Thomas Miles Murphy
Professor Emeritus of Pediatrics
AREAS OF RESEARCH INTEREST
1. Maturation of airway contractile responses
2. Ontogenesis of airways hyperresponsiveness caused by inflammation
3. Role of the high affinity IgE receptor on airway smooth muscle in regulating long term hyperresponsiveness and acute allergic contraction
4. Maturation and mechanisms of airway smooth muscle relaxation
5. Mechanical plasticity of airway smooth muscle and its development
6. Identity and function of airway smooth muscle NAD(P)H oxidase
7. Role of Gastrin Releasing Peptide in the Pathogenesis of Asthma and its Therapeutic Targetability
Our laboratory leads in developing important models of the contributions of airway smooth muscle (ASM) to airway hyperresponsiveness in the young. Five major paradigms are emerging from our work at Duke.
The first is that while active stress changes little with age, shortening of ASM in guinea pigs is maximal at a few weeks of age and then declines substantially during adulthood (Chitano et al, J Appl Physiol 88:1338-1345, 2000). The changes in shortening appear related to parallel changes in myosin light phosphorylation (Chitano et al, Pediatr Pulmonol 39:108-116, 2005) and the content of myosin light chain kinase (Chitano et al, Pediatr Pulmonol 38:456-464, 2005). Unpublished data suggest that an siRNA to MLCK genome suppresses MLCK content and shortening function without affecting active stress generation. Additional data suggest that there is also a secondary role for the content of the 7 amino acid insert + isoforms of myosin heavy chain (SMB1 and SMB2; the “fast” isoforms) that are expressed to a greater extent in the newborn.
The second paradigm examines the impact of early sensitization (3 subcutaneous injections of ovalbumin in the first week of life) on late effects on ASM shortening. Extensive preliminary data demonstrate no effect on active stress and a small effect on shortening in the young, but a late effect on shortening in adults that is substantial. This effect appears to arise jointly from an increase (rather than a decrease) in the adult age group in the content of myosin light chain kinase and a parallel decrease (rather than the normal increase) in the internal resistance to shortening. The changes in MLCK content appear to parallel changes in a cytoskeletal enzyme protein-activating kinase PAK1, which is related to the phosphorylation and fragmentation of the intermediate filament vimentin, which in its long form appears to function as a stress fiber. An increase in phospho-vimentin is postulated to facilitate shortening by decreasing cytoskeletal stress fiber formation. These first two paradigms formed the core of our latest NIH R01 application which was awarded a ranking at the 7th percentile.
The third paradigm involves the failure of ASM from young guinea pigs to relax as efficiently as adult ASM during sustained stimulation (Chitano et al, J Appl Physiol 92:1835-1842, 2002). Further work demonstrates that mechanisms involved in this failure of relaxation include differences in secretion of prostaglandins and differences in acetyl cholinesterase function.
The fourth paradigm involves the failure of newborn trachealis to relax in the fashion of adult ASM following stretch. (In fact, there is demonstrated the potentiation of active stress rather than relaxation following stretch.) Developmental changes in the ASM cytoskeleton play a role in these differences (Am J Physiol: Lung Cell Molec Physiol 289:L909-L915, 2005).
An Editorial Focus in the American Journal of Physiology (“The Importance of Maturational Studies in Airway Smooth Muscle, Am J Physiol: Lung Cell Mol Physiol 289: L898-L901, 2005) discussed elements of paradigms 1 and 4 and pointed to the importance of the work and emphasized that our laboratory was unique in pursuing and elucidating these questions.
A long-term collaboration with the laboratory of Dr. John Hoidal at the University of Utah has shed light on the identity and function of NAD(P)H oxidase in airway smooth muscle. A recent collaborative publication (Sturrock et al, Am J Physiol: Lung Cell Mol Physiol, 2007) provides evidence that the renal isoform of the nox paralog protein (NOX4) is constitutive in ASM NAD(P)H oxidase and is substantially upregulated by the growth factor transforming growth factor beta (TGFβ). Key functions of the ASM NAD(P)H oxidase are to regulate cell growth and hyperplasia and to modulate its contraction (Brar et al, J Biol Chem 28:20017-20026, 1999) through mechanisms involving the AP-1 transcription factor and NF kappa B (Brar et al, Am J Physiol: Lung Cell Molec Physiol 282:L782-L795, 2002). A locally supported project is designed to examine the potential contributions of excessive expression of TGFβ on the airways of patients with Down Syndrome. Using our background in TGFβ1 signaling we are currently examining its potential role in protecting the airways from excessive airway hyperresponsiveness via airway smooth muscle mechanisms.
The fifth paradigm, which is in an earlier stage of development, is that the high affinity IgE receptor of airway smooth muscle FcεRI, which binds non-specifidc and specific IgE molecules, appears to play a role both in generating long-term hyperresponsiveness and in mediating acute contraction from allergens. This work is being done in collaboration with Abdelilah Soussi Gounni PhD, an Associate Professor of Immunology at the University of Manitoba, who discovered the high affinity IgE receptor on human airway smooth muscle and described its upregulation in human asthma in 2005. The work also involves an active collaboration with Dr. Wesley Burks MD, chief of the Division of Allergy-Immunology at Duke. Other collaborators include Timothy Haystead PhD (proteomics); Weiguo Zhang PhD (expert on high affinity IgE receptors and adapter molecules); Jerry Eu MD (expert on mouse trachealis culture systems and calcium signaling). This collaboration will focus on the mechanisms of human airway smooth muscle sensitization and contractile provocation (i.e. asthmatic responses) caused by aeroallergens, food antigens or drugs. Model systems for investigation include human airway smooth muscle and airway smooth muscle from mice with targeted mutations or deletions of linker and adapter molecules assocatied with the high affinity IgE receptor.
The sixth paradigm is the participatory role of airway smooth muscle in the genesis of asthma pathophysiology resulting from secreted products of pulmonary neuroendocrine cells, especially gastrin releasing peptide (GRP). In work done in collaboration with the laboratory of Dr. Mary Sunday we recently discovered receptors for GRP and neuromedin B (NMB) on airway smooth muscle in mice and humans. The EC50 for GRP on mouse trachealis is approximately 10-9 molar. The specific small molecule inhibitor of GRP knocks down this response by 85%. Taken together with published (PNAS Feb 1, 2011) evidence of broad immune cellular inflammatory participation in asthmatic inflammation, we are working toward translation of this biology into potentially more powerful and better integrated therapy for asthma.
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