Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2-bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. / October 2016