Thromboxane receptor signaling and Rho GTPase activation on actin polymerization and contraction in hypoxic neonatal pulmonary arterial myocytes
| dc.contributor.author | Fediuk, Jena | |
| dc.contributor.examiningcommittee | Keijzer, Richard (Surgery) Halayko, Andrew (Physiology) Marshall, Aaron (Immunology) Seow, Chun (University of British Columbia) | en_US |
| dc.contributor.supervisor | Dakshinamurti, Shyamala (Physiology) | en_US |
| dc.date.accessioned | 2014-08-23T18:01:28Z | |
| dc.date.available | 2014-08-23T18:01:28Z | |
| dc.date.issued | 2012-01-01 | en_US |
| dc.degree.discipline | Physiology | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | INTRODUCTION: Persistent Pulmonary Hypertension of the Newborn (PPHN) is defined as the failure of normal circulatory relaxation in the lungs at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. Actin polymerization (APM), regulated by Rho GTPases, stabilizes force generation. We studied basal and thromboxane (TP)-induced APM and contraction in normoxic and hypoxic PA myocytes and rings. We also examined the downstream signaling pathways regulating hypoxia and TP-induced APM, and the role that actin plays in TP receptor internalization. METHODS: Smooth muscle myocytes from 2nd to 6th generation PAs of newborn piglets were cultured and exposed to hypoxia (10% O2) or normoxia (21% O2) for 72 hrs, then challenged with 10-6M TP-agonist U46619. APM was quantified by laser-scanning cytometry and stress fiber isolation. Downstream signaling pathways of TP receptor were studied by immunoprecipitation, Rhotekin-RBD and PAK-PBD affinity precipitation, Western blot, immunofluoresence and ELISA. Isometric force to serial concentrations of U46619 was measured in resistant PAs from PPHN and 3-day control swine. RESULTS: Hypoxia induced 2-fold APM via alpha- and gamma-actin isoforms, which contributed to increase U46619-induced contraction. Hypoxia decreased TP association with G12/13 in favor of Gαq. Basal RhoA and Cdc42 activity increased in hypoxia, while Rac activity decreased. U46619-challenge did not further alter RhoA activity in hypoxic cells, but increased Cdc42 and Rac activity. Hypoxia increased phosphorylation of LIMK and PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia, but increased after U46619 exposure. Jasplakinolide significantly stabilized gamma filaments, increasing force generation; cytochalasin D depolymerized all actin isoforms, which attenuated contractile force. Both actin-modifying agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. CONCLUSIONS: PA myocytes exhibit marked RhoA- and Rac-dependent APM in hypoxia. The additional APM response to U46619 challenge is independent of RhoA, but requires Cdc42 signaling. Hypoxia induces APM in PA myocytes, particularly causing an increase in filamentous alpha- and gamma-actin that contributes to increased U46619-induced force generation, a characteristic of PPHN. Dynamic actin also facilitates internalization of the TP receptor. Determining the mechanism that controls TP-mediated APM maybe beneficial as a potential target for PPHN. | en_US |
| dc.description.note | October 2014 | en_US |
| dc.identifier.citation | 302(1):L13-L26 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/23862 | |
| dc.language.iso | eng | en_US |
| dc.publisher | Am J Physiol Lung Cell Mol Physiol | en_US |
| dc.rights | open access | en_US |
| dc.subject | actin polymerization | en_US |
| dc.subject | actin isoforms | en_US |
| dc.subject | PPHN | en_US |
| dc.subject | hypoxia | en_US |
| dc.subject | thromboxane | en_US |
| dc.subject | receptor internalization | en_US |
| dc.subject | contraction | en_US |
| dc.subject | RhoA | en_US |
| dc.subject | Rac | en_US |
| dc.subject | Cdc42 | en_US |
| dc.title | Thromboxane receptor signaling and Rho GTPase activation on actin polymerization and contraction in hypoxic neonatal pulmonary arterial myocytes | en_US |
| dc.type | doctoral thesis | en_US |