Increased airway smooth muscle (ASM) mass contributes to airway hyperresponsiveness (AHR) in asthma and is orchestrated by growth factors, cytokines and chemokines. Airway contractile responses are influenced by neuromediators, such as acetylcholine, and glutamate released by parasympathetic and sympathetic airway nerves. Hyperactivity of these neural elements, termed neurogenic inflammation, is linked with hypercontractility and AHR. Glutamate is a non-essential amino acid derivative, and its physiological role is traditionally considered with respect to its being the primary excitatory neurotransmitter in brain, and regulation of neuronal development and memory. In allergic inflammation, immune cells including dendritic cells, neutrophils and eosinophils, constitutively synthesize and release glutamate, which signals through activation of glutamate receptors, most important among which are ionotrophic N-methyl D-aspartate receptors (NMDA-R). We hypothesized that glutamatergic signaling mediated through NMDA-Rs plays an important role in inducing functional Ca2+ responses in human (H) ASM cells that can underpin airway hypercontractility. We investigated the expression and function of NMDA-Rs in HASM cells, and assessed the effects of pro-inflammatory cytokines on NMDA-R expression and functional responses. Moreover, we measured airway responses to NMDA in mice, murine thin cut lung slice preparations, and floating collagen gels seeded with HASMs. Our data reveal that airway myocytes express multi-subunit NMDA-R complexes that function as receptor-operated calcium channels (ROCCs), mobilizing intracellular Ca2+ in ASM in vitro and airway contraction ex vivo. Individual airway myocytes treated with NMDA-R agonist exhibit disparate temporal patterns of intercellular Ca2+ flux that can be partitioned into four discrete function sub-groups. Further we show that tumor necrosis factor (TNF) exposure modulates NMDA-R subunit expression, and these changes are associated with a shift in the distribution of myocytes in individual Ca2+-mobilization sub-groups in vitro. Further, post-TNF exposure, NMDA-R agonists’ treatment induced Ca2+-dependent airway dilation in murine lung slice preparations, an effect that was prevented by co-treatment with inhibitors of nitric oxide synthase (NOS) or cyclooxygenase (COX). Taken together, we conclude that NMDA-R regulate HASM-mediated airway contraction and their role can be affected upon exposure to asthma-associated inflammatory mediators. Thus, NMDA-Rs are of relevance to mechanisms that determine airway narrowing and AHR associated with chronic respiratory diseases. / October 2015

<p>Asthma is a disease characterized by transient airway smooth muscle contraction leading to episodes of reversible airway narrowing. It affects over 300 million people worldwide and is implicated in over 250 000 deaths annually. The primary clinical features of asthma include airway inflammation, hyperresponsiveness, and remodeling. Generally, asthmatic patients experience exacerbations between periods of diminished symptoms. Interestingly, in addition to these above mentioned hallmarks, asthmatics have also been shown to react differently to ventilatory mechanical strain. This is most evident when assessing the effect of a deep inspiration (DI), clinically measured as a breath taken from functional residual capacity to total lung capacity, in healthy individuals <em>versus</em> asthmatics. These deep inspiratory efforts have been shown to produce a bronchodilatory response in healthy individuals, whereas in asthmatics, DIs are less effective in producing bronchodilation, can cause more rapid airway re-narrowing, and even bronchoconstriction in moderate to severe asthmatics. The mechanism by which a DI is able to cause bronchoconstriction remains ambiguous. Previous theories suggest that this phenomenon is intrinsic to airway smooth muscle (ASM) itself. However, the airway inflammation present in asthmatic airways may also add to the increased ASM contractility following stretch, by the release of mediators that can prime the contractile apparatus to react excessively in the presence of stretch.</p> <p>Thus, collectively, the studies contained in this thesis are linked to the general theme of greater characterization of the signalling mechanisms that regulate airway stretch-activated contractions using a pharmacological approach in intact bovine bronchial segments, with the hope of providing novel insights into the mechanisms that regulate the DI-induced bronchoconstriction seen in asthmatics.</p> / Doctor of Philosophy (Medical Science)