1 Université Paris 13, Sorbonne Paris Cité, Laboratoire Réponses Cellulaires et Fonctionnelles à l'Hypoxie (EA 2363), Bobigny, France.
Am J Respir Cell Mol Biol. 2014 Mar;50(3):526-37. doi: 10.1165/rcmb.2012-0518OC.
Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the β-Liddle mouse strain mice carrying a truncation of β-ENaC C-terminus abolishing the interaction between β-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated β-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.
通过肺泡上皮钠离子通道 (ENaC) 和 Na(+)，K(+)-ATP 酶进行跨上皮钠离子转运构成了清除肺泡水肿液的驱动力。与肺水肿相关的肺泡缺氧可能会通过降低肺泡上皮细胞 (AEC) 顶膜上 ENaC 亚基的表达来损害 ENaC 活性和肺泡钠离子吸收。在这里，我们在体内携带β-ENaC C 末端截断的β-Liddle 小鼠品系中研究了这一过程涉及的机制，该截断会破坏β-ENaC 与泛素蛋白连接酶 Nedd4-2 之间的相互作用，而 Nedd4-2 会将通道靶向内吞和降解；并在体外的大鼠 AEC 中进行了研究。缺氧（24 小时 8% O2）使野生型小鼠的氨苯蝶啶敏感的肺泡液体清除率降低了 69%，但在纯合突变的β-Liddle 同窝仔鼠中没有影响。在体外，急性暴露于缺氧（0.5-3% O2 持续 1-6 小时）可迅速降低 AEC 中的跨上皮钠离子转运，如等效短路电流 Ieq 和穿过顶膜的钠离子电流的氨苯蝶啶敏感成分所示，反映了 ENaC 活性。缺氧诱导 AEC 顶膜中 ENaC 亚基表达减少，而细胞内表达不变，并诱导α-ENaC 多泛素化增加 2 倍。用蛋白酶体抑制剂 MG132 和乳酰半胱氨酸或抗氧化剂 N-乙酰半胱氨酸预先孵育可完全防止缺氧抑制氨苯蝶啶敏感的 Ieq。我们的数据强烈表明，Nedd4-2 介导的 ENaC 泛素化导致顶端 Na(+) 通道的内吞和降解是缺氧抑制跨上皮肺泡钠离子转运的关键特征。
