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1.  Role of the Serotonergic System in Reduced Pulmonary Function after Exposure to Methamphetamine 
Although use of methamphetamine (MA) by smoking is the fastest growing method of administration, very limited data are available describing the effects of smoked MA. Using a murine inhalation exposure system, we explored the pulmonary effects of low-dose acute inhalation exposure to MA vapor (smoke). Inhalation of MA vapor resulted in transiently reduced pulmonary function, as measured by transpulmonary resistance, dynamic compliance, and whole-body plethysmography compared with unexposed control animals. These changes were associated with an approximately 34% reduction in serotonin (5-hydroxytryptamine [5-HT]) metabolism/inactivation to 5-hydroxyindolacetic acid, and a nearly 40% reduction in monoamine oxidase (MAO)-A activity in the lung. Pretreatment of mice with a selective 5-HT reuptake inhibitor completely ablated the MA-induced changes in pulmonary function, confirming a key role for the 5-HT transporter (serotonin transporter [SERT]) and the serotonergic system in this effect. Immunofluorescent staining of mouse lung tissue confirmed high expression of SERT in airway epithelial cells. Using mouse airway epithelial cell line, LA-4, and purified human MAO-A, it was demonstrated that MA impedes 5-HT metabolism through direct inhibition of MAO-A activity in vitro. Together, these data demonstrate that low-dose exposure to MA results in reduced pulmonary function mediated via SERT and subsequent perturbation of 5-HT metabolism in the lung. This supports a role for the serotonergic system in MA-mediated pulmonary effects.
PMCID: PMC2874440  PMID: 19541843
lung; monoamine oxidase A; selective serotonin reuptake inhibitor; serotonin transporter
2.  Elevated Asymmetric Dimethylarginine Alters Lung Function and Induces Collagen Deposition in Mice 
Increasing evidence suggests that lung mechanics and structure are maintained in part by an intimate balance between the L-arginine–metabolizing enzymes nitric oxide synthase (NOS) and arginase. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of NOS. The role of ADMA in the regulation of NOS and arginase in the airways has not yet been explored. Our objective was to investigate the role of ADMA in lung physiology. A murine model of continuous subcutaneous ADMA infusion via osmotic minipump was used for assessment of elevated ADMA in vivo, and primary lung fibroblasts were used for in vitro assessments. Two weeks after minipump placement, animals were anesthetized and mechanically ventilated, and lung mechanical responses were evaluated. Lungs were assessed histologically and biochemically for collagen content, arginase activity, and arginase protein levels. Lung lavage fluid was assessed for cellularity, nitrite, urea, and cytokine concentrations. ADMA infusion resulted in significantly enhanced lung resistance and decreased dynamic compliance in response to methacholine. These physiologic changes were associated with significantly increased lung collagen content in the absence of inflammation. Significant decreases in lung fluid nitrite were accompanied by elevated lung fluid urea and arginase activity in lung homogenates. These changes were reversed in mice 4 weeks after completion of ADMA administration. In addition, treatment of primary mouse lung fibroblasts with ADMA stimulated arginase activity and collagen formation in vitro. These data support the idea that ADMA may play a role in airway diseases, including asthma and pulmonary fibrosis, through NOS inhibition and enhancement of arginase activity.
PMCID: PMC2633140  PMID: 18703795
airway resistance; arginase; asthma; fibrosis; nitric oxide synthase
3.  Antigen-Presenting Cell Population Dynamics during Murine Silicosis 
Silicosis is an occupational lung disease resulting from the inhalation of silica particles over prolonged periods of time, which causes chronic inflammation and progressive pulmonary fibrosis. Alveolar macrophages (AM) are critical effector cells, while less is known about the role and function of pulmonary dendritic cells (DC) in silicosis. We hypothesize that a balance exists between the suppressive nature of AM and the stimulatory capacity of DC to regulate lung immunity, and that this equilibrium may be overcome by silica exposure in vivo. Our results demonstrate that in response to silica exposure, both the percent and absolute number of AM significantly decreased over time, with a concomitant significant increase in DC. Both AM and DC exhibited cellular activation in response to silica, indicated by increased expression of cell surface markers. In the absence of silica-induced AM apoptosis (TNFR 1/2–null and Gld mice), no change was observed in the percent or absolute number of either cell type. Furthermore, bone marrow–derived DC, but not bone marrow–derived macrophages, migrated from the alveoli into the lung parenchyma in response to silica, resulting in significantly increased numbers of activated T lymphocytes. Collectively, the results demonstrate that AM and DC are distinct antigen-presenting cells within the respiratory tract that respond to silica exposure in vivo in unique ways, with significant implications for immune reactivity of the lung in response to environmental pathogens.
PMCID: PMC2219550  PMID: 17641296
alveolar macrophage; pulmonary dendritic cell; silica; activation; inflammation
4.  Asymmetric Dimethylarginine Induces Oxidative and Nitrosative Stress in Murine Lung Epithelial Cells 
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Low L-arginine levels can result in the uncoupling of nitric oxide synthase (NOS) leading to production of both ROS and RNS. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of all NOS isoforms and has been demonstrated to inhibit NO formation and increase oxidative stress in vascular endothelial and smooth muscle cells. The effect of ADMA on inducible NOS (iNOS) activity in epithelial cells has not been explored. In this study, we investigated whether addition of exogenous ADMA alters the generation of NO and superoxide anion (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document}), leading to peroxynitrite (ONOO−) formation in a mouse epithelial cell line. In stimulated LA-4 cells, ADMA dose-dependently inhibited nitrite accumulation after 24 h of treatment. In addition, ADMA concentrations as low as 10 μM induced rapid increases in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document} production as measured by dihydroethidium oxidation. Furthermore, using dihydrorhodamine to monitor ONOO− formation, ADMA caused a dose-dependent increase in ONOO− after treatment for 24 h. Similar effects of ADMA were seen using purified iNOS protein in a cell-free system. Together, these data indicate that elevated ADMA may contribute to the production of ROS and RNS in airway inflammation.
PMCID: PMC1899333  PMID: 17158357
ADMA; epithelial cells; LA-4 cells; NOS; oxidative stress
5.  Silica-Directed Mast Cell Activation Is Enhanced by Scavenger Receptors 
Inhalation of crystalline silica results in pulmonary fibrosis and silicosis. It has been suggested that mast cells play a role in these conditions. How mast cells would influence pathology is unknown. We thus explored mast cell interactions with silica in vitro and in B6.Cg-kitW-sh mast cell–deficient mice. B6.Cg-kitW-sh mice did not develop inflammation or significant collagen deposition after instillation of silica, while C57Bl/6 wild-type mice did have these findings. Given this supporting evidence of a role for mast cells in the development of silicosis, we examined the ability of silica to activate mouse bone marrow–derived mast cells (BMMC), including degranulation (β-hexosaminidase release); production of reactive oxygen species (ROS) and inflammatory mediators; and the effects of silica on FcεRI-dependent activation. Silica did not induce mast cell degranulation. However, TNF-α, IL-13, monocyte chemotactic protein-1, protease activity, and production of ROS were dose-dependently increased after silica exposure, and production was enhanced after FcεRI stimulation. This mast cell activation was inhibited by anti-inflammatory compounds. As silica mediates some effects in macrophages through scavenger receptors (SRs), we first determined that mast cells express scavenger receptors; then explored the involvement of SR-A and macrophage receptor with colleagenous structure (MARCO). Silica-induced ROS formation, apoptosis, and TNF-α production were reduced in BMMC obtained from SR-A, MARCO, and SR-A/MARCO knockout mice. These findings demonstrate that silica directs mast cell production of inflammatory mediators, in part through SRs, providing insight into critical events in the pathogenesis and potential therapeutic targets in silicosis.
PMCID: PMC1899302  PMID: 16902192
B6.Cg-kitW-sh sash mouse; CD204; macrophage receptor with collagenous structure; mast cell; silicosis; SR-A

Results 1-5 (5)