Hypoxia inhibits myosin phosphatase in pulmonary arterial smooth muscle cells: role of Rho-kinase

Rho-kinase was recently found to phosphorylate the myosin-binding subunit (MBS) of myosin phosphatase (MP) and to regulate MP activity. Although myosin light chain (MLC) phosphorylation in pulmonary arterial smooth muscle cells (PASMCs) is thought to be the cellular/molecular basis for hypoxic pulmonary vasoconstriction (HPV), very little is known about the role that Rho-kinase/MP plays in HPV. Rat PASMCs were cultured and made hypoxic (PO2 = 23 +/- 2 mm Hg). Cells exposed to normoxia (PO2 approximately 148 mm Hg) served as controls. PASMCs exposed to hypoxia showed a significant increase in MLC and MBS phosphorylation, and a significant decrease in MP activity. Rho-kinase inhibitors (HA1077 or Y-27632) blocked hypoxia-induced MP inactivation and inhibited the hypoxia-induced MLC phosphorylation. Hypoxia was also found to induce stress fiber formation and actin polymerization in cultured PASMCs. In summary, these data show that MP inhibition in PASMCs is linked to activation of Rho-kinase, and that hypoxia inhibits the MP signaling pathway via Rho-kinase.     

人参皂甙Rb1通过Rho/Rho激酶通路影响低氧诱导大鼠肺动脉平滑肌细胞增殖及SERT和5-HT_(1B)R表达

目的:观察人参皂甙Rb1对低氧性大鼠肺动脉平滑肌细胞(PASMCs)5-羟色胺转运体(SERT)和5-羟色胺1B受体(5-HT_(1B)R)表达及细胞增殖的影响,并探讨Rho/Rho激酶通路在其中的作用。方法:分离并培养健康雄性SD大鼠PASMCs,随机分为常氧组(normal组)、低氧组(hypoxia组)以及低氧加50、100和200 mg/L人参皂甙Rb1组(HR50、HR100和HR200组),采用CCK-8、Brd U结合流式细胞术、Western blot及RT-PCR等方法,观察大鼠PASMCs的增殖程度以及SERT和5-HT_(1B)R的mRNA和蛋白表达变化;另取PASMCs分为normal组、hypoxia组、HR200组和低氧加Y-27632组(HY组),检测Rho激酶(ROCK1)mRNA表达和肌球蛋白磷酸酶目标亚单位1(MYPT1)磷酸化水平。结果:与normal组比较,hypoxia组PASMCs增殖明显(P0.01);与hypoxia组比较,人参皂甙Rb1可明显抑制PASMCs的增殖(P0.01),并浓度依赖性抑制SERT和5-HT_(1B)R的mRNA与蛋白表达(P0.05);HR200组可明显抑制ROCK1的mRNA表达和MYPT1磷酸化水平(P0.01),与HY组相比较,差异无统计学显著性。结论:低氧能诱导大鼠PASMCs增殖,上调SERT和5-HT_(1B)R表达;人参皂甙Rb1能浓度依赖性抑制这种作用,其机制可能与抑制Rho/Rho激酶通路表达有关。     

Ca2+ -independent phospholipase A2-dependent sustained Rho-kinase activation exhibits all-or-none response

Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca2+ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca2+ -independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response.     

RhoA调节失血性休克大鼠血管反应性的机制

目的： 探讨RhoA调节失血性休克大鼠血管反应性的机制。方法: 采用SD大鼠复制休克模型,取离体血管环,观察Rho激酶、肌球蛋白轻链磷酸酶（MLCP）、肌球蛋白轻链磷酸激酶（MLCK）对RhoA增加血管反应性的作用;同时取原代血管平滑肌细胞（VSMCs）,观察RhoA对缺氧后VSMC Rho激酶、MLCP和MLCK活性的调节作用以及对肌球蛋白轻链（MLC20）磷酸化水平的影响。结果: 失血性休克后大鼠肠系膜上动脉（SMA）对NE收缩反应性明显降低,RhoA的激动剂U-46619可明显升高休克后血管反应性,RhoA特异性抑制剂C3酶可拮抗U-46619所引起的血管收缩反应性的升高。Rho激酶抑制剂Y-27632可降低由U-46619所引起的血管反应性的升高,MLCP的抑制剂Calyculin可进一步增加由U-46619所引起的血管反应性的升高,而MLCK抑制剂对U-46619的作用影响不明显。缺氧后MLCK、Rho激酶活性以及MLC20磷酸化水平明显降低,MLCP活性明显升高,RhoA激动剂U-46619可明显升高缺氧后VSMC的MLC20磷酸化水平、Rho激酶活性和降低MLCP的活性,且U-46619的这一作用可被RhoA抑制剂C3酶所拮抗,调节RhoA的活性对MLCK活性无明显调节作用。结论: RhoA可通过Rho激酶调节MLCP活性和 MLC20磷酸化水平调节休克后血管反应性。     

Endothelial Rho and Rho kinase regulate neutrophil migration via endothelial myosin light chain phosphorylation

The transendothelial migration of neutrophils is a critical step in acute inflammation, which we previously showed to be regulated by endothelial myosin light chain (MLC) kinase. Recent studies suggest that Rho and Rho kinase are also key mediators of MLC phosphorylation, but their roles in neutrophil migration have not been investigated. In the present study, a transwell chamber migration assay system incorporating endothelial monolayer was used to examine the numbers of migrating neutrophils, endothelial F-actin and myosin II rearrangement, and endothelial MLC phosphorylation at selected times during the neutrophil migration in vitro. The results showed that pretreating endothelial cells with C3 (Rho inhibitor) or Y-27632 (Rho kinase inhibitor) significantly diminished neutrophil migration, actin polymerization, myosin II filament formation, and MLC phosphorylation normally associated with the migration. These data suggest that endothelial Rho and Rho kinase regulate transendothelial neutrophil migration by modulating the cytoskeletal events that mediate such migration.     

肿瘤细胞向血管外游走过程中内皮细胞调控机制的研究

目的：阐明肿瘤细胞向血管外游走过程中内皮细胞肌球蛋白轻链激酶(MLCK)及Rho／Rho激酶的调控作用。方法：利用肿瘤细胞向血管外游走的体外模型，观察肿瘤细胞的血管外游走状况，并测定肿瘤细胞游走过程中血管内皮单细胞层的电阻变化；通过Western blot评价肿瘤游走过程中内皮细胞肌球蛋白轻链(MLC)的磷酸化水平。结果：肿瘤细胞可以穿过血管内皮细胞游走至血管外；并且肿瘤细胞向血管外游走过程中跨血管内皮细胞层的电阻下降、内皮细胞MLC的磷酸化水平升高；而内皮细胞肌球蛋白轻链激酶抑制剂(ML-7)及Rho抑制剂(C3转移酶)、Rho激酶抑制剂(Y-27632)能够抑制上述变化。结论：内皮细胞MLCK及Rho／Rho激酶可以通过控制MLC的磷酸化水平来引起内皮细胞骨架蛋白的改变，从而调控肿瘤细胞穿过内皮细胞间缝隙向血管外游走。     

Rho-kinase and contractile apparatus proteins in murine airway hyperresponsiveness

Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Increased expression of smooth muscle contractile proteins or increased responsiveness of the contractile apparatus due to RhoA/Rho-kinase activation may contribute to AHR. BALB/c mice developed AHR following systemic sensitization by intraperitoneal injections of 20 microg ovalbumin (OVA) in presence of 2mg Al(OH)(3) on days 1 and 14, and airway challenge by 1% OVA-inhalation for 20 min each on days 28, 29 and 30. As assessed by Western blot, protein expression of RhoA, MLC (myosin light chain) and smMLCK (smooth muscle myosin light chain kinase) was increased in lungs of OVA/OVA-animals with AHR, as well as in lungs of OVA-sensitized and sham-challenged animals (OVA/PBS) without AHR, compared with lungs of PBS/PBS-animals. Pretreatment with the specific Rho-kinase inhibitor Y-27632 reduced MLC-phosphorylation and AHR. Contribution of Rho-kinase to bronchoconstriction was increased in lungs of OVA/OVA-animals compared with OVA/PBS- and PBS/PBS-animals, respectively. Furthermore, bronchoconstriction following MCh stimulation was significantly reduced after Y-27632 application. In conclusion, systemic allergen-sensitization increased pulmonary expression of proteins involved in smooth muscle contraction, which may contribute to development of AHR. However, this observation was independent from local allergen challenge, suggesting that additional cofactors may be required for the activation of Rho-kinase and thereby the induction of AHR. Rho-kinase may play an important role in murine AHR, and the bronchodilating action of Rho-kinase inhibition may offer a new therapeutic perspective in obstructive airway disease.     

The role of Rho/Rho-kinase pathway in the expression of ICAM-1 by linoleic acid in human aortic endothelial cells

Linoleic acid (LA), a dietary unsaturated fatty acid, has been known to increase the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) through the activation of nuclear factor-kappa B. Rho/Rho-kinase (ROCK) pathway mediates various cellular functions related to cardiovascular disease and affects the expression of ICAM-1. However, the exact mechanism underlying this action has not been fully elucidated. In this study, we aimed to find out the role of Rho/ROCK pathway in LA-induced ICAM-1 expression in human aortic endothelial cells (HAECs). We found that LA increased ICAM-1 expression and phosphorylation of ROCK and MYPT-1, a distal signal of ROCK. Y-27632, a ROCK inhibitor, suppressed ICAM-1 expression and phosphorylation of MYPT-1 induced by LA. The effect of LA on the increased phosphorylation of MYPT1 and expression of ICAM-1 was abolished by knocking down RhoA and ROCK2 protein level expression using small interfering RNA. LA increased NF-κB DNA-binding activity, which was inhibited with pretreatment with Y-27632. This study suggests that Rho/ROCK pathway plays a role in LA-induced ICAM-1 expression, which is possibly mediated by NF-κB in HAECs.     

ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery

Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 ^+/− and Rock2 ^+/− mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used.     

Localized phosphorylation of vimentin by rho-kinase in neuroblastoma N2a cells

BACKGROUND: Vimentin, which is one of the intermediate filaments, is the major cytoskeletal component in developing neurones or neuroblastoma cells. Rho-associated kinase (Rho-kinase), is rich in neurones and is found downstream of Rho. It is involved in the agonist-induced neurite retraction of neuronal cells, and phosphorylates vimentin at Ser-38 and Ser-71 resulting in in vitro disassembly of the filaments. RESULTS: We have investigated the distribution of vimentin phosphorylated by Rho-kinase in N2a neuroblastoma cells using site-specific phosphorylation-dependent antibodies. TM71 immunoreactivity, which specifically indicates Ser-71 phosphorylation on vimentin, was found in some neurites of dibutyryl cAMP-differentiated N2a cells. Transfection of the constitutively active form of Rho-kinase, CAT, significantly elevated TM71 immunoreactivity, and induced neurite retraction or cell rounding. Conversely, transfection of the dominant negative form of Rho-kinase, RB/PH(TT), or treatment of 10 microM Y-27632, a Rho-kinase specific inhibitor, abolished TM71 immuno-reactivity, and induced irregular neurite outgrowth. In contrast, 20 nM okadaic acid (OA) induced neurite retraction and specifically elevated TM71 immunoreactivity. In the OA-induced neurite retraction, tubulin disappeared in retracting neurites, where vimentin and actin remained co-localized. Furthermore, the OA-induced elevation of TM71 immunoreactivity and neurite retraction were completely blocked by pretreatment with 10 microM Y-27632, or by the ectopic expression of RB/PH(TT). CONCLUSIONS: This study suggests that the localized phosphorylation of vimentin by Rho-kinase in neurites was closely related with the cellular morphology of N2a cells, and that the Rho-kinase activity towards vimentin was balanced with OA-sensitive phosphatases.     

Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries

The present study was aimed at investigating whether, besides its pivotal role in Ca²⁺-independent contraction of smooth muscle, Rho-kinase is involved in the mechanisms underlying the Ca²⁺ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho-kinase inhibitor Y-27632 (10 μM) completely relaxed the contraction evoked by noradrenaline (1 μM) and simultaneously inhibited the Ca²⁺ signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), and the cell membrane depolarisation by 56 ± 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF₄⁻, while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca²⁺. The same effects were observed with another inhibitor of Rho-kinase (HA1077) but not with an inhibitor of protein kinase C (Ro-31-8220). Effects of Y-27632 were not prevented by incubating the artery in 25 mM KCl, with K⁺ channel blockers or with the Ca²⁺ channel blocker nimodipine. Y-27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca²⁺ from non-mitochondrial stores evoked by Ins P ₃ in permeabilised aortic cells, or the Ca²⁺ signals evoked by thapsigargin or caffeine. The capacitative Ca²⁺ entry activated by thapsigargin was not impaired by Y-27632, but the entry of Ba²⁺ activated by noradrenaline in the presence of nimodipine was blocked by 10 μM Y-27632. These results indicate that Rho-kinase is involved in noradrenaline activation of a Ca²⁺ entry distinct from voltage- or store-operated channels in rat arteries.     

Force and myosin light chain phosphorylation in dog airway smooth muscle activated in different ways

To assess activation mechanisms of dog trachealis muscle and test whether isometric force generation could be separated from myosin light-chain (MLC) phosphorylation, force and phosphorylation were measured in the presence of wortmannin (a light-chain kinase inhibitor) or Y-27632 (a rho-kinase inhibitor) during electrically stimulated tetani and sustained contractures induced by acetylcholine, KCl, or calyculin A, a light-chain phosphatase inhibitor which caused irreversible contractures and both di- and mono-phosphorylation of light chain. Phosphorylation was not much more than half under any circumstances. A nearly constant proportionality between steady force and phosphorylation existed over a 9-fold force range during contractures and 25-sec tetani, except that force correlated best with the di-phosphorylated light chain produced by calyculin A. Phosphorylation was disproportionately higher than force at the outset of tetani, and this disproportion was exaggerated by Y-27632. The results suggest that about half the light chain is sequestered from kinases and that mechanical activation is tightly linked to phosphorylation, except at the outset of stimulation.     

Attenuation of endotoxin-induced acute lung injury by the Rho-associated kinase inhibitor, Y-27632

A small GTPase, Rho, plays key roles in cell adhesion, motility, and contraction after stimulation. Among Rho effectors isolated, the family of Rho-associated coiled-coil-forming protein kinases (ROCK) is implicated in Rho-mediated cell adhesion and smooth muscle contraction. The effect of a specific inhibitor of ROCK, Y-27632, was evaluated in a murine model of acute lung injury induced by intravenous injection of Escherichia coli endotoxin (lipopolysaccharide [LPS]). Lung edema was evaluated by measuring extravascular leakage of radio-labeled serum albumin, and neutrophil emigration into the lung parenchyma by morphometric observation and measuring myeloperoxidase activity. Pretreatment with Y-27632 attenuated both lung edema and neutrophil emigration after LPS. We also measured albumin transfer through cultured endothelial cell monolayers on a porous filter. Tumor necrosis factor-alpha significantly increased albumin transfer, which was attenuated by pretreatment with Y-27632. Fluorescence microscopy revealed that morphologic changes in endothelial cells induced by tumor necrosis factor-alpha were inhibited by Y-27632. In contrast, the increased fraction of neutrophils with polymerized actin after formyl-methionyl-leucyl-phenylalanine was not altered by Y-27632. These data suggest that ROCK may play an important role in the pathogenesis of LPS-induced lung injury and that ROCK inhibition could attenuate cytoskeletal rearrangement of endothelial cells, leading to decreased neutrophil emigration into the lung parenchyma.     

Regulation of capacitative and noncapacitative receptor-operated Ca2+ entry by rho-kinase in tracheal smooth muscle

To determine the mechanisms of Ca2+ mobilization induced by receptor agonists, we examined the role of Rho-kinase on the sarcoplasmic reticulum (SR) Ca2+ stores-dependent and -independent Ca2+ influx in guinea pig tracheal smooth muscle (TSM). Isometric tension and intracellular Ca2+ concentration ([Ca2+]i) were simultaneously measured using fura-2-loaded tissues. Depletion of the SR Ca2+ stores by thapsigargin caused an increase in [Ca2+]i and contraction, demonstrating capacitative Ca2+ entry (CCE). Because CCE was not inhibited by nifedipine, voltage-operated Ca2+ channels are not involved in CCE. Under the condition that CCE is fully activated, methacholine (MCh) and histamine caused further increases in [Ca2+]i and tension, demonstrating noncapacitative receptor-operated Ca2+ entry (non-CCE). The Ca2+ influx and contraction via non-CCE was inhibited by Y-27632, a Rho-kinase inhibitor, in a concentration-dependent fashion. In contrast, Y-27632 did not affect thapsigargin-induced CCE. Cytochalasin D, which disrupts actin cytoskeleton, inhibited contraction induced by CCE or MCh with no change in [Ca2+]i. Our results indicate that not only CCE but also non-CCE exist in TSM and that the latter is regulated by Rho-kinase, independent of actin cytoskeleton. In conclusion, Ca2+ influx regulated by the RhoA/Rho-kinase pathway may play a functional role in contraction by agonists.     

Tonic protein kinase A activity maintains inactive beta2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase

Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.     

Differential regulation of human lung epithelial and endothelial barrier function by thrombin

Lung epithelial and endothelial barrier dysfunction is critical to the physiologic derangement observed in acute lung injury, but remains poorly understood. We utilized human alveolar epithelial (A549) and endothelial cells (EC) to study cytoskeletal remodeling, myosin light chain (MLC) phosphorylation and barrier regulation evoked by the edemagenic agent, thrombin. Thrombin-challenged human EC monolayers demonstrated increased MLC phosphorylation, actin stress fiber formation and loss of barrier integrity reflected by decreased transmonolayer electrical resistance (TER). In contrast, thrombin produced prominent circumferential localization of actin fibers, increased MLC phosphorylation and increased TER across epithelial monolayers, consistent with barrier protection. Reductions in MLC phosphorylation induced by cell pretreatment with pharmacological inhibitors of MLC kinase (ML-7) and Rho kinase (Y-27632) significantly attenuated thrombin-mediated TER changes and MLC phosphorylation in both lung cell types. Thrombin-produced, time-dependent activation of Rho GTPase in both epithelial and EC, whereas Rac GTPase activation was observed only in A549 cells. Molecular inhibition of Rac activity by adenoviral transfer of dominant-negative Rac mutant abolished thrombin-induced TER increases in alveolar epithelial cells. Finally, A549 cells, but not endothelium, demonstrated increased levels of tight junction proteins (ZO-1 and occludin) after thrombin at the cell-cell interface areas linked to thrombin-elicited barrier protection. These results demonstrate differential pulmonary endothelial and alveolar epithelial barrier regulation via unique actomyosin remodeling and cytoskeletal interactions with tight junction complexes, which confer selective barrier responses to edemagenic stimuli.     

Sema4D-plexin-B1 implicated in regulation of dendritic spine density through RhoA/ROCK pathway

Plexin-B1, Sema4D receptor, mediates retraction and extension signals in axon guidance by associating with PDZ-containing Rho guanine nucleotide exchange factors (PDZ-RhoGEFs) which can activate a small Rho GTPase RhoA. RhoA is implicated in spine formation by rearranging actin cytoskeleton. Exogenous application of Sema4D to cultured neurons caused activation of RhoA, increase of spine density and changes in spine shape. Sema4D-induced changes in spine density were blocked by either Rho-kinase (a downstream of RhoA, ROCK) inhibitor Y-27632 or by overexpression of plexin-B1 mutant lacking the C-terminus which no longer associates with PDZ-RhoGEFs. This study suggests that Sema4D-plexin-B1 play a crucial role in spine formation by regulating RhoA/ROCK pathway.     

Microtubule disruption modulates the Rho-kinase pathway in vascular smooth muscle

Microtubules constitute one of the main cytoskeletal components in eukaryotic cells. Recent studies have shown that microtubule disruption induced significant vasoconstriction or enhanced agonist-induced contraction in vascular smooth muscle. However, the underlying mechanisms are not clear. We hypothesize that microtubule disruption may affect contractile signaling in vascular smooth muscle and lead to the enhanced contraction. The present study demonstrates that both colchicine and nocodazole induced a small but sustained contraction (4–6% P ₀) in rat aortic rings. This microtubule disruption-induced contraction was abolished by co-treatment with either HA 1077 or Y-27632, both of which are relatively specific Rho-kinase inhibitors. However, co-treatment with ML-9, an inhibitor of myosin light chain kinase, (MLCK) did not have a significant effect on the colchicine-induced contraction. The enhanced KCl-induced contraction due to treatment with colchicine was also blocked by inhibition of Rho-kinase, but not by inhibition of MLCK. These results indicate that microtubule disruption modulates contractile signaling in vascular smooth muscle, mainly through the Rho-kinase pathway, but not MLCK. Interestingly, the colchicine-enhanced, phenylephrine-induced contraction was not completely blocked by inhibition of Rho-kinase suggesting that other signaling pathways might also be involved.     

大鼠肺动脉平滑肌细胞Rho激酶对 p-Smad1核迁移的作用及机制

目的: 研究Rho激酶对p-Smad1核迁移的作用及信号转导途径,探讨它们在肺血管重构中的作用机制。方法: 分离培养大鼠远端肺动脉平滑肌细胞,应用血小板源性生长因子BB(PDGF-BB)启动肺动脉平滑肌细胞Rho激酶信号通路,应用骨形态发生蛋白2(BMP-2)启动BMP信号通路,并用Rho激酶抑制剂Y-27632、MEK抑制剂U0126进行干预。培养细胞分成5组:(1)对照组;(2)BMP-2组;(3)BMP-2+PDGF-BB组;(4)BMP-2+PDGF-BB+Y-27632组;(5)BMP-2+PDGF-BB+U0126组。免疫荧光染色标记p-Smad1在细胞核内外的分布并计算p-Smad1核迁移阳性细胞百分数(即核迁移率)。分离平滑肌细胞核蛋白和胞浆蛋白,Western blotting分析p-Smad1在细胞内的总量和细胞核内外相对含量的变化。Kit-WST-8试剂盒检测细胞增殖。结果: BMP-2组p-Smad1在细胞内的总量、在细胞核中的相对含量和核迁移率明显高于对照组(P<0.05);BMP-2+PDGF-BB 组p-Smad1的核迁移率和在细胞核中的相对含量明显低于BMP-2组(P<0.05),但是细胞内p-Smad1总量无明显改变(P>0.05);BMP-2+PDGF-BB+ Y-27632组和BMP-2+PDGF-BB+U0126组细胞内p-Smad1的总量、在细胞核中的相对含量和核迁移率与BMP-2组基本相似(P>0.05)。BMP-2组的A值明显小于对照组(P<0.05);PDGF-BB+BMP-2组的A值明显大于BMP-2组(P<0.05)且大于对照组(P<0.05);BMP-2+PDGF-BB+Y-27632组和BMP-2+PDGF-BB+U0126组的A值均小于PDGF-BB+BMP-2组(P<0.05)。结论: 在大鼠肺动脉平滑肌细胞,PDGF-BB激活的Rho激酶通过MEK/ERK1/2抑制BMP-2引起的p-Smad1核迁移,促进平滑肌细胞增殖。     

Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase

Background

The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rho-kinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells.

Results

Here we examined whether Rho-kinase and myosin II are involved in neurite retraction in N1E-115 cells. We showed that the expression of constitutively active forms of Rho-kinase induced neurite retraction in N1E-115 cells and MLC phosphorylation in NIH 3T3 cells, whereas the expression of dominant negative forms of Rho-kinase inhibited the LPA-induced neurite retraction in N1E-115 cells and the serum-induced MLC phosphorylation in NIH 3T3 cells. The expression of mutant MLC^T18D,S19D (substitution of Thr and Ser by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chains in vitro, also promoted neurite retraction.

Conclusion

These results indicate that Rho-kinase is involved in the LPA-induced neurite retraction downstream of Rho, and that myosin II activation promotes neurite retraction downstream of Rho and Rho-kinase.