Abstract
We investigated the mechanisms by which chlorine (Cl(2)) and its reactive byproducts inhibit Na(+)-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na(+) channels (ENaC) by measuring AFC in mice exposed to Cl(2) (0-500 ppm for 30 min) and Na(+) and amiloride-sensitive currents (I(Na) and I(amil), respectively) across Xenopus oocytes expressing human alpha-, beta-, and gamma-ENaC incubated with HOCl (1-2000 microm). Both Cl(2) and HOCl-derived products decreased AFC in mice and whole cell and single channel I(Na) in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I(Na) in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I(Na) by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the gamma-ENaC subunit restored the ability of HOCl to inhibit I(Na). Finally, I(Na) of oocytes expressing wild type alpha- and gamma-ENaC and a mutant form of beta ENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.
Highlights
HL-31197, HL-51173, U01ES015676, and 1U54ES017218. 1 Both authors contributed to this work. 2 Present address: Dept. of Cardiology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China. 3 Present address: Dept. of Gastroenterology, The First People’s Hospital of Guangzhou, Guangzhou 510180, China. 4 To whom correspondence should be addressed: Dept. of Anesthesiology, selective amiloride-sensitive channels (ENaC)5 located at the apical membranes, their extrusion across the basolateral membranes by the electrogenic Naϩ-Kϩ-ATPase, and the passive movement of Kϩ ions through basolateral Kϩ channels
Because our results indicated that exposure to Cl2 decreased alveolar fluid clearance (AFC), we performed additional studies in Xenopus oocytes injected with cRNAs of the human epithelial Na؉ channels (ENaC) subunits (␣, , and ␥-ENaC) to identify the cellular and molecular mechanisms by which hypochlorous acid (HOCl) as well as reactive intermediates formed by the reaction of HOCl with components of the media decreased Naϩ- and amiloride-sensitive currents (INa and Iamil, respectively)
We investigate whether extracellular serine proteases reversed the inhibitory effects of HOCl and its reactive products formed by the interactions with components of ND96
Summary
HL-31197, HL-51173, U01ES015676, and 1U54ES017218. 1 Both authors contributed to this work. 2 Present address: Dept. of Cardiology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China. 3 Present address: Dept. of Gastroenterology, The First People’s Hospital of Guangzhou, Guangzhou 510180, China. 4 To whom correspondence should be addressed: Dept. of Anesthesiology, selective amiloride-sensitive channels (ENaC) located at the apical membranes, their extrusion across the basolateral membranes by the electrogenic Naϩ-Kϩ-ATPase, and the passive movement of Kϩ ions through basolateral Kϩ channels. Injury to either apical or basolateral pathways by partially reduced intermediates may lead to impairment of fluid reabsorption, which in turn may result in pulmonary edema, hypoxemia, and eventually death from respiratory failure (6 –9) One such specie is hypochlorous acid (HOCl), which may be generated either endogenously or exogenously. The effects of Cl2 inhalation on Naϩ-dependent alveolar fluid clearance (AFC) in vivo as well as the mechanisms by which Cl2 and HOCl damage ENaC have not been investigated This is an important area of research as damage to ENaC has been associated with abnormal fluid transport in various forms of lung injury [7, 21,22,23,24,25]. We concluded that HOCl inhibits ENaC by altering channel gating and preventing closed channels from opening
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