Regulation of Alveolar Epithelial Na⁺ Channels by ERK1/2 in Chlorine-Breathing Mice

Abstract

The mechanisms by which the exposure of mice to Cl 2 decreases vectorial Na 1 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 1 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. a-ENaC, g-ENaC, total and phosphorylated extracellular signalrelated 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 1 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 a-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.

Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury

“…In both cases there is increased generation of reactive intermediates, which lasts after the cessation of exposure. For example, we have previously shown the presence of reactive intermediates in lung epithelial cells exposed to Cl 2 and returned to room air using electron spin resonance and redox sensitive dyes (42). Increased levels of reactive intermediates were also documented in the lungs of mice and rats exposed to Cl 2 and returned to room air by measuring levels of isoprostanes (75) and products of lipid peroxidation (77).…”

ENaCs and ASICs as therapeutic targets

“…The picture is further complicated by the presence of more than one type of amiloride-sensitive channel in alveolar epithelium. A recent study by Lazrak et al (239) showed that two amiloride-sensitive channels, with single-channel conductances of 4.5 pS and 18 pS, could be recorded from ATI And ATII cells in lung slices from airbreathing mice. The 4.5 pS channel was highly selective for sodium and most likely corresponds to ␣␤␥ENaC, while the larger channel was less sodium selective and its molecular identity is currently unknown.…”

Postexposure aerosolized heparin reduces lung injury in chlorine-exposed mice

“…Exposures during the accidental release of Cl 2 have been modeled based on the Graniteville accident and have shown that Cl 2 levels during a 30-min exposure period were 6,868, 837, and 89 ppm at 0.2, 0.5, and 1 km downwind from the epicenter of the accident (5). Previous studies from our group and those of others have shown that mice exposed to 400 ppm Cl 2 for 30 min and returned to room air develop acute hypoxia, lung inflammation accompanied by perturbation of the integrity of the alveolar epithelial barrier, and significant lung oxidative stress (30,33,48,57,58). Moreover, there were also alterations in respiratory mechanics (34,45), compromised immune response to fungi (16), and impaired locomotion (13).…”

“…Furthermore, there is a large body of experimental evidence indicating that thrombin increases lung endothelial permeability via a protease-activated receptor-dependent mechanism (28) and alveolar epithelial permeability via an ␣ v ␤ 6 integrin-and TGF-␤-dependent mechanism (25) and compromises vectorial sodium transport via activation of PKC (50). In addition, it may act synergistically with reactive intermediates that are known to be generated both during and after exposure to Cl 2 both in vitro and in vivo (30,57), to activate the small GTPase RhoA and suppress Rac1, which also contribute to increased alveolar permeability and pulmonary edema (3,15). Heparin may abate these processes by preventing the continued formation of thrombin or by acting as an anti-inflammatory, which prevents epithelial damage and exposure of pulmonary thrombin to the systemic coagulation system.…”

“…Airway (12,45) and distal lung (30,46,58) injury is caused by increased concentrations of oxygen-nitrogen and chlorine-reactive intermediates that persist even when animals are returned to room air. Influx of activated inflammatory cells (mainly neutrophils), which secrete myeloperoxidase and reactive intermediates, exacerbate lung injury.…”