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A1215 - TRPA1 Relaxes Histamine and Methacholine Contracted Airways Through Complementary Mechanisms
Author Block: B. Marsh, A. D. Fryer, D. B. Jacoby, M. G. Drake; Pulmonary and Critical Care Medicine, Oregon Health and Science University, Portland, OR, United States.
Background: Bronchoconstriction is caused by many agents, acting through multiple pathways. Yet clinicians use a “one-size-fits-all” approach to treating bronchoconstriction. Multi-target approaches may improve bronchodilator efficacy. Stimulation of transient receptor potential ankyrin- 1 (TRPA1), a ligand- gated cation channel, causes vasodilation via multiple pathways. TRPA1 is expressed on endothelial cells, vascular smooth muscle cells, and sensory neurons, and its vasodilatory effects are the result of pathways induced by each of these cell types. Because TRPA1 is also expressed on airway epithelial cells, airway smooth muscle cells, and pulmonary sensory nerves, we reasoned that TRPA1 stimulation would similarly induce bronchodilation through multiple, complementary mechanisms. Methods: Isolated guinea pig tracheas, mouse tracheas, and post- mortem human trachealis muscle strips were suspended in an organ bath and contracted with methacholine (10uM, 10uM, or 20uM, respectively), histamine (20uM), or potassium chloride (40mM). Contraction strength was measured with a force transducer. Airways were then treated with the TRPA1 agonists allyl isothiocyanate or cinnamaldehyde (10-1000uM). Some airways were pre-treated with TRPA1 antagonists HC030031 or A967079, the nitric oxide synthase inhibitor L-NG-monomethyl arginine citrate (L-NMMA), the non-selective cyclooxygenase inhibitor indomethacin, or the M2 muscarinic receptor antagonist gallamine. Epithelium was removed from some airways prior to contraction. Airway responses were also assessed in guinea pigs in vivo. Animals were anesthetized and intubated. Bronchoconstriction was induced by electrical vagal stimulation (EVS), and calculated based on increases in inspiratory pressure. Animals then received intravenous or inhaled cinnamaldehyde, and EVS-induced contractions were repeated. Results: TRPA1 agonists relaxed pre-contracted human, mouse, and guinea pig tracheal segments in vitro, and blocked vagally-mediated bronchoconstriction in vivo. Relaxation of both histamine and methacholine contracted tissues was partially inhibited indomethacin. In methacholine contracted airways, relaxation required airway epithelium, whereas in histamine contracted airways, relaxation was epithelium-independent. Pretreatment with gallamine restored relaxation of methacholine-contracted airways that lacked epithelium. Pretreatment with L-NMMA partially inhibited relaxation of methacholine contracted airways. Conclusions: TRPA1 stimulation relaxed pre-contracted tracheal smooth muscle and prevented vagally-medicated bronchoconstriction. TRPA1 stimulation relaxed both histamine and methacholine contracted airways via non-epithelial prostaglandins. In addition, TRPA1 stimulation generated an epithelium-derived factor, likely the M2 muscarinic receptor inhibitor nitric oxide, that was specifically required for relaxation of methacholine contracted tissue. These studies demonstrate that TRPA1 stimulation relaxes bronchoconstriction via multiple mechanisms that target specific contractile pathways.