title: NMDA receptors mediate contractile responses in human airway smooth muscle cells.
abstract: Human airway smooth muscle (HASM) exhibits enhanced contractility in asthma. Inflammation is associated with airway hypercontractility, but factors that underpin these features are not fully elucidated. Glutamate toxicity associated with increased plasma glutamate concentrations was observed in airway inflammation, suggesting that multisubunit glutamate receptors, N-methyl-d-aspartate receptors (NMDA-R) contribute to airway hyperreactivity. We tested the hypothesis that HASM expresses NMDA-R subunits that can form functional receptors to mediate contractile responses to specific extracellular ligands. In cultured HASM cells, we measured NMDA-R subunit mRNA and protein abundance by quantitative PCR, immunoblotting, flow cytometry, and epifluorescence immunocytochemistry. We measured mRNA for a number of NMDA-R subunits, including the obligatory NR1 subunit, which we confirmed to be present as a protein. In vitro and ex vivo functional NMDA-R activation in HASM cells was measured using intracellular calcium flux (fura-2 AM), collagen gel contraction assays, and murine thin-cut lung slices (TCLS). NMDA, a pharmacological glutamate analog, induced cytosolic calcium mobilization in cultured HASM cells. We detected three different temporal patterns of calcium response, suggesting the presence of heterogeneous myocyte subpopulations. NMDA-R activation also induced airway contraction in murine TCLS and soft collagen gels seeded with HASM cells. Responses in cells, lung slices, and collagen gels were mediated by NMDA-R, as they could be blocked by (2R)-amino-5-phosphonopentanoate, a specific NMDA-R inhibitor. In summary, we reveal the presence of NMDA-R in HASM that mediate contractile responses via glutamatergic mechanisms. These findings suggest that accumulation of glutamate-like ligands for NMDA-R associated with airway inflammation contributes directly to airway hyperreactivity.
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Child Health Research Institute of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Vidyanand
authors.initials: V
authors.lastname: Anaparti
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Ramses
authors.initials: R
authors.lastname: Ilarraza
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Kanami
authors.initials: K
authors.lastname: Orihara
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Child Health Research Institute of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Stelmack
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Child Health Research Institute of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Oluwaseun O
authors.initials: OO
authors.lastname: Ojo
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Child Health Research Institute of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Mahood
authors.affiliation: Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; Section of Thoracic Surgery, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Helmut
authors.initials: H
authors.lastname: Unruh
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Child Health Research Institute of Manitoba, Winnipeg, Manitoba, Canada; andrew.halayko@umanitoba.ca.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Halayko
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Child Health Research Institute of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Redwan
authors.initials: R
authors.lastname: Moqbel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D009119:Muscle Contraction / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D012137:Respiratory System / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D002118:Calcium; D016257:Fura-2
keywords: Ca2+ mobilization; asthma; contraction; glutamate; inflammation