Cigarette smoke extract is a Nox agonist and regulates ENaC in alveolar type 2 cells

Charles A Downs,Abdel A Alli,Nicholle M Johnson,My N Helms

doi:10.3934/molsci.2016.3.439

Charles A Downs, Abdel A Alli + Show 2 more

Open Access

https://doi.org/10.3934/molsci.2016.3.439

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Abstract

There is considerable evidence that cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD), and that oxidative stress occurs in COPD with the family of tissue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes playing a significant role in lung pathogenesis. The purpose of this study was to determine the effects of cigarette smoke extract (CSE) on Nox signaling to epithelial sodium channels (ENaCs). Pre-treatment with diphenyleneiodonium (DPI), a pan-Nox inhibitor, prevented stimulatory effects of CSE on ENaC activity; open probability (Po) changed from 0.36 ± 0.09 to 0.11 ± 0.02; n = 10, p = 0.01 following CSE and DPI exposure. Likewise, Fulvene-5 (which inhibits Nox2 and Nox4 isoforms) decreased the number of ENaC per patch (from 2.75 ± 0.25 to 1 ± 0.5, n = 9, p = 0.002) and open probability (0.18 ± 0.08 to 0.02 ± 0.08, p = 0.04). Cycloheximide chase assays show that CSE exposure prevented α-ENaC subunit degradation, whereas concurrent CSE exposure in the presence of Nox inhibitor, Fulvene 5, resulted in normal proteolytic degradation of α-ENaC protein in primary isolated lung cells. In vivo, co-instillation of CSE and Nox inhibitor promoted alveolar flooding in C57Bl6 mice compared to accelerated rates of fluid clearance observed in CSE alone instilled lungs. Real-time PCR indicates that mRNA levels of Nox2 were unaffected by CSE treatment while Nox4 transcript levels significantly increased 3.5 fold in response to CSE. Data indicate that CSE is an agonist of Nox4 enzymatic activity, and that CSE-mediated Nox4 plays an important role in altering lung ENaC activity.

Highlights

Epithelial sodium channels (ENaCs) are composed of three homologous subunits (α, β and γ) and are expressed in tight epithelia lining the luminal surface of the kidney, colon, and lung [1]
The transcript data suggest that Nox2 is more abundantly transcribed in alveolar T2 cells, whereas Nox4 is more robustly expressed in T1 cells
Cigarette smoke and cigarette smoking are the primary etiology of chronic obstructive pulmonary disease (COPD), the underlying molecular mechanisms that lead to disease are not completely understood

Summary

Introduction

Epithelial sodium channels (ENaCs) are composed of three homologous subunits (α, β and γ) and are expressed in tight epithelia lining the luminal surface of the kidney, colon, and lung [1]. In the airways and alveoli, ENaC plays a critically important role in regulating the volume of fluid lining lung epithelia. The importance of studying lung ENaC and pulmonary pathogenesis has been shown in α-ENaC knockout mice that die within 40 hours of birth due to an inability to clear lung fluid [2,3]. This animal model highlights the sodium channel’s critically important role in edematous lung injury. We have previously shown that a family of Nox enzymes plays an important role in regulating ENaC NPo [7,8], the effects of Nox enzymatic activity remain unclear

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