Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor Dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPC) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness, and induce bronchial narrowing in murine thin cut lung slices (TCLS). OxPC activate many signaling pathways but mechanisms for these responses are unclear. We hypothesize that OxPC stimulate intracellular free Ca 2+ flux to trigger airway smooth muscle contraction. Intracellular Ca 2+ flux was assessed in Fura-2 loaded, cultured human airway smooth muscle (HASM) cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced ~3-fold increase in 20kDa myosin light chain phosphorylation. This correlated with a rapid peak in cytoplasmic Ca 2+ concentration ([Ca 2+ ] i )(143 nM), and a sustained plateau that included slow oscillations in [Ca 2+ ] i . Sustained [Ca 2+ ] i elevation was ablated in Ca 2+ -free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca 2+ ] i was unaffected in Ca 2+ -free buffer, TRPA1 inhibition, or by the IP3 receptor inhibition. Peak [Ca 2+ ] i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca 2+ release from sarcoplasmic reticulum. Inhibiting the upstream RyR activator cADPR with 8-Br-cADPR was sufficient to abolish OxPAPC-induced cytoplasmic Ca 2+ flux. OxPAPC induced ~15% bronchial narrowing in TCLS that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured HASM. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR mediated mobilization of intracellular and extracellular Ca 2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen challenged, and asthmatic subjects may contribute to airway hyperresponsiveness.