Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia

Chronic hypoxia (CH) augments basal and endothelin 1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction following CH. To test this hypothesis, both endpoints were monitored in pressurized, endothelium-disrupted pulmonary arteries (4th-5th order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous (F) and globular (G) actin ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor SMIFH2. Immunoblotting revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin, but not phospho-cofilin/total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity following CH in a ROS-, ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization following CH is dependent on mDia, but independent of changes in the phosphorylated/total cofilin ratio.