 trafficking, rather than by stimulation of hormone-dependent signaling.
钠钾 2 氯协同转运蛋白（NKCC2）介导 Henle 袢升支粗段（THAL）对 NaCl 的吸收。胞吐和胞吞作用调节大多数转运蛋白的表面表达。然而，在没有刺激激素的情况下，NKCC2 转运的机制知之甚少，以及这种机制是否有助于调节 THAL 中顶端 NKCC2 的稳态表面表达。我们测试了 NKCC2 是否经历组成性胞吞作用，从而调节 THAL 中稳态表面 NKCC2 和 NaCl 重吸收。我们通过表面生物素化和 Western blot 以及分离灌注大鼠 THAL 的共聚焦显微镜测量稳态表面 NKCC2 水平和 NKCC2 胞吞作用的速率。我们观察到 30 分钟内 NKCC2 的组成性胞吞作用，平均占表面池的 21.5±2.7%。然后，我们测试了甲基-β-环糊精（MβCD）是否阻断 NKCC2 内吞作用，MβCD 是一种通过螯合膜胆固醇抑制内吞作用的化合物。我们发现，30 分钟的 MβCD（5 mM）处理可阻断 NKCC2 内吞作用 81%（P<0.01）。MβCD 阻断内吞作用导致 NKCC2 在顶端膜上积聚，如在分离灌注的 THAL 中，稳态表面表达增加 60±16%（P<0.05）和增强顶端表面 NKCC2 免疫染色所示。急性 MβCD 处理不会改变 NKCC2 的总池。当 MβCD 在处理前与胆固醇形成复合物时，它不会影响 NKCC2 转运。用 MβCD 抑制内吞作用可使 NKCC2 依赖性 NaCl 内流增加 57±16%（P<0.05）。最后，我们观察到回收的 NKCC2 中有一部分在 30 分钟内重新循环到质膜（36±7%）。我们得出结论，组成性 NKCC2 转运维持 THAL 中的稳态表面 NKCC2，并调节 NaCl 重吸收。这些是通过改变组成性 NKCC2 转运速率而不是通过刺激激素依赖性信号来增加 THAL 中顶端膜 NKCC2 的首批数据。
