title: Chronic hypoxia augments depolarization-induced Ca2+ sensitization in pulmonary vascular smooth muscle through superoxide-dependent stimulation of RhoA.
abstract: 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.
authors.affiliation: Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
authors.forename: Brad R S
authors.initials: BR
authors.lastname: Broughton
authors.forename: Nikki L
authors.initials: NL
authors.lastname: Jernigan
authors.forename: Charles E
authors.initials: CE
authors.lastname: Norton
authors.forename: Benjimen R
authors.initials: BR
authors.lastname: Walker
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Resta
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D008168:Lung / Q000033:anatomy & histology / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011651:Pulmonary Artery / Q000033:anatomy & histology / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*; D014661:Vasoconstriction; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D013481:Superoxides; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D010100:Oxygen; D002118:Calcium