176 results on '"Emala CW"'
Search Results
2. Renal Protection with Isoflurane
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Emala Cw and Lee Ht
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Kidney ,business.industry ,Ischemia ,medicine.disease ,Anesthesiology and Pain Medicine ,medicine.anatomical_structure ,Isoflurane ,Anesthesia ,medicine ,Phosphorylation ,Renal protection ,business ,Reperfusion injury ,medicine.drug - Published
- 2006
3. GABA Transporters Are Expressed in Human Airway Smooth Muscle and Epithelial Cells.
- Author
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Zaidi, S, primary, Xu, D, additional, and Emala, CW, additional
- Published
- 2009
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4. TNF-a increases transcription of Gai-2 in human airway smooth muscle cells
- Author
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Hotta, K, primary, Hirshman, CA, additional, and Emala, CW, additional
- Published
- 2001
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5. Muscarinic receptor-mediated reorganization of the actin ctyoskeleton is coupled through the Giα 2 protein in human airway smooth muscle cells
- Author
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Emala, CW, primary, Togashi, H, additional, Shao, D, additional, and Hirshman, CA, additional
- Published
- 1999
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6. Case scenario: bronchospasm during anesthetic induction.
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Dewachter P, Mouton-Faivre C, Emala CW, Beloucif S, Dewachter, Pascale, Mouton-Faivre, Claudie, Emala, Charles W, and Beloucif, Sadek
- Published
- 2011
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7. Anaphylaxis and anesthesia: controversies and new insights.
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Dewachter P, Mouton-Faivre C, Emala CW, Dewachter, Pascale, Mouton-Faivre, Claudie, and Emala, Charles W
- Published
- 2009
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8. Inducible nitric oxide synthase promoter polymorphism affords protection against cognitive dysfunction after carotid endarterectomy.
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Yocum GT, Gaudet JG, Lee SS, Stern Y, Teverbaugh LA, Sciacca RR, Emala CW, Quest DO, McCormick PC, McKinsey JF, Morrissey NJ, Solomon RA, Connolly ES Jr, Heyer EJ, Yocum, Gene T, Gaudet, John G, Lee, Susie S, Stern, Yaakov, Teverbaugh, Lauren A, and Sciacca, Robert R
- Published
- 2009
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9. Endogenous gamma-aminobutyric acid modulates tonic guinea pig airway tone and propofol-induced airway smooth muscle relaxation.
- Author
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Gallos G, Gleason NR, Virag L, Zhang Y, Mizuta K, Whittington RA, Emala CW, Gallos, George, Gleason, Neil R, Virag, Laszlo, Zhang, Yi, Mizuta, Kentaro, Whittington, Robert A, and Emala, Charles W
- Published
- 2009
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10. Rapacuronium augments acetylcholine-induced bronchoconstriction via positive allosteric interactions at the M3 muscarinic receptor.
- Author
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Jooste EH, Sharma A, Zhang Y, Emala CW, Jooste, Edmund H, Sharma, Amit, Zhang, Yi, and Emala, Charles W
- Published
- 2005
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11. In Response.
- Author
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Emala CW, Tawfik VL, Lane-Fall MB, Toledo P, Wong CA, Vavilala MS, Fleisher LA, and Wood M
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- 2024
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12. The Anesthesiology Physician-Scientist Pipeline: Current Status and Recommendations for Future Growth-An Initiative of the Anesthesia Research Council.
- Author
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Emala CW Sr, Tawfik VL, Lane-Fall MB, Toledo P, Wong CA, Vavilala MS, Fleisher LA, and Wood M
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- United States, Female, Humans, Male, Benchmarking, Anesthesiology, Anesthesia, Physicians, Awards and Prizes
- Abstract
The limited number and diversity of resident physicians pursuing careers as physician-scientists in medicine has been a concern for many decades. The Anesthesia Research Council aimed to address the status of the anesthesiology physician-scientist pipeline, benchmarked against other medical specialties, and to develop strategic recommendations to sustain and expand the number and diversity of anesthesiology physician-scientists. The working group analyzed data from the Association of American Medical Colleges and the National Resident Matching Program to characterize the diversity and number of research-oriented residents from US and international allopathic medical schools entering 11 medical specialties from 2009 to 2019. Two surveys were developed to assess the research culture of anesthesiology departments. National Institutes of Health (NIH) funding information awarded to anesthesiology physician-scientists and departments was collected from NIH RePORTER and the Blue Ridge Medical Institute. Anesthesiology ranked eighth to tenth place of 11 medical specialties in the percent of "research-oriented" entering residents, defined as those with advanced degrees (Master's or PhDs) in addition to the MD degree or having published at least 3 research publications before residency. Anesthesiology ranked eighth of 11 specialties in the percent of entering residents who were women but ranked fourth of 11 specialties in the percent of entering residents who self-identified as belonging to an underrepresented group in medicine. There has been a 72% increase in both the total NIH funding awarded to anesthesiology departments and the number of NIH K-series mentored training grants (eg, K08 and K23) awarded to anesthesiology physician-scientists between 2015 and 2020. Recommendations for expanding the size and diversity of the anesthesiology physician-scientist pipeline included (1) developing strategies to increase the number of research intensive anesthesiology departments; (2) unifying the diverse programs among academic anesthesiology foundations and societies that seek to grow research in the specialty; (3) adjusting American Society of Anesthesiologists metrics of success to include the number of anesthesiology physician-scientists with extramural research support; (4) increasing the number of mentored awards from Foundation of Anesthesia Education and Research (FAER) and International Anesthesia Research Society (IARS); (5) supporting an organized and concerted effort to inform research-oriented medical students of the diverse research opportunities within anesthesiology should include the specialty being represented at the annual meetings of Medical Scientist Training Program (MSTP) students and the American Physician Scientist Association, as well as in institutional MSTP programs. The medical specialty of anesthesiology is defined by new discoveries and contributions to perioperative medicine which will only be sustained by a robust pipeline of anesthesiology physician-scientists., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 International Anesthesia Research Society.)
- Published
- 2023
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13. Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators.
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Webb DA, Meyer MJ, Medubi KM, Tylek AS, Yocum GT, Roni MSR, Zahn NM, Swartwout SA, Masoud AK, Emala CW, Stafford DC, and Arnold LA
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- Mice, Humans, Animals, Guinea Pigs, Ligands, Molecular Docking Simulation, gamma-Aminobutyric Acid, Receptors, GABA-A metabolism, Bronchodilator Agents pharmacology
- Abstract
Novel gamma-aminobutyric acid receptor (GABA
A R) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1 β3 γ2 GABAA R and correlated with in vitro binding data. The carboxylic acid containing GABAA R ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAA R ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAA Rs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAA Rs that was metabolically stable in the presence of human and mouse microsomes.- Published
- 2023
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14. Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.
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Perez-Zoghbi JF, Sajorda DR, Webb DA, Arnold LA, Emala CW, and Yocum GT
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- Animals, Calcium Signaling, Flumazenil metabolism, Inositol metabolism, Ligands, Lung metabolism, Methacholine Chloride pharmacology, Mice, Muscle Contraction, Muscle, Smooth metabolism, Picrotoxin metabolism, gamma-Aminobutyric Acid metabolism, Asthma drug therapy, Asthma metabolism, Calcium metabolism
- Abstract
Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABA
A R (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAA R is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAA Rs, affording a seemingly logical site of action, the mechanism(s) by which GABAA R ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction in vivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAA R-dependent, as it is not inhibited by the GABAA R antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2 , suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3 ). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3 Rs (IP3 receptors).- Published
- 2022
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15. Contribution of Trp63 CreERT2 -labeled cells to alveolar regeneration is independent of tuft cells.
- Author
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Huang H, Fang Y, Jiang M, Zhang Y, Biermann J, Melms JC, Danielsson JA, Yang Y, Qiang L, Liu J, Zhou Y, Wang M, Hu Z, Wang TC, Saqi A, Sun J, Matsumoto I, Cardoso WV, Emala CW, Zhu J, Izar B, Mou H, and Que J
- Subjects
- Animals, Biomarkers, Cell Differentiation, Cell Lineage, Epithelial Cells, Mice, Tamoxifen pharmacology, Trans-Activators, COVID-19
- Abstract
Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don't proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63
CreERT2 and KRT5-CreERT2 -labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63CreERT2 -labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3-/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells., Competing Interests: HH, YF, MJ, YZ, JB, JM, JD, YY, LQ, JL, YZ, MW, ZH, TW, AS, JS, IM, WC, CE, JZ, BI, HM, JQ No competing interests declared, (© 2022, Huang et al.)- Published
- 2022
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16. Pharmacokinetics of Gingerols, Shogaols, and Their Metabolites in Asthma Patients.
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Zhang S, DiMango E, Zhu Y, Saroya TK, Emala CW, and Sang S
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- Animals, Catechols chemistry, Cysteine metabolism, Fatty Alcohols chemistry, Humans, Ketones metabolism, Mice, Plant Extracts chemistry, Asthma drug therapy, Zingiber officinale chemistry
- Abstract
6-Gingerol and 6-shogaol are the most abundant gingerols and shogaols in ginger root and have been shown to reduce the asthmatic phenotype in murine models of asthma. Several studies have described the pharmacokinetics of gingerols and shogaols in humans following the oral ingestion of ginger, while little was known about the metabolism of these components in humans, particularly in patients with asthma. In this study, a dietary supplement of 1.0 g of ginger root extract was administered to asthma patients twice daily for 56 days and serum samples were drawn at 0.5-8 h on days 0, 28, and 56. The metabolic profiles of gingerols and shogaols in human plasma and the kinetic changes of gingerols, shogaols, and their metabolites in asthma patients collected on the three different visits were analyzed using liquid chromatography-mass spectrometry (LC-MS). Ketone reduction was the major metabolic pathway of both gingerols and shogaols. Gingerdiols were identified as the major metabolites of 6-, 8-, and 10-gingerols. M11 and M9 were identified as the double-bond reduction and both the double-bond and ketone reduction metabolites of 6-shogaol, respectively. Cysteine conjugation was another major metabolic pathway of 6-shogaol in asthma patients, and two cysteine-conjugated 6-shogaol, M1 and M2, were identified as the major metabolites of 6-shogaol. Furthermore, gingerols, shogaols, and their metabolites were quantitated in the human serum collected at different time points during each of the three visits using a very sensitive high-resolution LC-MS method. The results showed that one-third of 6-gingerol was metabolized to produce its reduction metabolites, 6-gingerdiols, and more than 90% of 6-shogaol was metabolized to its phase I and cysteine-conjugated metabolites, suggesting the importance of considering the contribution of these metabolites to the bioavailability and health beneficial effects of gingerols and shogaols. All gingerols, shogaols, and their metabolites reached their peak concentrations in less than 2 h, and their half-lives ( t
1/2 ) were from 0.6 to 2.4 h. Furthermore, long-term treatment of ginger supplements, especially after 56 days of treatment, increases the absorption of ginger compounds and their metabolites in asthma patients.- Published
- 2022
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17. Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.
- Author
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Rashid Roni MS, Zahn NM, Yocum GT, Webb DA, Mian MY, Meyer MJ, Tylek AS, Cook JM, Emala CW, Stafford DC, and Arnold LA
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- Animals, Azepines, Imidazoles, Mice, Rats, Receptors, GABA, Taurine, gamma-Aminobutyric Acid, Asthma drug therapy, Asthma metabolism
- Abstract
MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABA
A R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S., (© 2022 Wiley Periodicals LLC.)- Published
- 2022
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18. Development of Inhaled GABA A Receptor Modulators to Improve Airway Function in Bronchoconstrictive Disorders.
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Zahn NM, Roni MSR, Yocum GT, Meyer MJ, Webb DA, Mian MY, Cook JM, Stafford DC, Emala CW, and Arnold LA
- Abstract
We report the modification of MIDD0301, an imidazodiazepine GABA
A receptor (GABAA R) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting β2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABAA R binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1 β3 γ2 GABAA R structural data, suggests binding of the diazepine core between the α1+/γ2- interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 μM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 μM, with rapid onset (15 min) at 25 μM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally., Competing Interests: The authors declare the following competing financial interest(s): L.A.A. and D.C.S. are employees of Pantherics Incorporated. L.A.A. and D.C.S. have an ownership interest in Pantherics, which has acquired rights to the technology reported in this publication. Some of the research was funded by R41HL147658, which was awarded to Pantherics. Pantherics did not finance this research directly. The funders indicated in the acknowledgment section they had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2022 American Chemical Society.)- Published
- 2022
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19. Papper Event 2021: The COVID-19 Pandemic: Lessons Learned and the Way Forward.
- Author
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Sun LS, Brambrink A, Emala CW, Hua M, Lee HT, Levy RJ, Smiley RM, Whittington RA, and Narula JH
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- Humans, Pandemics, SARS-CoV-2, COVID-19
- Abstract
Competing Interests: The authors have no conflicts of interest to declare.
- Published
- 2022
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20. Melatonin MT 2 receptor is expressed and potentiates contraction in human airway smooth muscle.
- Author
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Sasaki H, Zhang Y, Emala CW Sr, and Mizuta K
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- Acetylcholine pharmacology, Adult, Animals, Antioxidants pharmacology, Colforsin pharmacology, Guinea Pigs, Humans, Male, Melatonin pharmacology, Middle Aged, Myocytes, Smooth Muscle cytology, Myocytes, Smooth Muscle drug effects, Receptor, Melatonin, MT2 antagonists & inhibitors, Respiratory System drug effects, Vasodilator Agents pharmacology, Cyclic AMP metabolism, Muscle Contraction, Muscle Relaxation, Myocytes, Smooth Muscle metabolism, Receptor, Melatonin, MT2 metabolism, Respiratory System metabolism
- Abstract
Nocturnal asthma is characterized by heightened bronchial reactivity at night, and plasma melatonin concentrations are higher in patients with nocturnal asthma symptoms. Numerous physiological effects of melatonin are mediated via its specific G protein-coupled receptors (GPCRs) named the MT
1 receptor, which couples to both Gq and Gi proteins, and the MT2 receptor, which couples to Gi . We investigated whether melatonin receptors are expressed on airway smooth muscle; whether they regulate intracellular cyclic AMP (cAMP) and calcium concentrations ([Ca2+ ]i ), which modulate airway smooth muscle tone; and whether they promote airway smooth muscle cell proliferation. We detected the mRNA and protein expression of the melatonin MT2 but not the MT1 receptor in native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by RT-PCR, immunoblotting, and immunohistochemistry. Activation of melatonin MT2 receptors with either pharmacological concentrations of melatonin (10-100 µM) or the nonselective MT1 /MT2 agonist ramelteon (10 µM) significantly inhibited forskolin-stimulated cAMP accumulation in HASM cells, which was reversed by the Gαi protein inhibitor pertussis toxin or knockdown of the MT2 receptor by its specific siRNA. Although melatonin by itself did not induce an initial [Ca2+ ]i increase and airway contraction, melatonin significantly potentiated acetylcholine-stimulated [Ca2+ ]i increases, stress fiber formation through the MT2 receptor in HASM cells, and attenuated the relaxant effect of isoproterenol in guinea pig trachea. These findings suggest that the melatonin MT2 receptor is expressed in ASM, and modulates airway smooth muscle tone via reduced cAMP production and increased [Ca2+ ]i .- Published
- 2021
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21. Ginger metabolites and metabolite-inspired synthetic products modulate intracellular calcium and relax airway smooth muscle.
- Author
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Luković E, Perez-Zoghbi JF, Zhang Y, Zhu Y, Sang S, and Emala CW
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- Animals, Asthma chemically induced, Asthma metabolism, Bronchoconstriction drug effects, Cyclic Nucleotide Phosphodiesterases, Type 4 metabolism, Humans, Isoproterenol pharmacology, Lung drug effects, Lung metabolism, Mice, Inbred C57BL, Muscle Relaxation physiology, Muscle, Smooth metabolism, Myocytes, Smooth Muscle metabolism, Mice, Calcium metabolism, Zingiber officinale metabolism, Muscle Relaxation drug effects, Myocytes, Smooth Muscle drug effects
- Abstract
Asthma affects millions of people worldwide and its prevalence is increasing. It is characterized by chronic airway inflammation, airway remodeling, and pathologic bronchoconstriction, and it poses a continuous treatment challenge with very few new therapeutics available. Thus, many asthmatics turn to plant-based complementary products, including ginger, for better symptom control, indicating an unmet need for novel therapies. Previously, we demonstrated that 6-shogaol (6S), the primary bioactive component of ginger, relaxes human airway smooth muscle (hASM) likely by inhibition of phosphodiesterases (PDEs) in the β-adrenergic (cyclic nucleotide PDEs), and muscarinic (phospholipase C, PLC) receptor pathways. However, oral 6S is extensively metabolized and it is unknown if the resulting metabolites remain bioactive. Here, we screened all the known human metabolites of 6S and several metabolite-based synthetic derivatives to better understand their mechanism of action and structure-function relationships. We demonstrate that several metabolites and metabolite-based synthetic derivatives are able to prevent Gq-coupled stimulation of intracellular calcium [Ca
2+ ]i and inositol trisphosphate (IP3 ) synthesis by inhibiting PLC, similar to the parent compound 6S. We also show that these compounds prevent recontraction of ASM after β-agonist relaxation likely by inhibiting PDEs. Furthermore, they potentiate isoproterenol-induced relaxation. Importantly, moving beyond cell-based assays, metabolites also retain the functional ability to relax Gq-coupled-contractions in upper (human) and lower (murine) airways. The current study indicates that, although oral ginger may be metabolized rapidly, it retains physiological activity through its metabolites. Moreover, we are able to use naturally occurring metabolites as inspiration to develop novel therapeutics for brochoconstrictive diseases.- Published
- 2021
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22. Opsin 3-G αs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.
- Author
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Wu AD, Dan W, Zhang Y, Vemaraju S, Upton BA, Lang RA, Buhr ED, Berkowitz DE, Gallos G, Emala CW, and Yim PD
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- Animals, Cells, Cultured, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Humans, Male, Mice, Mice, Inbred C57BL, Opsins metabolism, Signal Transduction physiology, G-Protein-Coupled Receptor Kinase 2 metabolism, Muscle Relaxation physiology, Muscle, Smooth metabolism, Myocytes, Smooth Muscle metabolism, Rod Opsins metabolism, Trachea metabolism
- Abstract
Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and G
α s using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS ( P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic β2 -agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gα s , which increases cAMP levels, activating PKA and modulated by GRK2.- Published
- 2021
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23. Nebulized MIDD0301 Reduces Airway Hyperresponsiveness in Moderate and Severe Murine Asthma Models.
- Author
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Zahn NM, Mikulsky BN, Roni MSR, Yocum GT, Mian MY, Knutson DE, Cook JM, Emala CW, Stafford DC, and Arnold LA
- Abstract
We report the relaxation of methacholine-constricted airways with nebulized MIDD0301, a positive allosteric γ-aminobutyric acid type A receptor (GABA
A R) modulator. The therapeutic efficacy of nebulized MIDD0301 in reducing airway resistance was investigated in spontaneous breathing mice using a whole-body plethysmograph and in unconscious mice using a forced oscillation technique. Prophylactic nebulized MIDD0301 reduced subsequent methacholine-induced bronchoconstriction in ovalbumin and house dust mite allergic asthma models and in normal mice. Nebulized MIDD0301 exhibited comparable or better therapeutic potency compared to nebulized albuterol and oral montelukast. Prophylactic nebulized MIDD0301 was also effective in reducing bronchoconstriction, comparable to nebulized albuterol or fluticasone, in a steroid resistant asthma mouse model induced by intratracheal installation of lipopolysaccharide and interferon-gamma. Oral dexamethasone was ineffective in this model. Nebulized MIDD0301 was also effective in reversing bronchospasm when dosed after methacholine challenge comparable to albuterol. Pharmacokinetic studies showed that about 0.06% of nebulized MIDD0301 entered the mouse lung when using a whole body plethysmograph and therapeutic levels were sustained in the lung for at least 25 min. Consistent with previous reports on orally dosed MIDD0301, high doses of nebulized MIDD0301 resulted in minimal brain exposure and thus no observable adverse sensorimotor or respiratory depression effects occurred. In addition, no adverse cardiovascular effects were observed following 100 mg/kg i.p. dosing. These results further demonstrate that charged imidazodiazepine MIDD0301 can selectively target lung GABAA R without adverse motor, cardiovascular, or respiratory effects and inhaled dosing is effective in reducing bronchoconstriction in allergen and infectious lung inflammation., Competing Interests: The authors declare the following competing financial interest(s): L.A.A., B.N.M., and D.C.S. are employees of Pantherics. L.A.A., D.C.S., and C.W.E. have an ownership interest in Pantherics, which has licensed the technology reported in this publication. Some of the research was funded by R41HL147658, which was awarded to Pantherics. Pantherics did not finance this research directly. The funders indicated in the Acknowledgment section had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results., (© 2020 American Chemical Society.)- Published
- 2020
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24. Reduced allergic lung inflammation and airway responsiveness in mice lacking the cytoskeletal protein gelsolin.
- Author
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Mikami M, Yocum GT, Heller NM, and Emala CW
- Abstract
Airway smooth muscle hyperresponsiveness associated with chronic airway inflammation leads to the typical symptoms of asthma including bronchoconstriction and wheezing. Asthma severity is associated with airway inflammation; therefore, reducing airway inflammation is an important therapeutic target. Gelsolin is an actin capping and severing protein that has been reported to be involved in modulation of the inflammatory response. Using mice genetically lacking gelsolin, we evaluated the role of gelsolin in the establishment of house dust mite (HDM) antigen-induced allergic lung inflammation. The genetic absence of gelsolin was found to be protective against HDM sensitization, resulting in reduced lung inflammation, inflammatory cytokines, and Muc5AC protein in bronchoalveolar lavage (BAL) fluid. The number of eosinophils, lymphocytes, and interstitial macrophages in the BAL were increased after HDM sensitization in wild-type mice but were attenuated in gelsolin-null mice. The observed attenuation of inflammation may be partly due to delayed migration of immune cells, because the reduced eosinophils in the BALs from gelsolin-null mice compared with controls occurred despite similar amounts of the chemoattractant eotaxin. Splenic T cells demonstrated similar proliferation rates, but ex vivo alveolar macrophage migration was delayed in gelsolin-null mice. In vivo, the reduced lung inflammation after HDM sensitization in gelsolin-null mice was associated with significantly diminished airway resistance to inhaled methacholine compared with HDM-treated wild-type mice. Our results suggest that modulation of gelsolin expression or function in selective inflammatory cell types that modulate allergic lung inflammation could be a therapeutic approach for asthma.
- Published
- 2020
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25. Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity.
- Author
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Heras AF, Veerappan A, Silver RB, Emala CW, Worgall TS, Perez-Zoghbi J, and Worgall S
- Subjects
- Alveolar Epithelial Cells drug effects, Alveolar Epithelial Cells metabolism, Bradykinin pharmacology, Calcium metabolism, Calcium Signaling drug effects, Fenretinide pharmacology, Glycine analogs & derivatives, Glycine pharmacology, Humans, Hydrazines pharmacology, Metabolome drug effects, Methacholine Chloride pharmacology, Muscle Contraction drug effects, Serine C-Palmitoyltransferase metabolism, Bronchial Hyperreactivity metabolism, Sphingolipids biosynthesis
- Abstract
Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides ( de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo . This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.
- Published
- 2020
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26. The short-chain free fatty acid receptor FFAR3 is expressed and potentiates contraction in human airway smooth muscle.
- Author
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Mizuta K, Sasaki H, Zhang Y, Matoba A, and Emala CW Sr
- Subjects
- Acetylcholine pharmacology, Adult, Calcium metabolism, Cells, Cultured, Colforsin pharmacology, Cyclic AMP metabolism, Fatty Acids, Volatile pharmacology, Humans, Isoproterenol pharmacology, Male, Middle Aged, Muscle Contraction drug effects, Muscle Relaxation drug effects, Muscle, Smooth drug effects, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, Lung physiology, Muscle Contraction physiology, Muscle, Smooth physiology, Receptors, G-Protein-Coupled metabolism
- Abstract
Emerging evidence suggests that gut microbiota-derived short-chain fatty acids (SCFAs; acetate, propionate, and butyrate) are important modulators of the inflammatory state in diseases such as asthma. However, the functional expression of the G
i protein-coupled free fatty acid receptors (FFAR2/GPR43 and FFAR3/GPR41) has not been identified on airway smooth muscle (ASM). Classically, acute activation of Gi -coupled receptors inhibits cyclic AMP (cAMP) synthesis, which impairs ASM relaxation and can also induce crosstalk between Gi - and Gq -signaling pathways, potentiating increases in intracellular Ca2+ concentration ([Ca2+ ]i ), favoring ASM contraction. In contrast, chronic activation of Gi -coupled receptors can sensitize adenylyl cyclase resulting in increased cAMP synthesis favoring relaxation. We questioned whether the Gi -coupled FFAR2 or FFAR3 is expressed in human ASM, whether they modulate cAMP and [Ca2+ ]i , and whether SCFAs modulate human ASM tone. We detected the protein expression of FFAR3 but not FFAR2 in native human ASM and primary cultured human airway smooth muscle (HASM) cells. In HASM cells, acute activation of FFAR3 with SCFAs inhibited forskolin-stimulated cAMP accumulation, but chronic activation did not sensitize cAMP synthesis. SCFAs induced [Ca2+ ]i increases that were attenuated by pertussis toxin, gallein, U73122, or xestospongin C. Acute treatment with SCFAs potentiated acetylcholine-stimulated [Ca2+ ]i increases and stress fiber formation in cells and contraction of ex vivo human airway tissues. In contrast, chronic pretreatment of human ASM with propionate did not potentiate airway relaxation. Together, these findings demonstrate that FFAR3 is expressed in human ASM and contributes to ASM contraction via reduced cAMP and increased [Ca2+ ]i .- Published
- 2020
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27. Agonism of the TMEM16A calcium-activated chloride channel modulates airway smooth muscle tone.
- Author
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Danielsson J, Kuforiji AS, Yocum GT, Zhang Y, Xu D, Gallos G, and Emala CW Sr
- Subjects
- Acetylcholine pharmacology, Animals, Anoctamin-1 genetics, Bronchial Hyperreactivity physiopathology, Bronchoconstriction drug effects, Calcium metabolism, Cells, Cultured, Guinea Pigs, Humans, Inositol Phosphates biosynthesis, Methacholine Chloride pharmacology, Muscle Contraction drug effects, Neoplasm Proteins genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Anoctamin-1 agonists, Anoctamin-1 metabolism, Lung metabolism, Muscle Tonus drug effects, Muscle, Smooth metabolism, Neoplasm Proteins agonists, Neoplasm Proteins metabolism
- Abstract
TMEM16A (anoctamin 1) is an important calcium-activated chloride channel in airway smooth muscle (ASM). We have previously shown that TMEM16A antagonists such as benzbromarone relax ASM and have proposed TMEM16A antagonists as novel therapies for asthma treatment. However, TMEM16A is also expressed on airway epithelium, and TMEM16A agonists are being investigated as novel therapies for cystic fibrosis. There are theoretical concerns that agonism of TMEM16A on ASM could lead to bronchospasm, making them detrimental as airway therapeutics. The TMEM16A agonist Eact induced a significant contraction of human ASM and guinea pig tracheal rings in an ex vivo organ bath model. Pretreatment with two different TMEM16A antagonists, benzbromarone or T16Ainh-A01, completely attenuated these Eact-induced contractions. Pretreatment with Eact alone augmented the maximum acetylcholine contraction. Pretreatment of A/J mice in vivo with nebulized Eact caused an augmentation of methacholine-induced increases in airway resistance measured by the forced oscillatory technique (flexiVent). Pretreatment with the TMEM16A antagonist benzbromarone significantly attenuated methacholine-induced increases in airway resistance. In in vitro cellular studies, TMEM16A was found to be expressed more abundantly in ASM compared with epithelial cells in culture (8-fold higher in ASM). Eact caused an increase in intracellular calcium in human ASM cells that was completely attenuated by pretreatment with benzbromarone. Eact acutely depolarized the plasma membrane potential of ASM cells, which was attenuated by benzbromarone or nifedipine. The TMEM16A agonist Eact modulates ASM contraction in both ex vivo and in vivo models, suggesting that agonism of TMEM16A may lead to clinically relevant bronchospasm.
- Published
- 2020
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28. Ginger and its bioactive component 6-shogaol mitigate lung inflammation in a murine asthma model.
- Author
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Yocum GT, Hwang JJ, Mikami M, Danielsson J, Kuforiji AS, and Emala CW
- Subjects
- Airway Resistance drug effects, Animals, Antigens, CD metabolism, Antigens, Dermatophagoides immunology, Asthma complications, Asthma immunology, Asthma physiopathology, Bronchial Hyperreactivity complications, Bronchial Hyperreactivity immunology, Bronchial Hyperreactivity physiopathology, Bronchoalveolar Lavage Fluid cytology, Catechols administration & dosage, Catechols pharmacology, Cell Count, Cyclic AMP metabolism, Disease Models, Animal, Female, Interleukin-4 metabolism, Lung pathology, Male, Mice, Inbred C57BL, NF-kappa B metabolism, Plant Extracts pharmacology, Plant Extracts therapeutic use, Pneumonia complications, Pneumonia immunology, Pneumonia pathology, Signal Transduction drug effects, T-Lymphocytes, Regulatory drug effects, Asthma drug therapy, Catechols therapeutic use, Zingiber officinale chemistry, Pneumonia drug therapy
- Abstract
Asthma, a common disorder associated with airway inflammation and hyperresponsiveness, remains a significant clinical burden in need of novel therapeutic strategies. Patients are increasingly seeking complementary and alternative medicine approaches to control their symptoms, including the use of natural products. Ginger, a natural product that we previously demonstrated acutely relaxes airway smooth muscle (ASM), has long been reported to possess anti-inflammatory properties, although a precise mechanistic understanding is lacking. In these studies, we demonstrate that chronic administration of whole ginger extract or 6-shogaol, a bioactive component of ginger, mitigates in vivo house dust mite antigen-mediated lung inflammation in mice. We further show that this decrease in inflammation is associated with reduced in vivo airway responsiveness. Utilizing in vitro studies, we demonstrate that 6-shogaol augments cAMP concentrations in CD4 cells, consistent with phosphodiesterase inhibition, and limits the induction of nuclear factor-κB signaling and the production of proinflammatory cytokines in activated CD4 cells. Sustained elevations in cAMP concentration are well known to inhibit effector T cell function. Interestingly, regulatory T cells (Tregs) utilize cAMP as a mediator of their immunosuppressive effects, and we demonstrate here that 6-shogaol augments the Treg polarization of naïve CD4 cells in vitro. Taken together with previous reports, these studies suggest that ginger and 6-shogaol have the potential to combat asthma via two mechanisms: acute ASM relaxation and chronic inhibition of inflammation.
- Published
- 2020
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29. Bile acids inhibit cholinergic constriction in proximal and peripheral airways from humans and rodents.
- Author
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Urso A, D'Ovidio F, Xu D, Emala CW Sr, Bunnett NW, and Perez-Zoghbi JF
- Subjects
- Animals, Bronchoconstrictor Agents pharmacology, Chenodeoxycholic Acid pharmacology, Electric Stimulation, Guinea Pigs, Humans, Inositol Phosphates biosynthesis, Lung drug effects, Male, Mice, Inbred C57BL, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, Receptors, G-Protein-Coupled metabolism, Receptors, Muscarinic metabolism, Serotonin pharmacology, Taurolithocholic Acid pharmacology, Trachea drug effects, Acetylcholine metabolism, Bile Acids and Salts pharmacology, Bronchoconstriction drug effects, Lung physiopathology
- Abstract
Duodenogastroesophageal reflux (DGER) is associated with chronic lung disease. Bile acids (BAs) are established markers of DGER aspiration and are important risk factors for reduced post-transplant lung allograft survival by disrupting the organ-specific innate immunity, facilitating airway infection and allograft failure. However, it is unknown whether BAs also affect airway reactivity. We investigated the acute effects of 13 BAs detected in post-lung-transplant surveillance bronchial washings (BW) on airway contraction. We exposed precision-cut slices from human and mouse lungs to BAs and monitored dynamic changes in the cross-sectional luminal area of peripheral airways using video phase-contrast microscopy. We also used guinea pig tracheal rings in organ baths to study BA effects in proximal airway contraction induced by electrical field stimulation. We found that most secondary BAs at low micromolar concentrations strongly and reversibly relaxed smooth muscle and inhibited peripheral airway constriction induced by acetylcholine but not by noncholinergic bronchoconstrictors. Similarly, secondary BAs strongly inhibited cholinergic constrictions in tracheal rings. In contrast, TC-G 1005, a specific agonist of the BA receptor Takeda G protein-coupled receptor 5 (TGR5), did not cause airway relaxation, and Tgr5 deletion in knockout mice did not affect BA-induced relaxation, suggesting that this receptor is not involved. BAs inhibited acetylcholine-induced inositol phosphate synthesis in human airway smooth muscle cells overexpressing the muscarinic M3 receptor. Our results demonstrate that select BAs found in BW of patients with lung transplantation can affect airway reactivity by inhibiting the cholinergic contractile responses of the proximal and peripheral airways, possibly by acting as antagonists of M3 muscarinic receptors.
- Published
- 2020
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30. Novel Expression of GABAA Receptors on Resistance Arteries That Modulate Myogenic Tone.
- Author
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Yim PD, Gallos G, Lee-Kong SA, Dan W, Wu AD, Xu D, Berkowitz DE, and Emala CW
- Subjects
- Animals, Arteries drug effects, Calcium Signaling, Cells, Cultured, Female, GABA-A Receptor Agonists pharmacology, GABA-A Receptor Antagonists pharmacology, Male, Membrane Potentials, Mice, Inbred C57BL, Muscle, Smooth, Vascular drug effects, Myocytes, Smooth Muscle drug effects, Receptors, GABA-A drug effects, Receptors, GABA-A genetics, Vasodilation, Arteries metabolism, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism, Omentum blood supply, Receptors, GABA-A metabolism, Tail blood supply, Vascular Resistance, Vasoconstriction
- Abstract
The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries., (© 2020 S. Karger AG, Basel.)
- Published
- 2020
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31. Correction to "A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABA A Receptors in the Lung".
- Author
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Forkuo GS, Nieman AN, Kodali R, Zahn NM, Li G, Roni MSR, Stephen MR, Harris TW, Jahan R, Guthrie ML, Yu OB, Fisher JL, Yocum GT, Emala CW, Steeber DA, Stafford DC, Cook JM, and Arnold LA
- Published
- 2019
- Full Text
- View/download PDF
32. Obesity-induced asthma: Role of free fatty acid receptors.
- Author
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Mizuta K, Matoba A, Shibata S, Masaki E, and Emala CW Sr
- Abstract
Obesity is a major risk factor for the development of asthma, and worsens the key features of asthma including airway hyperresponsiveness, inflammation, and airway remodeling. Although pro- and anti-inflammatory adipocytokines may contribute to the pathogenesis of asthma in obesity, the mechanistic basis for the relationship between asthma and obesity remains unclear. In obese individuals, the increased amount of adipose tissue results in the release of more long-chain free fatty acids as compared to lean individuals, causing an elevation in plasma long-chain free fatty acid concentrations. Recent findings suggest that the free fatty acid receptor 1 (FFAR1), which is a sensor of medium- and long-chain free fatty acids, is expressed on airway smooth muscle and plays a pivotal role in airway contraction and airway smooth muscle cell proliferation. In contrast, FFAR4, which is a sensor for long-chain n -3 polyunsaturated fatty acids and also expressed on airway smooth muscle, does not contribute to airway contraction and airway smooth muscle cell proliferation. Functional roles for short-chain fatty acid receptors FFAR2 and FFAR3 in the pathogenesis of asthma is still under debate. Taken together, adipose-derived long-chain free fatty acids may contribute to the pathogenesis of asthma in obesity through FFAR1.
- Published
- 2019
- Full Text
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33. Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells.
- Author
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Mori M, Furuhashi K, Danielsson JA, Hirata Y, Kakiuchi M, Lin CS, Ohta M, Riccio P, Takahashi Y, Xu X, Emala CW, Lu C, Nakauchi H, and Cardoso WV
- Subjects
- Acylation genetics, Animals, Blastocyst metabolism, Cell Differentiation genetics, DNA Methylation genetics, Disease Models, Animal, Histones genetics, Humans, Lung pathology, Lung Diseases pathology, Mice, Organogenesis genetics, Pluripotent Stem Cells transplantation, Receptor, Fibroblast Growth Factor, Type 2 genetics, beta Catenin genetics, Lung growth & development, Lung Diseases therapy, Pluripotent Stem Cells metabolism, Regeneration genetics
- Abstract
Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation
1 . Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3 . Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6 .- Published
- 2019
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34. Bitter Taste Receptors: Taking a Bigger Bite of Airway Smooth Muscle Pathophysiology.
- Author
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Emala CW Sr
- Subjects
- Mitogen-Activated Protein Kinase 3, Muscle, Smooth drug effects, Respiratory System drug effects, Bronchodilator Agents, Taste drug effects
- Published
- 2019
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- View/download PDF
35. A novel GABA A receptor ligand MIDD0301 with limited blood-brain barrier penetration relaxes airway smooth muscle ex vivo and in vivo.
- Author
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Yocum GT, Perez-Zoghbi JF, Danielsson J, Kuforiji AS, Zhang Y, Li G, Rashid Roni MS, Kodali R, Stafford DC, Arnold LA, Cook JM, and Emala CW Sr
- Subjects
- Animals, Guinea Pigs, Humans, Ligands, Lung drug effects, Lung metabolism, Male, Muscle, Smooth drug effects, Muscle, Smooth metabolism, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, Receptors, GABA-A metabolism, Trachea drug effects, Trachea metabolism, Asthma drug therapy, Blood-Brain Barrier drug effects, GABA Agents pharmacology, Receptors, GABA-A drug effects
- Abstract
Airway smooth muscle (ASM) cells express GABA A receptors (GABA
A Rs), and previous reports have demonstrated that GABAA R activators relax ASM. However, given the activity of GABAA Rs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAA R with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.- Published
- 2019
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36. Attenuation of murine and human airway contraction by a peptide fragment of the cytoskeleton regulatory protein gelsolin.
- Author
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Mikami M, Perez-Zoghbi JF, Zhang Y, and Emala CW Sr
- Subjects
- Animals, Gelsolin chemistry, Humans, Male, Mice, Muscle, Smooth pathology, Peptides chemistry, Phosphatidylinositol 4,5-Diphosphate metabolism, Trachea pathology, Bronchoconstriction drug effects, Gelsolin pharmacology, Muscle Contraction drug effects, Muscle, Smooth metabolism, Peptides pharmacology, Trachea metabolism
- Abstract
We have previously reported that mice genetically deficient in the actin binding protein gelsolin exhibit impaired airway smooth muscle (ASM) relaxation. Primary cultured ASM cells from these mice demonstrate enhanced inositol triphosphate (IP
3 ) synthesis and increased intracellular calcium in response to Gq -coupled agonists. We hypothesized that this was due to increased intracellular availability of unbound phosphatidylinositol 4,5-bisphosphate (PIP2 ), based on the fact that gelsolin contains a short peptide region that binds PIP2 , presumably making it a less available substrate. We now questioned whether a peptide that corresponds to the PIP2 binding region of gelsolin could modulate ASM signaling and contraction. The 10 amino acid sequence of the gelsolin peptide within the PIP2 -binding region was incubated with primary cultures of human ASM cells, and IP3 synthesis was measured in response to a Gq -coupled agonist. Gelsolin peptide-treated cells generated less IP3 under basal and bradykinin or acetylcholine (Gq -coupled) conditions. Acetylcholine-induced contractile force measured in isolated tracheal rings from mice and human tracheal muscle strips in organ baths was attenuated in the presence of the gelsolin peptide. The gelsolin peptide also attenuated methacholine-induced airway constriction in murine precision-cut lung slices. Furthermore, this peptide fragment delivered to the respiratory system of mice via nebulization attenuated subsequent methacholine-induced increases in airway resistance in vivo. The current study demonstrates that introduction of this small gelsolin peptide into the airway may be a novel therapeutic option in bronchoconstrictive diseases.- Published
- 2019
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37. Airway smooth muscle photorelaxation via opsin receptor activation.
- Author
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Yim PD, Gallos G, Perez-Zoghbi JF, Zhang Y, Xu D, Wu A, Berkowitz DE, and Emala CW
- Subjects
- Animals, Humans, Mice, Myocytes, Smooth Muscle cytology, Signal Transduction, Trachea cytology, Light, Muscle Relaxation, Myocytes, Smooth Muscle metabolism, Rod Opsins metabolism, Trachea metabolism
- Abstract
Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.
- Published
- 2019
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- View/download PDF
38. A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABA A Receptors in the Lung.
- Author
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Forkuo GS, Nieman AN, Kodali R, Zahn NM, Li G, Rashid Roni MS, Stephen MR, Harris TW, Jahan R, Guthrie ML, Yu OB, Fisher JL, Yocum GT, Emala CW, Steeber DA, Stafford DC, Cook JM, and Arnold LA
- Subjects
- Animals, Asthma metabolism, Brain drug effects, Brain metabolism, Bronchoalveolar Lavage Fluid chemistry, CD4-Positive T-Lymphocytes drug effects, CD4-Positive T-Lymphocytes metabolism, Constriction, Cytokines metabolism, Eosinophils drug effects, Eosinophils metabolism, Female, Guinea Pigs, Inflammation metabolism, Ligands, Lung metabolism, Macrophages drug effects, Macrophages metabolism, Male, Mice, Mice, Inbred BALB C, Muscle, Smooth metabolism, Ovalbumin metabolism, Respiratory Hypersensitivity metabolism, Asthma drug therapy, Inflammation drug therapy, Lung drug effects, Muscle, Smooth drug effects, Receptors, GABA-A metabolism
- Abstract
We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α
5 β3 γ2 selective GABAA receptor (GABAA R) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α1-3,5 β3 γ2 GABAA Rs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4+ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4+ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAA R ligands.- Published
- 2018
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39. The dopamine D 1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium.
- Author
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Matsuyama N, Shibata S, Matoba A, Kudo TA, Danielsson J, Kohjitani A, Masaki E, Emala CW, and Mizuta K
- Subjects
- Cell Line, Cells, Cultured, Dopamine Agonists pharmacology, Gene Expression, Humans, Mucin 5AC genetics, Phosphorylation drug effects, Phosphorylation physiology, Receptors, Dopamine D1 agonists, Receptors, Dopamine D1 genetics, Respiratory Mucosa drug effects, Cyclic AMP Response Element-Binding Protein metabolism, Mucin 5AC biosynthesis, Receptors, Dopamine D1 biosynthesis, Respiratory Mucosa metabolism
- Abstract
Background: Dopamine receptors comprise two subgroups, Gs protein-coupled “D1-like” receptors (D1, D5) and Gicoupled “D2-like” receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression., Methods: We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively., Results: The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells., Conclusions: These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms.
- Published
- 2018
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40. The free fatty acid receptor 1 promotes airway smooth muscle cell proliferation through MEK/ERK and PI3K/Akt signaling pathways.
- Author
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Matoba A, Matsuyama N, Shibata S, Masaki E, Emala CW Sr, and Mizuta K
- Subjects
- Adult, Animals, Cells, Cultured, Fatty Acids, Nonesterified metabolism, Humans, MAP Kinase Signaling System, Male, Middle Aged, Muscle, Smooth metabolism, Phosphorylation, Rats, Rats, Wistar, Respiratory System metabolism, Signal Transduction, Cell Proliferation, Extracellular Signal-Regulated MAP Kinases metabolism, Muscle, Smooth cytology, Phosphatidylinositol 3-Kinase metabolism, Proto-Oncogene Proteins c-akt metabolism, Receptors, G-Protein-Coupled metabolism, Respiratory System cytology
- Abstract
Obesity is a risk factor for asthma and influences airway hyperresponsiveness, which is in part modulated by airway smooth muscle proliferative remodeling. Plasma free fatty acids (FFAs) levels are elevated in obese individuals, and long-chain FFAs act as endogenous ligands for the free fatty acid receptor 1 (FFAR1), which couples to both G
q and Gi proteins. We examined whether stimulation of FFAR1 induces airway smooth muscle cell proliferation through classical MEK/ERK and/or phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. The long-chain FFAs (oleic acid and linoleic acid) and a FFAR1 agonist (GW9508) induced human airway smooth muscle (HASM) cell proliferation, which was inhibited by the MEK inhibitor U0126 and the PI3K inhibitor LY294002 . The long-chain FFAs and GW9508 increased phosphorylation of ERK, Akt, and p70S6K in HASM cells and freshly isolated rat airway smooth muscle. Downregulation of FFAR1 in HASM cells by siRNA significantly attenuated oleic acid-induced phosphorylation of ERK and Akt. Oleic acid-induced ERK phosphorylation was blocked by either the Gαi -protein inhibitor pertussis toxin or U0126 and was partially inhibited by either the Gαq -specific inhibitor YM-254890 or the Gβγ signaling inhibitor gallein. Oleic acid significantly inhibited forskolin-stimulated cAMP activity, which was attenuated by pertussis toxin. Akt phosphorylation was inhibited by pertussis toxin, the ras inhibitor manumycin A, the Src inhibitor PP1, or LY294002 . Phosphorylation of p70S6K by oleic acid or GW9508 was significantly inhibited by LY294002 , U0126, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin. In conclusion, the FFAR1 promoted airway smooth muscle cell proliferation and p70S6K phosphorylation through MEK/ERK and PI3K/Akt signaling pathways.- Published
- 2018
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41. Targeting brain tumors by intra-arterial delivery of cell-penetrating peptides: a novel approach for primary and metastatic brain malignancy.
- Author
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Joshi S, Cooke JRN, Ellis JA, Emala CW, and Bruce JN
- Subjects
- Administration, Intravenous, Animals, Brain drug effects, Brain metabolism, Brain pathology, Brain Neoplasms metabolism, Brain Neoplasms pathology, Cell Line, Tumor, Glioma metabolism, Glioma pathology, Humans, Injections, Intra-Arterial, Mice, Neoplasm Transplantation, Rats, Inbred F344, Antineoplastic Agents administration & dosage, Brain Neoplasms drug therapy, Cell-Penetrating Peptides administration & dosage, Drug Delivery Systems methods, Gene Products, tat administration & dosage, Glioma drug therapy
- Abstract
Computational modeling shows that intra-arterial delivery is most efficient when the delivered drugs rapidly and avidly bind to the target site. The cell-penetrating peptide trans-activator of transcription (TAT) is a candidate carrier molecule that could mediate such specificity for brain tumor chemotherapeutics. To test this hypothesis we first performed in vitro studies testing the uptake of TAT by one primary and three potentially metastatic brain cancer cell lines (9L, 4T-1, LLC, SKOV-3). Then we performed in vivo studies in a rat model where TAT was delivered either intra-arterially (IA) or intravenously (IV) to 9L brain tumors. We observed robust uptake of TAT by all tumor cell lines in vitro. Flow cytometry and confocal microscopy revealed a rapid uptake of fluorescein-labeled TAT within 5 min of exposure to the cancer cells. IA injections done under transient cerebral hypoperfusion (TCH) generated a four-fold greater tumor TAT concentration compared to conventional IV injections. We conclude that it is feasible to selectively target brain tumors with TAT-linked chemotherapy by the IA-TCH method.
- Published
- 2017
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42. GABA A receptor α 4 -subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model.
- Author
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Yocum GT, Turner DL, Danielsson J, Barajas MB, Zhang Y, Xu D, Harrison NL, Homanics GE, Farber DL, and Emala CW
- Subjects
- Animals, Asthma immunology, CD4-Positive T-Lymphocytes immunology, Cell Line, Disease Models, Animal, Lung immunology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Pneumonia immunology, Th2 Cells immunology, Asthma genetics, Lung pathology, Pneumonia genetics, Receptors, GABA-A genetics
- Abstract
Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABA
A R) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAA Rs, particularly isoforms containing α4 -subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAA R signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAA R α4 -subunit global knockout (KO; Gabra40/0 ) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAA R α4 -subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAA R α4 -subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation., (Copyright © 2017 the American Physiological Society.)- Published
- 2017
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43. Alleviation of Multiple Asthmatic Pathologic Features with Orally Available and Subtype Selective GABA A Receptor Modulators.
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Forkuo GS, Nieman AN, Yuan NY, Kodali R, Yu OB, Zahn NM, Jahan R, Li G, Stephen MR, Guthrie ML, Poe MM, Hartzler BD, Harris TW, Yocum GT, Emala CW, Steeber DA, Stafford DC, Cook JM, and Arnold LA
- Subjects
- Animals, Bronchoalveolar Lavage Fluid, Disease Models, Animal, Eosinophils metabolism, Flow Cytometry, Humans, Lung, Male, Mice, Mice, Inbred BALB C, Ovalbumin metabolism, Receptors, GABA metabolism, Respiratory Hypersensitivity metabolism, Swine, Asthma pathology
- Abstract
We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α
4 β3 γ2 GABAA R selective compound 1 and acidic α5 β3 γ2 selective GABAA R positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4+ T lymphocytes and directly modulated their transmembrane currents by acting on GABAA Rs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABAA Rs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAA R ligands.- Published
- 2017
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44. Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling.
- Author
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Yocum GT, Chen J, Choi CH, Townsend EA, Zhang Y, Xu D, Fu XW, Sanderson MJ, and Emala CW
- Subjects
- Acetylcholine pharmacology, Animals, Capsaicin analogs & derivatives, Capsaicin pharmacology, Guinea Pigs, Humans, Immunohistochemistry, Methacholine Chloride pharmacology, Mice, Muscle Contraction drug effects, Muscle, Smooth drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, TRPV Cation Channels genetics, Trachea drug effects, Calcium metabolism, Muscle, Smooth metabolism, TRPV Cation Channels metabolism, Trachea metabolism
- Abstract
Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca
2+ -ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine ( n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 ( n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction ( n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments ( n = 4-5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS ( n = 4-7, P = nonsignificant), it did inhibit calcium oscillations ( n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations., (Copyright © 2017 the American Physiological Society.)- Published
- 2017
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45. Impaired Relaxation of Airway Smooth Muscle in Mice Lacking the Actin-Binding Protein Gelsolin.
- Author
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Mikami M, Zhang Y, Danielsson J, Joell T, Yong HM, Townsend E, Khurana S, An SS, and Emala CW
- Subjects
- Actins metabolism, Animals, Biomechanical Phenomena drug effects, Cell Separation, Chloroquine pharmacology, Electric Impedance, Inositol Phosphates metabolism, Lung drug effects, Male, Mice, Inbred C57BL, Mice, Knockout, Muscle Relaxation drug effects, Muscle, Smooth drug effects, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled metabolism, Gelsolin metabolism, Lung physiology, Muscle Relaxation physiology, Muscle, Smooth physiology
- Abstract
Diverse classes of ligands have recently been discovered that relax airway smooth muscle (ASM) despite a transient increase in intracellular calcium concentrations ([Ca
2+ ]i ). However, the cellular mechanisms are not well understood. Gelsolin is a calcium-activated actin-severing and -capping protein found in many cell types, including ASM cells. Gelsolin also binds to phosphatidylinositol 4,5-bisphosphate, making this substrate less available for phospholipase Cβ-mediated hydrolysis to inositol triphosphate and diacylglycerol. We hypothesized that gelsolin plays a critical role in ASM relaxation and mechanistically accounts for relaxation by ligands that transiently increase [Ca2+ ]i . Isolated tracheal rings from gelsolin knockout (KO) mice showed impaired relaxation to both a β-agonist and chloroquine, a bitter taste receptor agonist, which relaxes ASM, despite inducing transiently increased [Ca2+ ]i . A single inhalation of methacholine increased lung resistance to a similar extent in wild-type and gelsolin KO mice, but the subsequent spontaneous relaxation was less in gelsolin KO mice. In ASM cells derived from gelsolin KO mice, serotonin-induced Gq-coupled activation increased both [Ca2+ ]i and inositol triphosphate synthesis to a greater extent compared to cells from wild-type mice, possibly due to the absence of gelsolin binding to phosphatidylinositol 4,5-bisphosphate. Single-cell analysis showed higher filamentous:globular actin ratio at baseline and slower cytoskeletal remodeling dynamics in gelsolin KO cells. Gelsolin KO ASM cells also showed an attenuated decrease in cell stiffness to chloroquine and flufenamic acid. These findings suggest that gelsolin plays a critical role in ASM relaxation and that activation of gelsolin may contribute to relaxation induced by ligands that relax ASM despite a transient increase in [Ca2+ ]i .- Published
- 2017
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46. Flow arrest intra-arterial delivery of small TAT-decorated and neutral micelles to gliomas.
- Author
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Nguyen J, Hossain SS, Cooke JRN, Ellis JA, Deci MB, Emala CW, Bruce JN, Bigio IJ, Straubinger RM, and Joshi S
- Subjects
- Animals, Blood-Brain Barrier drug effects, Blood-Brain Barrier metabolism, Brain Neoplasms metabolism, Cations, Cell Line, Tumor, Computer Simulation, Dose-Response Relationship, Drug, Endothelial Cells drug effects, Endothelial Cells metabolism, Glioma metabolism, Hemodynamics, Hydrogen-Ion Concentration, Injections, Intra-Arterial, Models, Biological, Nanoparticles administration & dosage, Nanoparticles chemistry, Particle Size, Rats, Brain Neoplasms drug therapy, Drug Delivery Systems, Gene Products, tat chemistry, Glioma drug therapy, Micelles
- Abstract
The cell-penetrating trans-activator of transcription (TAT) is a cationic peptide derived from human immunodeficiency virus-1. It has been used to facilitate macromolecule delivery to various cell types. This cationic peptide is capable of crossing the blood-brain barrier and therefore might be useful for enhancing the delivery of drugs that target brain tumors. Here we test the efficiency with which relatively small (20 nm) micelles can be delivered by an intra-arterial route specifically to gliomas. Utilizing the well-established method of flow-arrest intra-arterial injection we compared the degree of brain tumor deposition of cationic TAT-decorated micelles versus neutral micelles. Our in vivo and post-mortem analyses confirm glioma-specific deposition of both TAT-decorated and neutral micelles. Increased tumor deposition conferred by the positive charge on the TAT-decorated micelles was modest. Computational modeling suggested a decreased relevance of particle charge at the small sizes tested but not for larger particles. We conclude that continued optimization of micelles may represent a viable strategy for targeting brain tumors after intra-arterial injection. Particle size and charge are important to consider during the directed development of nanoparticles for intra-arterial delivery to brain tumors.
- Published
- 2017
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47. Dexmedetomidine's inhibitory effects on acetylcholine release from cholinergic nerves in guinea pig trachea: a mechanism that accounts for its clinical benefit during airway irritation.
- Author
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Mikami M, Zhang Y, Kim B, Worgall TS, Groeben H, and Emala CW
- Subjects
- Anesthetics pharmacology, Animals, Dose-Response Relationship, Drug, Electric Stimulation, Guinea Pigs, In Vitro Techniques, Lidocaine pharmacology, Male, Muscle Contraction drug effects, Muscle Contraction physiology, Muscle, Smooth drug effects, Muscle, Smooth physiology, Piperidines pharmacology, Remifentanil, Trachea drug effects, Acetylcholine metabolism, Cholinergic Neurons drug effects, Cholinergic Neurons metabolism, Dexmedetomidine pharmacology, Trachea innervation
- Abstract
Background: Airway instrumentation can evoke upper airway reflexes including bronchoconstriction and cough which can cause serious complications including airway trauma, laryngospasm or bronchospasm which may in turn lead to difficulty with ventilation and hypoxemia. These airway events are mediated in part by irritant-induced neuronal modulation of airway tone and cough responses. We investigated whether the commonly used anesthetic agents dexmedetomidine, lidocaine or remifentanil attenuated neuronal and airway smooth muscle responses in the upper airways of guinea pigs., Methods: The ability of dexmedetomidine, lidocaine or remifentanil to attenuate direct cholinergic nerve stimulation, C-fiber stimulation or direct smooth muscle contraction were studied using isolated tracheal rings from male guinea pigs under four paradigms; (1) the magnitude of contractile force elicited by cholinergic electrical field stimulation (EFS); (2) the amount of acetylcholine released during cholinergic EFS; (3) the direct airway smooth muscle relaxation of a sustained acetylcholine-induced contraction and (4) the magnitude of C-fiber mediated contraction., Results: Dexmedetomidine (1-100 μM) and lidocaine (1 mM) attenuated cholinergic 30Hz EFS-induced tracheal ring contraction while remifentanil (10 μM) had no effect. Dexmedetomidine at 10 μM (p = 0.0047) and 100 μM (p = 0.01) reduced cholinergic EFS-induced acetylcholine release while lidocaine (10 μM-1 mM) and remifentanil (0.1-10 μM) did not. Tracheal ring muscle force induced by the exogenous addition of the contractile agonist acetylcholine or by a prototypical C-fiber analogue of capsaicin were also attenuated by 100 μM dexmedetomidine (p = 0.0061 and p = 0.01, respectively). The actual tracheal tissue concentrations of dexmedetomidine achieved (0.54-26 nM) following buffer application of 1-100 μM of dexmedetomidine were within the range of clinically achieved plasma concentrations (12 nM)., Conclusions: The α2 adrenoceptor agonist dexmedetomidine reduced cholinergic EFS-induced contractions and acetylcholine release consistent with the presence of inhibitory α2 adrenoceptors on the prejunctional side of the postganglionic cholinergic nerve-smooth muscle junction. Dexmedetomidine also attenuated both exogenous acetylcholine-induced contraction and C-fiber mediated contraction, suggesting a direct airway smooth muscle effect and an underlying mechanism for cough suppression, respectively.
- Published
- 2017
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48. Ex vivo visualization of human ciliated epithelium and quantitative analysis of induced flow dynamics by using optical coherence tomography.
- Author
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Ling Y, Yao X, Gamm UA, Arteaga-Solis E, Emala CW, Choma MA, and Hendon CP
- Subjects
- Biopsy, Needle, Cilia pathology, Epithelium diagnostic imaging, Epithelium pathology, Humans, Immunohistochemistry, In Vitro Techniques, Mucociliary Clearance physiology, Respiratory System pathology, Sampling Studies, Sensitivity and Specificity, Tissue Culture Techniques, Image Processing, Computer-Assisted, Imaging, Three-Dimensional methods, Respiratory System diagnostic imaging, Tomography, Optical Coherence methods
- Abstract
Background and Objective: Cilia-driven mucociliary clearance is an important self-defense mechanism of great clinical importance in pulmonary research. Conventional light microscopy possesses the capability to visualize individual cilia and its beating pattern but lacks the throughput to assess the global ciliary activities and flow dynamics. Optical coherence tomography (OCT), which provides depth-resolved cross-sectional images, was recently introduced to this area., Materials and Methods: Fourteen de-identified human tracheobronchial tissues are directly imaged by two OCT systems: one system centered at 1,300 nm with 6.5 μm axial resolution and 15 μm lateral resolution, and the other centered at 800 nm with 2.72 μm axial resolution and 5.52 μm lateral resolution. Speckle variance images are obtained in both cross-sectional and volumetric modes. After imaging, sample blocks are sliced along the registered OCT imaging plane and processed with hematoxylin and eosin (H&E) stain for comparison. Quantitative flow analysis is performed by tracking the path-lines of microspheres in a fixed cross-section. Both the flow rate and flow direction are characterized., Results: The speckle variance images successfully segment the ciliated epithelial tissue from its cilia-denuded counterpart, and the results are validated by corresponding H&E stained sections. A further temporal frequency analysis is performed to extract the ciliary beat frequency (CBF) at cilia cites. By adding polyester microspheres as contrast agents, we demonstrate ex vivo imaging of the flow induced by cilia activities of human tracheobronchial samples., Conclusion: This manuscript presents an ex vivo study on human tracheobronchial ciliated epithelium and its induced mucous flow by using OCT. Within OCT images, intact ciliated epithelium is effectively distinguished from cilia-denuded counterpart, which serves as a negative control, by examining the speckle variance images. The cilia beat frequency is extracted by temporal frequency analysis. The flow rate, flow direction, and particle throughput are obtained through particle tracking. The availability of these quantitative parameters provides us with a powerful tool that will be useful for studying the physiology, pathophysiology and the effectiveness of therapies on epithelial cilia function, as well as serve as a diagnostic tool for diseases associated with ciliary dysmotility. Lasers Surg. Med. 49:270-279, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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49. Optimization of substituted imidazobenzodiazepines as novel asthma treatments.
- Author
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Jahan R, Stephen MR, Forkuo GS, Kodali R, Guthrie ML, Nieman AN, Yuan NY, Zahn NM, Poe MM, Li G, Yu OB, Yocum GT, Emala CW, Stafford DC, Cook JM, and Arnold LA
- Subjects
- Animals, Benzodiazepines chemistry, Benzodiazepines therapeutic use, Constriction, Pathologic drug therapy, Deuterium pharmacology, Drug Evaluation, Preclinical, Drug Stability, Guinea Pigs, Methacholine Chloride pharmacology, Mice, Respiratory Hypersensitivity drug therapy, Structure-Activity Relationship, Sulfuric Acid Esters pharmacology, Trachea drug effects, Trachea pathology, Asthma drug therapy, Benzodiazepines pharmacology, Receptors, GABA-A metabolism
- Abstract
We describe the synthesis of analogs of XHE-III-74, a selective α4β3γ2 GABA
A R ligand, shown to relax airway smooth muscle ex vivo and reduce airway hyperresponsiveness in a murine asthma model. To improve properties of this compound as an asthma therapeutic, a series of analogs with a deuterated methoxy group in place of methoxy group at C-8 position was evaluated for isotope effects in preclinical assays; including microsomal stability, cytotoxicity, and sensorimotor impairment. The deuterated compounds were equally or more metabolically stable than the corresponding non-deuterated analogs and increased sensorimotor impairment was observed for some deuterated compounds. Thioesters were more cytotoxic in comparison to other carboxylic acid derivatives of this compound series. The most promising compound 16 identified from the in vitro screens also strongly inhibited smooth muscle constriction in ex vivo guinea pig tracheal rings. Smooth muscle relaxation, determined by reduction of airway hyperresponsiveness with a murine ovalbumin sensitized and challenged model, showed that 16 was efficacious at low methacholine concentrations. However, this effect was limited due to suboptimal pharmacokinetics of 16. Based on these findings, further analogs of XHE-III-74 will be investigated to improve in vivo metabolic stability while retaining the efficacy at lung tissues involved in asthma pathology., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)- Published
- 2017
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50. Safety, feasibility, and optimization of intra-arterial mitoxantrone delivery to gliomas.
- Author
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Ellis JA, Cooke J, Singh-Moon RP, Wang M, Bruce JN, Emala CW, Bigio IJ, and Joshi S
- Subjects
- Animals, Brain drug effects, Brain metabolism, Cell Line, Tumor, Dose-Response Relationship, Drug, Drug Delivery Systems, Feasibility Studies, Female, Humans, Infusions, Intra-Arterial, Male, Rats, Xenograft Model Antitumor Assays, Antineoplastic Agents administration & dosage, Brain Neoplasms drug therapy, Glioma drug therapy, Mitoxantrone administration & dosage
- Abstract
Mitoxantrone is a highly cytotoxic antineoplastic drug, however, its poor penetration of the blood-brain barrier has limited its role in the treatment of brain cancers. We hypothesize that intra-arterial (IA) delivery of mitoxantrone may enhance its capacity for regional brain deposition thus expanding its potential as a brain tumor therapy agent. In this study we assessed the dose-response characteristics as well as the feasibility and safety of mitoxantrone delivery to the brain and specifically to gliomas in a rodent model. We show that delivery optimization utilizing the technique of intra-arterial transient cerebral hypoperfusion (IA-TCH) facilitates achieving the highest peak- and end- brain drug concentrations as compared to intravenous and IA delivery without hypoperfusion. Additionally, we observed significant tumor-specific uptake of mitoxantrone when delivered by the IA-TCH method. No untoward effects of IA-TCH delivery of mitoxantrone were observed. The IA-TCH method is shown to be a safely tolerated and feasible strategy for delivering mitoxantrone to tumors in the glioma model tested. Additional investigation is warranted to determine if IA-TCH delivery of mitoxantrone produces clinically relevant benefit.
- Published
- 2016
- Full Text
- View/download PDF
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