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

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(2+) 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 Ca(2+) 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 Ca(2+)-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 Ca(2+) 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 Ca(2+) sensitization and pulmonary vasoconstriction following CH.