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Regulation of alveolar epithelial Na+ channels by ERK1/2 in chlorine-breathing mice
Ahmed Lazrak1, Lan Chen, Asta Jurkuvenaite
1Department of Anesthesiology, School of Medicine, University of Alabama at Birmingham, BMR II 224, 901 19th St. South, Birmingham, AL 35205-3703, USA.
Abstract:
The mechanisms by which the exposure of mice to Cl(2) decreases vectorial Na(+) transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na(+) channels (ENaCs) after exposure to Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na(+) channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl(2), the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.
Insights
Chlorine gas (Cl(2)) exposure impairs lung epithelial sodium channel (ENaC) function by increasing reactive intermediates and activating ERK1/2 signaling. This reduces sodium transport and fluid clearance in the distal lung spaces.
Area of Science:
- Pulmonary Physiology
- Toxicology
- Molecular Biology
Background:
- Chlorine gas (Cl(2)) exposure is known to impair lung function, specifically reducing vectorial sodium (Na(+)) transport and fluid clearance in distal lung spaces.
- The precise mechanisms underlying Cl(2)-induced ENaC dysfunction remain poorly understood.
Purpose of the Study:
- To elucidate the biophysical, biochemical, and physiological mechanisms by which Cl(2) exposure alters rodent lung epithelial sodium channels (ENaCs).
- To identify the signaling pathways involved in Cl(2)-induced ENaC dysfunction.
Main Methods:
- Measurement of amiloride-sensitive short-circuit currents (I(amil)) across alveolar Type II (ATII) cell monolayers.
- Assessment of ENaC single-channel properties in ATII and ATI cells using patch-clamp techniques.
- Western blot analysis for α-ENaC, γ-ENaC, ERK1/2 phosphorylation, and lipid peroxidation products.
- Quantification of reactive intermediates using electron spin resonance (ESR).
Main Results:
- Cl(2) exposure decreased the open probability of amiloride-sensitive Na(+) channels in lung epithelial cells.
- Increased reactive intermediates and ERK1/2 phosphorylation were observed after Cl(2) exposure.
- Cl(2) exposure led to decreased I(amil) and α-ENaC concentrations.
- Antioxidant administration mitigated Cl(2)-induced reactive intermediate formation, ERK1/2 activation, and ENaC dysfunction.
- Inhibition of ERK1/2, proteasomes, or lysosomes prevented the Cl(2)-induced decrease in channel activity.
Conclusions:
- Reactive intermediates generated by Cl(2) exposure activate the ERK1/2 pathway in lung epithelial cells.
- Activated ERK1/2 leads to decreased ENaC concentrations and activity, impairing Na(+) transport and fluid clearance.
- Targeting oxidative stress and ERK1/2 signaling may offer therapeutic strategies for Cl(2)-induced lung injury.
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