Increased Myosin Light Chain Research Articles

A calcium‐independent PKC isoform mediates cerebral vasoconstriction through actin cytoskeleton reorganization (841.2)

The role of PKC in force production and vasoconstriction in resistance arterioles is hypothesized to be through several mechanisms including: increased calcium influx, calcium sensitization, and thin filament regulation. PKC involvement in these pathways was proposed based on pharmacological experiments without direct biochemical measurement of the phosphoproteins. We studied the effect of phorbol dibutyrate (PDBu, 1 microM) on rat middle cerebral artery in a pressure myography setup. A slow profound constriction was obtained as a result of PDBu treatment. The constriction was associated with increased myosin light chain (LC20) phosphorylation that was similar to that evoked by a maximal dose of 5‐HT producing a smaller constriction. PDBu constrictions were abolished by GF109302X, but neither with a calcium‐dependent PKC inhibitor, nor with calcium‐free Krebs’, though the latter reduced LC20 phosphorylation. There was no evidence of increased phosphorylation of caldesmon and calponin, ruling out a role for thin filament regulation. G‐actin content was greatly reduced in both regular and calcium‐free Krebs’ solution together with an increase in paxillin phosphorylation in both conditions implicating actin cytoskeleton reorganization as a potential mechanism contributing to force generation independent of LC20 phosphorylation following PKC activation.Grant Funding Source: Supported by CIHR MOP‐97988

P-212 A Role for Hypoxia-Inducible Factor (HIF) in Intestinal Epithelial Junctional Assembly and Barrier Function

The gastrointestinal mucosa constitutes the largest mucosal surface found in multicellular organisms, and provides the primary physical barrier against gut luminal contents. Intestinal epithelial cells that line the GI tract selectively permit flux of nutrients and water, while protecting against omnipresent luminal antigens. In mucosal diseases such as inflammatory bowel disease (IBD), hypoxia and inflammation occur coincidentally at the level of the epithelium. Adaptive transcriptional responses to oxygen deprivation are mediated primarily through the hypoxia-inducible factor complex (HIF), comprising an oxygen-labile “alpha” subunit and constitutively expressed “beta” subunit. A role for HIF has been implicated in orchestrating a signaling program that promotes intestinal epithelial function and homeostasis. Disruption of the epithelial barrier by proinflammatory cytokines such as IFN-gamma defines a major pathophysiological consequence of intestinal inflammation in IBD. We previously demonstrated that HIF activity and expression in intestinal epithelial cells (IECs) is prominently repressed at the transcriptional level by IFN-gamma. Given the concurrent expression of both HIF-1alpha and HIF-2alpha isoforms of the HIF transcriptional complex in IECs, we sought to define the relative contribution of HIF-1 and HIF-2 to epithelial barrier function. Human intestinal epithelial cell lines T84 and Caco-2 were used throughout. Stable knockdown lines targeting individual HIF subunits were generated using shRNA lentiviral transduction particles (MISSION TRC). To monitor apical junction assembly after calcium switch, T84 cells plated on transwell inserts were incubated for 16h in low Ca2+ medium (LCM) or 5 mins in HBSS with 2mM EDTA before switching to normal Ca2+ conditions (1.8 mM). Transepithelial resistance (TER) was measured over time using a voltohmeter, and expressed in ohm.cm2. Stable shRNA-mediated knockdown of HIF-1alpha, HIF-2alpha and HIF-1beta in model T84 epithelial cells resulted in significantly increased transcellular and paracellular permeability, as measured by transepithelial resistance and FITC-dextran flux assays. Immunolocalization studies of tight junction and adherens junction markers ZO-1 and E-cadherin, respectively, revealed non-uniform, undulating junctions in HIF-1alpha depleted T84 monolayers, consistent with attenuated intercellular tension and apical junction formation under conditions of altered HIF signaling. IFNgamma treatment of shControl IECs resulted in decreased barrier function and a junctional morphology similar to that observed in HIF-1alpha knockdown epithelia. Moreover, western blot analyses of shControl and knockdown lysates revealed increased myosin light chain (MLC) phosphorylation in HIF-1alpha knockdown IECs, indicative of an activated myosin II motor and increased perijunctional actomyosin contractility. Finally, inhibition of myosin light chain kinase (MLCK) or the Rho-associated protein kinases (ROCK) in HIF knockdown epithelia attenuated barrier defects and improved junctional morphology. These results define a role for HIF signaling in maintenance of apical IEC junctions at the level of the perijunctional actomyosin ring, and may in part explain the pathomechanism of IFN-gamma mediated loss of junctional integrity.

Differential Control of Calcium Homeostasis and Vascular Reactivity by Ca 2+ /Calmodulin-Dependent Kinase II

The multifunctional Ca 2+ /calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca 2+ channel current ( I Ca ), cytoplasmic and sarcoplasmic reticulum Ca 2+ , and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual I Ca 50 ms after peak amplitude, indicative of loss of L-type Ca 2+ channel–dependent I Ca facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca 2+ concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca 2+ concentration was abolished by pretreatment with nifedipine, an L-type Ca 2+ channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca 2+ content was reduced as a result of diminished sarcoplasmic reticulum Ca 2+ ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca 2+ ATPase inhibitor phospholamban. Despite the differences in intracellular Ca 2+ concentration, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin II, and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.

Targeted Overexpression of TGF-α in the Corneal Epithelium of Adult Transgenic Mice Induces Changes in Anterior Segment Morphology and Activates Noncanonical Wnt Signaling

Transforming growth factor-alpha (TGF-α) transduces its signal through the epidermal growth factor receptor and is essential for corneal epithelial homeostasis. Previous studies have demonstrated that overexpression of TGF-α in the developing eye leads to anterior segment dysgenesis. However, the underlying mechanisms remain unclear. Here we examined the effects of TGF-α overexpression on adult ocular surface homeostasis. Binary Tet-On transgenic Krt12(rtTA)/tet-O-TGF-α mice were subjected to doxycycline (Dox) induction to overexpress TGF-α in the corneal epithelium. Intraocular pressure (IOP) was measured by noninvasive tonometry. The enucleated eyes of the experimental mice were subjected to histopathology, immunohistochemistry, and biochemistry examination. Histologic and immunofluorescent examination showed that double-transgenic mice overexpressing TGF-α manifested peripheral anterior synechiae. Elevation of IOP, activation of glial cells, and loss of retinal ganglion cells were also observed. Quantitative real-time PCR revealed that the expressions of genes (RXRα, PITX2, and FOXC1) related to anterior segment dysgenesis were downregulated. Canonical Wnt signaling was suppressed, whereas noncanonical Wnt ligands (Wnt4 and Wnt5a) were upregulated. Increased myosin light chain phosphorylation suggested that noncanonical Wnt signaling is activated in affected eyes. Overexpression of TGF-α in the corneal epithelium induces changes in anterior segment morphology. Corneal endothelial abnormalities are associated with the activation of the noncanonical Wnt and RhoA/ROCK signaling axis, indicating a potential application of RhoA/ROCK inhibitors as a therapeutic strategy for certain types of secondary angle-closure glaucoma.

Iodinated contrast media cause endothelial damage leading to vasoconstriction of human and rat vasa recta

Contrast-induced acute kidney injury is an important clinical event with a worldwide increasing number of cases. Medullary hypoperfusion and hypoxia due to constriction of vasa recta are main factors in the pathophysiology of acute kidney injury. However, the mechanism of contrast media (CM)-induced vessel constriction is not known. We tested the hypothesis that vasa recta constriction is a consequence of endothelial dysfunction due to the cytotoxicity of CM. Human and rat descending vasa recta (DVR) were isolated and perfused with CM, and the luminal diameter was analyzed. For morphological analysis of the endothelium, renal arteries were CM perfused and then processed for electron microscopy. Transcellular electrical resistance was used to estimate CM-induced changes in the permeability of human umbilical vein endothelial cell (HUVEC) layers. Perfusion with CM constricted human and rat DRV (to 54.3 and 50.9% of initial diameter, respectively). This was blunted by adrenomedullin (77.7 and 77.1%, respectively). The ANG II response was enhanced by CM in rat DVR (reduction to 15.6 and 35.0% of initial diameter, respectively). Adrenomedullin blunted this effect (67.5%). CM led to endothelial damage of renal arteries characterized by a ragged surface, with sharply protruding intimal folds, spindle-like shape, and bulging in the lumen. These phenomena were reduced by adrenomedullin. The permeability of HUVEC cell layers was increased by CM, and this went along with increased myosin light chain phosporylation. Again, adremonedullin reduced the CM effect. Our study suggests that the constrictor effect of CM on the renal medullary microvasculature is a consequence of endothelial cell damage and the resulting endothelial dysfunction.

Endogenous Rho-Kinase Signaling Maintains Synaptic Strength by Stabilizing the Size of the Readily Releasable Pool of Synaptic Vesicles

Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCKα is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCKβ is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.

Rho-Associated Kinase 2 Polymorphism of Vasospastic Angina in Korean Population

Rho-Associated Kinase 2 Polymorphism of Vasospastic Angina in Korean Population

Vascular Remodeling in Hypertension

Over the past few years noninvasive and invasive techniques have allowed a better appreciation of vascular changes in hypertensive humans and experimental animals.1 Large arteries undergo outward hypertrophic remodeling and increased stiffness with aging,2–4 and in hypertension there may be an acceleration of this process leading to enhanced pulse pressure. A reduced aortic diameter in middle-aged hypertensive subjects may also play a role in increases in pulse pressure through increased specific impedance,5 contradicting the classic hypertensive aortic phenotype characterized by vascular wall degeneration and calcification and increased aortic diameter. In advanced hypertension, however, the elastic laminae undergo duplication and fragmentation, with increased deposition of collagen and fetal (EIIIA) fibronectin, contributing to increased stiffness. Classically the remodeling of small arteries in hypertension has been associated with increased media thickness, but recent studies have demonstrated that 2 types of remodeling are found, inward eutrophic or inward hypertrophic remodeling, depending on whether the media cross-sectional area is enlarged (true hypertrophy).6–9 Although eutrophic remodeling is usually found in essential (primary) hypertension in humans and spontaneously hypertensive rats (SHRs); in secondary hypertension such as in renovascular hypertension, primary aldosteronism, or in pheochromocytoma10; in hypertension associated with diabetes mellitus11,12; and in acromegaly,13 hypertrophic remodeling has been described. In mineralocorticoid hypertension in rodents14,15 and in salt-sensitive Dahl rats,16 in both of which the endothelin (ET) system is activated, remodeling of small arteries is also hypertrophic (see below). Thus, when the renin-angiotensin system is even mildly activated (primary hypertension and SHRs), remodeling is usually eutrophic. In salt-dependent hypertension, diabetes mellitus, and malignant hypertension, all conditions in which the ET system is activated, remodeling is hypertrophic.17 Hyperplasia of vascular smooth muscle cells (VSMCs) is found in small arteries of …

Delineating the receptor mechanisms underlying the rapid vascular contractile effects of aldosterone and estradiol

It is increasingly appreciated that steroid hormones such as aldosterone and estradiol can mediate important cardiovascular effects. Many of these effects occur over a time course not consistent with the genomic actions of these hormones acting through classical nuclear receptors / transcription factors. Further, multiple receptors have been implicated in mediating these rapid effects of both aldosterone and estradiol, including a newly appreciated G-protein-coupled receptor, GPR30. In previous studies we demonstrated that both aldosterone and estradiol mediate contraction in vascular smooth muscle cells, as assessed in single cell assays. However, the receptor mechanisms underlying these effects remained unclear. Therefore, we studied the actions of estradiol and aldosterone on rat aortic vascular smooth muscle cells. Both aldosterone and estradiol mediated a concentration-dependent increase in contraction, as assessed in substrate deformation assays with EC(50)s in the range of nanomoles per litre. These effects paralleled increased myosin light chain phosphorylation. The effects of aldosterone were inhibited by the mineralocorticoid selective antagonist eplerenone. Further, aldosterone's contractile effects were enhanced by increased expression of the mineralocorticoid receptor. The contractile effects of estradiol were inhibited by estrogen receptor (ER)-selective antagonists, tamoxifen, and ICI 182780, as well as eplerenone. Further, estradiol's effects were enhanced by the increased expression of both ERα and the mineralocorticoid receptor (MR). To assess the potential role of GPR30 in mediating the effects of aldosterone and estradiol, GPR30 was re-introduced, since these cells lose endogenous GPR30 expression in culture. Re-expression of GPR30 enhanced both estradiol- and aldosterone-mediated contraction. These studies demonstrate that in rat aortic vascular smooth muscle cells, both aldosterone and estradiol mediate vascular smooth muscle contraction and that these effects can be mediated by MR, ERα, and by GPR30.

Potentiation of vasoconstriction and pressor response by low concentration of monomethylarsonous acid (MMA III)

A close link between arsenic exposure and hypertension has been well-established through many epidemiological reports, yet the mechanism underlying it remains unclear. Here we report that nanomolar concentrations of monomethylarsonous acid (MMA III), a toxic trivalent methylated arsenic metabolite, can potentiate agonist-induced vasoconstriction and pressor responses. In freshly isolated rat aortic ring, exposure to nanomolar MMA III (100–500 nM) potentiated phenylephrine (PE)-induced vasoconstriction while at higher concentrations (≥2.5 μM), suppression of vasoconstriction and apoptosis of vascular smooth muscle were observed. Potentiation of agonist-induced vasoconstriction was also observed with other contractile agonists and it was retained in endothelium-denuded aortic rings, suggesting that these events are agonist-independent and smooth muscle cell dependent. Interestingly, exposure to MMA III resulted in increased myosin light chain phosphorylation while PE-induced Ca 2+ influx was not affected, reflecting that Ca 2+ sensitization is involved. In line with this, MMA III enhanced agonist-induced activation of small GTPase RhoA, a key contributor to Ca 2+ sensitization. Of note, treatment of MMA III to rats induced significantly higher pressor responses in vivo, demonstrating that this event can occur in vivo indeed. We believe that RhoA-mediated Ca 2+ sensitization and the resultant potentiation of vasoconstriction by MMA III may shed light on arsenic-associated hypertension.

Potential involvement of actin cytoskeleton reorganization in generation of the arterial myogenic response

The myogenic response of resistance arteries to intravascular pressure is due to cellular processes intrinsic to vascular smooth muscle cells. Myogenic constriction of cerebral arteries involves the activation and inhibition of myosin light chain kinase and phosphatase, respectively, to increase myosin light chain phosphorylation (pLC20) and permit cross‐bridge cycling. However, equivalent levels of pLC20 are detected at 80 mmHg when pressurized vessels flash‐frozen prior to or after spontaneous development of myogenic tone during the equilibration period are compared. Also, myogenic constriction evoked by a pressure step to 80 mmHg is observed following a maximal increase in pLC20 (~50%) due to 5‐HT stimulation, but there is no further increase in pLC20, or change in p‐caldesmon or p‐calponin. Latrunculin B (10 μM), that binds free G‐actin, had no effect on constriction due to 5‐HT or 80 mmHg alone, but it reversed contractions due to the combination of 5‐HT and 80 mmHg, or pressure alone at 120 mmHg, that are associated with maximal levels of pLC20. Our findings indicate that re‐establishment of myofilament‐actin cytoskeleton contacts during equilibration is necessary for myogenic constriction, and dynamic reorganization of the cytoskeleton may be required to faciltiate increased force generation in the myogenic response when peak levels of pLC20 are attained. (Supported by CIHR MOP‐97988 and AIHS).

Increased Myosin Light Chain 3f Content Restores Age‐Induced Slowing of Single Skeletal Muscle Fiber Contraction

Aging is characterized by a progressive loss of muscle mass and impaired contractile functions (e.g. decline in force, velocity and power). Although these phenomena in aging muscle are well described, the underlying molecular mechanisms responsible for a decrease in contraction speed are not defined. Evidence suggests that the slowing of muscle contraction, as demonstrated by slower unloaded shortening velocity (Vo), is correlated with a decrease in relative myosin light chain 3f (MLC3f) content. In the current study, we first established recombinant adenovirus (rAd) gene transfer technology to increase MLC3f content in semimembranosus muscles of aged rats. We hypothesized that 1) age-induced decline in Vo would be associated with a decrease in relative MLC3f content and 2) increasing MLC3f content via rAd would restore Vo in myosin heavy chain (MHC) type II fibers. Vo (3.2±0.2 vs. 2.2±0.2 FL/s), %MLC3f (13.6±1.0 vs. 3.7±0.3%) and MLC3f/MLC2f ratio (0.28±0.04 vs. 0.10±0.01) decreased with age in type II single fibers. Increasing %MLC3f to 8.0% and MLC3f/MLC2f ratio to 0.20 via rAd provided significant protection against age-induced reduction in Vo without causing muscle damage or influencing single fiber diameter, force and MHC composition. To the best of our knowledge, these are the first data to demonstrate affirmative effects of MLC3f against slowing of contraction in aged skeletal muscle. RO1AG017768

S1P2 receptor regulation of sphingosine-1-phosphate effects on conventional outflow physiology

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P₁ receptor on the inner wall of SC, we hypothesized that S1P₁ receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P₁-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P₂ antagonist JTE-013, but not the S1P₁ or S1P₁,₃ antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P₂ , and not S1P₁ or S1P₃, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.

RhoA-Mediated Signaling in Notch-Induced Senescence-Like Growth Arrest and Endothelial Barrier Dysfunction

Notch signaling has a critical role in vascular development and morphogenesis. Activation of Notch in endothelial cells led to a senescence-like phenotype with loss of barrier function. Our objective was to understand the molecular pathways mediating this phenotype. Human primary endothelial cells increase expression of Notch receptors and ligands during propagation in vitro toward natural senescence. This senescence was induced at low passage with Notch activation. We characterized the pathways activated downstream of Notch signaling. Notch was activated by Delta-like 4 ligand or constitutively active Notch receptors and measured for cell proliferation, migration, and sprouting. Notch signaling triggered early senescence in low-passage cells, characterized by increased p53 and p21 expression. The senescence phenotype was associated with hyperpermeability of the monolayer, with disrupted vascular endothelial cadherin and β-catenin levels and localization. Consistent with changes in cell shape and contact, we demonstrated that Notch activation increases myosin light chain phosphorylation by activating Rho kinase. Inhibition of Rho abrogated Notch-induced myosin light chain phosphorylation and led to enhanced barrier function by reorganizing F-actin to β-catenin-containing cell-cell adherens junctions. Our findings show that RhoA/Rho kinase regulation by Notch signaling in endothelial cells triggers a senescence phenotype associated with endothelial barrier dysfunction.

Rho GTPase‐mediated cytoskeletal organization in Schlemm's canal cells play a critical role in the regulation of aqueous humor outflow facility

The increased intraocular pressure (IOP) has been considered to be an increased resistance of the aqueous humor outflow through the inner wall of Schlemm's canal (SC) and/or the juxtacanalicular tissue (JCT). The Rho GTPase-regulated actomyosin organization appears to be an important mechanistic determinant of aqueous humor outflow facility. Therefore, in this study, we have evaluated the effects of modulating Rho GTPase activity on actomyosin cytoskeletal organization, monolayer permeability/barrier function of human SC cells, and aqueous humor outflow facility in enucleated porcine eyes ex vivo. Human SC cells, isolated from cadaver eyes, were treated with either Rho GTPase activators such as thrombin and lysophosphatidic acid (LPA), or a specific inhibitor (C3-exoenzyme) of Rho GTPases. Treatment of SC cells with thrombin and LPA led to increased formation of stress fibers, focal adhesion, and increased myosin light chain phosphorylation, whereas treatment with C3-exoenzyme showed the opposite effects like H-7 and ECA, known for increasing the outflow facility in porcine eyes. The findings presented here suggest that LPA and thrombin, presumably through activation of Rho GTPase-mediated actomyosin cytoskeletal reorganization in SC cells, cause a decrease in monolayer permeability of SC cells as well as a decrease in outflow facility of porcine eyes in ex vivo. Our results suggest that decrease in aqueous humor outflow may be correlated better with the changes in cytoskeletal organizations of SC, which could be the prime locus of the outflow resistance.

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences.

Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET(B) ) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction. ET(B) receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca(2+) , F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A(2) and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively. Cigarette smoke extract dose-dependently increased ET(B) receptor expression in HPAECs after 24h incubation. CSE-induced ET(B) expression was attenuated by bosentan, the ET(B) receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET(B) receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET(B) receptor expression, mimicking the effect of CSE. CSE-induced ET(B) receptor overexpression caused greater cell contraction; increased intracellular Ca(2+) ; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA(2) and ROS production; and decreased NO after acute ET-1 (10nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine. Cigarette smoke extract induced ET(B) receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET(B) receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.

Epac as a novel relaxant factor in airway smooth muscle

Obstructive airway diseases are characterized by airway narrowing and progressive lung function decline. Elevations of cAMP by β2‐adrenoceptors reverse airway smooth muscle contraction, a process classically assigned to protein kinase A (PKA). Here we report on a novel cAMP mechanism which regulates airway smooth muscle relaxation. Specific activation of Epac (exchange protein directly activated by cAMP) relaxes pre‐contracted guinea pig tracheal open‐rings independent of PKA. Importantly, human airway smooth muscle also expresses Epac and activated Epac relaxes pre‐contracted human airway smooth muscle. Contraction of smooth muscle cells is believed to depend on Rho/Rac balancing. Regulation of cytoskeleton dynamics by Rho increases myosin light chain (MLC) phosphorylation and stress fiber formation, whereas activation of Rac has the opposite effect. Toxin B‐1470‐inhibited Rac1 attenuated in guinea pig tracheal rings Epac‐mediated airway smooth muscle relaxation, suggesting a key role for Rac1 in the relaxing properties of Epac. Indeed, activation of Epac induced GTP‐loading Rac1 and minimized metacholine‐induced stress fiber formation and MCL phosphorylation in airway smooth muscle. We identify Epac as a novel relaxing factor in airway smooth muscle, most likely by shifting the Rho/Rac balance towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.

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 &amp; 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.

A rapid, PPAR-γ-dependent effect of pioglitazone on the phosphorylation of MYPT

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, thiazolidinediones, have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO; 20 muM) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit of myosin phosphatase (MYPT). PIO decreased the phosphorylation of Thr(697) on MYPT within 15 min, and the inhibition was maintained up to 6 h. The PPAR-gamma antagonist GW-9662 (5 microM) abrogated the inhibition of Thr(697) phosphorylation mediated by PIO. Because longer-term PIO treatment inhibits RhoA/Rho kinase (ROCK) signaling and Thr(697) phosphorylation, we tested the effect of the ROCK inhibitor Y-27632 (10 muM) on the inhibition of Thr(697) phosphorylation by PIO. Y-27632 alone inhibited Thr(697) phosphorylation, and there was an additive effect with PIO. In addition, up to 1 h of PIO treatment did not affect RhoA localization or decrease ROCK-dependent phosphorylation of Thr(855). These results suggest that the effect of PIO is independent of inhibition of RhoA/ROCK. PIO increased the phosphorylation of Ser(696) in the same time course as its effect on Thr(697). Ser(696) has been shown to be phosphorylated by PKA and PKG. PKA inhibitor H-89 (10 microM) and PKG inhibitor KT-5823 (0.5 microM) abrogated the effect of PIO on both Thr(697) and Ser(696) phosphorylation. The constitutive turnover of phosphorylation of Thr(697) is rapid, suggesting that the decreased phosphorylation of Thr(697) by PIO is due to enhanced phosphorylation of Ser(696). This is supported by the finding that PIO blocks ANG II-stimulated phosphorylation of Thr(697) but not ANG II-stimulated RhoA translocation. Therefore, the effect of shorter-term PIO apparently is to increase myosin light chain phosphatase activity, thereby desensitizing the vascular smooth muscle to agonist signaling.

Mechanisms of C-Reactive Protein-Induced Blood–Brain Barrier Disruption

Increased mortality after stroke is associated with brain edema formation and high plasma levels of the acute phase reactant C-reactive protein (CRP). The aim of this study was to examine whether CRP directly affects blood-brain barrier stability and to analyze the underlying signaling pathways. We used a cell coculture model of the blood-brain barrier and the guinea pig isolated whole brain preparation. We could show that CRP at clinically relevant concentrations (10 to 20 microg/mL) causes a disruption of the blood-brain barrier in both approaches. The results of our study further demonstrate CRP-induced activation of surface Fcgamma receptors CD16/32 followed by p38-mitogen-activated protein kinase-dependent reactive oxygen species formation by the NAD(P)H-oxidase. The resulting oxidative stress increased myosin light chain kinase activity leading to an activation of the contractile machinery. Blocking myosin light chain phosphorylation prevented the CRP-induced blood-brain barrier breakdown and the disruption of tight junctions. Our data identify a previously unrecognized mechanism linking CRP and brain edema formation and present a signaling pathway that offers new sites of therapeutic intervention.