Acute Effects of Aerosolized S-Nitrosoglutathione in Cystic Fibrosis

This is the publisher's version, also available electronically from http://www.atsjournals.org/doi/abs/10.1164/ajrccm.165.7.2105032#.VJCPDXs4d41.S-Nitrosoglutathione (GSNO), a naturally occurring constituent of airway lining fluid, enhances ciliary motility, relaxes airway smooth muscle, inhibits airway epithelial amiloride-sensitive sodium transport, and prevents pathogen replication. Remarkably, airway levels of GSNO are low in patients with cystic fibrosis (CF). We hypothesized that replacement of airway GSNO would improve gas exchange in CF. In a double-blind, placebo controlled study, we administered 0.05 ml/kg of 10 mM GSNO or phosphate buffered saline by aerosol to patients with CF and followed oxygen saturation, spirometry, respiratory rate, blood pressure, heart rate, and expired nitric oxide (NO). Nine patients received GSNO and 11 placebo. GSNO inhalation was associated with a modest but sustained increase in oxygen saturation at all time points. Expired NO increased in the low ppb range with GSNO treatment, peaking at 5 minutes but remaining above baseline at 30 minutes. There were no adverse effects. We conclude that GSNO is well tolerated in patients with CF and improves oxygenation through a mechanism that may be independent of free NO. Further, GSNO breakdown increases expired NO. We suggest that therapy aimed at restoring endogenous GSNO levels in the CF airway may merit study

Nitric Oxide, NOC-12, and S -Nitrosoglutathione Modulate the Skeletal Muscle Calcium Release Channel/Ryanodine Receptor by Different Mechanisms: AN ALLOSTERIC FUNCTION FOR O2INS-NITROSYLATION OF THE CHANNEL

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) contains approximately 50 thiols per subunit. These thiols have been grouped according to their reactivity/responsiveness toward NO, O(2), and glutathione, but the molecular mechanism enabling redox active molecules to modulate channel activity is poorly understood. In the case of NO, very low concentrations (submicromolar) activate RyR1 by S-nitrosylation of a single cysteine residue (Cys-3635), which resides within a calmodulin binding domain. S-Nitrosylation of Cys-3635 only takes place at physiological tissue O(2) tension (pO(2); i.e. approximately 10 mm Hg) but not at pO(2) approximately 150 mm Hg. Two explanations have been offered for the loss of RyR1 responsiveness to NO at ambient pO(2), i.e. Cys-3635 is oxidized by O(2) versus O(2) subserves an allosteric function (Eu, J. P., Sun, J. H., Xu, L., Stamler, J. S., and Meissner, G. (2000) Cell 102, 499-509). Here we report that the NO donors NOC-12 and S-nitrosoglutathione both activate RyR1 by release of NO but do so independently of pO(2). Moreover, NOC-12 activates the channel by S-nitrosylation of Cys-3635 and thereby reverses channel inhibition by calmodulin. In contrast, S-nitrosoglutathione activates RyR1 by oxidation and S-nitrosylation of thiols other than Cys-3635 (and calmodulin is not involved). Our results suggest that the effect of pO(2) on RyR1 S-nitrosylation is exerted through an allosteric mechanism

Classes of Thiols That Influence the Activity of the Skeletal Muscle Calcium Release Channel

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O(2) (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of approximately 10 RyR1 thiols (from approximately 48 to approximately 38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to approximately 23/subunit, whereas more extensive oxidation (to approximately 13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions

S-Nitrosylation of Surfactant Protein-D Controls Inflammatory Function

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional “bunch of flowers” arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity

Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface

Regulation of the Cardiac Muscle Ryanodine Receptor by O 2 Tension and S -Nitrosoglutathione †

The cardiac and skeletal muscle sarcoplasmic reticulum ryanodine receptor Ca2+ release channels contain thiols that are potential targets of endogenously produced reactive oxygen and nitrogen intermediates. Previously, we showed that the skeletal muscle ryanodine receptor (RyR1) has O2-sensitive thiols; only when these thiols are in the reduced state (pO2 ~ 10 mmHg) can physiological concentrations of NO (nanomolar) activate RyR1. Here, we report that cardiac muscle ryanodine receptor (RyR2) activity also depends on pO2, but unlike RyR1, RyR2 was not activated or S-nitrosylated directly by NO. Rather, activation and S-nitrosylation of RyR2 required S-nitrosoglutathione. The effects of peroxynitrite were indiscriminate on RyR1 and RyR2. Our results indicate that both RyR1 and RyR2 are pO2-responsive yet point to different mechanisms by which NO and S-nitrosoglutathione influence cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ release

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function

Home-based isometric exercise training induced reductions resting blood pressure

Purpose: Isometric exercise training (IET) reduces resting blood pressure (BP). Most previous protocols impose exercise barriers which undermine its effectiveness as a potential physical therapy for altering BP. An inexpensive, home-based programme would promote IET as a valuable tool in the fight against hypertension. The aims of this study were: (a) to investigate whether home-based wall squat training could successfully reduce resting BP, and (b) to explore the physiological variables that might mediate a change in resting BP. Methods: Twenty-eight healthy normotensive males were randomly assigned to a control and a 4 week home-based IET intervention using a crossover design with a 4 week ‘washout’ period in-between. Wall squat training was completed 3x weekly over 4 weeks with 48 hours between sessions. Each session comprised 4x 2 minute bouts of wall squat exercise performed at a participant-specific knee joint angle relative to a target HR of 95% HRpeak, with 2 minutes rest between bouts. Resting heart rate, BP, cardiac output, total peripheral resistance and stroke volume were taken at baseline and post each condition. Results: Resting BP (systolic = -4 ± 5, diastolic = -3 ± 3 and mean arterial = -3 ± 3 mmHg), cardiac output (-0.54 ± 0.66 L∙min-1) and heart rate (-5 ± 7 beats∙min-1) were all reduced following IET, with no change in total peripheral resistance or stroke volume compared to the control. Conclusion: These findings suggest the wall squat provides an effective method for reducing resting BP in the home resulting primarily from a reduction in resting heart rate

Fiber Type-Specific Nitric Oxide Protects Oxidative Myofibers against Cachectic Stimuli

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia