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.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement

2014 ◽  
Vol 306 (11) ◽  
pp. H1485-H1494 ◽  
Author(s):  
Rheure Alves Moreira Lopes ◽  
Karla Bianca Neves ◽  
Cezar Rangel Pestana ◽  
André Lima Queiroz ◽  
Camila Ziliotto Zanotto ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Smooth Muscle ◽  
Cytochrome C ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Annexin V ◽  
Ros Generation ◽  
Oxygen Species ◽  
Apoptotic Pathway

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10−7 mol/l) or vehicle (2–12 h) in the presence of flutamide (10−5 mol/l), CCCP (10−6 mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10−5 mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10−5 mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome- c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2ΔΔCt, 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

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

2010 ◽  
Vol 298 (2) ◽  
pp. L232-L242 ◽  
Author(s):  
Brad R. S. Broughton ◽  
Nikki L. Jernigan ◽  
Charles E. Norton ◽  
Benjimen R. Walker ◽  
Thomas C. Resta
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Chronic Hypoxia ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Membrane Depolarization ◽  
Dependent Manner ◽  
Rock Inhibitor ◽  
Rhoa Activation

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 Ca2+ sensitization. On the basis of separate lines of evidence that CH increases pulmonary arterial superoxide (O2−) generation and that O2− stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O2− generation mediates enhanced RhoA-dependent Ca2+ sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor HA-1077 and the O2−-specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca2+-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 O2− generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O2− to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca2+ 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 O2− generation, and augmented depolarization-dependent RhoA activation in a O2−-dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O2− generation, and RhoA activation that mediates enhanced myofilament Ca2+ sensitization and pulmonary vasoconstriction following CH.

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.

Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling

2004 ◽  
Vol 287 (6) ◽  
pp. L1220-L1229 ◽  
Author(s):  
Nikki L. Jernigan ◽  
Benjimen R. Walker ◽  
Thomas C. Resta
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Chronic Hypoxia ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Free Calcium ◽  
No Donor ◽  
Pulmonary Vasodilation ◽  
Spermine Nonoate

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca2+]i). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca2+ desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca2+-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-μm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca2+ sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca2+ desensitization through inhibition of RhoA/ROK.

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.

scholarly journals The role of mitochondrial bioenergetics and reactive oxygen species in coronary collateral growth

2013 ◽  
Vol 305 (9) ◽  
pp. H1275-H1280 ◽  
Author(s):  
Yuh Fen Pung ◽  
Wai Johnn Sam ◽  
James P. Hardwick ◽  
Liya Yin ◽  
Vahagn Ohanyan ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Protein Synthesis ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Energy Production ◽  
Phenotypic Switching ◽  
Oxygen Species ◽  
Coronary Collateral ◽  
Collateral Growth

Coronary collateral growth is a process involving coordination between growth factors expressed in response to ischemia and mechanical forces. Underlying this response is proliferation of vascular smooth muscle and endothelial cells, resulting in an enlargement in the caliber of arterial-arterial anastomoses, i.e., a collateral vessel, sometimes as much as an order of magnitude. An integral element of this cell proliferation is the process known as phenotypic switching in which cells of a particular phenotype, e.g., contractile vascular smooth muscle, must change their phenotype to proliferate. Phenotypic switching requires that protein synthesis occurs and different kinase signaling pathways become activated, necessitating energy to make the switch. Moreover, kinases, using ATP to phosphorylate their targets, have an energy requirement themselves. Mitochondria play a key role in the energy production that enables phenotypic switching, but under conditions where mitochondrial energy production is constrained, e.g., mitochondrial oxidative stress, this switch is impaired. In addition, we discuss the potential importance of uncoupling proteins as modulators of mitochondrial reactive oxygen species production and bioenergetics, as well as the role of AMP kinase as an energy sensor upstream of mammalian target of rapamycin, the master regulator of protein synthesis.

Sign in / Sign up

Export Citation Format

Share Document