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.