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Lee RJ, Adappa ND, Palmer JN. Akt activator SC79 stimulates antibacterial nitric oxide generation in human nasal epithelial cells in vitro. Int Forum Allergy Rhinol 2024; 14:1147-1162. [PMID: 38197521 PMCID: PMC11219270 DOI: 10.1002/alr.23318] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2023] [Revised: 12/05/2023] [Accepted: 12/26/2023] [Indexed: 01/11/2024]
Abstract
BACKGROUND The role of Akt in nasal immunity is unstudied. Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) expressed in epithelial ciliated cells. Nitric oxide (NO) production by ciliated cells can have antibacterial and antiviral effects. Increasing nasal NO may be a useful antipathogen strategy in chronic rhinosinusitis (CRS). We previously showed that small-molecule Akt activator SC79 induces nasal cell NO production and suppresses IL-8 via the transcription factor Nrf-2. We hypothesized that SC79 NO production may additionally have antibacterial effects. METHODS NO production was measured using fluorescent dye DAF-FM. We tested effects of SC79 during co-culture of Pseudomonas aeruginosa with primary nasal epithelial cells, using CFU counting and live-dead staining to quantify bacterial killing. Pharmacology determined the mechanism of SC79-induced NO production and tested dependence on Akt. RESULTS SC79 induced dose-dependent, Akt-dependent NO production in nasal epithelial cells. The NO production required eNOS and Akt. The NO released into the airway surface liquid killed P. aeruginosa. No toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed over 24 h. CONCLUSIONS Together, these data suggest multiple immune pathways are stimulated by SC79, with antipathogen effects. This in vitro pilot study suggests that a small-molecule Akt activator may have clinical utility in CRS or respiratory other infection settings, warranting future in vivo studies.
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Affiliation(s)
- Robert J. Lee
- Department of Otorhinolaryngology, University of Pennsylvania Perelman School of Medicine
- Department of Physiology, University of Pennsylvania Perelman School of Medicine
| | - Nithin D. Adappa
- Department of Otorhinolaryngology, University of Pennsylvania Perelman School of Medicine
| | - James N. Palmer
- Department of Otorhinolaryngology, University of Pennsylvania Perelman School of Medicine
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2
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Tan DX, Reiter RJ, Zimmerman S, Hardeland R. Melatonin: Both a Messenger of Darkness and a Participant in the Cellular Actions of Non-Visible Solar Radiation of Near Infrared Light. BIOLOGY 2023; 12:89. [PMID: 36671781 PMCID: PMC9855654 DOI: 10.3390/biology12010089] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/10/2022] [Revised: 12/25/2022] [Accepted: 01/04/2023] [Indexed: 01/07/2023]
Abstract
Throughout the history of melatonin research, almost exclusive focus has been on nocturnally-generated pineal melatonin production, which accounts for its circadian rhythm in the blood and cerebrospinal fluid; these light/dark melatonin cycles drive the daily and seasonal photoperiodic alterations in organismal physiology. Because pineal melatonin is produced and secreted primarily at night, it is referred to as the chemical expression of darkness. The importance of the other sources of melatonin has almost been ignored. Based on current evidence, there are at least four sources of melatonin in vertebrates that contribute to the whole-body melatonin pool. These include melatonin produced by (1) the pineal gland; (2) extrapineal cells, tissues, and organs; (3) the microbiota of the skin, mouth, nose, digestive tract, and vagina as well as (4) melatonin present in the diet. These multiple sources of melatonin exhibit differentially regulated mechanisms for its synthesis. Visible light striking the retina or an intense physical stimulus can suppress nocturnal pineal melatonin levels; in contrast, there are examples where extrapineal melatonin levels are increased during heavy exercise in daylight, which contains the whole range of NIR radiation. The cumulative impact of all cells producing augmented extrapineal melatonin is sufficient to elevate sweat concentrations, and potentially, if the exposure is sustained, to also increasing the circulating values. The transient increases in sweat and plasma melatonin support the premise that extrapineal melatonin has a production capacity that exceeds by far what can be produced by the pineal gland, and is used to maintain intercellular homeostasis and responds to rapid changes in ROS density. The potential regulatory mechanisms of near infrared light (NIR) on melatonin synthesis are discussed in detail herein. Combined with the discovery of high levels of melanopsin in most fat cells and their response to light further calls into question pineal centric theories. While the regulatory processes related to microbiota-derived melatonin are currently unknown, there does seem to be crosstalk between melatonin derived from the host and that originating from microbiota.
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Affiliation(s)
- Dun-Xian Tan
- Department of Cell Systems and Anatomy, UT Health San Antonio, Long School of Medicine, San Antonio, TX 78229, USA
| | - Russel J. Reiter
- Department of Cell Systems and Anatomy, UT Health San Antonio, Long School of Medicine, San Antonio, TX 78229, USA
| | | | - Ruediger Hardeland
- Johann Friedric Blumenbach Institute of Zoology and Anthropology, University of Göttingen, D-37073 Göttingen, Germany
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Carey RM, Palmer JN, Adappa ND, Lee RJ. Loss of CFTR function is associated with reduced bitter taste receptor-stimulated nitric oxide innate immune responses in nasal epithelial cells and macrophages. Front Immunol 2023; 14:1096242. [PMID: 36742335 PMCID: PMC9890060 DOI: 10.3389/fimmu.2023.1096242] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2022] [Accepted: 01/03/2023] [Indexed: 01/19/2023] Open
Abstract
Introduction Bitter taste receptors (T2Rs) are G protein-coupled receptors identified on the tongue but expressed all over the body, including in airway cilia and macrophages, where T2Rs serve an immune role. T2R isoforms detect bitter metabolites (quinolones and acyl-homoserine lactones) secreted by gram negative bacteria, including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis (CF). T2R activation by bitter bacterial products triggers calcium-dependent nitric oxide (NO) production. In airway cells, the NO increases mucociliary clearance and has direct antibacterial properties. In macrophages, the same pathway enhances phagocytosis. Because prior studies linked CF with reduced NO, we hypothesized that CF cells may have reduced T2R/NO responses, possibly contributing to reduced innate immunity in CF. Methods Immunofluorescence, qPCR, and live cell imaging were used to measure T2R localization, calcium and NO signaling, ciliary beating, and antimicrobial responses in air-liquid interface cultures of primary human nasal epithelial cells and immortalized bronchial cell lines. Immunofluorescence and live cell imaging was used to measure T2R signaling and phagocytosis in primary human monocyte-derived macrophages. Results Primary nasal epithelial cells from both CF and non-CF patients exhibited similar T2R expression, localization, and calcium signals. However, CF cells exhibited reduced NO production also observed in immortalized CFBE41o- CF cells and non-CF 16HBE cells CRISPR modified with CF-causing mutations in the CF transmembrane conductance regulator (CFTR). NO was restored by VX-770/VX-809 corrector/potentiator pre-treatment, suggesting reduced NO in CF cells is due to loss of CFTR function. In nasal cells, reduced NO correlated with reduced ciliary and antibacterial responses. In primary human macrophages, inhibition of CFTR reduced NO production and phagocytosis during T2R stimulation. Conclusions Together, these data suggest an intrinsic deficiency in T2R/NO signaling caused by loss of CFTR function that may contribute to intrinsic susceptibilities of CF patients to P. aeruginosa and other gram-negative bacteria that activate T2Rs.
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Affiliation(s)
- Ryan M Carey
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - James N Palmer
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Nithin D Adappa
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Robert J Lee
- Department of Otorhinolaryngology-Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.,Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
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4
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Lewis SA, Doratt BM, Sureshchandra S, Jankeel A, Newman N, Shen W, Grant KA, Messaoudi I. Ethanol Consumption Induces Nonspecific Inflammation and Functional Defects in Alveolar Macrophages. Am J Respir Cell Mol Biol 2022; 67:112-124. [PMID: 35380939 PMCID: PMC9273227 DOI: 10.1165/rcmb.2021-0346oc] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Accepted: 04/05/2022] [Indexed: 11/24/2022] Open
Abstract
Chronic alcohol drinking is associated with increased susceptibility to viral and bacterial respiratory pathogens. In this study, we use a rhesus macaque model of voluntary ethanol self-administration to study the effects of long-term alcohol drinking on the immunological landscape of the lung. We report a heightened inflammatory state in alveolar macrophages (AMs) obtained from ethanol (EtOH)-drinking animals that is accompanied by increased chromatin accessibility in intergenic regions that regulate inflammatory genes and contain binding motifs for transcription factors AP-1, IRF8, and NFKB p-65. In line with these transcriptional and epigenetic changes at the basal state, AMs from EtOH-drinking animals generate elevated inflammatory mediator responses to lipopolysaccharides and respiratory syncytial virus. However, the transcriptional analysis revealed an inefficient induction of interferon-stimulated genes with EtOH in response to the respiratory syncytial virus, suggesting disruption of antimicrobial defenses. Correspondingly, AMs from EtOH-drinking animals exhibited transcriptional shifts indicative of increased oxidative stress and oxidative phosphorylation, which was coupled with higher cytosolic reactive oxygen species and mitochondrial potential. This heightened oxidative stress state was accompanied by decreased ability to phagocytose bacteria. Bulk RNA and assay for transposase-accessible chromatin sequencing data further revealed reduced expression and chromatin accessibility of loci associated with tissue repair and maintenance with chronic EtOH drinking. Similarly, analysis of single-cell RNA sequencing data revealed shifts in cell states from tissue maintenance to inflammatory responses with EtOH. Collectively, these data provide novel insight into mechanisms by which chronic EtOH drinking increases susceptibility to infection in patients with alcohol use disorders.
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Affiliation(s)
- Sloan A. Lewis
- Department of Molecular Biology and Biochemistry
- Institute for Immunology, and
| | - Brianna M. Doratt
- Department of Molecular Biology and Biochemistry
- Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, Kentucky; and
| | - Suhas Sureshchandra
- Department of Molecular Biology and Biochemistry
- Institute for Immunology, and
| | | | - Natali Newman
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon
| | - Weining Shen
- Department of Statistics, University of California, Irvine, Irvine, California
| | - Kathleen A. Grant
- Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon
| | - Ilhem Messaoudi
- Department of Molecular Biology and Biochemistry
- Institute for Immunology, and
- Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, Kentucky; and
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Carey RM, Hariri BM, Adappa ND, Palmer JN, Lee RJ. HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages. Cells 2022; 11:1478. [PMID: 35563784 PMCID: PMC9101439 DOI: 10.3390/cells11091478] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 04/22/2022] [Accepted: 04/26/2022] [Indexed: 02/04/2023] Open
Abstract
Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two innate immune cell types are activated by bitter products, including those secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells. NO also increases phagocytosis by macrophages. Using biochemistry and live-cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). Immunofluorescence showed that H441 cells express eNOS and T2Rs and that the bitter agonist denatonium benzoate activates NO production in a Ca2+- and HSP90-dependent manner in cells grown either as submerged cultures or at the air-liquid interface. In primary sinonasal epithelial cells, we determined that HSP90 inhibition reduces T2R-stimulated NO production and ciliary beating, which likely limits pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves as an innate immune modulator by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream Ca2+ signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases such as chronic rhinosinusitis, asthma, or cystic fibrosis.
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Affiliation(s)
- Ryan M. Carey
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - Benjamin M. Hariri
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - Nithin D. Adappa
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - James N. Palmer
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
| | - Robert J. Lee
- Department of Otorhinolaryngology—Head and Neck Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; (B.M.H.); (N.D.A.); (J.N.P.)
- Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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6
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Salvatori P. The rationale of ethanol inhalation for disinfection of the respiratory tract in SARS-CoV-2-positive asymptomatic subjects. Pan Afr Med J 2021; 40:201. [PMID: 35136464 PMCID: PMC8783302 DOI: 10.11604/pamj.2021.40.201.31211] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Accepted: 09/01/2021] [Indexed: 12/20/2022] Open
Abstract
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health concern and is affecting the socio-economic lives. As other highly contagious diseases, it is of outmost importance to early identify and treat the healthy carriers or positive asymptomatic subjects (PAS). SARS-CoV-2 entry points are mainly in the respiratory tract. No specific virucidal treatments against SARS-CoV-2 are currently available. Monoclonal antibodies are under evaluation, but high cost and possible ineffectiveness against virus variants could limit its use. Resorting nonspecific drugs is an alternative approach. Among them, ethanol (EtOH) is known to be a powerful, cost-effective and abundant virucidal agent, now advised for surgical hand and surfaces disinfection. The paper aims to determine the potential role of inhaled ethanol to disinfect SARS-CoV-2 PAS, taking into account the dimension of the problem, ethanol efficiency and other beneficial effects on the respiratory tract, ethanol local and general toxicity and ethanol therapeutic window; consequently, to propose a study in order to verify this hypothesis. Together with the consolidated knowledge, an extensive review of the medical literature has been carried out looking for sound data able to support (or discard) the rationale on which a study could be built up. Evident data supporting the inhaled ethanol potential role on SARS-CoV-2 PAS disinfection have been found and discussed. A clinical trial to test the hypothesis that inhaled ethanol could be rapidly efficient in lowering or eradicating SARS-CoV-2 from the respiratory tract in PAS is advisable. Individual and public health benefits are stressed, together with socio-economic positive fallouts.
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Affiliation(s)
- Pietro Salvatori
- Retired Head & Neck Surgeon, Former Head of Ear, Nose and Throat (ENT) Department at the Humanitas San Pio X Hospital, Milan, Italy
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7
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Neuropeptide Y Reduces Nasal Epithelial T2R Bitter Taste Receptor-Stimulated Nitric Oxide Production. Nutrients 2021; 13:nu13103392. [PMID: 34684394 PMCID: PMC8538228 DOI: 10.3390/nu13103392] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 09/22/2021] [Accepted: 09/24/2021] [Indexed: 12/30/2022] Open
Abstract
Bitter taste receptors (T2Rs) are G-protein-coupled receptors (GPCRs) expressed on the tongue but also in various locations throughout the body, including on motile cilia within the upper and lower airways. Within the nasal airway, T2Rs detect secreted bacterial ligands and initiate bactericidal nitric oxide (NO) responses, which also increase ciliary beat frequency (CBF) and mucociliary clearance of pathogens. Various neuropeptides, including neuropeptide tyrosine (neuropeptide Y or NPY), control physiological processes in the airway including cytokine release, fluid secretion, and ciliary beating. NPY levels and/or density of NPYergic neurons may be increased in some sinonasal diseases. We hypothesized that NPY modulates cilia-localized T2R responses in nasal epithelia. Using primary sinonasal epithelial cells cultured at air–liquid interface (ALI), we demonstrate that NPY reduces CBF through NPY2R activation of protein kinase C (PKC) and attenuates responses to T2R14 agonist apigenin. We find that NPY does not alter T2R-induced calcium elevation but does reduce T2R-stimulated NO production via a PKC-dependent process. This study extends our understanding of how T2R responses are modulated within the inflammatory environment of sinonasal diseases, which may improve our ability to effectively treat these disorders.
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8
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Gongola A, Bradshaw JC, Jin J, Jensen HK, Bhavaraju A, Margolick J, Sexton KW, Robertson R, Kalkwarf KJ. Retrospective study on rib fractures: smoking and alcohol matter for mortality and complications. Trauma Surg Acute Care Open 2021; 6:e000732. [PMID: 34212116 PMCID: PMC8207992 DOI: 10.1136/tsaco-2021-000732] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Accepted: 05/23/2021] [Indexed: 11/17/2022] Open
Abstract
Background Rib fractures and substance use are both common in trauma patients, but there is little data on how smoking and alcohol use may be associated with outcomes in these patients. We assessed the association between smoking or alcohol use disorder (AUD) and outcomes in patients with rib fractures. Methods We used institutional databases to conduct a retrospective review of patients with rib fractures at the only American College of Surgeons-verified adult level 1 trauma center in a rural state between 2015 and 2019. The key exposure variables were smoking and AUD. The key outcome variables were mortality and pulmonary complications (pneumonia, adult respiratory distress syndrome, and pneumothorax). We used multivariable regression for analysis and directed acyclic graphs to identify variables for adjustment. Results We identified 1880 eligible patients with rib fractures, including 693 (37%) who were smokers and 204 (11%) who had AUD. Compared with non-smokers, smokers were younger, more often male, and had lower mortality rates. Regression showed that smokers had a lower likelihood of mortality (OR 0.48; 95% CI 0.27 to 0.87; p=014). Likelihood of pneumonia, ARDS, and pneumothorax was not different between smokers and non-smokers. Compared with patients without AUD, patients with AUD were older, more often male, and had higher likelihood of pneumonia and lower likelihood of pneumothorax. Regression showed that patients with AUD had higher likelihood of pneumonia (OR 1.82; 95% CI 1.24 to 2.68; p=0.002) and lower likelihood of pneumothorax (OR 0.51; 95% CI 0.33 to 0.75; p=0.002). Discussion In trauma patients with rib fractures treated at a level 1 trauma center over 5 years, smoking was associated with decreased risk of mortality. These findings have implications for risk stratification and clinical decision-making for patients with rib fractures. Level of evidence III
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Affiliation(s)
| | - Jace C Bradshaw
- University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Jing Jin
- University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Hanna K Jensen
- Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Avi Bhavaraju
- Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Joseph Margolick
- Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Kevin W Sexton
- Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Ronald Robertson
- Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Kyle J Kalkwarf
- Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
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9
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Mikhailik A, Michurina TV, Dikranian K, Hearn S, Maxakov VI, Siller SS, Takemaru KI, Enikolopov G, Peunova N. nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea. Life Sci Alliance 2021; 4:4/5/e202000981. [PMID: 33653689 PMCID: PMC8008965 DOI: 10.26508/lsa.202000981] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2021] [Revised: 02/08/2021] [Accepted: 02/09/2021] [Indexed: 11/24/2022] Open
Abstract
Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.
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Affiliation(s)
- Anatoly Mikhailik
- Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, USA.,Department of Anesthesiology, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, USA
| | - Tatyana V Michurina
- Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, USA.,Department of Anesthesiology, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, USA
| | - Krikor Dikranian
- Department of Neuroscience, Washington University, St. Louis, MO, USA
| | - Stephen Hearn
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
| | - Vladimir I Maxakov
- Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, USA.,Department of Anesthesiology, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, USA
| | - Saul S Siller
- Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA
| | - Ken-Ichi Takemaru
- Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA
| | - Grigori Enikolopov
- Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, USA.,Department of Anesthesiology, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, USA
| | - Natalia Peunova
- Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, USA .,Department of Anesthesiology, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, USA
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10
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NMR Profiling of Exhaled Breath Condensate Defines Different Metabolic Phenotypes of Non-Cystic Fibrosis Bronchiectasis. Int J Mol Sci 2020; 21:ijms21228600. [PMID: 33202684 PMCID: PMC7698311 DOI: 10.3390/ijms21228600] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 11/06/2020] [Accepted: 11/10/2020] [Indexed: 12/16/2022] Open
Abstract
Nuclear-magnetic-resonance (NMR) profiling of exhaled breath condensate (EBC) provides insights into the pathophysiology of bronchiectasis by identifying specific biomarkers. We evaluated whether NMR-based metabolomics discriminates the EBC-derived metabolic phenotypes (“metabotypes”) of 41 patients with non-cystic fibrosis (nCF) bronchiectasis of various etiology [24 subjects with Primary Ciliary Dyskinesia (PCD); 17 patients with bronchiectasis not associated with PCD (nCF/nPCD)], who were compared to 17 healthy subjects (HS). NMR was used for EBC profiling, and Orthogonal Projections to Latent Structures with partial least-squares discriminant analysis (OPLS-DA) was used as a classifier. The results were validated by using the EBC from 17 PCD patients not included in the primary analysis. Different statistical models were built, which compared nCF/nPCD and HS, PCD and HS, all classes (nCF/nPCD-PCD-HS), and, finally, PCD and nCF/nPCD. In the PCD-nCF/nPCD model, four statistically significant metabolites were able to discriminate between the two groups, with only a minor reduction of the quality parameters. In particular, for nCF/nPCD, acetone/acetoin and methanol increased by 21% and 18%, respectively. In PCD patients, ethanol and lactate increased by 25% and 28%, respectively. They are all related to lung inflammation as methanol is found in the exhaled breath of lung cancer patients, acetone/acetoin produce toxic ROS that damage lung tissue in CF, and lactate is observed in acute inflammation. Interestingly, a high concentration of ethanol hampers cilia beating and can be associated with the genetic defect of PCD. Model validation with 17 PCD samples not included in the primary analysis correctly predicted all samples. Our results indicate that NMR of EBC discriminates nCF/nPCD and PCD bronchiectasis patients from HS, and patients with nCF/nPCD from those with PCD. The metabolites responsible for between-group separation identified specific metabotypes, which characterize bronchiectasis of a different etiology.
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11
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Choi MR, Han JS, Chai YG, Jin YB, Lee SR, Kim DJ. Gene expression profiling in the hippocampus of adolescent rats after chronic alcohol administration. Basic Clin Pharmacol Toxicol 2019; 126:389-398. [PMID: 31628824 DOI: 10.1111/bcpt.13342] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2019] [Accepted: 10/15/2019] [Indexed: 11/29/2022]
Abstract
In South Korea, the average age of onset of alcohol drinking is 13.3 years and half of adolescents drink alcohol more than once a month; 8.45% of the Korean adolescent population become future high-risk alcohol drinkers. Chronic alcohol abuse causes physical and psychiatric health problems such as alcohol addiction, liver disease, stroke and cognitive impairments. This study aimed to investigate the effect of alcohol on gene expression and their function in the hippocampus of adolescent rats. After chronic alcohol administration in male (control, n = 6; alcohol, n = 6) Sprague-Dawley rats for 6 weeks, we analysed up- or down-regulated genes using RNA-sequencing technology. We found 83 genes more than 1.5-fold up- or down-regulated in the alcohol-treated group. Among them, genes (Dnai1, Cfap206 and Dnah1) associated with cilium movement were up-regulated in the alcohol-treated group. Mlf1, related to cell cycle arrest, was also up-regulated in the alcohol-treated group. On the other hand, genes (Smad3 and Plk5) involved in negative regulation of cell proliferation were down-regulated in the hippocampus by chronic alcohol administration. In addition, expression levels of genes associated with oxidative stress (Krt8 and Car3) and migration (Vim) were changed by chronic alcohol administration. These results pave a path for a better understanding of the neuromolecular mechanisms mediated by chronic alcohol exposure in the hippocampus of adolescents and negative pathology due to chronic alcohol abuse.
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Affiliation(s)
- Mi Ran Choi
- Department of Psychiatry, Seoul St. Mary's Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea
| | - Jasmin Sanghyun Han
- Department of Psychiatry, Seoul St. Mary's Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea
| | - Young Gyu Chai
- Department of Molecular and Life Sciences, Hanyang University, Ansan, Korea
| | - Yeung-Bae Jin
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Korea
| | - Sang-Rae Lee
- National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Korea
| | - Dai-Jin Kim
- Department of Psychiatry, Seoul St. Mary's Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea
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12
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Price ME, Gerald CL, Pavlik JA, Schlichte SL, Zimmerman MC, DeVasure JM, Wyatt TA, Sisson JH. Loss of cAMP-dependent stimulation of isolated cilia motility by alcohol exposure is oxidant-dependent. Alcohol 2019; 80:91-98. [PMID: 30291947 DOI: 10.1016/j.alcohol.2018.09.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Revised: 09/18/2018] [Accepted: 09/26/2018] [Indexed: 02/07/2023]
Abstract
Alcohol exposure is associated with decreased mucociliary clearance, a key innate defense essential to lung immunity. Previously, we identified that prolonged alcohol exposure results in dysfunction of airway cilia that persists at the organelle level. This dysfunction is characterized by a loss of 3',5'-cyclic adenosine monophosphate (cAMP)-mediated cilia stimulation. However, whether or not ciliary dysfunction develops intrinsically at the organelle level has not been explored. We hypothesized that prolonged alcohol exposure directly to isolated demembranated cilia (axonemes) causes ciliary dysfunction. To test this hypothesis, we exposed isolated axonemes to alcohol (100 mM) for 1-24 h and assessed ciliary beat frequency (CBF) in response to cAMP at 1, 3, 4, 6, and 24 h post-exposure. We found that after 1 h of alcohol exposure, cilia axonemes do not increase CBF in response to cAMP. Importantly, by 6 h after the initial exposure to alcohol, cAMP-mediated CBF was restored to control levels. Additionally, we found that thioredoxin reverses ciliary dysfunction in axonemes exposed to alcohol. Finally, we identified, using a combination of a xanthine oxidase oxidant-generating system, direct application of hydrogen peroxide, and electron paramagnetic resonance, that hydrogen peroxide versus superoxide, is likely the key oxidant species driving alcohol-induced ciliary dysfunction in isolated axonemes. These data highlight the role of alcohol to stimulate local production of oxidants in the axoneme to cause ciliary dysfunction. Additionally, these data specifically add hydrogen peroxide as a potential therapeutic target in the treatment or prevention of alcohol-associated ciliary dysfunction and subsequent pneumonia.
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Affiliation(s)
- Michael E Price
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, United States; Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, United States
| | - Carresse L Gerald
- Department of Environmental, Earth and Geospatial Science, North Carolina Central University, Durham, NC, United States
| | - Jacqueline A Pavlik
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, United States
| | - Sarah L Schlichte
- Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, United States
| | - Matthew C Zimmerman
- Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, United States
| | - Jane M DeVasure
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, United States
| | - Todd A Wyatt
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, United States; Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, NE, United States; Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Research Service, Omaha, NE, United States
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, United States.
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13
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Price ME, Sisson JH. Redox regulation of motile cilia in airway disease. Redox Biol 2019; 27:101146. [PMID: 30833143 PMCID: PMC6859573 DOI: 10.1016/j.redox.2019.101146] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2019] [Revised: 02/14/2019] [Accepted: 02/15/2019] [Indexed: 02/07/2023] Open
Abstract
Motile cilia on airway cells are necessary for clearance of mucus-trapped particles out of the lung. Ciliated airway epithelial cells are uniquely exposed to oxidants through trapping of particles, debris and pathogens in mucus and the direct exposure to inhaled oxidant gases. Dynein ATPases, the motors driving ciliary motility, are sensitive to the local redox environment within each cilium. Several redox-sensitive cilia-localized proteins modulate dynein activity and include Protein Kinase A, Protein Kinase C, and Protein Phosphatase 1. Moreover, cilia are rich in known redox regulatory proteins and thioredoxin domain-containing proteins that are critical in maintaining a balanced redox environment. Importantly, a nonsense mutation in TXNDC3, which contains a thioredoxin motif, has recently been identified as disease-causing in Primary Ciliary Dyskinesia, a hereditary motile cilia disease resulting in impaired mucociliary clearance. Here we review current understanding of the role(s) oxidant species play in modifying airway ciliary function. We focus on oxidants generated in the airways, cilia redox targets that modulate ciliary beating and imbalances in redox state that impact health and disease. Finally, we review disease models such as smoking, asthma, alcohol drinking, and infections as well as the direct application of oxidants that implicate redox balance as a modulator of cilia motility.
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Affiliation(s)
- Michael E Price
- University of Nebraska Medical Center, Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, NE, USA; University of Nebraska Medical Center, Department of Cellular & Integrative Physiology, Omaha, NE, USA.
| | - Joseph H Sisson
- University of Nebraska Medical Center, Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, NE, USA.
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14
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Price ME, Case AJ, Pavlik JA, DeVasure JM, Wyatt TA, Zimmerman MC, Sisson JH. S-nitrosation of protein phosphatase 1 mediates alcohol-induced ciliary dysfunction. Sci Rep 2018; 8:9701. [PMID: 29946131 PMCID: PMC6018795 DOI: 10.1038/s41598-018-27924-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 06/01/2018] [Indexed: 01/13/2023] Open
Abstract
Alcohol use disorder (AUD) is a strong risk factor for development and mortality of pneumonia. Mucociliary clearance, a key innate defense against pneumonia, is perturbed by alcohol use. Specifically, ciliated airway cells lose the ability to increase ciliary beat frequency (CBF) to β-agonist stimulation after prolonged alcohol exposure. We previously found that alcohol activates protein phosphatase 1 (PP1) through a redox mechanism to cause ciliary dysfunction. Therefore, we hypothesized that PP1 activity is enhanced by alcohol exposure through an S-nitrosothiol-dependent mechanism resulting in desensitization of CBF stimulation. Bronchoalveolar S-nitrosothiol (SNO) content and tracheal PP1 activity was increased in wild-type (WT) mice drinking alcohol for 6-weeks compared to control mice. In contrast, alcohol drinking did not increase SNO content or PP1 activity in nitric oxide synthase 3-deficient mice. S-nitrosoglutathione induced PP1-dependent CBF desensitization in mouse tracheal rings, cultured cells and isolated cilia. In vitro expression of mutant PP1 (cysteine 155 to alanine) in primary human airway epithelial cells prevented CBF desensitization after prolonged alcohol exposure compared to cells expressing WT PP1. Thus, redox modulation in the airways by alcohol is an important ciliary regulatory mechanism. Pharmacologic strategies to reduce S-nitrosation may enhance mucociliary clearance and reduce pneumonia prevalence, mortality and morbidity with AUD.
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Affiliation(s)
- Michael E Price
- From the Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA
- Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA
| | - Adam J Case
- Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA
| | - Jacqueline A Pavlik
- From the Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA
| | - Jane M DeVasure
- From the Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA
| | - Todd A Wyatt
- From the Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA
- Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, NE, USA
- Nebraska-Western Iowa VA Healthcare System, Research Service, Omaha, NE, USA
| | - Matthew C Zimmerman
- Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA
| | - Joseph H Sisson
- From the Department of Internal Medicine, Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA.
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15
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Freund JR, Mansfield CJ, Doghramji LJ, Adappa ND, Palmer JN, Kennedy DW, Reed DR, Jiang P, Lee RJ. Activation of airway epithelial bitter taste receptors by Pseudomonas aeruginosa quinolones modulates calcium, cyclic-AMP, and nitric oxide signaling. J Biol Chem 2018; 293:9824-9840. [PMID: 29748385 DOI: 10.1074/jbc.ra117.001005] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2017] [Revised: 04/17/2018] [Indexed: 12/12/2022] Open
Abstract
Bitter taste receptors (taste family 2 bitter receptor proteins; T2Rs), discovered in many tissues outside the tongue, have recently become potential therapeutic targets. We have shown previously that airway epithelial cells express several T2Rs that activate innate immune responses that may be important for treatment of airway diseases such as chronic rhinosinusitis. It is imperative to more clearly understand what compounds activate airway T2Rs as well as their full range of functions. T2R isoforms in airway motile cilia (T2R4, -14, -16, and -38) produce bactericidal levels of nitric oxide (NO) that also increase ciliary beating, promoting clearance of mucus and trapped pathogens. Bacterial quorum-sensing acyl-homoserine lactones activate T2Rs and stimulate these responses in primary airway cells. Quinolones are another type of quorum-sensing molecule used by Pseudomonas aeruginosa To elucidate whether bacterial quinolones activate airway T2Rs, we analyzed calcium, cAMP, and NO dynamics using a combination of fluorescent indicator dyes and FRET-based protein biosensors. T2R-transfected HEK293T cells, several lung epithelial cell lines, and primary sinonasal cells grown and differentiated at the air-liquid interface were tested with 2-heptyl-3-hydroxy-4-quinolone (known as Pseudomonas quinolone signal; PQS), 2,4-dihydroxyquinolone, and 4-hydroxy-2-heptylquinolone (HHQ). In HEK293T cells, PQS activated T2R4, -16, and -38, whereas HHQ activated T2R14. 2,4-Dihydroxyquinolone had no effect. PQS and HHQ increased calcium and decreased both baseline and stimulated cAMP levels in cultured and primary airway cells. In primary cells, PQS and HHQ activated levels of NO synthesis previously shown to be bactericidal. This study suggests that airway T2R-mediated immune responses are activated by bacterial quinolones as well as acyl-homoserine lactones.
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Affiliation(s)
- Jenna R Freund
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | | | | | - Nithin D Adappa
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | - James N Palmer
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | - David W Kennedy
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and
| | - Danielle R Reed
- the Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104
| | - Peihua Jiang
- the Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104
| | - Robert J Lee
- From the Departments of Otorhinolaryngology-Head and Neck Surgery and .,Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104 and
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16
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Carson JL, Zhou L, Brighton L, Mills KH, Zhou H, Jaspers I, Hazucha M. Temporal structure/function variation in cultured differentiated human nasal epithelium associated with acute single exposure to tobacco smoke or E-cigarette vapor. Inhal Toxicol 2017; 29:137-144. [PMID: 28470140 PMCID: PMC5545111 DOI: 10.1080/08958378.2017.1318985] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Revised: 03/09/2017] [Accepted: 04/10/2017] [Indexed: 01/08/2023]
Abstract
OBJECTIVE Mucociliary clearance sustains a baseline functionality and an "on demand" capability to upregulate clearance upon irritant exposure involving mucus hypersecretion and accelerated ciliary beat frequency (CBF) modulated by nitric oxide (NO). This study characterized these elements as well as cellular and exogenous NO concentrations subsequent to a single exposure to tobacco smoke (TS) or e-cigarette vapor (EV) on cultured human airway epithelium. MATERIALS AND METHODS Air-liquid interface (ALI) airway epithelial cultures per nonsmoking human subjects were subjected to single TS or EV exposures. Measures of ciliary function and secretion were performed and cellular and exogenous NO concentrations under control and experimental conditions were assessed. RESULTS Both TS and EV exposures resulted similar patterns of decline in CBF within 1 min of the completion of exposure followed by a gradual return often exceeding baseline within 1 h. Post-exposure examination of exposed cultures suggested morphologic differences in secretory function relative to controls. The relative NO concentrations of TS and EV chamber air were sharply different with EV NO being only slightly elevated relative to cellular NO production. DISCUSSION AND CONCLUSIONS Epithelial remodeling and mucociliary dysfunction have been clearly associated with TS exposure. However, information contrasting epithelial structure/function following a single acute TS or EV exposure is limited. This study demonstrates a similar pattern of epithelial response to acute TS or EV exposure. Inasmuch as NO may contribute to an inflammatory milieu and generation of toxic metabolites, it is plausible that recurrent exposures over time may be contributory to chronic pathologies.
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Affiliation(s)
- Johnny L. Carson
- The Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- The Department of Pediatrics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Laura Zhou
- The Department of Biostatistics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Luisa Brighton
- The Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Katherine H. Mills
- The Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Haibo Zhou
- The Department of Biostatistics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Ilona Jaspers
- The Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- The Department of Pediatrics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Milan Hazucha
- The Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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17
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Price ME, Pavlik JA, Liu M, Ding SJ, Wyatt TA, Sisson JH. Alcohol drives S-nitrosylation and redox activation of protein phosphatase 1, causing bovine airway cilia dysfunction. Am J Physiol Lung Cell Mol Physiol 2017; 312:L432-L439. [PMID: 28062487 DOI: 10.1152/ajplung.00513.2016] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2016] [Revised: 12/29/2016] [Accepted: 01/05/2017] [Indexed: 11/22/2022] Open
Abstract
Individuals with alcohol (ethanol)-use disorders are at increased risk for lung infections, in part, due to defective mucociliary clearance driven by motile cilia in the airways. We recently reported that isolated, demembranated bovine cilia (axonemes) are capable of producing nitric oxide (∙NO) when exposed to biologically relevant concentrations of alcohol. This increased presence of ∙NO can lead to protein S-nitrosylation, a posttranslational modification signaling mechanism involving reversible adduction of nitrosonium cations or ∙NO to thiolate or thiyl radicals, respectively, of proteins forming S-nitrosothiols (SNOs). We quantified and compared SNO content between isolated, demembranated axonemes extracted from bovine tracheae, with or without in situ alcohol exposure (100 mM × 24 h). We demonstrate that relevant concentrations of alcohol exposure shift the S-nitrosylation status of key cilia regulatory proteins, including 20-fold increases in S-nitrosylation of proteins that include protein phosphatase 1 (PP1). With the use of an ATP-reactivated axoneme motility system, we demonstrate that alcohol-driven S-nitrosylation of PP1 is associated with PP1 activation and dysfunction of axoneme motility. These new data demonstrate that alcohol can shift the S-nitrothiol balance at the level of the cilia organelle and highlight S-nitrosylation as a novel signaling mechanism to regulate PP1 and cilia motility.
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Affiliation(s)
- Michael E Price
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska.,Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Jacqueline A Pavlik
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Miao Liu
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Shi-Jian Ding
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
| | - Todd A Wyatt
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska.,Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska; and.,Research Service, Nebraska-Western Iowa VA Healthcare System, Omaha, Nebraska
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska;
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18
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Gerald CL, Romberger DJ, DeVasure JM, Khazanchi R, Nordgren TM, Heires AJ, Sisson JH, Wyatt TA. Alcohol Decreases Organic Dust-Stimulated Airway Epithelial TNF-Alpha Through a Nitric Oxide and Protein Kinase-Mediated Inhibition of TACE. Alcohol Clin Exp Res 2016; 40:273-83. [PMID: 26842246 DOI: 10.1111/acer.12967] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 11/18/2015] [Indexed: 12/24/2022]
Abstract
BACKGROUND Farm workers in rural areas consume more alcohol than those who reside in urban areas. Occupational exposures such as agricultural work can pose hazards on the respiratory system. It is established that hog barn dust induces inflammation in the airway, including the release of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8. We have shown that alcohol alters airway epithelial innate defense through changes in both nitric oxide (NO) and cAMP-dependent protein kinase A (PKA). Simultaneous exposure to hog barn dust and alcohol decreases inflammatory mediators, TNF-α, IL-6, and IL-8, in mice. Previously, mice exposed to both alcohol and hog barn dust showed a depleted amount of lymphocytes compared to mice exposed only to hog barn dust. Weakening of the innate immune response could lead to enhanced susceptibility to disease. In addition, mice that were co-exposed to hog barn dust and alcohol also experienced increased mortality. METHODS Because we recently demonstrated that PKA activation inhibits the TNF-α sheddase, TNF-α-converting enzyme (TACE), we hypothesized that an alcohol-mediated PKA pathway blocks TACE activity and prevents the normative inflammatory response to hog barn dust exposure. To delineate these effects, we used PKA pathway inhibitors (adenylyl cyclase [AC], cAMP, and PKA) to modulate the effects of alcohol on dust-stimulated TNF-α release in the bronchial epithelial cell line, BEAS-2B. Alcohol pretreatment blocked TACE activity and TNF-α release in hog barn dust-treated cells. RESULTS Alcohol continued to block hog barn dust-mediated TNF-α release in the presence of the particulate AC inhibitor, SQ22,536. The soluble adenylyl cyclase inhibitor, KH7, however, significantly increased the inflammatory response to hog barn dust. phosphodiesterase 4 inhibitors significantly elevated cAMP and enhanced alcohol-mediated inhibition of dust-stimulated TNF-α release. In addition, the NO synthase inhibitor, l-NMMA, also reversed the alcohol-blocking effect on dust-stimulated TNF-α. CONCLUSIONS These data suggest that alcohol requires a soluble cyclase-generated cAMP-PKA pathway that is dependent upon the action of NO to inhibit TACE and TNF-α release. These findings support our observations that alcohol functions through a dual NO and PKA pathway in bronchial epithelial cells.
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Affiliation(s)
- Carresse L Gerald
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska
| | - Debra J Romberger
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska.,Department of Veterans Affairs Medical Center, VA Nebraska-Western Iowa Health Care System Research Service, Omaha, Nebraska
| | - Jane M DeVasure
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska
| | - Rohan Khazanchi
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska
| | - Tara M Nordgren
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska
| | - Art J Heires
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska
| | - Todd A Wyatt
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska.,Department of Veterans Affairs Medical Center, VA Nebraska-Western Iowa Health Care System Research Service, Omaha, Nebraska.,Department of Environmental, Agricultural, and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska
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19
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Yeligar SM, Chen MM, Kovacs EJ, Sisson JH, Burnham EL, Brown LAS. Alcohol and lung injury and immunity. Alcohol 2016; 55:51-59. [PMID: 27788778 DOI: 10.1016/j.alcohol.2016.08.005] [Citation(s) in RCA: 82] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Revised: 07/07/2016] [Accepted: 08/24/2016] [Indexed: 02/06/2023]
Abstract
Annually, excessive alcohol use accounts for more than $220 billion in economic costs and 80,000 deaths, making excessive alcohol use the third leading lifestyle-related cause of death in the US. Patients with an alcohol-use disorder (AUD) also have an increased susceptibility to respiratory pathogens and lung injury, including a 2-4-fold increased risk of acute respiratory distress syndrome (ARDS). This review investigates some of the potential mechanisms by which alcohol causes lung injury and impairs lung immunity. In intoxicated individuals with burn injuries, activation of the gut-liver axis drives pulmonary inflammation, thereby negatively impacting morbidity and mortality. In the lung, the upper airway is the first checkpoint to fail in microbe clearance during alcohol-induced lung immune dysfunction. Brief and prolonged alcohol exposure drive different post-translational modifications of novel proteins that control cilia function. Proteomic approaches are needed to identify novel alcohol targets and post-translational modifications in airway cilia that are involved in alcohol-dependent signal transduction pathways. When the upper airway fails to clear inhaled pathogens, they enter the alveolar space where they are primarily cleared by alveolar macrophages (AM). With chronic alcohol ingestion, oxidative stress pathways in the AMs are stimulated, thereby impairing AM immune capacity and pathogen clearance. The epidemiology of pneumococcal pneumonia and AUDs is well established, as both increased predisposition and illness severity have been reported. AUD subjects have increased susceptibility to pneumococcal pneumonia infections, which may be due to the pro-inflammatory response of AMs, leading to increased oxidative stress.
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Affiliation(s)
- Samantha M Yeligar
- Department of Medicine, Emory University and Atlanta Veterans Affairs Medical Center, Decatur, GA 30033, USA
| | - Michael M Chen
- Burn and Shock Trauma Research Institute, Alcohol Research Program, Integrative Cell Biology Program, Loyola University Chicago Stritch School of Medicine, Maywood, IL 60153, USA
| | - Elizabeth J Kovacs
- Department of Surgery, University of Colorado School of Medicine, Aurora, CO 80045, USA
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Ellen L Burnham
- Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine, Aurora, CO 80045, USA
| | - Lou Ann S Brown
- Department of Pediatrics, Emory University, Atlanta, GA 30322, USA.
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20
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Afshar M, Poole JA, Cao G, Durazo R, Cooper RC, Kovacs EJ, Sisson JH. Exhaled Nitric Oxide Levels Among Adults With Excessive Alcohol Consumption. Chest 2016; 150:196-209. [PMID: 26905362 DOI: 10.1016/j.chest.2016.02.642] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2015] [Revised: 02/08/2016] [Accepted: 02/09/2016] [Indexed: 11/26/2022] Open
Abstract
BACKGROUND More than one-quarter of the US population qualify as excessive alcohol consumers. Alcohol use impacts several lung diseases, and heavy consumption has been associated with poor clinical outcomes. The fractional excretion of exhaled nitric oxide (Feno) has clinical implications in multiple airways diseases. We hypothesized that excessive alcohol intake is associated with lower Feno levels. METHODS To test this hypothesis, we examined a sample consisting of 12,059 participants, aged 21 to 79 years, interviewed between 2007 and 2012 from the National Health and Examination Survey. Two valid Feno measurements that were reproducible were recorded. Alcohol questionnaire data were used to define the following alcohol groups: never drinkers, nonexcessive drinkers, excessive drinkers, and former excessive drinkers. The natural logarithm of Feno values [ln(Feno)] as well as blood eosinophil count and C-reactive protein were used as dependent variables to test the association with alcohol groups including multivariable linear regression models with adjustment for predictors of Feno. RESULTS Excessive alcohol consumption comprised 3,693 (26.9%) of the US sample population. Controlling for all other factors, excessive alcohol consumption had a negative association and was an independent predictor for ln(Feno) levels in comparison with the never-drinker group (-0.11; 95% CI, -0.17 to -0.06; P < .001). ln(Feno) levels decreased across categories of increasing alcohol use (P < .001). CONCLUSIONS Accounting for alcohol use in the interpretation of Feno levels should be an additional consideration, and further investigations are warranted to explore the complex interaction between alcohol and nitric oxide in the airways.
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Affiliation(s)
- Majid Afshar
- Division of Pulmonary and Critical Care Medicine, Loyola University Chicago Health Sciences Campus, Maywood, IL; Department of Public Health Sciences, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL; Alcohol Research Program, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL.
| | - Jill A Poole
- Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Nebraska Medical Center, Omaha, NE
| | - Guichan Cao
- Department of Public Health Sciences, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL
| | - Ramon Durazo
- Department of Public Health Sciences, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL
| | - Richard C Cooper
- Department of Public Health Sciences, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL
| | - Elizabeth J Kovacs
- Department of Surgery, Loyola University Chicago Health Sciences Campus, Maywood, IL; Alcohol Research Program, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL; Burn and Shock Trauma Research Institute, Stritch School of Medicine, Loyola University Chicago Health Sciences, Maywood, IL
| | - Joseph H Sisson
- Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Nebraska Medical Center, Omaha, NE
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21
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Mehta AJ. Alcoholism and critical illness: A review. World J Crit Care Med 2016; 5:27-35. [PMID: 26855891 PMCID: PMC4733453 DOI: 10.5492/wjccm.v5.i1.27] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2015] [Revised: 10/07/2015] [Accepted: 12/04/2015] [Indexed: 02/06/2023] Open
Abstract
Alcohol is the most commonly used and abused drug in the world, and alcohol use disorders pose a tremendous burden to healthcare systems around the world. The lifetime prevalence of alcohol abuse in the United States is estimated to be around 18%, and the economic consequences of these disorders are staggering. Studies on hospitalized patients demonstrate that about one in four patients admitted to critical care units will have alcohol-related issues, and unhealthy alcohol consumption is responsible for numerous clinical problems encountered in intensive care unit (ICU) settings. Patients with alcohol use disorders are not only predisposed to developing withdrawal syndromes and other conditions that often require intensive care, they also experience a considerably higher rate of complications, longer ICU and hospital length of stay, greater resource utilization, and significantly increased mortality compared to similar critically ill patients who do not abuse alcohol. Specific disorders seen in the critical care setting that are impacted by alcohol abuse include delirium, pneumonia, acute respiratory distress syndrome, sepsis, gastrointestinal hemorrhage, trauma, and burn injuries. Despite the substantial burden of alcohol-induced disease in these settings, critical care providers often fail to identify individuals with alcohol use disorders, which can have significant implications for this vulnerable population and delay important clinical interventions.
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Cho DY, Skinner D, Zhang S, Fortenberry J, Sorscher EJ, Dean NR, Woodworth BA. Cystic fibrosis transmembrane conductance regulator activation by the solvent ethanol: implications for topical drug delivery. Int Forum Allergy Rhinol 2015; 6:178-84. [PMID: 26869199 DOI: 10.1002/alr.21638] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2015] [Revised: 07/28/2015] [Accepted: 08/04/2015] [Indexed: 02/03/2023]
Abstract
BACKGROUND Decreased cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl) secretion across mucosal surfaces contributes to the development of airway disease by depleting airway surface liquid, increasing mucus viscosity and adhesion, and consequently hindering mucociliary clearance. We serendipitously discovered during testing of drugs solubilized in low concentrations ethanol (0.25%, 43 mM) that the control vehicle produced robust activation of CFTR-mediated Cl(-) transport. The objective of the current study is to investigate low concentrations of ethanol for effects on Cl(-) secretion and ciliary beat frequency (CBF). METHODS Wild-type (WT) and transgenic CFTR(-/-) primary murine nasoseptal epithelial (MNSE) cultures and WT and F508del/F508del human sinonasal epithelial (HSNE) cultures were subjected to transepithelial ion transport measurements using pharmacologic manipulation in Ussing chambers. CBF activation was also monitored. Murine nasal potential difference (NPD) was measured in vivo. RESULTS Ussing chamber tracings revealed ethanol activated CFTR-mediated Cl transport in a dose-dependent fashion in WT MNSE (n = 4, p < 0.05) and HSNE (n = 4, p < 0.05). Ethanol also significantly increased CBF (fold change) in WT MNSE cultures in a dose-dependent fashion (phosphate-buffered saline [PBS], 1.33 ± 0.04; 0.25% ethanol, 1.37 ± 0.09; 0.5% ethanol, 1.53 ± 0.06 [p < 0.05]; 1% ethanol, 1.62 ± 0.1 [p < 0.05]). Lack of stimulation in CFTR(-/-) and F508del/F508del cultures indicated activity was dependent on the presence of intact functional CFTR. Ethanol perfusion (0.5%) resulted in a significant -3.5-mV mean NPD polarization when compared to control solution (p < 0.05). CONCLUSION The observation that brief exposure of ethanol stimulated Cl(-) secretion via CFTR-mediated pathways indicates its possible use as topical aerosol delivered alone or in combination with other CFTR activators for diseases of dysfunctional mucociliary clearance (MCC) in chronic rhinosinusitis (CRS).
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Affiliation(s)
- Do-Yeon Cho
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Birmingham, AL
| | - Daniel Skinner
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Shaoyan Zhang
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - James Fortenberry
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Birmingham, AL
| | - Eric J Sorscher
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Birmingham, AL.,Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Birmingham, AL.,Department of Medicine, University of Alabama at Birmingham, Birmingham, Birmingham, AL
| | - Nichole R Dean
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Bradford A Woodworth
- Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Birmingham, AL
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Abstract
Motile cilia of the lungs respond to environmental challenges by increasing their ciliary beat frequency in order to enhance mucociliary clearance as a fundamental tenant of innate defense. One important second messenger in transducing the regulable nature of motile cilia is cyclic guanosine 3′,5′-monophosphate (cGMP). In this review, the history of cGMP action is presented and a survey of the existing data addressing cGMP action in ciliary motility is presented. Nitric oxide (NO)-mediated regulation of cGMP in ciliated cells is presented in the context of alcohol-induced cilia function and dysfunction.
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Affiliation(s)
- Todd A Wyatt
- VA Nebraska-Western Iowa Health Care System, Research Service, Department of Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, NE 68105, USA.
- Department of Environmental, Agricultural, and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198-5910, USA.
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, 985910 Nebraska Medical Center, Omaha, NE 68198-5910, USA .
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Yang F, Pavlik J, Fox L, Scarbrough C, Sale WS, Sisson JH, Wirschell M. Alcohol-induced ciliary dysfunction targets the outer dynein arm. Am J Physiol Lung Cell Mol Physiol 2015; 308:L569-76. [PMID: 25595647 DOI: 10.1152/ajplung.00257.2014] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Alcohol abuse results in an increased incidence of pulmonary infection, in part attributable to impaired mucociliary clearance. Analysis of motility in mammalian airway cilia has revealed that alcohol impacts the ciliary dynein motors by a mechanism involving altered axonemal protein phosphorylation. Given the highly conserved nature of cilia, it is likely that the mechanisms for alcohol-induced ciliary dysfunction (AICD) are conserved. Thus we utilized the experimental advantages offered by the model organism, Chlamydomonas, to determine the precise effects of alcohol on ciliary dynein activity and identify axonemal phosphoproteins that are altered by alcohol exposure. Analysis of live cells or reactivated cell models showed that alcohol significantly inhibits ciliary motility in Chlamydomonas via a mechanism that is part of the axonemal structure. Taking advantage of informative mutant cells, we found that alcohol impacts the activity of the outer dynein arm. Consistent with this finding, alcohol exposure results in a significant reduction in ciliary beat frequency, a parameter of ciliary movement that requires normal outer dynein arm function. Using mutants that lack specific heavy-chain motor domains, we have determined that alcohol impacts the β- and γ-heavy chains of the outer dynein arm. Furthermore, using a phospho-threonine-specific antibody, we determined that the phosphorylation state of DCC1 of the outer dynein arm-docking complex is altered in the presence of alcohol, and its phosphorylation correlates with AICD. These results demonstrate that alcohol targets specific outer dynein arm components and suggest that DCC1 is part of an alcohol-sensitive mechanism that controls outer dynein arm activity.
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Affiliation(s)
- Fan Yang
- University of Mississippi Medical Center, Department of Biochemistry, Jackson, Mississippi
| | - Jacqueline Pavlik
- University of Nebraska Medical Center, Department of Internal Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, Omaha, Nebraska; and
| | - Laura Fox
- Emory University School of Medicine, Department of Cell Biology, Atlanta, Georgia
| | - Chasity Scarbrough
- University of Mississippi Medical Center, Department of Biochemistry, Jackson, Mississippi
| | - Winfield S Sale
- Emory University School of Medicine, Department of Cell Biology, Atlanta, Georgia
| | - Joseph H Sisson
- University of Nebraska Medical Center, Department of Internal Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, Omaha, Nebraska; and
| | - Maureen Wirschell
- University of Mississippi Medical Center, Department of Biochemistry, Jackson, Mississippi;
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Price ME, Pavlik JA, Sisson JH, Wyatt TA. Inhibition of protein phosphatase 1 reverses alcohol-induced ciliary dysfunction. Am J Physiol Lung Cell Mol Physiol 2015; 308:L577-85. [PMID: 25575517 DOI: 10.1152/ajplung.00336.2014] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Airway mucociliary clearance is a first-line defense of the lung against inhaled particles and debris. Among individuals with alcohol use disorders, there is an increase in lung diseases. We previously identified that prolonged alcohol exposure impairs mucociliary clearance, known as alcohol-induced ciliary dysfunction (AICD). Cilia-localized enzymes, known as the ciliary metabolon, are key to the pathogenesis of AICD. In AICD, cyclic nucleotide-dependent ciliary kinases, which modulate phosphorylation to regulate cilia beat, are desensitized. We hypothesized that alcohol activates cilia-associated protein phosphatase 1 (PP1) activity, driving phosphorylation changes of cilia motility regulatory proteins. To test this hypothesis we identified the effects of prolonged alcohol exposure on phosphatase activity, cilia beat, and kinase responsiveness and cilia-associated phosphorylation targets when stimulated by β-agonist or cAMP. Prolonged alcohol activated PP1 and blocked cAMP-dependent cilia beat and protein kinase A (PKA) responsiveness and phosphorylation of a 29-kDa substrate of PKA. Importantly, prolonged alcohol-induced phosphatase activation was inhibited by the PP1 specific inhibitor, inhibitor-2 (I-2), restoring cAMP-stimulated cilia beat and PKA responsiveness and phosphorylation of the 29-kDa substrate. The I-2 inhibitory effect persisted in tissue, cell, and isolated cilia-organelle models, highlighting the association of ciliary metabolon-localized enzymes to AICD. Prolonged alcohol exposure drives ciliary metabolon-localized PP1 activation. PP1 activation modifies phosphorylation of a 29-kDa protein related to PKA activity. These data reinforce our previous findings that alcohol is acting at the level of the ciliary metabolon to cause ciliary dysfunction and identifies PP1 as a therapeutic target to prevent or reverse AICD.
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Affiliation(s)
- Michael E Price
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Jacqueline A Pavlik
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska
| | - Joseph H Sisson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska;
| | - Todd A Wyatt
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska; Veterans Affairs Nebraska-Western Iowa Healthcare System, Research Service, Omaha, Nebraska; and Department of Environmental, Agricultural, and Occupational Health, University of Nebraska Medical Center, Omaha, Nebraska
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26
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Simet SM, Sisson JH. Alcohol's Effects on Lung Health and Immunity. Alcohol Res 2015; 37:199-208. [PMID: 26695745 PMCID: PMC4590617] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
It has long been known that people with alcohol use disorder (AUD) not only may develop physical dependence but also may experience devastating long-term health problems. The most common and identifiable alcohol-associated health problems include liver cirrhosis, pancreatitis, cardiomyopathies, neuropathies, and dementia. However, the lung also is adversely affected by alcohol abuse, a fact often overlooked by clinicians and the public. Individuals with AUD are more likely to develop pneumonia, tuberculosis (TB), respiratory syncytial virus (RSV) infection, and acute respiratory distress syndrome (ARDS). Increased susceptibility to these and other pulmonary infections is caused by impaired immune responses in people with AUD. The key immune cells involved in combating pulmonary conditions such as pneumonia, TB, RSV infection, and ARDS are neutrophils, lymphocytes, alveolar macrophages, and the cells responsible for innate immune responses. Researchers are only now beginning to understand how alcohol affects these cells and how these effects contribute to the pathophysiology of pulmonary diseases in people with AUD.
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Jackson CL, Lucas JS, Walker WT, Owen H, Premadeva I, Lackie PM. Neuronal NOS localises to human airway cilia. Nitric Oxide 2014; 44:3-7. [PMID: 25460324 DOI: 10.1016/j.niox.2014.11.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2014] [Revised: 10/21/2014] [Accepted: 11/03/2014] [Indexed: 01/13/2023]
Abstract
BACKGROUND Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. METHODS AND RESULTS Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. CONCLUSIONS We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function.
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Affiliation(s)
- Claire L Jackson
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK.
| | - Jane S Lucas
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
| | - Woolf T Walker
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
| | - Holly Owen
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Irnthu Premadeva
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK
| | - Peter M Lackie
- Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
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28
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Abstract
A characteristic feature of the human airway epithelium is the presence of ciliated cells bearing motile cilia, specialized cell surface projections containing axonemes composed of microtubules and dynein arms, which provide ATP-driven motility. In the airways, cilia function in concert with airway mucus to mediate the critical function of mucociliary clearance, cleansing the airways of inhaled particles and pathogens. The prototypical disorder of respiratory cilia is primary ciliary dyskinesia, an inherited disorder that leads to impaired mucociliary clearance, to repeated chest infections, and to the progressive destruction of lung architecture. Numerous acquired lung diseases are also marked by abnormalities in both cilia structure and function. In this review we summarize current knowledge regarding airway ciliated cells and cilia, how they function to maintain a healthy epithelium, and how disorders of cilia structure and function contribute to inherited and acquired lung disease.
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Montuschi P, Paris D, Montella S, Melck D, Mirra V, Santini G, Mores N, Montemitro E, Majo F, Lucidi V, Bush A, Motta A, Santamaria F. Nuclear Magnetic Resonance–based Metabolomics Discriminates Primary Ciliary Dyskinesia from Cystic Fibrosis. Am J Respir Crit Care Med 2014; 190:229-33. [DOI: 10.1164/rccm.201402-0249le] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
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Asymmetric dimethylarginine blocks nitric oxide-mediated alcohol-stimulated cilia beating. Mediators Inflamm 2013; 2013:592892. [PMID: 24307761 PMCID: PMC3836567 DOI: 10.1155/2013/592892] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2013] [Revised: 09/16/2013] [Accepted: 09/18/2013] [Indexed: 01/29/2023] Open
Abstract
The airway epithelium is exposed to alcohol during drinking through direct exhalation of volatized ethanol from the bronchial circulation. Alcohol exposure leads to a rapid increase in the cilia beat frequency (CBF) of bronchial epithelial cells followed by a chronic desensitization of cilia stimulatory responses. This effect is governed in part by the nitric oxide regulation of cyclic guanosine and adenosine monophosphate-dependent protein kinases (PKG and PKA) and is not fully understood. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is implicated in the pathogenesis of several pulmonary disorders. We hypothesized that the inhibition of nitric oxide synthase by ADMA blocks alcohol-stimulated increases in CBF. To test this hypothesis, ciliated primary bovine bronchial epithelial cells (BBEC) were preincubated with ADMA (100 µM) and stimulated with 100 mM ethanol. CBF was measured and PKA assayed. By 1 hr, ethanol activated PKA, resulting in elevated CBF. Both alcohol-induced PKA activation and CBF were inhibited in the presence of ADMA. ADMA alone had no effect on PKA activity or CBF. Using a mouse model overexpressing the ADMA-degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), we examined PKA and CBF in precision-cut mouse lung slices. Alcohol-stimulated increases in lung slice PKA and CBF were temporally enhanced in the DDAH mice versus control mice.
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31
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Abstract
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.
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Simet SM, Pavlik JA, Sisson JH. Proteomic analysis of bovine axonemes exposed to acute alcohol: role of endothelial nitric oxide synthase and heat shock protein 90 in cilia stimulation. Alcohol Clin Exp Res 2012; 37:609-15. [PMID: 23078267 DOI: 10.1111/acer.12014] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2012] [Accepted: 08/17/2012] [Indexed: 12/17/2022]
Abstract
BACKGROUND Cilia are finger-like motor-driven organelles, which propel inhaled particles and mucus from the lung and airways. We have previously shown that brief alcohol exposure stimulates ciliary motility through an endothelial nitric oxide synthase (eNOS)-dependent pathway localized in the ciliary metabolon. However, the signaling molecules of the ciliary metabolon involved in alcohol-triggered ciliary beat frequency (CBF) stimulation upstream of eNOS activation remain unknown. METHODS We hypothesized that brief alcohol exposure alters threonine and serine phosphorylation of proteins involved in stimulating CBF. Two-dimensional electrophoresis indicated both increases and decreases in the serine and threonine phosphorylation states of several proteins. One of the proteins identified was heat shock protein 90 (HSP90), which undergoes increased threonine phosphorylation after brief alcohol exposure. Because HSP90 has been shown to associate with eNOS in lung tissue, we hypothesized that HSP90 is a key component in alcohol-triggered eNOS activation and that these 2 proteins co-localize within the ciliary metabolon. RESULTS Immunofluorescence experiments demonstrate that eNOS and HSP90 co-localize within basal bodies of the ciliary metabolon and partially translocate to the axoneme upon brief alcohol exposure. Pretreatment with geldanamycin, which disrupts HSP90 chaperone functions, prevented eNOS-HSP90 association and prevented the translocation of eNOS from the ciliary metabolon to the axoneme. Functional cilia motility studies revealed that geldanamycin blocked alcohol-stimulated ciliary motility in bovine bronchial epithelial cells and mouse tracheal rings. CONCLUSIONS On the basis of the HSP90 localization with eNOS, alcohol activation of HSP90 phosphorylation, and geldanamycin's ability to inhibit HSP90-eNOS association, prevent eNOS translocation to the axoneme, and block alcohol-stimulated ciliary motility, we conclude that alcohol-induced cilia stimulation occurs through the increased association of HSP90 with eNOS. These data help further elucidate the mechanism through which brief alcohol exposure stimulates CBF.
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Affiliation(s)
- Samantha M Simet
- Pulmonary, Critical Care, Sleep, and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA
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Navarrette CR, Sisson JH, Nance E, Allen-Gipson D, Hanes J, Wyatt TA. Particulate matter in cigarette smoke increases ciliary axoneme beating through mechanical stimulation. J Aerosol Med Pulm Drug Deliv 2012; 25:159-68. [PMID: 22280523 PMCID: PMC3377952 DOI: 10.1089/jamp.2011.0890] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2011] [Accepted: 11/20/2011] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND The lung's ability to trap and clear foreign particles via the mucociliary elevator is an important mechanism for protecting the lung against respirable irritants and microorganisms. Although cigarette smoke (CS) exposure and particulate inhalation are known to alter mucociliary clearance, little is known about how CS and nanoparticles (NPs) modify cilia beating at the cytoskeletal infrastructure, or axonemal, level. METHODS We used a cell-free model to introduce cigarette smoke extract (CSE) and NPs with variant size and surface chemistry to isolated axonemes and measured changes in ciliary motility. We hypothesized that CSE would alter cilia beating and that alterations in ciliary beat frequency (CBF) due to particulate matter would be size- and surface chemistry-dependent. Demembranated axonemes were isolated from ciliated bovine tracheas and exposed to adenosine triphosphate (ATP) to initiate motility. CBF was measured in response to 5% CSE, CSE filtrate, and carboxyl-modified (COOH), sulphate (SO(4))-modified (sulfonated), or PEG-coated polystyrene (PS) latex NPs ranging in size from 40 nm to 500 nm. RESULTS CSE concentrations as low as 5% resulted in rapid, significant stimulation of CBF (p<0.05 vs. baseline control). Filtering CSE through a 0.2-μm filter attenuated this effect. Introduction of sulphate-modified PS beads ~300 nm in diameter resulted in a similar increase in CBF above baseline ATP levels. Uncharged, PEG-coated beads had no effect on CBF regardless of size. Similarly, COOH-coated particles less than 200 nm in diameter did not alter ciliary motility. However, COOH-coated PS particles larger than 300 nm increased CBF significantly and increased the number of motile points. CONCLUSIONS These data show that NPs, including those found in CSE, mechanically stimulate axonemes in a size- and surface chemistry-dependent manner. Alterations in ciliary motility due to physicochemical properties of NPs may be important for inhalational lung injury and efficient drug delivery of respirable particles.
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Affiliation(s)
- Chelsea R. Navarrette
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
| | - Joseph H. Sisson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
| | - Elizabeth Nance
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University School of Engineering, Baltimore, Mayland
| | - Diane Allen-Gipson
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
| | - Justin Hanes
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University School of Engineering, Baltimore, Mayland
- Center for Nanomedicine and Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Todd A. Wyatt
- Pulmonary, Critical Care, Sleep & Allergy Division, Department of Internal Medicine, Omaha, Nebraska
- VA Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska
- Department of Environmental, Agricultural, and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska
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Oldenburg PJ, Poole JA, Sisson JH. Alcohol reduces airway hyperresponsiveness (AHR) and allergic airway inflammation in mice. Am J Physiol Lung Cell Mol Physiol 2011; 302:L308-15. [PMID: 22114149 DOI: 10.1152/ajplung.00077.2011] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
There is very limited knowledge about the effects of alcohol on airway hyperresponsiveness and inflammation in asthma. Historical accounts of alcohol administration to patients with breathing problems suggest that alcohol may have bronchodilating properties. We hypothesized that alcohol exposure will alter airway hyperresponsiveness (AHR) and pulmonary inflammation in a mouse model of allergic asthma. To test this hypothesis, BALB/c mice were fed either 18% alcohol or water and then sensitized and challenged with ovalbumin (OVA). AHR was assessed by means of ventilation or barometric plethysmography and reported as either total lung resistance or enhanced pause, respectively. Airway inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluid (BALF), cytokine levels in BALF, lung histology, and serum immunoglobulin E (IgE) levels. Alcohol feeding significantly blocked methacholine-induced increases in AHR compared with water-fed controls. Alcohol feeding significantly reduced total cell numbers (64%) as well as the number of eosinophils (84%) recruited to the lungs of these mice. Modest changes in lung pathology were also observed. Alcohol exposure led to a reduction of IgE in the serum of the EtOH OVA mice. These data demonstrate that alcohol exposure blunts AHR and dampens allergic airway inflammation indices in allergic mice and suggest that there may be an important role for alcohol in the modulation of asthma. These data provide an in vivo basis for previous clinical observations in humans substantiating the bronchodilator properties of alcohol and for the first time demonstrates an alcohol-induced reduction of allergic inflammatory cells in a mouse model of allergic asthma.
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Affiliation(s)
- Peter J Oldenburg
- Nebraska Medical Center, Univ. of Nebraska Medical Ctr., Omaha, NE 68198-5910, USA
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Jain R, Ray JM, Pan JH, Brody SL. Sex hormone-dependent regulation of cilia beat frequency in airway epithelium. Am J Respir Cell Mol Biol 2011; 46:446-53. [PMID: 22033264 DOI: 10.1165/rcmb.2011-0107oc] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Previous studies have demonstrated a female disadvantage in airway diseases, such as asthma and bronchiectasis. The basis for this sex disparity is unknown. We hypothesized that the female sex hormone, progesterone (P4), inhibits functions of the normal airway mucociliary apparatus. P4 receptor (PR) expression was evaluated in human lung and cultured primary human airway epithelial cells isolated from male and female lung transplant donors. PR expression was restricted to the proximal region of the cilia of airway epithelia, and was similar in men and women. Expression of isoform PR-B was more abundant than PR-A in cells from both sexes. Airway epithelial cell exposure to P4 decreased cilia beat frequency (CBF) by 42.3% (±7.2). Inhibition of CBF was prevented by coadministration of P4 with the active form of estrogen, 17β-estradiol, or the PR antagonist, mifepristone. P4 inhibition was time and dose dependent, with a significant decrease by 8 hours and maximal effect at 24 hours, accompanied by translocation of PR from the cilia to the nucleus. Inhibition of cilia beat was also prevented by treatment of cells with actinomycin D, suggesting that CBF inhibition is a transcriptionally mediated event. Together, these findings indicate that sex hormones influence the function of a key component of the mucociliary apparatus. These mechanisms may contribute to the sex disparity present in airway diseases and provide therapeutic targets for the treatment of these debilitating airway diseases.
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Affiliation(s)
- Raksha Jain
- Department of Internal Medicine, Division of Pulmonary and Critical Care, Washington University School of Medicine, St. Louis, Missouri, USA.
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Jiao J, Wang H, Lou W, Jin S, Fan E, Li Y, Han D, Zhang L. Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells. Exp Cell Res 2011; 317:2548-53. [PMID: 21787770 DOI: 10.1016/j.yexcr.2011.07.007] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2011] [Revised: 07/07/2011] [Accepted: 07/09/2011] [Indexed: 11/30/2022]
Abstract
OBJECTIVES Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells. METHODS We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods. RESULTS L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N(G)-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium. CONCLUSION NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS-NO-sGC β-cGMP-PKG I pathway.
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Affiliation(s)
- Jian Jiao
- Key Laboratory of Otolaryngology, Head and Neck Surgery (Ministry of Education), Beijing Institute of Otolaryngology, China
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Tresguerres M, Levin LR, Buck J. Intracellular cAMP signaling by soluble adenylyl cyclase. Kidney Int 2011; 79:1277-88. [PMID: 21490586 DOI: 10.1038/ki.2011.95] [Citation(s) in RCA: 161] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cyclic adenosine 3',5' monophosphate (cAMP). sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems that are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells.
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Affiliation(s)
- Martin Tresguerres
- Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, USA
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Bailey KL, Robinson JE, Sisson JH, Wyatt TA. Alcohol decreases RhoA activity through a nitric oxide (NO)/cyclic GMP(cGMP)/protein kinase G (PKG)-dependent pathway in the airway epithelium. Alcohol Clin Exp Res 2011; 35:1277-81. [PMID: 21410486 DOI: 10.1111/j.1530-0277.2011.01463.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
BACKGROUND Alcohol has been shown to have a number of harmful effects on the lung, including increasing the risk of pneumonia and bronchitis. How alcohol increases the risk of these diseases is poorly defined. RhoA is a small guanosine triphosphate (GTP)ase that plays an integral role in many basic functions of airway epithelial cells. It is not known how alcohol affects RhoA activity in the airway epithelium. We hypothesized that brief alcohol exposure modulates RhoA activity in the airway epithelium through a nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG)-dependent pathway. METHODS Primary airway epithelial cells were cultured and exposed to ethanol at various concentrations and times. The cell layers were harvested and RhoA activity was measured. RESULTS Alcohol induced a time- and concentration-dependent decrease in RhoA activity in airway epithelial cells. We were able to block this decrease in activity using Nω-nitro-l-arginine methyl ester (L-NAME) hydrochloride, a nitric oxide synthase (NOS) inhibitor. Likewise, we were able to demonstrate the same decrease in RhoA activation using 0.1 μM sodium nitroprusside, an NO donor. To determine the role of cGMP/PKG, we pretreated the cells with a cGMP antagonist analog, Rp-8Br-cGMPS. This blocked the decrease in RhoA activity caused by alcohol, suggesting that alcohol exerts its effect on RhoA activity through cGMP/PKG. CONCLUSIONS Alcohol decreases airway epithelial RhoA activity through an NO/cGMP/PKG-dependent pathway. RhoA activity controls many aspects of basic cellular function, including cell morphology, tight junction formation, and cell cycle progression and gene regulation. Dysregulation of RhoA activity can potentially have several consequences, including dysregulation of inflammation. This may partially explain how alcohol increases the risk of pneumonia and bronchitis.
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Affiliation(s)
- Kristina L Bailey
- Pulmonary, Critical Care, Sleep & Allergy Division, University of Nebraska Medical Center, Omaha, NE 68198, USA.
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Lavogina D, Nickl CK, Enkvist E, Raidaru G, Lust M, Vaasa A, Uri A, Dostmann WR. Adenosine analogue-oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIalpha). BIOCHIMICA ET BIOPHYSICA ACTA 2010; 1804:1857-68. [PMID: 20406699 PMCID: PMC3071016 DOI: 10.1016/j.bbapap.2010.04.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/19/2010] [Revised: 04/12/2010] [Accepted: 04/13/2010] [Indexed: 10/24/2022]
Abstract
INTRODUCTION Type I cGMP-dependent protein kinase (PKGIalpha) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIalpha is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIalpha would lead to advances in the treatment of a variety of cardiovascular diseases. AIM Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIalpha to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. RESULTS Structure-activity profiling of ARCs with PKGIalpha in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC(50) values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1alpha-induced vasodilation.
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Affiliation(s)
- Darja Lavogina
- Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
| | - Christian K. Nickl
- Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Erki Enkvist
- Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
| | - Gerda Raidaru
- Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
| | - Marje Lust
- Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
| | - Angela Vaasa
- Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
| | - Asko Uri
- Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
| | - Wolfgang R. Dostmann
- Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA
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Dean GE, Finnell DS, Scribner M, Wang YJ, Steinbrenner LM, Gooneratne NS. Sleep in Lung Cancer. J Addict Nurs 2010. [DOI: 10.3109/10884601003777620] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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Oldenburg PJ, Wyatt TA, Sisson JH. Ethanol attenuates contraction of primary cultured rat airway smooth muscle cells. Am J Respir Cell Mol Biol 2009; 43:539-45. [PMID: 19933378 DOI: 10.1165/rcmb.2009-0252oc] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Airway smooth muscle cells are the main effector cells involved in airway narrowing and have been used to study the signaling pathways involved in asthma-induced airway constriction. Our previous studies demonstrated that ethanol administration to mice attenuated methacholine-stimulated increases in airway responsiveness. Because ethanol administration attenuates airway responsiveness in mice, we hypothesized that ethanol directly blunts the ability of cultured airway smooth muscle cells to shorten. To test this hypothesis, we measured changes in the size of cultured rat airway smooth muscle (RASM) cells exposed to ethanol (100 mM) after treatment with methacholine. Ethanol markedly attenuated methacholine-stimulated cell shortening (methacholine-stimulated length change = 8.3 ± 1.2% for ethanol versus 43.9 ± 1.5% for control; P < 0.001). Ethanol-induced inhibition of methacholine-stimulated cell shortening was reversible 24 hours after removal of alcohol. To determine if ethanol acts through a cGMP-dependent pathway, incubation with ethanol for as little as 15 minutes produced a doubling of cGMP-dependent protein kinase (PKG) activity. Furthermore, treatment with the PKG antagonist analog Rp-8Br-cGMPS (10 μM) inhibited ethanol-induced kinase activation when compared with control-treated cells. In contrast to the effect of ethanol on PKG, ethanol pretreatment did not activate a cAMP-dependent protein kinase. These data demonstrate that brief ethanol exposure reversibly prevents methacholine-stimulated RASM cell contraction. In addition, it appears that this effect is the result of activation of the cGMP/PKG kinase pathway. These findings implicate a direct effect of ethanol on airway smooth muscle cells as the basis for in vivo ethanol effects.
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Affiliation(s)
- Peter J Oldenburg
- Department of Internal Medicine, Pulmonary, Critical Care, Sleep, & Allergy Division, University of Nebraska Medical Center, Omaha, Nebraska 68198-5910, USA
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