Nitric Oxide Inhibits Highly Selective Sodium Channels and the Na+/K+-ATPase in H441 Cells

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

Cigarette Smoke-Induced Emphysema and Pulmonary Hypertension Can Be Prevented by Phosphodiesterase 4 and 5 Inhibition in Mice

<div><p>Rationale</p><p>Chronic obstructive pulmonary disease (COPD) is a widespread disease, with no curative therapies available. Recent findings suggest a key role of NO and sGC-cGMP signaling for the pathogenesis of the disease. Previous data suggest a downregulation/inactivation of the cGMP producing soluble guanylate cyclase, and sGC stimulation prevented cigarette smoke-induced emphysema and pulmonary hypertension (PH) in mice. We thus aimed to investigate if the inhibition of the cGMP degrading phosphodiesterase (PDE)5 has similar effects. Results were compared to the effects of a PDE 4 inhibitor (cAMP elevating) and a combination of both.</p><p>Methods</p><p>C57BL6/J mice were chronically exposed to cigarette smoke and in parallel either treated with Tadalafil (PDE5 inhibitor), Piclamilast (PDE4 inhibitor) or both. Functional measurements (lung compliance, hemodynamics) and structural investigations (alveolar and vascular morphometry) as well as the heart ratio were determined after 6 months of tobacco smoke exposure. In addition, the number of alveolar macrophages in the respective lungs was counted.</p><p>Results</p><p>Preventive treatment with Tadalafil, Piclamilast or a combination of both almost completely prevented the development of emphysema, the increase in lung compliance, tidal volume, structural remodeling of the lung vasculature, right ventricular systolic pressure, and right ventricular hypertrophy induced by cigarette smoke exposure. Single, but not combination treatment prevented or reduced smoke-induced increase in alveolar macrophages.</p><p>Conclusion</p><p>Cigarette smoke-induced emphysema and PH could be prevented by inhibition of the phosphodiesterases 4 and 5 in mice.</p></div

Effect of Piclamilast and/or Tadalafil on cigarette smoke-induced lung emphysema development assessed by structural parameters.

<p>Mice were exposed to cigarette smoke for 6 months and treated in parallel with Piclamilast (10 mg/kg body weight/day) and/or Tadalafil (10 mg/kg body weight/day). Time matched controls received solvent only. <b>(A)</b> Air space, <b>(B)</b> septal wall thickness and <b>(C)</b> mean linear intercept. <b>(D)</b> Representative histology from mice lung sections stained with hematoxylin and eosin. Data are given as mean ± SEM from n = 5–6, each. Bars indicate significant differences using ANOVA followed by Student-Newman-Keuls post hoc test (*<i>P</i><0.05;** <i>P</i><0.01; ***<i>P</i><0.001).</p

Systemic effects of smoke exposure and treatment with Piclamilast and/or Tadalafil in mice.

<p>Mice were exposed to cigarette smoke for 6 months and treated in parallel with Piclamilast (10 mg/kg body weight/day) and/or Tadalafil (10 mg/kg body weight/day). Time matched controls were received solvent only. Systemic arterial pressure determined by <i>A</i>. <i>carotis</i> catheterization and quantified in anesthetized animals. Data are given as mean ± SEM from n = 5–8, each. Bars indicate significant differences using ANOVA followed by Student-Newman-Keuls post hoc test (**<i>P</i><0.01; ***<i>P</i><0.001).</p

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette smokeinduced pulmonary emphysema in mice and is upregulated in patients with COPD

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein Sestrin 2 (Sesn2) as a repressor of PDGFRβ signalling and PDGFRβ signalling as an upstream regulator of alveolar maintenance programs. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2-). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of patients with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2/PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD

Effect of Piclamilast and/or Tadalafil on cigarette smoke-induced pulmonary hypertension in mice assessed by functional parameters.

<p>Mice were exposed to cigarette smoke for 6 months and treated in parallel with Piclamilast (10 mg/kg body weight/day) and/or Tadalafil (10 mg/kg body weight/day). Time matched controls received solvent only. <b>(A)</b> Right ventricular systolic pressure quantified by right heart catheterization in anesthetized animals. <b>(B)</b> Right heart hypertrophy, given as the ratio of right ventricular (RV) mass to left ventricular plus septum (LV+S) mass, from dried heart tissue; left ventricular weights did not differ between the different groups. Data are given as mean ± SEM from n = 5–8, each. Bars indicate significant differences using ANOVA followed by Student-Newman-Keuls post hoc test (**<i>P</i><0.01; ***<i>P</i><0.001).</p

Effect of Piclamilast and/or Tadalafil on cigarette smoke-induced pulmonary vascular remodeling in mice—histology.

<p>Representative histology from lung sections stained with antibodies against α-smooth muscle actin and von Willebrand factor.</p

Effect of Piclamilast and/or Tadalafil on cigarette smoke-induced lung emphysema development assessed by <i>in vivo</i> lung functional parameters.

<p>Mice were exposed to cigarette smoke for 6 months and treated in parallel with Piclamilast (10 mg/kg body weight/day) and/or Tadalafil (10 mg/kg body weight/day). Time matched controls received solvent only. <b>(A)</b> dynamic lung compliance, <b>(B)</b> tidal volume and <b>(C)</b> airway resistance. Data are given as mean ± SEM from n = 6–9, each. Bars indicate significant differences using ANOVA followed by Student-Newman-Keuls post hoc test (* <i>P</i><0.05**; <i>P</i><0.01; ***<i>P</i><0.001).</p

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD

SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD