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 (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca²⁺ flux to trigger airway smooth muscle contraction. Intracellular Ca²⁺ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) (143 nM) and a sustained plateau that included slow oscillations in [Ca²⁺]_i. Sustained [Ca²⁺]_i elevation was ablated in Ca²⁺-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca²⁺]_i was unaffected in Ca²⁺-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca²⁺]_i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca²⁺ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca²⁺ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca²⁺ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.