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

<jats:p>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 Ca<jats:sup>2+</jats:sup>sensitization. On the basis of separate lines of evidence that CH increases pulmonary arterial superoxide (O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>) generation and that O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>generation mediates enhanced RhoA-dependent Ca<jats:sup>2+</jats:sup>sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor HA-1077 and the O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>-specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca<jats:sup>2+</jats:sup>-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<jats:sub>2</jats:sub><jats:sup>−</jats:sup>generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca<jats:sup>2+</jats:sup>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<jats:sub>2</jats:sub><jats:sup>−</jats:sup>generation, and augmented depolarization-dependent RhoA activation in a O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>-dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O<jats:sub>2</jats:sub><jats:sup>−</jats:sup>generation, and RhoA activation that mediates enhanced myofilament Ca<jats:sup>2+</jats:sup>sensitization and pulmonary vasoconstriction following CH.</jats:p>