5' CArG degeneracy in smooth muscle alpha-actin is required for injury-induced gene suppression in vivo

CC(A/T)6GG-dependent (CArG-dependent) and serum response factor-dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle alpha-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.     

In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle

It is well established that mutations in specific structural elements of the motor protein myosin are directly linked to debilitating diseases involving malfunctioning striated muscle cells. A potential way to study the relationship between myosin structure and function is to express exogenous myosin in vivo and determine contractile properties of the transgenic muscle cells. However, in vivo expression of functional levels of contractile proteins using transient transgenesis in skeletal muscle has not been demonstrated. Presently, we used in vivo gene transfer to express high levels of full-length myosin light chain (MLC) in skeletal muscle fibers of Rana pipiens. Anterior tibialis (AT) muscles were injected with cardiotoxin to cause degeneration and then injected at various stages of regeneration with plasmid expression vectors encoding full-length MLC1(f). In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1(f) expression averaged 22-43% of the endogenous MLC1(f). Trans-MLC1(f) expression was the same whether a small epitope tag was placed on the C- or N-terminus and was highly variable along individual fibers. Confocal microscopy of skinned fibers showed correct sarcomeric incorporation of trans-MLC1(f). The expression profile of myosin heavy chain isoforms 21 days after transfection was similar to normal AT muscle. These data demonstrate the feasibility of using in vivo gene transfer to probe the structural basis of contractile protein function in skeletal muscle. Based on these promising results, we discuss how further improvements in the level and consistency of myosin transgene expression may be achieved in future studies, and the therapeutic potential of plasmid gene transfer in regenerating muscle.     

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.     

Inhibition of smooth muscle myosin as a novel therapeutic target for hypertension

We examined a novel therapeutic approach for hypertension, a small-molecule direct inhibitor of smooth muscle myosin, CK-2018448 (CK-448), which is an N,N'-alkylurea (U.S. Patent Publication 2009-0275537 A1) in conscious dogs with renal hypertension and compared its efficacy with that of a calcium channel blocker, amlodipine. Dogs were instrumented with a miniature left ventricular pressure gauge, an aortic pressure catheter, and ultrasonic flow probes in the ascending aorta and renal and iliac arteries for measurement of cardiac output and regional blood flow. In the hypertensive state, mean arterial pressure increased from 101 ± 3.8 to 142 ± 1.9 mm Hg. At the doses selected, CK-448 and amlodipine increased cardiac output similarly (30 ± 11% versus 33 ± 6.4%) and similarly reduced mean arterial pressure (-22 ± 3.6% versus -16 ± 3.4%) and total peripheral resistance (-36 ± 5.9% versus -37 ± 5.8%). CK-448 had the greatest vasodilator effect in the renal bed, where renal blood flow increased by 46 ± 9.0%, versus 11 ± 3.4% for amlodipine (p < 0.01). CK-488 produced significantly less vasodilation in the limb, where iliac blood flow did not change; in contrast, it rose by 48 ± 12% with amlodipine (p < 0.01). The minimal effects on limb blood flow could limit the development of peripheral edema, an adverse side effect of Ca(2+) channel blockers. In addition, in a rodent model of hypertension, oral administration of a smooth muscle myosin inhibitor resulted in a sustained antihypertensive effect. Thus, the smooth muscle myosin inhibitor's preferential effect on renal blood flow makes this drug mechanism particularly appealing, because many patients with hypertension have renal insufficiency, and patients with heart failure could benefit from afterload reduction coupled with enhanced renal blood flow.     

Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester-induced stress and myosin light chain phosphorylation in swine carotid medial smooth muscle

The mechanisms by which activators of protein kinase C (PKC) stimulate contractile responses in arterial smooth muscle is not known. In this study, we assessed the relative contribution of CA(++)-dependent and independent pathways in mediating phorbol ester-induced 20 kdalton myosin light chain (MLC)-phosphorylation and force in medial smooth muscle strips from swine carotid artery. Phorbol 12,13-dibutyrate (PDB; 10(-7)M)-stimulated stress development was associated with a significant increase in the fraction of phosphorylated MLC, from 0.08 +/- 0.02 to 0.24 +/- 0.02 after 30 min of stimulation. Under conditions of Ca++ depletion, which normally do not support Ca++/calmodulin-dependent activation of myosin light chain kinase (MLCK) by physiological stimuli, PDB-induced contractile responses were reduced significantly. However, after Ca2++ depletion, PDB (10(-6) M; 30 min) still caused an increase in MLC-phosphorylation from 0.10 +/- 0.02 at rest to 0.19 +/- 0.03. Preincubation with nifedipine (10(-7) M) had no significant effect on contractile responses to PDB, indicating that Ca++ influx through nifedipine-sensitive voltage channels did not contribute significantly to the observed Ca++ dependency of the PDB responses. Staurosporine (0.1-0.3 microM), a putative PKC inhibitor, significantly inhibited PDB-induced contractile and MLC phosphorylation responses. Tonic histamine (3 microM)- and KCl-induced contractile and MLC-phosphorylation responses were inhibited by the same concentrations of staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines.

Scavenger receptor (ScR)-mediated uptake of modified lipoproteins may contribute to the transformation of smooth muscle cells into lipid-laden foam cells during atherogenesis. This study examined the in vivo expression of ScRs in aortas, with or without balloon injury, taken from hypercholesterolemic or normocholesterolemic rabbits. Numerous intimal cells in the rabbit aortic lesions expressed ScRs as detected by immunocytochemical staining with a goat anti-rabbit ScR antibody. Single immunostaining for cell identification markers in serial sections, as well as double staining, confirmed the expression of ScRs by both intimal smooth muscle cells and macrophages. To explore potential inducers of ScR expression by smooth muscle cells in vivo, we studied the regulation of ScR expression in vitro by cytokines known to be present in atherosclerotic lesions. Tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) increased ScR mRNA levels, protein expression, and AcLDL degradative activity in cultured rabbit aortic smooth muscle cells. The induction of ScR expression in intimal smooth muscle cells in vivo could be a useful marker of smooth muscle cell activation during atherogenesis and may contribute to foam cell formation by this cell type following balloon injury and/or hypercholesterolemia. Cytokines, such as TNF-alpha or IFN-gamma, may stimulate some of the phenotypic changes that characterize the alteration in gene expression of intimal smooth muscle cells in rabbit atherosclerotic lesions.     

Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle

The Ca dependence of contraction and myosin phosphorylation was investigated in canine tracheal smooth muscle stimulated with carbachol, K or serotonin. Previous studies of tracheal muscle showed carbachol concentration-response curves for contraction and myosin phosphorylation were superposable. In contrast, there was a striking difference in the Ca++ sensitivities of tension and myosin phosphorylation when Ca++ concentration-response curves were constructed in the presence of 10(-7) M carbachol. Significant phosphorylation (greater than 0.3 moles phosphate/mole 20,000 dalton myosin light chain) was observed in the absence of active tension. In the present study, carbachol (10(-7) and 10(-6) M) and serotonin (10(-5) M) also induced significant myosin phosphorylation in low Ca++ solutions (0-0.025 mM CaCl2) without proportional increases in tension. K+ depolarization in Ca++-free physiological salt solution (60 mM KCl, 10(-6) M atropine) yielded phosphorylation not significantly different from basal levels. All stimulants induced active stress after readmission of Ca. The Ca++ dependence curve for myosin phosphorylation in muscles stimulated with carbachol was shifted up and to the left of the force curve. Atropine (10(-6) M) significantly reduced phosphorylation induced by carbachol in Ca++-free solutions, as did 3 X 10(-6) M nifedipine and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Phorbol 12-myristate, 13-acetate or phorbol 12,13-dibutyrate did not increase basal phosphorylation or phosphorylation in low Ca++ solutions, suggesting that protein kinase C did not phosphorylate myosin in this case. Myosin phosphorylation under these conditions is not sufficient to support contraction, and is reduced by treatments that decrease Ca++ entry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium dependence of phorbol 12,13-dibutyrate-induced force and myosin light chain phosphorylation in arterial smooth muscle

Phorbol dibutyrate (PDB) is an activator of protein kinase C and has been observed to cause a slow developing contraction in vascular smooth muscle. The mechanism of phorbol ester-induced contraction is unknown. We studied the Ca++-dependence of, and the degree of myosin light chain phosphorylation (MLC-P), during PDB-induced contractions in rabbit aortic rings. PDB elicited concentration-dependent contractions (3 X 10(-8) to 10(-6) M) in rabbit aortic rings incubated in normal (1.6 mM Ca++) physiologic salt solution (PSS). Addition of the Ca++-channel blocker nifedipine (0.1 microM) to PSS or removal or Ca++ from PSS significantly reduced the contractile responses to PDB. Depletion of Ca++ by repeated washes in O Ca++-PSS containing 10(-3) M ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid reduced, but did not eliminate, the responses to PDB. In PSS, PDB significantly increased the fraction of phosphorylated MLC/total MLC to 0.33 from a resting value of 0.20. Ca++ depletion reduced the resting fraction (MLC-P/MLC) to 0.14. PDB-stimulated contractions in Ca++-depleted tissues occurred in the absence of significant increases in MLC-P. Sodium nitroprusside partially relaxed PDB-induced contractions by approximately 50% whether elicited in the presence of 1.6 mM Ca++ or after Ca++ depletion. In both cases relaxation occurred in the absence of statistically significant decreases in MLC phosphorylation. Ca++-dependent MLC phosphorylation may account for a component of the PDB contractile response in rabbit aorta. Studies in the absence of Ca++ suggest that PDB may activate contraction without concomitant MLC-P.     

非钙依赖性调节肌球蛋白活性对维持平滑肌张力的影响

目的:揭示肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)的一个活性片段对肌球蛋白功能的非钙依赖性调节作用,进一步完善平滑肌收缩的非钙依赖性调节机制研究,并为平滑肌系统疾病的治疗以及新药开发提供理论基础。方法:实验于2004-01/05在大连医科大学生化药理实验室完成。用胰蛋白酶(trypsin)水解MLCK,再经离子交换层析分离纯化而获得MLCK的活性片段(myosinlightchainkinasefragment,MLCKF,61ku);用west-ernblot检测MLCKF与完整MLCK的同源性;用SDS-PAGE及ScoinImage扫描软件检测肌球蛋白轻链的磷酸化程度;用分光光度法测定肌球蛋白Mg2+-ATP酶的活性。结果:在无Ca2+/CaM条件下,61kuMLCKF能轻度磷酸化20ku肌球蛋白轻链,并提高肌球蛋白Mg2+-ATP酶活性;其磷酸化效力强于无Ca2+/CaM时MLCK的磷酸化效力,但弱于有Ca2+/CaM时MLCK的磷酸化效力,且持续时间长,较稳定,不易被MLCK抑制剂ML-9抑制。结论:MLCKF能以非钙依赖性方式轻度磷酸化肌球蛋白轻链,并提高肌球蛋白Mg2+-ATP酶活性,对低钙时平滑肌张力的维持可能起一定作用。     

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1beta or TNF-alpha (tumour necrosis factor-alpha) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with p22(phox)antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.     

Potent inhibition of arterial smooth muscle tonic contractions by the selective myosin II inhibitor, blebbistatin

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC(50) = 0.5-5 microM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC(50) approximately 80 microM). We found that blebbistatin potently (IC(50) approximately 3 microM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC(50) approximately 5 microM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited beta-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC(50) approximately 20 microM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.     

Colitis-induced alterations in adrenergic control of circular smooth muscle in vitro in rats

The present study investigated inflammation-induced changes in adrenergic regulation of smooth muscle. Colitis was induced in rats by intrarectal administration of trinitrobenzenesulfonic acid in ethanol. After 4 h (acute) or 7 days (chronic), in vitro isometric tension was measured in strips of circular smooth muscle taken from the distal colon. In controls, the major inhibitory control of smooth muscle responses to nerve stimulation was mediated by nitric oxide and beta adrenergic receptors. There was less evidence of alpha adrenergic control. Studies with the beta3 receptor antagonist cyanopindolol and the beta3 receptor agonist BRL37344 revealed that beta adrenergic regulation of spontaneous contractions and responses to nerve stimulation were mediated primarily by the beta3 adrenoreceptor. Both acute and chronic colitis significantly increased responses to electrical field stimulation. This effect was attributed to a loss of inhibitory nitrergic regulation as well as to selective changes in the beta adrenergic control of colonic circular smooth muscle. Inflammation did not alter alpha adrenergic control. Chronic colitis also decreased the sensitivity to nerve stimulation and pharmacological contractile agents. Acute and chronic inflammation reduced the ability of BRL37344 to inhibit contractions in response to nerve stimulation. In addition, in inflamed colon, BRL37344 was less effective in relaxing carbachol-induced precontractions. Finally, inflammation resulted in a loss of the ability of the cyanopindolol to increase the amplitude of both spontaneous contractions and contractions in response to nerve stimulation. These effects indicated that colitis induced a down-regulation of inhibitory beta3 adrenergic control of colonic smooth muscle function. This loss of adrenergic regulation may contribute to the diarrhea in inflammatory bowel disease.     

前列腺素受体在低氧诱导的肺动脉平滑肌细胞中的表达研究

目的:探讨前列腺素受体家族在低氧诱导的肺动脉高压平滑肌细胞中的表达变化。方法:体内实验,以低氧(10%O2)3周诱导建立小鼠肺动脉高压模型,分离肺动脉;体外实验,分别对小鼠源性和人源性的肺动脉平滑肌细胞进行低氧(1%O2)24 h的诱导。提取相应组织或细胞的RNA,以反转录-聚合酶链反应(RT-PCR)扩增不同前列腺素受体基因片段,并将产物进行琼脂糖凝胶电泳。结果:无论是小鼠源性还是人源性的肺动脉平滑肌细胞中,前列腺素受体基因基础表达值均以血栓素A2受体TP和前列腺素E受体1(E-prostanoid receptor 1,EP1)为最高,EP2、EP3、EP4和前列腺素I2受体(IP)居中,前列腺素D1受体、前列腺素D2受体和前列腺素F2α受体最少。在低氧诱导后,小鼠肺动脉平滑肌细胞血栓素A2受体基因和EP3受体基因表达上调显著,而前列腺素D1受体、EP1和前列腺素I2受体则明显下调。结论:在低氧诱导下,前列腺素受体基因表达发生不同变化,对筛选不同前列腺素用于肺动脉高压的疗效研究和新药开发及探究其病理机制有重要意义。     

Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo

We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.