Michel Aubier

Aminophylline improves diaphragmatic contractility.

In an attempt to explain the clinical efficacy of aminophylline, we studied its effect on diaphragmatic function in eight normal subjects. The relation between the electrical activity of the diaphragm and the pressure generated by the diaphragm was assessed during voluntary contractions before and after aminophylline infusion. Aminophylline shifted the electrical activity/pressure curve to the left; the pressure at a given electrical activity increased an average of 15 per cent (P less than 0.001). In four subjects, pressure was also measured during stimulation of the phrenic nerve at various frequencies before and after diaphragmatic fatigue was produced by resistive breathing, with and without aminophylline infusion. Pressure increased after fatigue at all stimulation frequencies with aminophylline, as compared with the pressure after identical fatigue runs at the same stimulation frequencies without aminophylline. The mean plasma aminophylline concentration associated with these responses was 13 +/- 0.9 mg per liter. We conclude that aminophylline improves the diaphragms contractility and renders it less susceptible to fatigue.

Myocardial Cell Death in Fibrillating and Dilated Human Right Atria

OBJECTIVES The aim of the present study was to determine if myocytes can die by apoptosis in fibrillating and dilated human atria. BACKGROUND The cellular remodeling that occurs during atrial fibrillation (AF) may reflect a degree of dedifferentiation of the atrial myocardium, a process that may be reversible. METHODS We examined human right atrial myocardium specimens (n = 50) for the presence of apoptotic myocytes. We used immunohistochemical and Western blotting analysis to examine the expression of a final effector of programmed cell death, caspase-3 (CASP-3) and of regulatory proteins from the BCL-2 family. RESULTS Sections from atria in AF contained a high percentage of large myocytes with a disrupted sarcomeric apparatus replaced by glycogen granules (64.4 +/- 6.3% vs. 12.2 +/- 5.8%). These abnormal myocytes, which also predominated in atria from hearts with decreased left ventricular ejection fraction (42.3 +/- 10.1%), contained large nuclei, most of which were TUNEL positive, indicating a degree of DNA breakage. None of these abnormal myocytes expressed the proliferative antigen Ki-67. A small percentage of the enlarged nuclei (4.2 +/- 0.8%) contained condensed chromatin and were strongly TUNEL positive. Both the pro- and activated forms of CASP-3 were detected in diseased myocardial samples, which also showed stronger CASP-3 expression than controls. Expression of the antiapoptotic BCL-2 protein was decreased in diseased atria, whereas that of the proapoptotic BAX protein remained unchanged. CONCLUSIONS In fibrillating and dilated atria, apoptotic death of myocytes with myolysis contributes to cellular remodeling, which may not be entirely reversible.

Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production

The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1beta release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o-) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1β release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.

Induction of Heme Oxygenase-1 Inhibits NAD(P)H Oxidase Activity by Down-regulating Cytochrome b558 Expression via the Reduction of Heme Availability

Heme-oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has powerful anti-oxidant properties related to the production of the reactive oxygen species scavenger bilirubin. However, some data suggest that HO-1 could also inhibit the cellular production of reactive oxygen species. Therefore, we investigated whether the anti-oxidant properties of HO-1 could be mediated by modulation of the activity and/or expression of the heme-containing NAD(P)H oxidase, the main source of the superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{O}_{{\dot{2}}}^{-}\) \end{document}) in phagocytic cells. Increasing HO-1 expression in RAW 264.7 macrophages effectively decreased NAD(P)H oxidase activity and expression of gp91phox, its heme-containing catalytic component, because of deficient protein maturation and increased degradation. Loading cells with heme reversed the decrease in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{O}_{{\dot{2}}}^{-}\) \end{document} production and gp91phox expression induced by HO-1 overexpression. Similar results were obtained in vivo in rat alveolar macrophages after pharmacological modulation of HO-1 expression or activity. These results show that a decrease in heme content due to HO-1 activation limits heme availability for maturation of the gp91phox subunit and assembly of the functional NAD(P)H oxidase. This study provides a new mechanism to explain HO-1 anti-oxidant properties.

Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia

It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysac‐charide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady‐state concentration of 0.5 µM NO in the muscle associated with increased detection of hydrogen peroxide (H2O2). In vitro, the same NO concentration produced a marked increase in H2O2 production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O2−·). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H2O2 production associated with uncoupling and decreased phosphor‐ylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L‐NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O2−· that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility.—Boczkowski, J., Lis‐dero, C. L., Lanone, S., Samb, A., Carreras, M. C., Boveris, A., Aubier, M., Poderoso, J. J. Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia. FASEB J. 13, 1637–1647 (1999)

Myosin heavy chain gene expression changes in the diaphragm of patients with chronic lung hyperinflation

In striated muscle, chronic increases in workload result in changes in myosin phenotype. The aim of this study was to determine whether such changes occur in the diaphragm of patients with severe chronic obstructive pulmonary disease, a situation characterized by a chronic increase in respiratory load and lung volume. Diaphragm biopsies were obtained from 22 patients who underwent thoracic surgery. Myosin was characterized with electrophoresis in nondenaturing conditions, SDS-glycerol PAGE, and Western blotting with monoclonal antibodies specific for slow and fast myosin heavy chain (MHC) isoforms. Flow volume curves, total lung capacity, and functional residual capacity were measured before surgery in 20 patients. We found that the human diaphragm is composed of at least four myosin isoforms, one slow and three fast, resulting from the combination of three MHC species. Chronic overload was associated with an increase in the slow beta-MHC species at the expense of the fast species (beta-MHC, 78.2 +/- 4.6 and 50.0 +/- 6.5% in emphysematous and control patients, respectively; P < 0.005). Linear correlations were found between beta-MHC percentage and forced expiratory volume in 1 s (r = -0.52; P < 0.02), total lung capacity (r = 0.44; P < 0.05), and functional residual capacity (r = 0.65; P < 0.003). The human adult diaphragm is composed of a balanced proportion of slow and fast myosin isoforms. In patients with chronic obstructive pulmonary disease, the proportion of fast myosins decreases, whereas that of slow myosin increases. This increase appears to be closely related to lung hyperinflation and may reflect an adaptation of the diaphragm to the new functional requirements.

Changes in airway inflammation following nasal allergic challenge in patients with seasonal rhinitis

Background:  Seasonal allergic rhinitis could predispose to the development of chronic bronchial inflammation as observed in asthma. However, direct links between nasal inflammation, bronchial inflammation and airway responsiveness in patients with seasonal allergic rhinitis and without asthma are not fully understood. The aim of this study was to analyse the changes induced by allergic nasal challenge outside the pollen season in airway responsiveness and bronchial inflammation of patients with seasonal allergic rhinitis.

Association of lung function decline with the heme oxygenase-1 gene promoter microsatellite polymorphism in a general population sample. Results from the European Community Respiratory Health Survey (ECRHS), France

Inducible heme oxygenase (HO-1) acts against oxidants that are thought to play a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD), characterised by impaired lung function. A (GT)n repeat polymorphism in the HO-1 gene promoter can modulate the gene transcription in response to oxidative stress. We hypothesised that this polymorphism could be associated with the level of lung function and decline in subjects exposed to oxidative agression (smokers). We genotyped 749 French subjects (20–44 years, 50% men, 40% never smokers) examined in both 1992 and 2000 as part of the ECRHS. Lung function was assessed by forced expiratory volume in 1 second (FEV1) and FEV1/forced ventilatory capacity (FVC) ratio. We compared long (L) allele carriers ((GT)n ⩾33 repeats for one or two alleles) to non-carriers. Cross sectionally, in 2000, L allele carriers showed lower FEV1/FVC than non-carriers. During the 8 year period, the mean annual FEV1 and FEV1/FVC declines were −30.9 (31.1) ml/year and −1.8 (6.1) U/year, respectively. FEV1/FVC decline was steeper in L allele carriers than in non-carriers (−2.6 (5.5) v −1.5 (6.4), p = 0.07). There was a strong interaction between the L allele and smoking. In 2000, the L allele was associated with lower FEV1 and FEV1/FVC in heavy smokers (⩾20 cigarettes/day) only (p for interaction = 0.07 and 0.002 respectively). Baseline heavy smokers carrying the L allele showed the steepest FEV1 decline (−62.0 (29.5 ml/year) and the steepest FEV1/FVC decline (−8.8 (5.4 U/year) (p for interaction = 0.009 and 0.0006).These results suggest that a long (L) HO-1 gene promoter in heavy smokers is associated with susceptibility to develop airway obstruction.

Predominant role of A1 adenosine receptors in mediating adenosine induced vasodilatation of rat diaphragmatic arterioles: involvement of nitric oxide and the ATP‐dependent K+ channels

We investigated, by intravital microscopy in rats, the role of the subtypes of adenosine receptors A1 (A1/AR) and A2 (A2AR) in mediating adenosine‐induced vasodilatation of second and third order arterioles of the diaphragm. Adenosine, and the A1AR selective agonists R(−)‐N6‐(2‐phenylisopropyl)‐adenosine (R‐PIA) and N6‐cyclo‐pentyl‐adenosine (CPA) induced a similar concentration‐dependent dilatation of diaphragmatic arterioles. The non selective A2AR subtype agonist N6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl) ethyl]adenosine (DPMA) also dilated diaphragmatic arterioles but induced a significantly smaller dilatation than adenosine. By contrast the selective A2aAR subtype agonist 2‐[p‐(2‐carboxyethyl)phenyl amino]‐5′‐N‐ethyl carboxamido adenosine (CGS 21680) did not modify diaphragmatic arteriolar diameter. The non selective adenosine receptor antagonist 1,3‐dipropyl‐8‐p‐sulphophenylxanthine (SPX, 100 μM) and the selective A1AR antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX, 50 nM) significantly attenuated adenosine‐induced dilatation of diaphragmatic arterioles. By contrast, adenosine significantly dilated diaphragmatic arterioles in the presence of A2AR antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX, 10 μM). The dilatation induced by adenosine was unchanged by the mast cell stabilizing agent sodium cromoglycate (cromolyn, 10 μM). The nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG, 300 μM) attenuated the dilatation induced by adenosine, and by the A1AR and A2AR agonists. The ATP‐dependent K+ channel blocker glibenclamide (3 μM) significantly attenuated diaphragmatic arteriolar dilatation induced by adenosine and by the A1AR agonists R‐PIA and CPA. By contrast, glibenclamide did not significantly modify arteriolar dilatation induced by the A2AR agonist DPMA. These findings suggest that adenosine‐induced dilatation of diaphragmatic arterioles in the rat is predominantly mediated by the A1AR, via the release of NO and activation of the ATP‐dependent K+ channels.

Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.

Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity.