scholarly journals Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity after chronic hypoxia

2018 ◽  
Vol 314 (5) ◽  
pp. H1011-H1021 ◽  
Author(s):  
Laura Weise-Cross ◽  
Michelle A. Sands ◽  
Joshua R. Sheak ◽  
Brad R. S. Broughton ◽  
Jessica B. Snow ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Actin Polymerization ◽  
Chronic Hypoxia ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Reactive Oxygen ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Pulmonary Arterial ◽  
Arterial Constriction

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

2004 ◽  
Vol 287 (4) ◽  
pp. H1495-H1500 ◽  
Author(s):  
Liming Jin ◽  
Zhekang Ying ◽  
R. Clinton Webb
Keyword(s):  
Reactive Oxygen Species ◽  
Signaling Pathway ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Rho Kinase ◽  
Reactive Oxygen ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Kinase Signaling ◽  
Kinase Signaling Pathway

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H2O2 are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 μM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-( R)- trans-4-(1-aminoethyl- N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca2+-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca2+ sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca2+-independent PKC.

scholarly journals Pulmonary Arterial Responses to Reactive Oxygen Species Are Altered in Newborn Piglets With Chronic Hypoxia-Induced Pulmonary Hypertension

2011 ◽  
Vol 70 (2) ◽  
pp. 136-141 ◽  
Author(s):  
Candice D Fike ◽  
Judy L Aschner ◽  
James C Slaughter ◽  
Mark R Kaplowitz ◽  
Yongmei Zhang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Pulmonary Hypertension ◽  
Chronic Hypoxia ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Newborn Piglets ◽  
Pulmonary Arterial ◽  
Arterial Responses

scholarly journals PKCβ and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats

2020 ◽  
Vol 318 (2) ◽  
pp. H470-H483 ◽  
Author(s):  
Joshua R. Sheak ◽  
Simin Yan ◽  
Laura Weise-Cross ◽  
Rosstin Ahmadian ◽  
Benjimen R. Walker ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Chronic Hypoxia ◽  
Primary Cultures ◽  
Pulmonary Arteries ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Neonatal Rats ◽  
Basal Tone

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCβ and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCβ and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCβ inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline -perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCβ-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCβ in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCβ activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure. NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCβ and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

scholarly journals Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia

2008 ◽  
Vol 295 (3) ◽  
pp. L515-L529 ◽  
Author(s):  
Nikki L. Jernigan ◽  
Benjimen R. Walker ◽  
Thomas C. Resta
Keyword(s):  
Reactive Oxygen Species ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Chronic Hypoxia ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Ros Generation ◽  
Oxygen Species

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 ([Ca2+]i). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca2+ sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca2+ 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, Ca2+-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 Ca2+ sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca2+ sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1Thr696 phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca2+ sensitization following CH. We conclude that CH augments ET-1-induced Ca2+ sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

2003 ◽  
Vol 285 (2) ◽  
pp. L322-L333 ◽  
Author(s):  
J. Q. Liu ◽  
J. S. K. Sham ◽  
L. A. Shimoda ◽  
P. Kuppusamy ◽  
J. T. Sylvester
Keyword(s):  
Reactive Oxygen Species ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Pulmonary Arteries ◽  
Reactive Oxygen ◽  
Spin Adduct ◽  
Internal Diameter ◽  
Oxygen Species ◽  
Paramagnetic Resonance ◽  
Alkyl Radicals ◽  
Pulmonary Arterial

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endotheliumdenuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.

scholarly journals Mechanisms of NFATc3 activation by increased superoxide and reduced hydrogen peroxide in pulmonary arterial smooth muscle

2014 ◽  
Vol 307 (10) ◽  
pp. C928-C938 ◽  
Author(s):  
Juan Manuel Ramiro-Diaz ◽  
Wieslawa Giermakowska ◽  
John M. Weaver ◽  
Nikki L. Jernigan ◽  
Laura V. Gonzalez Bosc
Keyword(s):  
Reactive Oxygen Species ◽  
Smooth Muscle ◽  
Actin Polymerization ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Arterial Smooth Muscle ◽  
Activated T Cells ◽  
Intracellular Ca ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle

We recently demonstrated increased superoxide (O2·−) and decreased H2O2 levels in pulmonary arteries of chronic hypoxia-exposed wild-type and normoxic superoxide dismutase 1 (SOD1) knockout mice. We also showed that this reciprocal change in O2·− and H2O2 is associated with elevated activity of nuclear factor of activated T cells isoform c3 (NFATc3) in pulmonary arterial smooth muscle cells (PASMC). This suggests that an imbalance in reactive oxygen species levels is required for NFATc3 activation. However, how such imbalance activates NFATc3 is unknown. This study evaluated the importance of O2·− and H2O2 in the regulation of NFATc3 activity. We tested the hypothesis that an increase in O2·− enhances actin cytoskeleton dynamics and a decrease in H2O2 enhances intracellular Ca2+ concentration, contributing to NFATc3 nuclear import and activation in PASMC. We demonstrate that, in PASMC, endothelin-1 increases O2·− while decreasing H2O2 production through the decrease in SOD1 activity without affecting SOD protein levels. We further demonstrate that O2·− promotes, while H2O2 inhibits, NFATc3 activation in PASMC. Additionally, increased O2·−-to-H2O2 ratio activates NFATc3, even in the absence of a Gq protein-coupled receptor agonist. Furthermore, O2·−-dependent actin polymerization and low intracellular H2O2 concentration-dependent increases in intracellular Ca2+ concentration contribute to NFATc3 activation. Together, these studies define important and novel regulatory mechanisms of NFATc3 activation in PASMC by reactive oxygen species.

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