title: Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.
abstract: In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms regulating thromboxane-mediated actin polymerization are potential targets for future PPHN pharmacotherapy.
authors.affiliation: Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; Department of Physiology University of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Jena
authors.initials: J
authors.lastname: Fediuk
authors.affiliation: Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; Department of Physiology University of Manitoba, Winnipeg, Manitoba, Canada;
authors.forename: Anurag S
authors.initials: AS
authors.lastname: Sikarwar
authors.affiliation: Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada;
authors.forename: Nora
authors.initials: N
authors.lastname: Nolette
authors.affiliation: Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; Department of Physiology University of Manitoba, Winnipeg, Manitoba, Canada; Department of Pediatrics, University of Manitoba, Winnipeg, Manitoba, Canada dakshina@cc.umanitoba.ca.
authors.forename: Shyamala
authors.initials: S
authors.lastname: Dakshinamurti
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology*; D000199:Actins / Q000096:biosynthesis / Q000378:metabolism*; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D009119:Muscle Contraction / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010547:Persistent Fetal Circulation Syndrome / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D011651:Pulmonary Artery / Q000187:drug effects* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D013552:Swine; D013931:Thromboxanes / Q000494:pharmacology*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000199:Actins; D000634:Aminoquinolines; C487513:NSC 23766; D019384:Nucleic Acid Synthesis Inhibitors; D020033:Protein Isoforms; D011743:Pyrimidines; D013931:Thromboxanes; D014662:Vasoconstrictor Agents; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D015638:Cytochalasin D; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein
keywords: Cdc42; Rac; actin isoforms; actin polymerization; hypoxia; persistent pulmonary hypertension of the newborn; thromboxane