Oxidized phosphatidylcholines inhibit airway smooth muscle β2-adrenergic receptors via protein kinase C

Understanding mechanisms for β2 adrenergic receptor (β2AR) insensitivity in asthma is incomplete. We discovered that the accumulation of oxidized phosphatidylcholines (OxPCs) in the lung correlates with airway hyperresponsiveness and that OxPCs induce contraction and cytokine synthesis in human airway smooth muscle (HASM) cells. Here, we test whether OxPCs impair β2AR agonist bronchodilator responses and related mechanism(s) for β2AR insensitivity. Using tracheal rings from BALB/c mice, we assayed the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on β2AR agonist (isoproterenol [Iso])-induced relaxation of methacholine-contracted airways. OxPAPC preexposure attenuated Iso-induced airway relaxation, increasing Iso half-maximal effective concentration (EC50) 4.3-fold and reducing maximum relaxation by 12.2%. OxPAPC did not affect adenylyl cyclase-mediated relaxation induced by forskolin. OxPAPC significantly inhibited Iso-induced bronchodilation in murine precision-cut lung slices. Intranasal OxPAPC prechallenge significantly inhibited albuterol-mediated suppression of methacholine-induced respiratory resistance in BALB/c mice. In cultured HASM cells, OxPAPC dose-dependently inhibited Iso (but not forskolin)-induced phosphorylation of the protein kinase A substrate VASP by up to 53%. Live cell cyclic adenosine monophosphate (cAMP) difference detector in situ assay for intracellular cAMP confirmed that OxPAPC dose-dependently reduces Iso-elicited cAMP generation up to 50%. OxPAPC did not reduce the abundance of cell surface hemagglutinin-tagged human β2AR in HEK293 cells. However, inhibition of protein kinase C prevented the suppressive effects of OxPAPC on both Iso-induced relaxation of murine tracheal rings and cAMP signaling in cultured HASM cells. In summary, OxPAPC impairs β2AR agonist-induced bronchodilation by PKC-dependent suppression of β2AR-mediated cAMP signaling upstream of adenylyl cyclase in HASM cells. These observations reveal a new mechanism for bronchodilator insensitivity.