title: Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia.
abstract: Recent evidence supports a prominent role for Rho kinase (ROK)-mediated pulmonary vasoconstriction in the development and maintenance of chronic hypoxia (CH)-induced pulmonary hypertension. Endothelin (ET)-1 contributes to the pulmonary hypertensive response to CH, and recent studies by our laboratory and others indicate that pulmonary vascular reactivity following CH is largely independent of changes in vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca(2+)](i)). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca(2+) sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined the effect of pharmacological inhibitors of ROK, myosin light chain kinase (MLCK), tyrosine kinase (TK), and PKC on ET-1-induced vasoconstriction in endothelium-denuded, Ca(2+)-permeabilized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Further experiments examined ET-1-mediated, ROK-dependent phosphorylation of the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1. Finally, we measured ET-1-induced ROS generation in dihydroethidium-loaded small pulmonary arteries and investigated the role of ROS in mediating ET-1-induced, RhoA/ROK-dependent Ca(2+) sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca(2+) sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1(Thr696) phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca(2+) sensitization following CH. We conclude that CH augments ET-1-induced Ca(2+) sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.
authors.affiliation: Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, MSC 08-4750, 1 Univ. of New Mexico, Albuquerque, NM 87131-0001, USA. njernigan@salud.unm.edu
authors.forename: Nikki L
authors.initials: NL
authors.lastname: Jernigan
authors.forename: Benjimen R
authors.initials: BR
authors.lastname: Walker
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Resta
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002908:Chronic Disease; D019332:Endothelin-1 / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism* / Q000503:physiopathology; D066298:In Vitro Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology; D054645:Protein Phosphatase 1 / Q000378:metabolism; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D019332:Endothelin-1; D017382:Reactive Oxygen Species; D054460:rho-Associated Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D020742:rhoA GTP-Binding Protein