Decrease In Myosin Light Chain Phosphorylation Research Articles

Mechanical Load and Piezo1 Channel Regulated Myosin II Activity in Mouse Lenses.

The cytoarchitecture and tensile characteristics of ocular lenses play a crucial role in maintaining their transparency and deformability, respectively, which are properties required for the light focusing function of ocular lens. Calcium-dependent myosin-II-regulated contractile characteristics and mechanosensitive ion channel activities are presumed to influence lens shape change and clarity. Here, we investigated the effects of load-induced force and the activity of Piezo channels on mouse lens myosin II activity. Expression of the Piezo1 channel was evident in the mouse lens based on immunoblot and immufluorescence analyses and with the use of a Piezo1-tdT transgenic mouse model. Under ex vivo conditions, change in lens shape induced by the load decreased myosin light chain (MLC) phosphorylation. While the activation of Piezo1 by Yoda1 for one hour led to an increase in the levels of phosphorylated MLC, Yoda1 treatment for an extended period led to opacification in association with increased calpain activity and degradation of membrane proteins in ex vivo mouse lenses. In contrast, inhibition of Piezo1 by GsMTx4 decreased MLC phosphorylation but did not affect the lens tensile properties. This exploratory study reveals a role for the mechanical load and Piezo1 channel activity in the regulation of myosin II activity in lens, which could be relevant to lens shape change during accommodation.

Low-Dose Lithium Stabilizes Human Endothelial Barrier by Decreasing MLC Phosphorylation and Universally Augments Cholinergic Vasorelaxation Capacity in a Direct Manner.

Lithium at serum concentrations up to 1 mmol/L has been used in patients suffering from bipolar disorder for decades and has recently been shown to reduce the risk for ischemic stroke in these patients. The risk for stroke and thromboembolism depend not only on cerebral but also on general endothelial function and health; the entire endothelium as an organ is therefore pathophysiologically relevant. Regardless, the knowledge about the direct impact of lithium on endothelial function remains poor. We conducted an experimental study using lithium as pharmacologic pretreatment for murine, porcine and human vascular endothelium. We predominantly investigated endothelial vasorelaxation capacities in addition to human basal and dynamic (thrombin-/PAR-1 receptor agonist-impaired) barrier functioning including myosin light chain (MLC) phosphorylation (MLC-P). Low-dose therapeutic lithium concentrations (0.4 mmol/L) significantly augment the cholinergic endothelium-dependent vasorelaxation capacities of cerebral and thoracic arteries, independently of central and autonomic nerve system influences. Similar concentrations of lithium (0.2–0.4 mmol/L) significantly stabilized the dynamic thrombin-induced and PAR-1 receptor agonist-induced permeability of human endothelium, while even the basal permeability appeared to be stabilized. The lithium-attenuated dynamic permeability was mediated by a reduced endothelial MLC-P known to be followed by a lessening of endothelial cell contraction and paracellular gap formation. The well-known lithium-associated inhibition of inositol monophosphatase/glycogen synthase kinase-3-β signaling-pathways involving intracellular calcium concentrations in neurons seems to similarly occur in endothelial cells, too, but with different down-stream effects such as MLC-P reduction. This is the first study discovering low-dose lithium as a drug directly stabilizing human endothelium and ubiquitously augmenting cholinergic endothelium-mediated vasorelaxation. Our findings have translational and potentially clinical impact on cardiovascular and cerebrovascular disease associated with inflammation explaining why lithium can reduce, e.g., the risk for stroke. However, further clinical studies are warranted.

Active components of ginger potentiate β-agonist-induced relaxation of airway smooth muscle by modulating cytoskeletal regulatory proteins.

β-Agonists are the first-line therapy to alleviate asthma symptoms by acutely relaxing the airway. Purified components of ginger relax airway smooth muscle (ASM), but the mechanisms are unclear. By elucidating these mechanisms, we can explore the use of phytotherapeutics in combination with traditional asthma therapies. The objectives of this study were to: (1) determine if 6-gingerol, 8-gingerol, or 6-shogaol potentiate β-agonist-induced ASM relaxation; and (2) define the mechanism(s) of action responsible for this potentiation. Human ASM was contracted in organ baths. Tissues were relaxed dose dependently with β-agonist, isoproterenol, in the presence of vehicle, 6-gingerol, 8-gingerol, or 6-shogaol (100 μM). Primary human ASM cells were used for cellular experiments. Purified phosphodiesterase (PDE) 4D or phospholipase C β enzyme was used to assess inhibitory activity of ginger components using fluorescent assays. A G-LISA assay was used to determine the effects of ginger constituents on Ras homolog gene family member A activation. Significant potentiation of isoproterenol-induced relaxation was observed with each of the ginger constituents. 6-Shogaol showed the largest shift in isoproterenol half-maximal effective concentration. 6-Gingerol, 8-gingerol, or 6-shogaol significantly inhibited PDE4D, whereas 8-gingerol and 6-shogaol also inhibited phospholipase C β activity. 6-Shogaol alone inhibited Ras homolog gene family member A activation. In human ASM cells, these constituents decreased phosphorylation of 17-kD protein kinase C-potentiated inhibitory protein of type 1 protein phosphatase and 8-gingerol decreased myosin light chain phosphorylation. Isolated components of ginger potentiate β-agonist-induced relaxation in human ASM. This potentiation involves PDE4D inhibition and cytoskeletal regulatory proteins. Together with β-agonists, 6-gingerol, 8-gingerol, or 6-shogaol may augment existing asthma therapy, resulting in relief of symptoms through complementary intracellular pathways.

In This Issue

HomeCirculation ResearchVol. 113, No. 5In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published16 Aug 2013https://doi.org/10.1161/RES.0b013e3182a71457Circulation Research. 2013;113:479Dyssynchrony in Cytosolic Ca2+ Decay (p 527)Download figureDownload PowerPointHeart cells clear up their cytoplasmic calcium somewhat dyssynchronously, say Hohendanner et al, and this dyssynchrony gets worse in failing hearts.Calcium is the major intracellular regulator of cardiomyocyte contraction. It is released from its intracellular storage site, the sarcoplasmic reticulum, into the cytoplasm where it then activates the contractile machinery. But after each contraction, the cell must relax—a process that requires the prompt and efficient removal of calcium from the cytosol. This combined release and clear up of calcium is known as a calcium transient. It has been shown that the release of calcium across the cell is synchronized to ensure coordinated control of the contractile machinery, but less is known about the clean-up, or decay phase. Hohendanner and colleagues now show that calcium removal occurs at different microdomains throughout the cell and that the speed of decay varies from domain to domain. Furthermore, the rate of relaxation at adjacent contractile machinery varies in concordance with the differing decay rates of the domains. They also found that the degree of dyssynchrony is vastly exaggerated in failing human hearts as well as in mice with hypertrophy and pigs with chronic myocardial ischemia. This, increased dyssynchrony slows down the overall rate of relaxation and, the authors suggest that it might contribute to contractile dysfunction associated with cardiac hypertrophy and heart failure.Bone-Derived Stem Cells Repair the Heart (p 539)Download figureDownload PowerPointBelieve it or not, bone stem cells are better than cardiac stem cells for repairing a damaged heart, say Duran et al.While stem cell therapy is a promising approach for repairing damaged hearts, the results obtained to-date have been modest. Part of the problem is that different types of stem cells yield different results and the precise mechanisms underlying the beneficial effects of stem cells remain unclear. Thus, Duran and colleagues examined two mechanisms of repair—differentiation of stem cells into cardiac cell types and secretion of paracrine factors—in two different stem cell types, one from cortical bone and another from the heart. They found that when injected into damaged mouse hearts, the bone cells were actually more adept at differentiating into cardiac cell types—including cardiomyocytes, vascular endothelial cells, and smooth muscle cells—than the heart-derived cells, which only differentiated into immature cardiomyocytes. Cells from the bone also displayed sustained expression of pro-angiogenic paracrine factors, which correlated with better neovascularization of the damaged hearts. Cardiac stem cells, on the other hand, produced similar paracrine factors but only for one day after injection. Importantly, the better performance by the bone cells translated to improved heart function and increased survival for the mice, suggesting that the use of these cells might improve outcomes of cell therapy.IKK2 is a Myosin Light Chain Kinase (p 562)Download figureDownload PowerPointYing et al identify a possible new target for treating hypertension.Constriction of vascular smooth muscle cells (VSMCs), which contributes to hypertension, is controlled in part by phosphorylation of the myosin light chain (MLC). This post-translational modification enables actin and myosin molecules of the contractile machinery to interact and initiate contraction. MLC is known to be phosphorylated by MLCK but recent work suggests that at least one other kinase may be involved. For example, it has been observed that inhibition of MLCK does not lead to the complete dephosphorylation of MLC and that in MLCK-deficient mice, blood vessels remain responsive to vasoconstrictors. In addition, previous work has shown that the kinase IKK2 regulates migration and morphology in mouse embryonic fibroblasts, and that both of these processes depend on MLC phosphorylation. This led Ying and colleagues to ask whether IKK2 could be an MLC kinase in VSMCs. They found that not only was MLC phosphorylated by IKK2 in vitro and but also overexpression of IKK2 in VSMCs led to increased MLC phosphorylation. Conversely, inhibition of IKK2 decreased MLC phosphorylation and attenuated vasoconstrictive responses of isolated rat aortas. Furthermore, mice that lacked IKK2 in their VSMCs showed a decreased hypertensive response to three different vasoconstrictors. These results suggest that IKK2 inhibition could be a novel approach for decreasing hypertension. Previous Back to top Next FiguresReferencesRelatedDetails August 16, 2013Vol 113, Issue 5 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e3182a71457 Originally publishedAugust 16, 2013 PDF download Advertisement

Hypoxia-Inducible Factor-1α in Pulmonary Artery Smooth Muscle Cells Lowers Vascular Tone by Decreasing Myosin Light Chain Phosphorylation

Hypoxia-inducible factor-1α (HIF-1α), an oxygen (O2)-sensitive transcription factor, mediates transcriptional responses to low-O2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1α in modulating pulmonary vascular tone. To investigate the role of smooth muscle cell (SMC)-specific HIF-1α in regulating pulmonary vascular tone. Mice with an SMC-specific deletion of HIF-1α (SM22α-HIF-1α(-/-)) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1α modulates pulmonary vascular tone and the response to hypoxia. SM22α-HIF-1α(-/-) mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22α-HIF-1α(-/-) mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1α decreased myosin light chain phosphorylation in HIF-1α-null SMCs. In both normoxia and hypoxia, PASMC HIF-1α maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1α expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury

BackgroundSevere burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.Methodology/Principal FindingsMale balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.Conclusions/SignificanceThe MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)-elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP-mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre-contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine-induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine-induced RhoA activation, measured by both stress fibre formation and pull-down assay whereas the same Epac activation prevented methacholine-induced Rac1 inhibition measured by pull-down assay. Epac-driven inhibition of both methacholine-induced muscle contraction by Toxin B-1470, and MLC phosphorylation by the Rac1-inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac-mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre-contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.

The effects of Type 1 diabetes on colon smooth muscle

Approximately 76% of diabetic patients develop gastrointestinal (GI) symptoms such as constipation. Our study investigated the effects of diabetes on smooth muscle (SM) of the GI tract. Mice were treated with low-dose streptozotocin once a day for 5 days to induce hyperglycemia (>200 mg/dl). Colon motility was examined by CT scan after 7 weeks of hyperglycemia and revealed decreased overall GI tract motility. To directly measure the contractility of colonic SM, colons were dissected, cut into circular rings, hung in a myograph and contraction was elicited by KCl-induced depolarization. In short term (1 week) and long term (7 weeks) diabetic mice there was decreased colon SM contractility that was associated with an attenuated Ca2+ increase, as measured by ratiometric imaging of fura-2 fluorescence of colonic SM strips. This attenuated Ca2+ increase resulted in decreased myosin light chain phosphorylation thus explaining the decreased contractility of the colon. Long term diabetes was also associated with increased basal Ca2+levels. Western blotting and qRT-PCR analysis revealed significant changes in Ca2+handling protein and mRNA levels following 7 weeks of hyperglycemia. After 1 week of hyperglycemia we also observed attenuated expression of Cav1.2b, SERCA2b and PMCA4. These results suggest that type 1 diabetes in mice leads to decreased colon motility in part due to altered Ca2+ handling. NIH RO1 DK61130

INOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow due to arterial vasodilation and augmented intrahepatic vascular resistance due to sinusoidal constriction. In this study, we examined the possible involvement of resident macrophages in the tone regulation of splanchnic blood vessels using bile duct ligated (BDL) portal hypertensive rats and an in vitro organ culture method. In BDL cirrhosis, the number of ED2-positive resident macrophages increased by two- to fourfold in the vascular walls of the mesenteric artery and extrahepatic portal vein compared with those in sham-operated rats. Many ED1-positive monocytes were also recruited into this area. The expression of inducible nitric oxide (NO) synthase (iNOS) mRNA was increased in the vascular tissues isolated from BDL rats, and accordingly, nitrate/nitrite production was increased. Immunohistochemistry revealed that iNOS was largely expressed in ED1-positive and ED2-positive cells. We further analyzed the effect of iNOS expression on vascular smooth muscle contraction using an in vitro organ culture system. iNOS mRNA expression and nitrate production significantly increased in vascular tissues (without endothelium) incubated with 1 μg/ml lipopolysaccharide (LPS) for 6 h. Immunohistochemistry indicated that iNOS was largely expressed in ED2-positive resident macrophages. α-Adrenergic-stimulated contractility of the mesenteric artery was greatly suppressed by LPS treatment and was restored by N(G)-nitro-L-arginine methyl ester (NO synthase inhibitor); in contrast, portal vein contractility was largely unaffected by LPS. Sodium nitroprusside (NO donor) and 8-bromo-cGMP showed greater contractile inhibition in the mesenteric artery than in the portal vein with decreasing myosin light chain phosphorylation. In the presence of an α-adrenergic agonist, the mesenteric artery cytosolic Ca(2+) level was greatly reduced by sodium nitroprusside; however, the portal vein Ca(2+) level was largely unaffected. These results suggest that the induction of iNOS in monocytes/macrophages contributes to a hypercirculatory state in the cirrhosis model rat in which the imbalance of the responsiveness of visceral vascular walls to NO (mesenteric artery >> portal vein) may account for the increased portal venous flow in portal hypertension.

The small GTPase RhoA Stimulates the Contraction of Airway Smooth Muscle (ASM) by Regulating Actin Polymerization rather than Myosin Light Chain (MLC) Phosphorylation

Activation of RhoA has been shown to inhibit MLC phosphatase activity, thus increasing MLC phosphorylation and force in some smooth muscle. In ASM, actin polymerization is required for active contraction in addition to MLC phosphorylation. RhoA can regulate cytoskeletal processes and actin polymerization. We evaluated whether RhoA regulated the contraction of ASM primarily through its effects on MLC phosphorylation or actin polymerization. RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, or by treating tissues with the cell permeant RhoA inhibitor, C3 exoenzyme. RhoA inactivation inhibited ACh‐induced contractile force by 60–70%, inhibited ACh‐induced actin polymerization, and also caused a small (10–20%) decrease in MLC phosphorylation. Tissues were treated with the phosphatase inhibitor, calyculin A, by inactivate MLC phosphatase. Calyculin A induced MLC phosphorylation and contraction in the absence of ACh and potentiated MLC phosphorylation induced by ACh, but it did not affect ACh stimulated contraction or actin polymerization. However, inhibition of MLC phosphatase by calyculin A did not prevent the depression of force caused by the RhoA inactivation. This suggests RhoA activation regulates ASM contraction primarily through its effects on actin dynamics.Supported by HL29289, HL074099, ALA, and NIH T32 Grant GM12453‐03

Nuclear factor-κB activation by edema inhibits intestinal contractile activity

To investigate the molecular mechanisms leading to edema-induced decreases in intestinal smooth muscle myosin light-chain phosphorylation. Intestinal interstitial edema often develops during abdominal surgery and after fluid resuscitation in trauma patients. Intestinal edema causes decreased intestinal contractile activity via decreased intestinal smooth muscle myosin light-chain phosphorylation, leading to slower intestinal transit. Interstitial edema development is a complex phenomenon, resulting in many changes to the interstitial environment surrounding intestinal smooth muscle cells. Thus, the mechanism(s) by which intestinal edema development causes intestinal dysfunction are likely to be multifactorial. Randomized animal study. University laboratory. Male Sprague-Dawley rats, weighing 250-350 g. Studies were performed in a rat model in which a combination of mesenteric venous hypertension and administration of resuscitative fluids induces intestinal edema, mimicking the clinical setting of damage control resuscitation. Microarray analysis of edematous intestinal smooth muscle combined with an in silico search for overrepresented transcription factor binding sites revealed the involvement of nuclear factor-kappaB in edema-induced intestinal dysfunction. Nuclear factor-kappaB deoxyribonucleic acid binding activity was significantly increased in edematous intestinal smooth muscle compared with controls. Inhibition of nuclear factor-kappaB activation blocked edema-induced decreases in basal intestinal contractile activity. Inhibition of nuclear factor-kappaB activation also attenuated edema-induced decreases in myosin light-chain phosphorylation. We conclude that intestinal edema activates nuclear factor-kappaB, which, in turn, triggers a gene regulation program that eventually leads to decreased myosin light-chain phosphorylation and, thus, decreased intestinal contractile activity.

Abstract 5648: Epigenetic Basis of Nitrate Tolerance: Fundamental Role of Protein Acetylation

INTRODUCTION : We recently demonstrated that nitric oxide (NO) plays a key role regulating epigenetic enzymes, including histone acetylases (HAT) and deacetylases (HDAC). In this context, we hypothesized that during nitrate tolerance the HDAC/HAT balance could be altered and that drugs capable of modulate HDAC and/or HAT function could have an impact on this alteration. METHODS & RESULTS : Adult male rats (n=6, each group) were treated for 72 hours with subcutaneous nitroglycerin (NTG) alone (50 mg/kg) or in combination with different HDAC inhibitors (DI) including trichostatin A (TSA, 0.6 mg/kg), suberoylanilide hydroxamic acid (SAHA, 0.6 mg/kg), MS-27–275 (0.6 mg/kg) and valproic acid (VPA, 5 mg/kg). Other animals received the HAT inhibitor anacardic acid (ANAC, 2.5 mg/kg) or equivalent amounts of solvent. Ex vivo experiments performed on phenylephrine-contracted aortic rings (AR) showed that the induction of tolerance (-logEC=5.9±0.2 in tolerant vs -logEC=7.6±0.1 in control) was reversed by TSA, SAHA (-logEC=7.6±0.1) and VPA (-logEC=7.4±0.2). ANAC treatment displayed value similar to nitrotolerant (-logEC=6.2±0.1). Moreover, our data revealed that in AR from controls animals a single NTG administration induced relaxation paralleled by a rapid increase in HAT activity. Nitrate tolerant rings, however, exhibited a higher HDAC activity and failed to up-regulate HATs’ function. Remarkably, in AR from animals treated with DI in combination with NTG, nitrate tolerance was prevented while animals treated with ANAC developed resistance to both arterial relaxing or contracting stimuli suggesting that the intracellular level of HAT activity is important for arterial adaptive responses. Mechanistically, in isolated smooth muscle cells the NTG increased myosin light chain (MLC) and smooth muscle actin (SMA) acetylation paralleled by a decreased MLC phosphorylation and contractile protein association. These effects were rescued to normal by DI treatment. CONCLUSIONS : an alteration in HAT/HDAC functional balance occurs during nitrate tolerance in which the content of acetylated contractile proteins is markedly reduce. DIs, preventing this effect, may be envisaged as novel tools for the treatment of nitrate tolerance.

Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to "pull" the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha.

Activation of Endothelial Nitric Oxide Synthase by a Vanadium Compound Ameliorates Pressure Overload-Induced Cardiac Injury in Ovariectomized Rats

We here investigated the effect of bis(1-oxy-2-pyridinethiolato) oxovanadium (IV), [VO(OPT)], against myocardial hypertrophy and cardiac functional recovery in pressure overload-induced hypertrophy in ovariectomized female rats and defined mechanisms underlying its cardioprotective action. Wistar rats subjected to bilateral ovariectomy were further treated with abdominal aortic stenosis. VO(OPT) (containing 1.25 and 2.50 mg of vanadium per kg) was administered orally once a day for 14 days starting from 2 weeks after aortic banding. Treatment with VO(OPT) significantly inhibited pressure overload-induced increase both in the heart weight:body weight ratio and the lung weight:body weight ratio. VO(OPT) also attenuated hypertrophy-induced impaired left ventricular end-diastolic pressure, left ventricular developed pressure, and left ventricular contractility (+/-dp/dt(max)). VO(OPT) treatment significantly restored pressure overload-induced impaired endothelial NO synthase activity with concomitant increased phosphorylation of endothelial NO synthase (Ser1179). Moreover, VO(OPT) treatment significantly restored pressure overload-induced reduced Akt activity, as indicated by increased phosphorylation at Ser473 and at Thr308. Treatment with VO(OPT) also secondarily inhibited calpastatin and dystrophin breakdown and decreased myosin light chain phosphorylation. Finally, VO(OPT) treatment significantly attenuated mortality after repeated isoproterenol administration in pressure overloaded-ovariectomized rats. Taken together, VO(OPT) attenuates cardiac myocytes hypertrophy in vivo in pressure overload-induced hypertrophy in ovariectomized rats and prevents the process from hypertrophy to heart failure. These effects are mediated by inhibition of calpastatin and dystrophin breakdown in addition to increased Akt and endothelial NO synthase activities.

A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

Myosin II phosphorylation-dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3beta to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3beta overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3beta inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3beta overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase-dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

CGMP-Dependent Relaxation of Smooth Muscle Is Coupled With the Change in the Phosphorylation of Myosin Phosphatase

Nitric oxide/cGMP pathway induces vasodilatation, yet the underlying mechanism is obscure. In the present study, we studied the mechanism of cGMP-induced relaxation of the smooth muscle contractile apparatus using permeabilized rabbit femoral arterial smooth muscle. 8-Br-cGMP-induced relaxation was accompanied with a decrease in myosin light chain (MLC) phosphorylation. MLC phosphatase (MLCP) activity, once decreased by agonist-stimulation, recovered to the resting level on addition of 8-Br-cGMP. Because MLCP activity is regulated by the phosphorylation of a MLCP-specific inhibitor, CPI17 at Thr38 and MBS (myosin binding subunit of MLCP) at Thr696, we examined the effect of 8-Br-cGMP on the phosphorylation of these MLCP modulators. Whereas CPI17 phosphorylation was unchanged after addition of 8-Br-cGMP, MBS phosphorylation at Thr696 was significantly decreased by 8-Br-cGMP. We found that 8-Br-cGMP markedly increased MBS phosphorylation at Ser695 in the fiber pretreated with phenylephrine. MBS phosphorylation of Thr696 phosphorylated MBS at Ser695 partially resumed MLCP activity inhibited by Thr696 phosphorylation. Whereas Ser695 phosphorylation was markedly increased, the extent of diphosphorylated MBS at Ser695 and Thr696 in fibers was unchanged after cGMP-stimulation. We found that MBS phosphatase activity in arteries for both diphosphorylated MBS and monophosphorylated MBS at Thr696 significantly increased by 8-Br-cGMP, whereas MBS kinase activity was unchanged. These results suggest that the phosphorylation at Ser640 induced by cGMP shifted the equilibrium of the Thr641 phosphorylation toward dephosphorylation, thus increasing MLCP activity. This results in the decrease in MLC phosphorylation and smooth muscle relaxation.

Regulation of α1-Adrenoceptor-Mediated Contractions of the Uterine Artery by Protein Kinase C: Role of the Thick- and Thin-Filament Regulatory Pathways

Previously we demonstrated that activation of protein kinase C (PKC) enhanced alpha(1)-adrenoceptor-induced contractions in nonpregnant uterine arteries (NPUA) by increasing the Ca(2+) sensitivity but that it inhibited the contractions in pregnant uterine arteries (PUA) by decreasing intracellular Ca(2+) mobilization. The present study tested the hypothesis that PKC activation differentially regulated the thick- and thin-filament regulatory pathways in alpha(1)-adrenoceptor-induced contractions of NPUA and PUA in sheep. Simultaneous measurements of contractions and phosphorylation levels of 20-kDa regulatory myosin light chain (LC(20)) in the same tissue revealed that the PKC activator phorbol-12,13-dibutyrate (PDBu) inhibited phenylephrine-induced phosphorylation of LC(20) and contractions in PUA. In NPUA, PDBu significantly potentiated phenylephrine-induced contractions without significantly changing phosphorylation levels of LC(20). Further studies in NPUA demonstrated that PDBu-mediated potentiation of phenylephrine-induced contractions was associated with a significant increase in phosphorylation levels of extracellular signal-regulated kinase (ERK(42/44)) and caldesmon-Ser(789), measured simultaneously with the tension in the same tissue. In addition, the ERK(42/44) inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] and the actin polymerization inhibitor cytochalasin B produced a concentration-dependent inhibition of PDBu-mediated potentiation of phenylephrine-induced contractions in NPUA. The results suggest that activation of PKC inhibits alpha(1)-adrenoceptor-mediated contractions in PUA through down-regulation of the thick-filament pathway and decreased myosin light chain phosphorylation, but that it enhances the contractions in NPUA through its effect on the thin-filament regulatory pathway and activation of ERK/caldesmon and actin polymerization.

Intestinal edema decreases intestinal contractile activity via decreased myosin light chain phosphorylation

The purpose of this study was to investigate the effects of interstitial edema on intestinal contractile activity. Randomized animal study. University laboratory. Male Sprague-Dawley rats. Intestinal edema was induced in rats by a combination of fluid infusion and mesenteric venous hypertension. Rats were divided into four groups: CONTROL, sham; RESUS, saline infusion only; RESUS+VH, saline infusion and venous hypertension; and VH, venous hypertension only. Edema development, basal contractile activity, maximum agonist-induced contractile response (measured as total force generation during the first 2 mins after carbachol treatment), and myosin light chain phosphorylation were measured in the distal small intestine. The amount of interstitial fluid, indicated by the wet-to-dry ratio, increased significantly in both the RESUS and RESUS+VH groups as early as 30 mins after surgery compared with the CONTROL group. Whereas the tissue fluid remained significantly elevated in the RESUS+VH group up to 6 hrs after surgery, the RESUS group wet-to-dry ratios returned to CONTROL group levels by 2 hrs after surgery. Basal contractile activity was significantly less in the RESUS+VH group compared with either the RESUS group or the CONTROL group 6 hrs after surgery. Maximum contractile response decreased significantly in the RESUS+VH group compared with the RESUS group. Basal contractile activity and maximum contractile response did not change significantly in the VH group compared with the CONTROL group. The phosphorylated fraction of myosin light chain was significantly lower in the RESUS+VH group compared with the CONTROL group at 0.5, 2, and 6 hrs after surgery. We conclude that edema decreases myosin light chain phosphorylation, leading to decreased intestinal contractile activity.

Increased basal phosphorylation of detrusor smooth muscle myosin in alloxan-induced diabetic rabbit is mediated by upregulation of Rho-kinase β and CPI-17

Urinary bladder dysfunction caused by the alteration of detrusor smooth muscle (DSM) is one of the complications of diabetes. It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca(2+) via myosin light chain (MLC) phosphorylation. However, recent studies have shown the modulation of MLC phosphorylation without a rise in Ca(2+) in smooth muscle and that two key molecules (Rho-kinase and CPI-17) are involved in the regulation of calcium sensitization. This study investigates the effect of diabetes on DSM calcium sensitization. Diabetes was induced by alloxan in New Zealand White rabbits, and age-matched rabbits given 5% sucrose in the drinking water served as control for diuresis. Two-dimensional gel electrophoresis showed that basal MLC phosphorylation level was significantly higher in diabetic animals than normal or diuretic controls, and Rho-kinase-specific inhibitor, Y-27632, decreased MLC phosphorylation level. Adding Y-27632 to bethanechol-precontracted DSM strips can induce muscle relaxation, but it occurred much more slowly in diabetic samples compared with controls. RT-PCR, Western blot analysis, and immunohistochemistry revealed the overexpression of Rho-kinase beta and CPI-17 at both mRNA and protein levels in response to diabetes. In conclusion, our results demonstrate that Rho-kinase contributes to DSM MLC phosphorylation and there is a higher basal MLC phosphorylation level in diabetic DSM. Our results also suggest that this high basal MLC phosphorylation may be due to the upregulation of Rho-kinase and CPI-17. Thus Rho-kinase- and CPI-17-mediated Ca(2+) sensitization might play a role in diabetes-induced alteration of the detrusor contractility and bladder dysfunction.