Keywordspersistent pulmonary hypertension of the newborn; hypoxia; prostanoid; prostacyclin; phosphodiesterase inhibitor ---------------------------------------------------------------- BACKGROUND AND PURPOSENeonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACHWe examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca 2+ response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTSLigand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONSTP receptor sensitivity and EC50 for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca 2+ mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. AbbreviationsEP, prostaglandin E receptor; IP, prostacyclin receptor; IP3, inositol-1,4,5-trisphosphate; NECA, adenosine-5′-N-ethylcarboxamide; PDE, phosphodiesterase; PKA, protein kinase A; TP, thromboxane receptor
Pulmonary arterial vasoconstriction is a hallmark of persistent pulmonary hypertension of the newborn (PPHN). We reported increased calcium responses to thromboxane and selectively increased thromboxane prostanoid (TP) association with Gαq in hypoxic pulmonary artery. Palmitoylation of Gαq is important for efficient receptor-Gαq-phospholipase-C interactions. TPα receptor is not itself amenable to palmitoylation. We studied the role of Gαq palmitoylation in constriction of hypoxic pulmonary artery using pharmacological palmitoylation inhibition, the effects of hypoxia on palmitoylation, and the effects of site-specific cysteine substitution mutations of Gαq on Gαq membrane targeting, TPα association, and calcium dose-response curve to a TP agonist. PPHN pulmonary artery and HEK293T cells expressing TPα were exposed to irreversible palmitoylation inhibitor 2-bromopalmitate before challenge with TP agonist U46619. Palmitate uptake was studied in hypoxic and normoxic myocytes. Wild-type Gαq and Gαq cysteine-to-alanine mutants C9A, C10A, and C9A/C10A were transiently coexpressed in HEK293T cells stably expressing TPα. We examined membrane localization of Gαq, TP receptor-Gαq association by coimmunoprecipitation, and Ca(2+) responses to U46619 in hypoxic and normoxic cells. Gαq palmitoylation is essential for the Ca(2+) response to TPα stimulation. Inhibition of palmitoylation reduces contractile force to thromboxane in PPHN but not in control pulmonary artery. Hypoxia increases palmitoylation of Gαq; the hypoxic. but not the normoxic, response to thromboxane is palmitoylation sensitive. Palmitoylation of one N-terminal cysteine is required for physical association of Gαq with the TPα receptor. Palmitoylation of both cysteines is required for Gαq membrane localization and Ca(2+) mobilization. Depalmitoylation of any one Gαq cysteine reduces the hypoxic response to thromboxane challenge to equal that of normoxic cells.
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