Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-ß1 in alveolar epithelial cells.

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²âº chelation and protein kinase C (PKCα-ß1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²âº-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²âº/PKCα-ß1 pathway independent of ATP signaling.

Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma.

BACKGROUND: Exposure to chlorine (Cl2) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl2-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury. METHODS: Balb/C mice were exposed to Cl2 gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl2, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl2 exposure. RESULTS: Mice exposed to Cl2 had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl2 exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl2-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl2 prevented lipid peroxidation in the lung. Following Cl2 exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl2 exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU. CONCLUSION: Our data show that the anti-oxidant DMTU is effective in attenuating Cl2 induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.

Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle.

Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (nu(max)) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of nu(max) versus [MgADP] was significantly greater for tonic (-0.51+/-0.04) than phasic muscle myosin (-0.15+/-0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092+/-0.022 pN for phasic muscle and 0.084+/-0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.

Nitration of respiratory epithelial cells by myeloperoxidase depends on extracellular nitrite.

To investigate peroxidase induced 3'-nitrotyrosine (3NT) formation, neutrophil derived myeloperoxidase (MPO) (0.025 microM) was directly administered to A549 epithelial cells with or without H(2)O(2) (150 microM). Little evidence of 3NT was found. In contrast, there was a dose dependent increase in intracellular NO (p<0.001, n=8) following MPO (0.025 microM) treatment, which was further enhanced (p<0.0003, n=8) by addition of H(2)O(2). Extracellular NO also increased after MPO (p<0.002, n=5) and with MPO and H(2)O(2) (p<0.004, n=5). Substantial 3NT formation was only detected following addition of nitrite (NO(2)(-), > or =100 microM), which induced a dose dependent increase in epithelial 3NT. In contrast, protein carbonyl formation and increased GSSG/GSH ratios were associated with MPO treatment even in the absence of NO(2)(-). Co-culture of A549 epithelial cells with polymorphonuclear leukocytes (PMN) (10(6)/ml) led to immunocytochemical detection of epithelial 3NT and induction of nitric oxide synthase (NOS2). However, in a Transwell system direct contact between PMN and A549 cells was necessary for immunodetection of 3NT but not of NOS2 consistent with a role for high local nitrite concentrations. These findings demonstrate dissociation between epithelial endogenous NO production and 3NT formation. Although MPO can influence cellular oxidative stress, particularly in the presence of H(2)O(2), 3NT formation requires the presence of high concentrations of NO(2)(-) in the milieu.

Nitric oxide production in the ventilatory muscles in response to acute resistive loading.

The effect of muscle activation on muscle nitric oxide (NO) production remains controversial. Whereas NO release increases in in vitro activated muscles and in vivo limb muscles, diaphragmatic NO synthase (NOS) activity declines after 3 h of inspiratory resistive loading (IRL). We tested in this study the hypotheses that acute IRL decreases diaphragmatic NO derivatives levels and reduces protein expression of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases, as well as 3-nitrotyrosine formation. Anesthetized, tracheostomized, spontaneously breathing adult rats were subjected to IRL (50% of the maximum inspiratory pressure) for 1, 3, or 6 h. Quietly breathing rats served as controls. After 3 h of IRL, muscle eNOS and nNOS protein levels rose by 80 and 60% of control values, respectively. Whereas eNOS expression did not change any further, nNOS expression reached 550% of control values after 6 h of IRL. Strong iNOS protein expression was detected in the diaphragms after 6 h of IRL. Total NO derivatives levels in the diaphragm declined during IRL as a result of reduction in nitrate, nitrite, and nitrosothiols. Diaphragmatic protein tyrosine nitration decreased in response to IRL, and this reduction was mainly due to reduced tyrosine nitration of enolase and aldolase. We conclude that diaphragmatic NO derivatives levels decline in response to IRL and that the rise in diaphragmatic NOS protein expression may be a compensatory response designed to counterbalance the decline in NOS activity.

Airway inflammation assessed by invasive and noninvasive means in severe asthma: eosinophilic and noneosinophilic phenotypes.

BACKGROUND: Airway inflammation assessed by bronchial biopsies demonstrates distinct eosinophilic and noneosinophilic phenotypes in severe asthma, but their relationship to other biomarkers of disease (induced sputum and nitric oxide [NO]) is not clear. OBJECTIVES: We sought to compare airway inflammation using noninvasive (induced sputum, exhaled NO), and invasive (bronchial biopsies) methods in moderate and severe asthma and to assess whether induced sputum and exhaled NO would allow the identification of eosinophilic and noneosinophilic phenotypes in severe asthma. METHODS: We performed a cross-sectional study of 32 subjects with severe asthma and 35 subjects with moderate asthma, from whom we obtained bronchial biopsies, induced sputum, and exhaled NO measurements. RESULTS: Among subjects with severe asthma, we identified eosinophilic and noneosinophilic phenotypes using both bronchial biopsies and sputum cell counts. However, the vast majority of subjects with high sputum eosinophil counts did not have high mucosal eosinophil counts. Exhaled NO was increased in the eosinophilic phenotype as judged from bronchial biopsy findings, but not on the basis of induced sputum. Subjects with high sputum eosinophil counts experienced more asthma exacerbations than the subjects with low sputum eosinophil counts. In contrast, we did not find any differences in the clinical characteristics between eosinophilic and noneosinophilic phenotypes that were identified by bronchial biopsies. CONCLUSION: The use of sputum cell counts allowed the identification of a subgroup of subjects with severe asthma who were at risk of more frequent asthma exacerbations. CLINICAL IMPLICATIONS: Monitoring sputum eosinophil counts in subjects with severe asthma may allow identifying the subjects with the greatest disease activity.

Sarcolemmal damage in dystrophin deficiency is modulated by synergistic interactions between mechanical and oxidative/nitrosative stresses.

Dystrophin deficiency is the cause of Duchenne muscular dystrophy, but the precise physiological basis for muscle necrosis remains unclear. To determine whether dystrophin-deficient muscles are abnormally susceptible to oxidative and nitric oxide (NO)-driven tissue stress, a hindlimb ischemia/reperfusion (I/R) model was used. Dystrophic mdx mice exhibited abnormally high levels of lipid peroxidation and protein nitration, which were preceded by exaggerated NO production during ischemia. Visualization of NO with the fluorescent probe 4,5-diaminofluorescein diacetate suggested that excess NO production during ischemia occurred within a subset of mdx fibers. In mdx muscles only, prior exposure to I/R dramatically increased the level of sarcolemmal damage resulting from stretch-mediated mechanical stress, indicating greatly exacerbated hyperfragility of the dystrophic fiber membrane. Treatment with NO synthase inhibitors (l-N(G)-nitroarginine methyl ester hydrochloride or 7-nitroindazol) effectively blocked the synergistic interaction between I/R and mechanical stress-mediated sarcolemmal damage under these conditions. Taken together, our findings provide direct ex-perimental evidence that several prevailing hy-potheses regarding the cause of muscle fiber damage in dystrophin-deficient muscle can be integrated into a common pathophysiological framework involving interactions between oxidative stress, ab-normal NO regulation, and hyperfragility of the sarcolemma.

A role for endoglin in coupling eNOS activity and regulating vascular tone revealed in hereditary hemorrhagic telangiectasia.

Decreased endothelial NO synthase (eNOS)-derived NO bioavailability and impaired vasomotor control are crucial factors in cardiovascular disease pathogenesis. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular disorder associated with ENDOGLIN (ENG) haploinsufficiency and characterized by venous dilatations, focal loss of capillaries, and arteriovenous malformations (AVMs). We report that resistance arteries from Eng+/- mice display an eNOS-dependent enhancement in endothelium-dependent dilatation and impairment in the myogenic response, despite reduced eNOS levels. We have found that eNOS is significantly reduced in endoglin-deficient endothelial cells because of decreased eNOS protein half-life. We demonstrate that endoglin can reside in caveolae and associate with eNOS, suggesting a stabilizing function of endoglin for eNOS. After Ca2+-induced activation, endoglin-deficient endothelial cells have reduced eNOS/Hsp90 association, produce less NO, and generate more eNOS-derived superoxide (O2-), indicating that endoglin also facilitates eNOS/Hsp90 interactions and is an important regulator in the coupling of eNOS activity. Treatment with an O2- scavenger reverses the vasomotor abnormalities in Eng(+/-) arteries, suggesting that uncoupled eNOS and resulting impaired myogenic response represent early events in HHT1 pathogenesis and that the use of antioxidants may provide a novel therapeutic modality.

Harvesting airway surface liquid: a comparison of two techniques.

The quantity and composition of airway surface liquid (ASL) are essential to host defense. To date, attempts to harvest ASL and measure its composition have yielded conflicting results. We investigated the physical principles underlying two techniques that were proposed for harvesting ASL: filter paper pledgets and polyethylene catheters. We compared the force and pressure generation and the kinematics of capillarity-induced fluid uptake with both techniques. Both have significant limitations for harvesting ASL, generating physiologically significant pressures (filter paper, 60.4 Pa; polyethylene, 14.3 Pa) that could potentially compromise epithelial integrity. Furthermore, filter paper generates a force 85-fold higher than the polyethylene catheter, which is associated with a very high rate of uptake of liquid and a large total amount of liquid relative to ASL thickness. While the PE catheter harvests liquid more gently, it is only effective when ASL surface tension is below 31 mN/m. These limitations likely account for some of the variability in reported ASL composition, and highlight the need for improved methods for harvesting ASL.