Chronic hypoxia augments depolarization-induced Ca2+ sensitization in pulmonary vascular smooth muscle through superoxide-dependent stimulation of RhoA

Broughton BR, Jernigan NL, Norton CE, Walker BR, Resta TC. Chronic hypoxia augments depolarization-induced Ca2+ sensitization in pulmonary vascular smooth muscle through superoxide-dependent stimulation of RhoA. Am J Physiol Lung Cell Mol Physiol 298: L232-L242, 2010. First published November 6, 2009; doi: 10.1152/ajplung.00276.2009.-Rho kinase (ROCK)-dependent vasoconstriction has been implicated as a major factor in chronic hypoxia (CH)-induced pulmonary hypertension. This component of pulmonary hypertension is associated with arterial myogenicity and increased vasoreactivity to receptor-mediated agonists and depolarizing stimuli resulting from ROCK-dependent myofilament Ca2+ sensitization. On the basis of separate lines of evidence that CH increases pulmonary arterial superoxide (O-2(-)) generation and that O-2(-) stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O-2(-) generation mediates enhanced RhoA-dependent Ca2+ sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor HA-1077 and the O-2(-)-specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca2+-permeabilized, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Using the same vessel preparation, we examined effects of CH on KCl-dependent VSM membrane depolarization and O-2(-) generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O-2(-) to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca2+ sensitization mechanism that was inhibited by HA-1077 and tiron. Furthermore, CH caused VSM membrane depolarization that persisted with increasing concentrations of KCl, enhanced KCl-induced O-2(-) generation, and augmented depolarization-dependent RhoA activation in a O-2(-)-dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O-2(-) generation, and RhoA activation that mediates enhanced myofilament Ca2+ sensitization and pulmonary vasoconstriction following CH.