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Nasal nitric oxide concentrations are extremely low in primary ciliary dyskinesia (PCD), and measurement of this nasal gas is recommended as a PCD diagnostic test in cooperative patients aged 5 years and older. However, nasal nitric oxide measurements must be performed with chemiluminescence analyzers using a standardized protocol to ensure proper results, because nasal nitric oxide values can be influenced by various internal and external factors. Repeat nasal nitric oxide testing on separate visits is required to ensure that low diagnostic values are persistent and consistent with PCD. This technical paper presents the standard operating procedures for nasal nitric oxide measurement used by the PCD Foundation Clinical and Research Centers Network at various specialty centers across North America. Adherence to this document ensures reliable nasal nitric oxide testing and high diagnostic accuracy when employed in a population with appropriate clinical phenotypes for PCD.

Significance Although resistance to glucocorticoids is a major clinical problem, the underlying mechanisms are unknown. It is known that glucocorticoid use can suppress adrenal androgen production. In population studies, animal models, and cell culture experiments, androgens are associated with several benefits in asthma, but neither androgen use in glucocorticoid-resistant asthma nor the genetic determinants of androgen responsiveness have been studied in humans. A missense-encoding variant in HSD3B1 is known to regulate conversion from adrenal precursors to potent androgens and clinical outcomes in prostate cancer. This is the first genetic evidence to our knowledge that implicates an androgen synthesis variant in resistance to glucocorticoids for asthma or any other inflammatory disease. Furthermore, this study demonstrates an adverse consequence of adrenal androgen suppression with glucocorticoid therapy., Asthma resistance to glucocorticoid treatment is a major health problem with unclear etiology. Glucocorticoids inhibit adrenal androgen production. However, androgens have potential benefits in asthma. HSD3B1 encodes for 3β-hydroxysteroid dehydrogenase-1 (3β-HSD1), which catalyzes peripheral conversion from adrenal dehydroepiandrosterone (DHEA) to potent androgens and has a germline missense-encoding polymorphism. The adrenal restrictive HSD3B1(1245A) allele limits conversion, whereas the adrenal permissive HSD3B1(1245C) allele increases DHEA metabolism to potent androgens. In the Severe Asthma Research Program (SARP) III cohort, we determined the association between DHEA-sulfate and percentage predicted forced expiratory volume in 1 s (FEV1PP). HSD3B1(1245) genotypes were assessed, and association between adrenal restrictive and adrenal permissive alleles and FEV1PP in patients with (GC) and without (noGC) daily oral glucocorticoid treatment was determined (n = 318). Validation was performed in a second cohort (SARP I&II; n = 184). DHEA-sulfate is associated with FEV1PP and is suppressed with GC treatment. GC patients homozygous for the adrenal restrictive genotype have lower FEV1PP compared with noGC patients (54.3% vs. 75.1%; P < 0.001). In patients with the homozygous adrenal permissive genotype, there was no FEV1PP difference in GC vs. noGC patients (73.4% vs. 78.9%; P = 0.39). Results were independently confirmed: FEV1PP for homozygous adrenal restrictive genotype in GC vs. noGC is 49.8 vs. 63.4 (P < 0.001), and for homozygous adrenal permissive genotype, it is 66.7 vs. 67.7 (P = 0.92). The adrenal restrictive HSD3B1(1245) genotype is associated with GC resistance. This effect appears to be driven by GC suppression of 3β-HSD1 substrate. Our results suggest opportunities for prediction of GC resistance and pharmacologic intervention.

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

Introduction Among individuals with severe asthma, FEV1 is low in individuals with low dehydroepiandrosterone (DHEA) sulfate (DHEAS) levels. In the Severe Asthma Research Program (SARP), no women with DHEAS > 200 μg/dL had an FEV1 1 in asthmatic women with low DHEAS. Methods Premenopausal, nonsmoking, otherwise healthy women, 18-50 years old, with mild or moderate asthma and baseline FEV1 > 60% predicted received 100 mg DHEA orally every 12 h for 2 weeks. Spirometry and DHEAS were measured at the initial visit and 2 weeks later, after completion of DHEA treatment. Based on our previous work, the primary outcome variable for this pilot study was post-albuterol spirometry in the low-DHEAS group. Subjects also continued their other routine asthma management. Results Serum DHEAS increased with DHEA treatment in women with starting DHEAS n = 10; p = 0.0001). The increase in the high-DHEAS group was smaller. Post-albuterol FEV1 increased by 51 mL, from 3.026 ± 0.5 to 3.077 ± 0.49 L (n = 10; p = 0.034 by paired t test, significant after Bonferroni), in women with low DHEAS. In the high-DHEAS group (baseline DHEAS ≥ 200 µg/dl), post-albuterol FEV1 did not change significantly (n = 3, p = NS). Three subjects were excluded: one had comorbid COPD, one could not perform spirometry, and one did not take the DHEA. There were no adverse effects of DHEA treatment in this trial. Conclusions Endocrine treatments (corticosteroids) are a mainstay of anti-inflammatory management for moderate and severe asthma. Their use has improved asthma outcomes. Androgens also reduce airway inflammation and promote airway smooth muscle relaxation, but are rarely used clinically for asthma treatment. Our results suggest that the over-the-counter steroid DHEA may improve lung function in asthma outcomes among women with DHEAS

This study investigates whether androgen receptor deficiency is associated with increased asthma risk.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase–independent (sGC-independent) effects are stereoselective — that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) — and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays — followed by unbiased proteomic analysis — to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO–Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance., Two complementary affinity chromatography assays, followed by unbiased proteomic analysis, identified voltage-gated K+ channel (Kv) proteins as binding partners for S-nitroso-l-cysteine (L-CSNO).

Context: Exogenous nitrogen oxides must be made bioavailable to sustain normal physiology because nitric oxide synthase (NOS) deficient mice are viable. In the stomach, S-nitrosoglutathione (GSNO) is formed from ingested nitrite and high levels of airway glutathione (GSH) that are cleared and swallowed. However, gastric GSNO may be broken down by nutrients like ascorbic acid (AA) before it is absorbed. Objective: To study the effect of AA on GSNO formation and stability. Materials and methods: GSH and nitrite were reacted with or without 5 mM AA or Resiston (5 mM AA with retinoic acid and α-tocopherol). GSNO was measured by reduction/chemiluminescence and HPLC. AA and reduced thiols were measured colorimetrically. O-Nitrosoascorbate and AA were measured by gas chromatography–mass spectrometry (GC–MS). Results: GSNO was formed in saline and gastric samples (pH ∼4.5) from physiological levels of GSH and nitrite. Neither AA nor Resiston decreased [GSNO] at pH >3; rather, they increased [GSNO] (0.12 ± 0.19 μM without AA; 0.42 ± 0.35 μM with AA; and 0.43 ± 0.23 μM with Resiston; n = 4 each; p ≤ 0.05). However, AA compounds decreased [GSNO] at lower pH and with incubation >1 h. Mechanistically, AA, but not dehydroascorbate, increased GSNO formation; and the O-nitrosoascorbate intermediate was formed. Conclusions: AA, with or without other antioxidants, did not deplete GSNO formed from physiological levels of GSH and nitrite at pH >3. In fact, it favoured GSNO formation, likely through O-nitrosoascorbate. Gastric GSNO could be a NOS-independent source of bioavailable nitrogen oxides.

The mechanisms by which transepithelial pressure changes observed during exercise and airway clearance can benefit lung health are challenging to study. Here, we have studied 117 mature, fully ciliated airway epithelial cell filters grown at air-liquid interface grown from 10 cystic fibrosis (CF) and 19 control subjects. These were exposed to cyclic increases in apical air pressure of 15 cmH2O for varying times. We measured the effect on proteins relevant to lung health, with a focus on the CF transmembrane regulator (CFTR). Immunoflourescence and immunoblot data were concordant in demonstrating that air pressure increased F508Del CFTR expression and maturation. This effect was in part dependent on the presence of cilia, on Ca2+ influx, and on formation of nitrogen oxides. These data provide a mechanosensory mechanism by which changes in luminal air pressure, like those observed during exercise and airway clearance, can affect epithelial protein expression and benefit patients with diseases of the airways.

OBJECTIVES Oxygen tension affects the biology of aerobic and denitrifying organisms. Using a novel, fast-response sensor, we developed a noninvasive procedure to measure pO2 in distal human airways. We hypothesized that distal pO2 would be low in cystic fibrosis (CF) airways. MATERIALS AND METHODS We measured the fraction of expired oxygen (FEO2 ) in real time using a fast laser diode analyzer in healthy subjects and in patients with CF, asthma, and primary ciliary dyskinesia (PCD). Subjects slowly exhaled to residual volume (RV), where the nadir of FEO2 (NFO) was recorded. Values were compared to peripheral oxygen saturation (Sa O2 ), expired CO2 at RV, FEV1 , FEV1 /FVC, and FEF25-75 . We also measured the effect of supplemental oxygen on FEO2 . RESULTS Seventy-four subjects completed the study. Seven additional subjects could not perform the maneuver. Mean (±SD) NFO values for controls (n = 29), CF patients (n = 23), asthma patients (n = 15), and PCD patients (n = 7) were 13.4 ± 1.1%, 12.4 ± 1.2%, 13.3 ± 1.1%, 14.4 ± 0.6%, respectively. NFO in CF was lower than in controls (P = 0.0162), and NFO in PCD was higher than in CF (P = 0.0007). Asthma results were heterogeneous. Oxygen caused a dose-dependent increase in NFO (P

The versatile chemistry of nitrogen is important to pulmonary physiology. Indeed, almost all redox forms of nitrogen are relevant to pulmonary physiology and to pathophysiology. Here we review the relevance to pulmonary biology of (a) elemental nitrogen; (b) reduced forms of nitrogen such as amines, ammonia, and hydroxylamine; and (c) oxidized forms of nitrogen such as the nitroxyl anion, the nitric oxide free radical, and S-nitrosothiols. Our focus is on oxidized nitrogen in the form of S-nitrosothiol bond–containing species, which are now appreciated to be important to every type of cell-signaling process in the lung. We also review potential clinical applications of nitrogen oxide biochemistry. These principles are being translated into clinical practice as diagnostic techniques and therapies for a range of pulmonary diseases including asthma, cystic fibrosis, adult respiratory distress syndrome, primary ciliary dyskinesia, and pulmonary hypertension.

Nasal nitric oxide (nNO) measurement is a diagnostic test for primary ciliary dyskinesia (PCD). Here, we have shown the development of unilateral PCD-like symptoms associated with low nNO. A 60-year-old man had been previously healthy but developed unilateral, severe pansinusitis. He required surgical drainage of all left sinuses, and biopsies showed loss of the ciliated epithelium. At 4 weeks, he had unilateral (left-sided), profuse, clear rhinorrhea characteristic of PCD, and his surgical ostia were all patent endoscopically. His left-sided nNO was less than the right side by 37 ± 1.2 nL/min; this difference decreased to 18 ± 0.87 nL/min at 5 weeks and was gone by 6 weeks when his symptoms resolved. Measurements of 2- and 10-second measurements, in addition to standard nNO measurements, identified this discordance. We conclude that nNO reflects, in part, the production of NO by the ciliated epithelium, not just in the absence or occlusion of sinuses. Early (nasal/sinus volume) measures may be better for diagnosing PCD in than standard, steady-state assays in certain populations.

S-Nitrosoglutathione is an endogenous airway smooth muscle relaxant. Increased airway S-nitrosoglutathione breakdown occurs in some asthma patients. We asked whether patients with increased airway catabolism of this molecule had clinical features that distinguished them from other asthma patients.We measured S-nitrosoglutathione reductase expression and activity in bronchoscopy samples taken from 66 subjects in the Severe Asthma Research Program. We also analysed phenotype and genotype data taken from the program as a whole.Airway S-nitrosoglutathione reductase activity was increased in asthma patients (p=0.032). However, only a subpopulation was affected and this subpopulation was not defined by a “severe asthma” diagnosis. Subjects with increased activity were younger, had higher IgE and an earlier onset of symptoms. Consistent with a link between S-nitrosoglutathione biochemistry and atopy: 1) interleukin 13 increased S-nitrosoglutathione reductase expression and 2) subjects with an S-nitrosoglutathione reductase single nucleotide polymorphism previously associated with asthma had higher IgE than those without this single nucleotide polymorphism. Expression was higher in airway epithelium than in smooth muscle and was increased in regions of the asthmatic lung with decreased airflow.An early-onset, allergic phenotype characterises the asthma population with increased S-nitrosoglutathione reductase activity.

Background S-Nitrosothiols are made by nitric oxide synthases and other metalloproteins. Unlike nitric oxide, S-nitrosothiols are involved in localized, covalent signaling reactions in specific cellular compartments. These reactions are enzymatically regulated. Scope S-Nitrosylation affects interactions involved in virtually every aspect of normal cell biology. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. Major Conclusions and Significance S-Nitrosylation is a regulated signaling reaction.

S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

In this issue of the Journal, Grasemann and coworkers (pp. 1363–1368) report that levels of asymmetric dimethyl arginine (ADMA) are increased in cystic fibrosis (CF) airways (1). ADMA inhibits cellular arginine uptake and nitric oxide (NO) synthase (NOS) activity. Levels of ADMA decrease during antibiotic therapy in association with improved lung function. This observation may prove to have therapeutic relevance. However, it is important to note that increased ADMA in CF airways may be both beneficial (through inhibition of NO production) and harmful (through inhibition of S-nitrosothiol production). Nitric oxide in the concentrations measured in exhaled air (parts per billion) is generally irrelevant to normal lung physiology (2). However, NO can be relevant to lung pathology. It interacts with oxygen, superoxide, and other molecules to injure airway epithelium. Products of these reactions, such as nitrous acid and peroxynitrous acid, are potent cytotoxins downstream of inducible NOS activity (3). Nitric oxide can also affect bacteria in the CF airway (4, 5). Indeed, the CF airway is a complex ecosystem in which nitrogen oxides, oxygen, protons, and more complex chemical species are exchanged between prokaryotic and eukaryotic cells (Figure 1; 4–7). For example, levels of NO are lower than normal in the CF airway, in part because NO is consumed by Pseudomonas, Aspergillus, and other denitrifying organisms (4). NO can serve both as an electron acceptor (dissimilatory denitrification, ultimately forming ammonia) and as a precursor for amino acid formation (assimilatory denitrification). Airway NO levels rise and NH3 levels fall with antibiotic therapy in CF (4). Levels of oxidized NO in the form of nitrate are high in the CF airway (6); and nitrate, like NO, can feed denitrifying organisms. Together, the effects of NOS products to cause cytotoxicity and promote prokaryotic growth suggest that the high levels of ADMA should be advantageous for patients with CF, and that a decrease in ADMA levels with antibiotic therapy might be disadvantageous. Figure 1. Cystic fibrosis airway nitrogen redox ecology. Nitric oxide (NO) synthase (NOS) isoforms in the cystic fibrosis (CF) lung produce both NO and S-nitrosothiols. NO is oxidized to nitrite (NO2−) and nitrate (NO3−). In this issue of the Journal ... However, NOS also produces S-nitrosothiols in concentrations two log orders higher than NO (8–10). S-nitrosothiols are antimicrobial. They augment ciliary beat frequency (2). They relax human airway smooth muscle and prevent tachyphylaxis to β2-adrenergic agonists (2, 10, 11). They inhibit amiloride-sensitive sodium transport (12). They augment expression, maturation and function of delF508 CF transmembrane conductance regulator through inhibition of the expression of Hsp70/Hsp90-organizing protein (13). S-nitrosothiol levels are decreased in the CF airway (14), consistent with high ADMA levels (1); indeed, S-nitrosothiol replacement therapy improves oxygenation in CF (9). Prevention of S-nitrosothiol formation is therefore likely to be an important disadvantage of having high levels of ADMA in the CF airway (1). We do not know why ADMA levels are high in CF. One possible mechanism is based on what we know about the CF airway ecosystem. Anti-pseudomonal therapy decreases ADMA levels in patients with CF (1). Biochemically, ADMA levels are decreased by dimethylarginine dimethylaminohydrolases (DDAHs). DDAHs are inhibited by S-nitrosylation, or physiological protein modification by NO (15). S-nitrosylation is driven both by NOS activation and—in relatively acidic conditions in the CF airway ecosystem (5)—by nitrite protonation; at baseline, this should increase ADMA levels. Antimicrobial therapy can decrease bacterial conversion of abundant airway nitrate to nitrite. Nitrite depletion would decrease DDAH S-nitrosylation, thereby increasing ADMA breakdown during the course of therapy. However, this proposed mechanism is speculative. Much work remains to be done. The CF airway is dark, damp, and largely anaerobic (7). It is also surprisingly well-suited to denitrifying prokaryotic species, given that NOS expression is decreased and ADMA levels are increased. To understand why—and to learn how to use nitrogen oxide redox ecology to therapeutic advantage—we need to get beyond simply modeling NO radical diffusion (2, 3). The Cystic Fibrosis Foundation has shown the benefit of promoting interactions among scientists across disciplines. The work of Grasemann and coworkers suggests that there might be much to gain by organizing a small working group to bring together nitrogen balance ecologists, biochemists, CF airway epithelial biologists, and mathematicians experienced in biochemical modeling. This group could model prokaryotic and eukaryotic outcomes of specific CF interventions to identify strategies to optimize therapeutic development in CF. The elegant insights of Grasemann and coworkers serve to alert us that both the benefits and toxicities of airway nitrogen oxides need to be better understood to improve clinical outcomes in CF.

See related article, pages 545–553 Erythrocytes dilate peripheral blood vessels as a function of oxyhemoglobin desaturation.1 This effect increases regional blood flow to hypoxic tissues. The mechanisms underlying the peripheral vasodilatory effects of desaturating erythrocytes are incompletely understood but do not involve activation of local, endothelial NO synthase (eNOS). Indeed, eNOS-derived NO itself primarily relaxes large vessels and does that primarily only in the absence of blood. In this issue of Circulation Research , Diesen et al confirm that thiols carrying a nitrosonium (NO+) equivalent signal cyclic GMP-dependent vascular smooth muscle relaxation during erythrocytic oxyhemoglobin desaturation.2 These data support paradigm-changing work demonstrating that nitrogen oxides are transported by circulating erythrocytes to signal oxyhemoglobin desaturation through serial NO/NO+ thiol equilibria and transfer reactions (transnitrosation) and that these reactions normally take place at sites remote from NOS activity.1–3 These new data show clearly that this signaling is independent of local NOS activity, of cyclooxygenase, of ATP, and of the effects of hypoxia itself on vascular smooth muscle. Erythrocytes are endogenously “preloaded” with nitrogen oxides for delivery to vessels in conditions of oxyhemoglobin desaturation.1–3 This signaling links delivery of erythrocytic NO/NO+ groups to oxyhemoglobin desaturation, permitting augmented blood flow to hypoxic tissue. Three mechanisms have been proposed by which transitions in hemoglobin (Hb) conformation may result in nitrogen oxide transfer to blood vessels. (1) In the originally proposed mechanism, Hb deoxygenation (R-to-T transition) permits transnitrosation from Hb β-chain cysteine 93 (βCys93) to erythrocytic carrier thiols (Figure).1–3 This concept is supported by the data obtained by Diesen et al.2 (2) At one time, NO radical was proposed to diffuse away from the Fe (II) heme iron in T state Hb. Although there was an initial enthusiasm for this construct as an NO radical–based …

BACKGROUND Children with severe asthma have poor symptom control and elevated markers of airway oxidative and nitrosative stress. Paradoxically, they have decreased airway levels of S-nitrosothiols (SNOs), a class of endogenous airway smooth muscle relaxants. This deficiency results from increased activity of an enzyme that both reduces SNOs to ammonia and oxidizes formaldehyde to formic acid, a volatile carboxylic acid that is more easily detected in exhaled breath condensate (EBC) than SNOs. We therefore hypothesize that depletion of airway SNOs is related to asthma pathology, and breath formate concentration may be a proxy measure of SNO catabolism. METHODS AND FINDINGS We collected EBC samples from children and adolescents, including 38 with severe asthma, 46 with mild-to-moderate asthma and 16 healthy adolescent controls, and the concentration of ionic constituents was quantified using ion chromatography. The concentrations of EBC components with volatile conjugates were log-normally distributed. Formate was the principal ion that displayed a significant difference between asthma status classifications. The mean EBC formate concentration was 40% higher in samples collected from all asthmatics than from healthy controls (mean = 5.7 microM, mean+/-standard deviation = 3.1-10.3 microM vs. 4.0, 2.8-5.8 microM, p = 0.05). EBC formate was higher in severe asthmatics than in mild-to-moderate asthmatics (6.8, 3.7-12.3 microM vs. 4.9, 2.8-8.7 microM, p = 0.012). In addition, formate concentration was negatively correlated with methacholine PC(20) (r = -0.39, p = 0.002, asthmatics only), and positively correlated with the NO-derived ion nitrite (r = 0.46, p

The endogenous signaling molecule S-nitrosoglutathione (GSNO) and other S-nitrosylating agents can cause full maturation of the abnormal gene product DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR). However, the molecular mechanism of action is not known. Here we show that Hsp70/Hsp90 organizing protein (Hop) is a critical target of GSNO, and its S-nitrosylation results in DeltaF508 CFTR maturation and cell surface expression. S-nitrosylation by GSNO inhibited the association of Hop with CFTR in the endoplasmic reticulum. This effect was necessary and sufficient to mediate GSNO-induced cell-surface expression of DeltaF508 CFTR. Hop knockdown using siRNA recapitulated the effect of GSNO on DeltaF508 CFTR maturation and expression. Moreover, GSNO acted additively with decreased temperature, which promoted mutant CFTR maturation through a Hop-independent mechanism. We conclude that GSNO corrects DeltaF508 CFTR trafficking by inhibiting Hop expression, and that combination therapies--using differing mechanisms of action--may have additive benefits in treating CF.

Background Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. Principal Findings Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. Significance These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.