Regulation of the expression of soluble guanylyl cyclase by reactive oxygen species

Background and purpose: Superoxide anions produced during vascular disease scavenge nitric oxide (NO), thereby reducing its biological activity. The aim of the present study was to investigate whether reactive oxygen species (ROS) have a direct effect on soluble guanylyl cyclase (sGC) subunit levels and function and to ascertain the mechanism(s) involved. Experimental approach: Rat aortic smooth muscle cells (RASM) or freshly isolated vessels were exposed to reactive oxygen species (ROS)-generating agents and sGC subunit expression was determined at the mRNA and/or protein level. cGMP accumulation was also determined in RASM exposed to ROS. Key results: Incubation of smooth muscle cells with H 2O2, xanthine/xanthine oxidase (X/XO) or menadione sodium bisulphite (MSB) significantly decreased protein levels of α1 and β1 subunits of sGC and reduced SNP-induced cGMP formation. Similarly, sGC expression was reduced in freshly isolated vessels exposed to ROS-generating agents. The ROS-triggered inhibition of α1 and β1 levels was not blocked by proteasome inhibitors, suggesting that decreased sGC protein was not due to protein degradation through this pathway. Real time RT-PCR analysis demonstrated a 68% reduction in steady state mRNA levels for the α1 subunit following exposure to H2O2. In addition, α1 promoter-driven luciferase activity in RASM decreased by 60% after H 2O2 treatment. Conclusion and implications: We conclude that oxidative stress triggers a decrease in sGC expression and activity that results from reduced sGC steady state mRNA levels. Altered sGC expression is expected to contribute to the changes in vascular tone and remodeling observed in diseases associated with ROS overproduction. © 2007 Nature Publishing Group All rights reserved

Interleukin-18 in induced sputum: Association with lung function in chronic obstructive pulmonary disease

Background: It has been shown that interleukin (IL)-18 levels in induced sputum are reduced in asthmatic and healthy smokers. However, in chronic obstructive pulmonary disease (COPD) patients, recent data show an overproduction in the lungs and increased serum levels of IL-18, suggesting that IL-18 may be involved in the pathogenesis of COPD. Method: In order to assess the relation of IL-18 with pulmonary function and airway inflammation in COPD, IL-18, tumour necrosis factor-α, and IL-8 levels were measured by ELISA in sputum supernatants obtained from patients with bronchitis type COPD (n = 28), and healthy subjects (18 smokers and 17 non-smokers). Cellular localization of IL-18 was assessed by immunocytochemistry. Results: The levels of IL-18 were significantly higher in sputum supernatants of COPD patients compared to healthy smokers and non-smokers (p < 0.05). IL-18 production was localized to sputum macrophages. IL-18 levels were inversely correlated with FEV1 (% predicted) (r = -0.572, p = 0.002) and FEV1/FVC ratio in COPD smokers (r = -0.608, p = 0.001). No correlations were found between IL-18 levels and inflammatory markers studied in induced sputum obtained from COPD patients, healthy smokers and non-smokers. Conclusion: In patients with COPD, increased levels of IL-18 in induced sputum were associated with airflow limitation, suggesting that IL-18 may be implicated in the pathogenesis of COPD. © 2009

IL-18 in induced sputum and airway hyperresponsiveness in mild asthmatics: Effect of smoking

Interleukin 18 (IL-18) is a pro-inflammatory cytokine, which has been shown to be implicated in the induction of airway hyperresponsiveness (AHR) in murine asthma models. The association of IL-18 with AHR in human bronchial asthma is not clear as yet. As cigarette smoking modifies airway inflammation we aimed to assess the relationship of IL-18 with airway hyperresponsiveness (AHR) in non-smoking versus smoking asthmatics. IL-18 was measured in sputum supernatants obtained from asthmatic (24 smokers and 22 non-smokers) and healthy subjects (16 smokers and 17 non-smokers). All subjects were assessed by spirometry, skin-prick tests to common aeroallergens and bronchial provocation to methacholine (Mch). There was no significant difference in IL-18 levels between healthy and asthmatic smokers and between healthy and asthmatic non-smokers. IL-18 levels in sputum were significantly lower in healthy smokers compared to non-smokers (p = 0.048); similarly, in asthmatic smokers as compared to non-smokers (p = 0.037). An inverse correlation was found between IL-18 levels, FEV1 (% pred) (r = -0.495, p = 0.043), and PD20Mch in non-smoking asthmatics (r = -0.621, p = 0.024). A positive correlation was found in smoking asthmatics between IL-18 levels in sputum and FEV1 (% pred) (r = 0.627, p = 0.002), FVC (% pred) (r = 0.460, p = 0.031), and PD20Mch (r = 0.809, p = 0.005). Cigarette smoking reduced IL-18 levels in induced sputum in healthy and asthmatic smokers. IL-18 levels were correlated with airway obstruction and AHR in an inverse way in smoking and non-smoking asthmatics. These results suggest the implication of IL-18 in airway hyperresponsiveness characterizing bronchial asthma, which is modified by smoking. © 2009 Elsevier Ltd. All rights reserved

Interaction between the 90-kDa heat shock protein and soluble guanylyl cyclase: Physiological significance and mapping of the domains mediating binding

The 90-kDa heat shock protein (hsp90) regulates the stability and function of many client proteins, including members of the NO-cGMP signaling pathway. Soluble guanylyl cyclase (sGC), an NO receptor, was recently reported to be an hsp90-interacting partner. In the present study, we show that hsp90 binds to both subunits of the most common sGC form (alpha(1)beta(1)) when these are expressed individually but only interacts with beta(1) in the heterodimeric form of the enzyme. Characterization of the region of hsp90 required to bind each subunit in immunoprecipitation experiments revealed that residues 310 to 456 of hsp90 interact with the sGC subunits. The region of beta(1) responsible for binding to hsp90 beta was mapped using in vitro binding assays and immunoprecipitation experiments and was found to lie in the regulatory domain. The physiological importance of the hsp90/sGC interaction was investigated by treating rat smooth muscle cells with the hsp90 inhibitors radicicol and geldanamycin (GA) and determining both sGC activity and protein levels. Long-term ( 24 or 48 h) inhibition of hsp90 resulted in a strong decrease of both alpha(1) and beta(1) protein levels and sGC activity. Moreover, incubation of smooth muscle cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked the GA-induced down-regulation of sGC. We conclude that the N-terminal region of the beta(1) subunit mediates binding of the heterodimeric form of sGC to hsp90 and that this interaction involves the M domain of hsp90. Hsp90 binding to sGC regulates the pool of active enzymes by affecting the protein levels of the two subunits

Regulation of the expression of soluble guanylyl cyclase by reactive oxygen species

Background and purpose: Superoxide anions produced during vascular disease scavenge nitric oxide (NO), thereby reducing its biological activity. The aim of the present study was to investigate whether reactive oxygen species (ROS) have a direct effect on soluble guanylyl cyclase (sGC) subunit levels and function and to ascertain the mechanism(s) involved. Experimental approach: Rat aortic smooth muscle cells (RASM) or freshly isolated vessels were exposed to reactive oxygen species (ROS)-generating agents and sGC subunit expression was determined at the mRNA and/or protein level. cGMP accumulation was also determined in RASM exposed to ROS. Key results: Incubation of smooth muscle cells with H2O2, xanthine/xanthine oxidase (X/XO) or menadione sodium bisulphite (MSB) significantly decreased protein levels of α1 and β1 subunits of sGC and reduced SNP-induced cGMP formation. Similarly, sGC expression was reduced in freshly isolated vessels exposed to ROS-generating agents. The ROS-triggered inhibition of α1 and β1 levels was not blocked by proteasome inhibitors, suggesting that decreased sGC protein was not due to protein degradation through this pathway. Real time RT-PCR analysis demonstrated a 68% reduction in steady state mRNA levels for the α1 subunit following exposure to H2O2. In addition, α1 promoter-driven luciferase activity in RASM decreased by 60% after H2O2 treatment. Conclusion and implications: We conclude that oxidative stress triggers a decrease in sGC expression and activity that results from reduced sGC steady state mRNA levels. Altered sGC expression is expected to contribute to the changes in vascular tone and remodeling observed in diseases associated with ROS overproduction

Functional Analysis of the SIM1 Variant p.G715V in 2 Patients With Obesity

CONTEXT: Single-minded homologue 1 (SIM1) is a transcription factor with several physiological and developmental functions. Haploinsufficiency of SIM1 is associated with early-onset obesity with or without Prader-Willi-like (PWL) features and may exhibit incomplete penetrance. CASE DESCRIPTION: Next-generation sequencing was performed for 2 male patients with obesity, including 1 man presenting with intellectual disability (ID), body mass index (BMI) of 47.4, and impulse-control disorder, and the other man with early obesity (BMI of 36); sequencing revealed a missense variant in SIM1 (c.2144G>T; p.G715V) in both individuals. Previous studies have identified several disease-associated variants that fall near the p.G715V variant within the C-terminal domain of SIM1. We examined p.G715V variant stability and activity in a doxycycline-inducible stable cell line transfected with an artificial reporter construct and either ARNT or ARNT2 as a partner protein. CONCLUSIONS: Functional testing of the p.G715V variant revealed a significant reduction in SIM1-mediated transcriptional activity. We also generated the first ab initio hybrid protein model for full-length SIM1 to show the predicted spatial relationship between p.G715V and other previously described variants in this region and identified a putative mutation hotspot within the C-terminus. Significant clinical heterogeneity has been observed in patients with SIM1 variants, particularly with regards to the PWL phenotype. In the patient with ID, a second variant of uncertain significance in CHD2 was identified that may contribute to his ID and behavioral disturbances, emphasizing the role of additional genetic modifiers

Soluble guanylate cyclase stimulation reduces oxidative stress in experimental Chronic Obstructive Pulmonary Disease

OBJECTIVE: Soluble guanylate cyclase (sGC) is a key enzyme of the nitric oxide-cyclic guanosine 3',5'-monophosphate (NO-cGMP) signaling pathway, and its pharmacological stimulation has been shown to prevent the development of emphysema and pulmonary vascular remodeling in animal models of chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate the effects of sGC stimulation on oxidative stress in the plasma of guinea pigs chronically exposed to cigarette smoke (CS). METHODS AND RESULTS: Guinea pigs were exposed to CS or sham for three months, and received either the sGC stimulator BAY 41-2272 or vehicle. Body weight was measured weekly; and markers of oxidative stress in plasma, and airspace size and inflammatory cell infiltrate in lung tissue were analyzed at the end of the study. Compared to sham-exposed guinea pigs, CS-exposed animals gained less body weight and showed higher plasma levels of nitrated tyrosine residues (3-NT), 4-hydroxynonenal (4-HNE), and 8-hydroxydeoxyguanosine (8-OHdG). Treatment with the sGC stimulator led to a body weight gain in the CS-exposed guinea pigs similar to non-exposed and attenuated the increase in 3-NT and 4-HNE. Plasma levels of 3-NT correlated with the severity of inflammatory cell infiltrate in the lung. CONCLUSION: Stimulation of sGC prevents oxidative stress induced by CS exposure and is associated with an attenuated inflammatory response in the lung