Nitric Oxide Inhibits Highly Selective Sodium Channels and the Na+/K+-ATPase in H441 Cells
Nitric Oxide Inhibits Highly Selective Sodium Channels and the Na+/K+-ATPase in H441 Cells
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DOI：
10.1165/2009-0335oc
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发表时间：
2011-01-01
影响因子：
6.4
通讯作者：
Morty, Rory E.
中科院分区：
文献类型：
--
作者：
Althaus, Mike;Pichl, Alexandra;Morty, Rory E.
一氧化氮（NO）是肺上皮细胞对Na+重吸收的重要调节剂，因此也是肺泡液体清除的重要调节剂。NO影响上皮细胞离子转运的机制知之甚少，并且因模型而异。在这项研究中，NO对钠重吸收的H441细胞单层的影响进行了研究在一个Ussing室。两种NO供体（Z）-1-[N-（3-氨基丙基）-N-（正丙基）氨基]二氮烯-1-鎓-1，2-二醇盐和二乙基铵（Z）-1-（N，N-二乙基氨基）二氮烯-1-鎓-1，2-二醇盐）可快速、可逆和剂量依赖性地降低阿米洛利敏感的H441细胞单层短路电流。这种作用被NO清除剂血红蛋白中和，并且在不活跃的NO供体中没有观察到。NO的作用不被8-溴鸟苷-3'，5'-环磷酸或可溶性鸟苷酸环化酶抑制剂（亚甲蓝和1H-[1，2，4]恶二唑并[4，3-a]喹喔啉-1-酮）阻断，因此不依赖于可溶性鸟苷酸环化酶信号传导。NO靶向顶端，高选择性，阿米洛利敏感的Na+通道在基底侧透性H441细胞单层。NO对爪蟾卵母细胞中异源表达的人上皮钠通道的活性没有影响。NO降低H 441细胞Na +/K +-ATP酶活性。NO对Na +/K +-ATP酶活性的抑制作用可被汞逆转，并被N-乙基马来酰亚胺模拟，它们分别是逆转和模拟NO与巯基反应的试剂。与这些数据相一致，S-NO组检测到的Na +/K +-ATP酶a亚基响应NO供体的应用，使用生物素开关的方法耦合到蛋白质印迹。这些数据表明，在H441细胞模型中，NO通过干扰高选择性Na+通道和Na +/K +-ATP酶的活性来损害Na+重吸收。
Nitric oxide (NO) is an important regulator of Na+ reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl) amino]diazen-1-ium-1,2-diolate and diethylammonium(Z)-1-(N, N-diethylamino) diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na+ channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na+/K+-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na+/K+-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na+/K+-ATPase a subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na+ reabsorption by interfering with the activity of highly selective Na+ channels and the Na+/K+-ATPase.