Author:
Hinton Martha,Gutsol Alex,Dakshinamurti Shyamala
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 TxA2hypersensitivity. Here, we examined TxA2prostanoid receptor (TP-R) localization and kinetics following hypoxia to determine the mechanism of hypoxia-induced TxA2hypersensitivity. 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% FiO2for 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 using3H-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 μm. 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 Kdand 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 Gαqcoupling 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 Gαq; 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.
Publisher
American Physiological Society
Subject
Cell Biology,Physiology (medical),Pulmonary and Respiratory Medicine,Physiology
Cited by
26 articles.
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