title: Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.
abstract: We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, Canada; Department of Internal Medicine, University of Manitoba, Winnipeg, Canada; and.
authors.forename: Vidyanand
authors.initials: V
authors.lastname: Anaparti
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, Canada;
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Pascoe
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, Canada;
authors.forename: Aruni
authors.initials: A
authors.lastname: Jha
authors.affiliation: Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, Canada;
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Mahood
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Canada;
authors.forename: Ramses
authors.initials: R
authors.lastname: Ilarraza
authors.affiliation: Department of Internal Medicine, University of Manitoba, Winnipeg, Canada; and Section of Thoracic Surgery, University of Manitoba, Winnipeg, Canada.
authors.forename: Helmut
authors.initials: H
authors.lastname: Unruh
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, Canada;
authors.forename: Redwan
authors.initials: R
authors.lastname: Moqbel
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Canada; Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Canada; Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, Canada; Department of Internal Medicine, University of Manitoba, Winnipeg, Canada; and andrew.halayko@umanitoba.ca.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Halayko
mesh_terms: D000818:Animals; D001980:Bronchi / Q000502:physiology; D016084:Bronchoconstriction; D020013:Calcium Signaling; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D008807:Mice, Inbred BALB C; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha
keywords: Ca2+ mobilization; asthma; bronchodilation; glutamate; inflammation