Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity

2005 ◽  
Vol 98 (5) ◽  
pp. 1940-1948 ◽  
Author(s):  
Natalia I. Gokina ◽  
Kristen M. Park ◽  
Keara McElroy-Yaggy ◽  
George Osol
Keyword(s):  
Smooth Muscle ◽  
Cerebral Artery ◽  
Rho Kinase ◽  
Oscillatory Activity ◽  
Comparable Efficacy ◽  
Myogenic Tone ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase Inhibition ◽  
Kinase Pathway

Several recent studies have implicated the RhoA-Rho kinase pathway in arterial myogenic behavior. The goal of this study was to determine the effects of Rho kinase inhibition (Y-27632) on cerebral artery calcium and diameter responses as a function of transmural pressure. Excised segments of rat posterior cerebral arteries (100–200 μm) were cannulated and pressurized in an arteriograph at 37°C. Increasing pressure from 10 to 60 mmHg triggered an elevation of cytosolic calcium concentration ([Ca2+]i) from 113 ± 9 to 199 ± 12 nM and development of myogenic tone. Further elevation of pressure to 120 mmHg induced only a minor additional increase in [Ca2+]i and constriction. Y-27632 (0.3–10 μM) inhibited myogenic tone in a concentration-dependent manner at 60 and 120 mmHg with comparable efficacy; conversely, sensitivity was decreased at 120 vs. 60 mmHg (50% inhibitory concentration: 2.5 ± 0.3 vs. 1.4 ± 0.1 μM; P < 0.05). Dilation was accompanied by further increases in [Ca2+]i and an enhancement of Ca2+ oscillatory activity. Y-27632 also effectively dilated the vessels permeabilized with α-toxin in a concentration-dependent manner. However, dilator effects of Y-27632 at low concentrations were larger at 60 vs. 100 mmHg. In summary, the results support a significant role for RhoA-Rho kinase pathway in cerebral artery mechanotransduction of pressure into sustained vasoconstriction (myogenic tone and reactivity) via mechanisms that augment smooth muscle calcium sensitivity. Potential downstream events may involve inhibition of myosin phosphatase and/or stimulation of actin polymerization, both of which are associated with increased smooth muscle force production.

Sevoflurane Inhibits Guanosine 5′-[γ-thio]triphosphate–stimulated, Rho/Rho-kinase–mediated Contraction of Isolated Rat Aortic Smooth Muscle

2003 ◽  
Vol 99 (3) ◽  
pp. 646-651 ◽  
Author(s):  
Jingui Yu ◽  
Koji Ogawa ◽  
Yasuyuki Tokinaga ◽  
Yoshio Hatano
Keyword(s):  
Smooth Muscle ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Rho Kinase ◽  
Force Transducer ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Aortic Smooth Muscle ◽  
Gamma S ◽  
Kinase Pathway

Background The Rho/Rho-kinase signaling pathway plays an important role in mediating Ca2+ sensitization of vascular smooth muscle. The effect of anesthetics on Rho/Rho-kinase-mediated vasoconstriction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane on the Rho/Rho-kinase pathway by measuring guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)-stimulated contraction and translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol to the membrane in rat aortic smooth muscle. Methods GTP gamma S-induced contraction of rat aortic endothelium-denuded rings was measured using an isometric force transducer, and GTP gamma S-stimulated membrane translocation of RhoA and Rock-2 in smooth muscle cells was detected with Western blotting in the presence and absence of sevoflurane. Results GTP gamma S (10(-4) m) induced a sustained contraction, which was significantly inhibited by the Rho-kinase inhibitor, Y27632 (3 x 10(-6) m). Before treatment with GTP gamma S, RhoA and Rock-2 were detected primarily in the cytosolic fraction. GTP gamma S (10(-4) m) stimulated the translocation of RhoA and Rock-2 from the cytosol to the membrane, which was sustained for more than 60 min. Sevoflurane (1.7, 3.4, and 5.1%) concentration dependently inhibited the GTP gamma S-induced constriction of rat aortic smooth muscle with a reduction of constriction of 52-75% (P &lt; 0.01, n = 8), and attenuated the translocation of RhoA and Rock-2 by 31-66% and 34-78%, respectively (P &lt; 0.05-0.01, respectively; n = 4). Conclusion The current findings show that sevoflurane depresses the GTP gamma S-stimulated contraction and translocation of both Rho and Rho-kinase from the cytosol in a concentration-dependent manner, indicating that sevoflurane is able to inhibit vasoconstriction mediated by the Rho/Rho-kinase pathway in rat aortic smooth muscle.

Effects of ω-hydroxylase product on distal human pulmonary arteries

2008 ◽  
Vol 294 (3) ◽  
pp. H1435-H1443 ◽  
Author(s):  
Caroline Morin ◽  
Christelle Guibert ◽  
Marco Sirois ◽  
Vincent Echave ◽  
Marcio M. Gomes ◽  
...  
Keyword(s):  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Dependent Manner ◽  
Response Curves ◽  
Inhibitory Protein ◽  
Concentration Dependent Manner ◽  
Intracellular Process ◽  
Free Arachidonic Acid ◽  
Kinase Pathway

The aim of the present study was to provide a mechanistic insight into how 20-hydroxyeicosatetraenoic acid (20-HETE) relaxes distal human pulmonary arteries (HPAs). This compound is produced by ω-hydroxylase from free arachidonic acid. Tension measurements, performed on either fresh or 1 day-cultured pulmonary arteries, revealed that the contractile responses to 1 μM 5-hydroxytryptamine were largely relaxed by 20-HETE in a concentration-dependent manner (0.01–10 μM). Iberiotoxin pretreatments (10 nM) partially decreased 20-HETE-induced relaxations. However, 10 μM indomethacin and 3 μM SC-560 pretreatments significantly reduced the relaxations to 20-HETE in these tissues. The relaxing responses induced by the eicosanoid were likely related to a reduced Ca2+ sensitivity of the myofilaments since free Ca2+ concentration ([Ca2+])-response curves performed on β-escin-permeabilized cultured explants were shifted toward higher [Ca2+]. 20-HETE also abolished the tonic responses induced by phorbol-ester-dibutyrate (a PKC-sensitizing agent). Western blot analyses, using two specific primary antibodies against the PKC-potentiated inhibitory protein CPI-17 and its PKC-dependent phosphorylated isoform pCPI-17, confirmed that 20-HETE interferes with this intracellular process. We also investigated the effect of 20-HETE on the activation of Rho-kinase pathway-induced Ca2+ sensitivity. The data demonstrated that 20-HETE decreased U-46619-induced Ca2+ sensitivity on arteries. Hence, this observation was correlated with an increased staining of p116Rip, a RhoA-binding protein. Together, these results strongly suggest that the 20-hydroxyarachidonic acid derivative is a potent modulator of tone in HPAs in vitro.

Inhibition of geranylgeranyltransferase inhibits bronchial smooth muscle hyperresponsiveness in mice

2009 ◽  
Vol 297 (5) ◽  
pp. L984-L991 ◽  
Author(s):  
Yoshihiko Chiba ◽  
Shunsuke Sato ◽  
Motohiko Hanazaki ◽  
Hiroyasu Sakai ◽  
Miwa Misawa
Keyword(s):  
Smooth Muscle ◽  
Rho Kinase ◽  
Selective Inhibition ◽  
Dependent Manner ◽  
Bronchial Smooth Muscle ◽  
Concentration Dependent Manner ◽  
Characteristic Features ◽  
Novel Strategy

Recent studies revealed an involvement of RhoA/Rho-kinase in the contraction of bronchial smooth muscle (BSM), and this pathway has now been proposed as a new target for asthma therapy. A posttranslational geranylgeranylation of RhoA is required for its activation. Thus selective inhibition of geranylgeranyltransferase may be a novel strategy for treatment of the BSM hyperresponsiveness in asthmatics. To test this hypothesis, we investigated the effect of a geranylgeranyltransferase inhibitor, GGTI-2133, on antigen-induced BSM hyperresponsiveness by using mice with experimental asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also were treated with GGTI-2133 (5 mg/kg ip) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused a BSM hyperresponsiveness to acetylcholine with the increased expressions of RhoA and the anti-farnesyl-positive 21-kDa proteins, probably geranylgeranylated RhoA. The in vivo GGTI-2133 treatments significantly inhibited BSM hyperresponsiveness induced by antigen exposure. In another series of experiments, BSM tissues isolated from the repeatedly antigen-challenged mice were cultured for 48 h in the absence or presence of GGTI-2133. Under these conditions, the putative geranylgeranylated RhoA was decreased in a GGTI-2133 concentration-dependent manner. The in vitro incubation with GGTI-2133 also inhibited BSM hyperresponsiveness induced by antigen exposure. These findings suggest that GGTI-2133 inhibits antigen-induced BSM hyperresponsiveness, probably by reducing downstream signal transduction of RhoA. Selective geranylgeranyltransferase inhibitors may be beneficial for the treatment of airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway

2003 ◽  
Vol 89 (05) ◽  
pp. 904-914 ◽  
Author(s):  
Natalia Tkachuk ◽  
Hermann Haller ◽  
Inna Dumler ◽  
Ioulia Kiian
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Vascular Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Janus Kinase ◽  
Immunocytochemical Staining ◽  
Boyden Chamber ◽  
Dependent Manner

SummaryUrokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.

scholarly journals Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

2013 ◽  
Vol 304 (5) ◽  
pp. G527-G535 ◽  
Author(s):  
Senthilkumar Rajagopal ◽  
Divya P. Kumar ◽  
Sunila Mahavadi ◽  
Sayak Bhattacharya ◽  
Ruizhe Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Kinase Activity ◽  
Muscle Relaxation ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Receptor Activation ◽  
Gastric Smooth Muscle ◽  
Selective Ligand ◽  
Gastric Muscle ◽  
Kinase Pathway

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5−/− mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2′-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

scholarly journals Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle

2015 ◽  
Vol 308 (1) ◽  
pp. L1-L10 ◽  
Author(s):  
Bo Lan ◽  
Linhong Deng ◽  
Graham M. Donovan ◽  
Leslie Y. M. Chin ◽  
Harley T. Syyong ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Initial Phase ◽  
Rho Kinase ◽  
Myosin Filament ◽  
Second Phase ◽  
Minimal Effect ◽  
Kinase Inhibition ◽  
Myosin Filaments ◽  
Force Maintenance

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation.

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