title: Thromboxane hypersensitivity in hypoxic pulmonary artery myocytes: altered TP receptor localization and kinetics.
abstract: Hypoxia-induced neonatal persistent pulmonary hypertension (PPHN) is characterized by sustained vasospasm and increased thromboxane (TxA2)-to-prostacyclin ratio. We previously demonstrated that moderate hypoxia induces myocyte TxA2 hypersensitivity. Here, we examined TxA2 prostanoid receptor (TP-R) localization and kinetics following hypoxia to determine the mechanism of hypoxia-induced TxA2 hypersensitivity. Primary cultured neonatal pulmonary artery myocytes were exposed to 10% O2 (hypoxic myocytes; HM) or 21% O2 (normoxic myocytes; NM) for 3 days. PPHN was induced in neonatal piglets by in vivo exposure to 10% FiO2 for 3 days. TP-R was studied in whole lung sections from pigs with hypoxic PPHN- and age-matched controls; intracellular localization was studied by immunocytochemistry. TP-R affinity was studied in cultured myocytes by saturation binding kinetics using 3H-SQ-29548 and competitive binding kinetics by coincubation with U-46619. Phosphorylation and coupling were examined in immunoprecipitated TP-R. We report distal propagation of TP-R expression in PPHN, extending to pulmonary arteries <50 microm. In HM, intracellular TP-R moves towards the perinuclear region, mirroring a change in endoplasmic reticulum (ER) morphology. TP-R kinetics also alter in HM membranes, with decreased Kd and Bmax (maximal binding sites). Additionally, in hypoxia, 3H-SQ-29548 is displaced at lower concentration of U-46619 than in normoxia, suggesting increased agonist affinity. Phosphorylation of serine residues on HM TP-R was significantly decreased compared with NM; this difference correlated with increased Galphaq coupling in hypoxia and was ablated by incubation with PKA. We conclude that the TP-R is normally desensitized in the neonatal pulmonary circuit by PKA-mediated regulatory phosphorylation, decreasing ligand affinity and coupling to Galphaq; this protection is lost following hypoxic exposure. Also, the appearance of TP-R in resistance arteries after development of hypoxic PPHN may contribute to increased pulmonary arterial pressure.
authors.affiliation: Department of Physiology, University of Manitoba, Manitoba Institute of Child Health, Manitoba, Canada.
authors.forename: Martha
authors.initials: M
authors.lastname: Hinton
authors.forename: Alex
authors.initials: A
authors.lastname: Gutsol
authors.forename: Shyamala
authors.initials: S
authors.lastname: Dakshinamurti
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004195:Disease Models, Animal; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D000860:Hypoxia / Q000503:physiopathology*; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D007700:Kinetics; D008024:Ligands; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000378:metabolism*; D013552:Swine; D013928:Thromboxane A2 / Q000378:metabolism*; D014661:Vasoconstriction / Q000187:drug effects*
chemical_list: D008024:Ligands; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013928:Thromboxane A2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium