心肌细胞和血管平滑肌细胞收缩调控机制的研究进展

心脏和血管构成体内的心血管系统,两者都具有收缩性。心脏收缩要求在很短的时间内升高室内压,因此要求细胞收缩快速和有力,这就需要细胞的收缩结构和钙调控过程能满足其要求。血管收缩缓慢而持久,其收缩结构及机制也正好与之功能相适应。本文从细胞水平讨论了心脏和血管的收缩结构和收缩机制,以及钙调控机制,并分别对两者之间的异同点作了介绍。     

平滑肌收缩中Ca^2＋敏感性调节的机理

多种激动剂增加细胞器对Ｃａ＾２＋的敏感性,即Ｃａ＾２＋的敏感性,即Ｃａ＾２＋敏感化作用。激动剂的这种作用可能是通过Ｇ蛋白,经信号分子花生四烯酸和二酰基甘油,反暹号传递到肌球蛋白轻链磷酸酶（ＭＬＣＰ）,增加肌球蛋白磷酸化。细胞内游离Ｃａ＾２＋升高到一定程度,ｃａｌｍｏｄｕｌｉｎ激酶Ⅱ活化,导致ＭＬＣＫ磷酸化,降低了其对Ｃａ＾２＋－ｃａｌｍｏｄｕｌｉｎ亲和力,ＭＬＣＫ对Ｃａ＾２＋敏感性降低,即Ｃａ＾２     

平滑肌收缩调节的信号转导

平滑肌细胞内信号转导主要有肌球蛋白轻链激酶（ＭＬＣＫ）和蛋白激酶Ｃ（ＰＫＣ）途径。前者通过肌浆内Ｃａ＾２＋浓度升高,激活钙调蛋白（ＣａＭ）依赖性ＭＬＣＫ,催化肌球蛋白轻链丝氨酸（Ｓｅｒ）－１９磷酸化,肌球蛋白ＡＴＰ酶活性增加,肌丝滑行,肌肉收缩。肌浆内Ｃａ＾２＋浓度的恢复使ＭＬＣＫ失活,肌球蛋白轻链磷酸酶（ＭＬＣＰ）使肌球蛋白脱磷酸化,肌肉舒张。近来有证据表明ＰＫＣ信号转导途径通过影响细肌丝相关蛋     

RyR反义寡核苷酸对气道平滑肌细胞增殖及细胞内钙离子浓度的影响

目的:观察RyR(Ryanodme受体)反义寡核苷酸(ASODN)对大鼠ASMCs(airway smooth muscle cells,气道平滑肌细胞)增殖的抑制作用及对细胞内钙离子浓度的影响.方法:采用胶原酶消化法培养大鼠ASMCs,利用LipofectamineTM2000将正义、反义RyR寡核苷酸导入大鼠ASMCs,MTS/PES法检测不同寡核苷酸对大鼠ASMCs增殖的抑制作用,RT-PCR检测大鼠ASMCs中RyR的mRNA表达,流式细胞仪测定不同寡核苷酸对细胞内钙离子浓度的影响.结果:RyR反义寡核苷酸可抑制大鼠ASMCs的增殖,降低其RyR受体mRNA的表达,并能降低兴奋后的细胞内钙离子浓度的升高.结论:RyR反义寡核苷酸可能通过降低兴奋后的细胞内钙离子浓度来抑制大鼠ASMCs的增殖.     

ERK信号通道调控大鼠气道平滑肌细胞的增殖与凋亡

 为了了解ERK信号通道对正常大鼠气道平滑肌细胞(airway smooth muscle cells, ASMCs)增殖与凋亡的调控. 通过对正常大鼠ASMCs原代培养,4～7代用于实验,以ERK激动剂表皮生长因子（EGF）和抑制剂PD98059干预ASMCs生长,采用RT-PCR和免疫荧光染色观察ASMCs上ERK mRNA和蛋白的表达,MTT法、^３Ｈ-TdR掺入法检测ASMC增殖,Hoechst染色和Annexin-Ⅴ FITC PI双染色法检测细胞凋亡,Western免疫印迹检测ERK1/2、磷酸化ERK1/2和procaspase-3蛋白的表达.结果发现ASMCs上存在ERK mRNA和蛋白的表达,与空白对照组比较,PD98059干预后ASMCs的Ａ_４９０值和细胞DNA合成量均减少(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均增高(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均降低, procaspase-3蛋白的表达增高.EGF干预后ASMCs的Ａ_４９０值和细胞DNA合成量均增高(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均下降(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均增高, procaspase-3蛋白的表达降低.P+E组无明显差异(P>0.05).ERK信号通道参与大鼠ASMCs增殖和凋亡的调控,ERK对大鼠ASMCs凋亡的调控与procaspase-3蛋白有关,这一发现将有助于对哮喘ASMCs异常增殖调控机制的深入研究.     

基于细胞三维培养的气道平滑肌细胞收缩效应检测方法的建立与应用

气道平滑肌细胞(airway smooth muscle cells,ASMCs)是引起哮喘患者气道收缩狭窄、呼吸阻力增加的主要效应细胞。ASMCs收缩效应检测是研究哮喘病理生理机制、评估或研发新的支气管舒张药物的重要实验依据。而常用的活体组织张力检测以及单层培养ASMCs显微镜形态观察等方法存在样品取材或测量误差等问题。该研究利用胶原蛋白凝胶构建气道平滑肌细胞的三维立体培养模型,将凝胶与培养孔壁分离,在不同的时间点记录细胞收缩力作用下凝胶面积的变化值,以此反映ASMCs的收缩效应。结果显示,0.1,1,10 mmol/L的乙酰胆碱(acetylcholine,Ach)刺激后,凝胶面积均显著缩小,随着剂量增加ASMCs的收缩反应增强。提前加入肌球蛋白ATP酶抑制剂BDM(butanedione monoxime),能明显抑制Ach诱导的ASMCs收缩反应,说明该方法的可重复性和准确性好。     

SM22α对血管平滑肌细胞骨架及收缩功能的影响

SM22α(smooth muscle 22 alpha,SM22α)是血管平滑肌细胞(vascular smooth muscle cells,VSMC)的标志蛋白,为了探讨该蛋白与VSMC表型和功能的关系,利用血清饥饿法诱导VSMC由合成型向收缩型转变,用RT—PCR对不同表型VSMC的SM22α表达活性进行检测,并通过转染反义SM22α表达载体,观察SM22α表达对VSMC细胞骨架和收缩功能的影响。结果显示,在VSMC由合成型逆转为收缩型的过程中,SM22α和平滑肌α-肌动蛋白(smooth muscle α—actin,SMα—actin)的表达分别被显诱导和轻度上调,与此同时,细胞骨架由稀疏的网格状变成均匀、致密的束状,VSMC重新获得收缩功能。用反义SM22α抑制该基因表达后,血清饥饿诱导的VSMC细胞骨架重构受阻,乙酰胆碱刺激引发的细胞收缩消失。结果提示,SM22α参与VSMC细胞骨架的构成及调节细胞的收缩功能,对维持VSMC处于收缩表型具有重要作用。     

胃动素对大鼠胃平滑肌细胞收缩活动的作用

本研究用大鼠游离的胃平滑肌细胞,观察胃动素对胃平滑肌细胞的收缩作用。结果表明：（１）胃动素明显增强单个胃平滑肌细胞收缩活动,在生理剂量１０（－１１）─１０（－１０）ｍｏｌ范围内,呈剂量依赖性。（２）不同胃分区平滑肌细胞对冒动素兴奋反应不同,胃动素对胃窦平滑肌细胞收缩强度大于胃体和幽门。（３）给予抗胃动素血清可以完全取消胃动素对胃肌细胞的收缩反应,而阿托品、ＴＴＸ、甲氰米胍、ｌｏｘｉｇｌｕｍｉｄｅ均不影响胃动素的作用。（４）给予胞内钙释放阻断剂ＴＭＢ－８可抑制胃动素对目肌细胞的收缩作用。上述结果提示,胃动素对胃平滑肌细胞的直接作用是由胃动素受体所介导,且与胞内Ｃａ（２＋）释放起重要作用。     

第二信使系统与消化道平滑肌收缩活动的调节

本主要从三方面介绍了第二信使系统对消化道平滑肌运动的调节作用：（１）ｃＡＭＰ、ｃＧＭＰ作用机制和某些胃肠激素经受体活化后的信息传递机制;（２）ＩＰ３、ＤＡＧ对肌收缩的作用特点和双向调节作用;（３）胞内信使间在调控活动中的相互关系。     

Lysophosphatidic acid enhances contractility of isolated airway smooth muscle

Toews, M. L., E. E. Ustinova, and H. D. Schultz.Lysophosphatidic acid enhances contractility of isolated airwaysmooth muscle. J. Appl. Physiol.83(4): 1216-1222, 1997.The effects of the simple phospholipidmediator lysophosphatidic acid (LPA) on the contractile responsivenessof isolated tracheal rings from rabbits and cats were assessed. In bothspecies, LPA increased the contractile response to the muscarinicagonist methacholine, but LPA did not induce contraction on its own.Conversely, LPA decreased the relaxation response to the-adrenergic-agonist isoproterenol in both species. Concentrations ofLPA as low as 10⁸ M wereeffective, and the effects of LPA were rapidly reversed on washing.Phosphatidic acid was much less effective, requiring higherconcentrations and producing only a minimal effect. Contractions induced by serotonin and by substance P also were enhanced by LPA, butKCl-induced contractions were unaffected. LPA inhibited theisoproterenol-induced relaxation of KCl-precontracted rings, similar toits effects on methacholine-precontracted rings, and relaxation inducedby the direct adenylyl cyclase activator forskolin was inhibited in amanner similar to that induced by isoproterenol. Epithelium removal didnot alter the contraction-enhancing effect of LPA. The ability of LPAto both enhance contraction and inhibit relaxation of airway smoothmuscle suggests that LPA could contribute to airway hypercontractilityin asthma, airway inflammation, or other types of lung injury.

     

MLC-kinase/phosphatase control of Ca2+ signal transduction in airway smooth muscles

In airway smooth muscles, kinase/phosphatase-dependent phosphorylation and dephosphorylation of the myosin light chain (MLC) have been revealed by many authors as important steps in calcium (Ca²⁺) signalling pathway from the variation of Ca²⁺ concentration in cytosol to the force development. Here, a theoretical analysis of the control action of MLC-kinase (MLCK) and MLC-phosphatase (MLCP) in Ca²⁺ signalling is presented and related to the general control principles of these enzymes, which were previously studied by Reinhart Heinrich and his co-workers. The kinetic scheme of the mathematical model considers interactions among Ca²⁺, calmodulin (CaM) and MLCK and the well-known 4-state actomyosin latch bridge model, whereby a link between them is accomplished by the conservation relation of all species of MLCK. The mathematical model predicts the magnitude and velocity of isometric force in smooth muscles upon transient biphasic Ca²⁺ signal. The properties of signal transduction in the system such as the signalling time, signal duration and signal amplitude, which are reflected in the properties of force developed, are studied by the principles of the metabolic control theory. The analysis of our model predictions confirms as shown by Reinhart Heinrich and his co-workers that MLCK controls the amplitude of signal more than its duration, whereas MLCP controls both. Finally, the simulations of elevated total content of MLCK, a typical feature of bronchial muscles of asthmatic subjects and spontaneously hypertensive rats as well as potentiation of MLCP catalytic activity, are carried out and are discussed in view of an increase in the force magnitude.     

Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.     

Intracellular signal systems in the epithelium- and endothelium-dependent relaxation of smooth muscles

The research of mechanisms of a regulation electrical and contractile of properties of unstriated muscles of an internals remains by an actual problem of modern physiology and medicine. Already now it is possible to state that the efficacy of means of correction of distresses of an internals depends on a degree of a level of scrutiny of these mechanisms. Among physiologically active substances effecting on smooth muscle cells (SM), the special relaxing factor synthesized by endotheliocytes, epithelial cells and SM. Identified by the majority of the explorers as oxide of nitrogen (NO), relaxing factor responds for exhibiting of many myogenic responses of pots and pneumatic routes. The mechanisms of synthesis and implementation of effects of this factor in SM cells up to the extremity are not clarified. The considerable advance in learning mechanisms of operation relaxing factor on SM is connected to discovering of ability of some nitro compounds to replicate NO-dependent relaxing effects in these cells. The main systems of intracellular regulation are involved in mechanisms of implementation endothelial and epithelial local regulatory effects on SM. The majority of the explorers bind an epithelium-dependent release phenomenon SM to an activation of a solvable fraction guanilatcyclase, found in the majority of cells, and effects of cGMP-dependent protein kinases. There are reports on ability of inhibitors NO-sintase to depress a release phenomenon SM of pots and bronchuses, about dependence of a mechanical strain SM of pots and respiratory tract from a contents cGMP in cells. However there are datas giving establishments to guess, that alongside with guanilatciclase in a release phenomenon SM, induced relaxing factors or nitro compounds, the immediate involvement is accepted by cAMP-dependent protein kinases. The most probable point of interaction cAMP and cGMP-dependent processes is phospodiesterase of cyclic nucleotides. It citosolium the enzyme labilized by calmodulin, is capable to carry out a hydrolysis of both cyclic nucleotides, and the affinity native phospodiesterase to cGMP exceeds affinity to cAMP more, than on the order. It is impossible to eliminate immediate interference of NO-dependent processes in a regulation of activity contractile proteins. The ability cGMP-dependent processes to depressing mechanisms of phosphorylation and intensifying of a dephosphorylization of contraktion proteins SM is shown. At these processes can variate and affinity of the acto-miosin complex to ions of calcium, producing a release phenomenon of smooth muscles. On all visibility, production relaxing of the factor and the implementation is epithelial and endothelium-SM of mutual relation in a respiratory tract and pots comes true by modulating influence at the calcium signal system of other systems. For example, production relaxing of the factor by an epithelium and endothelium, being calcium-dependent process, is regulated at involvement calmodulin-similar Ca(2+)-connecting proteins and protein kinase C. Control of tone SM through change of membrane potential relaxing factor carries out by paravariation of potassium conduction of a membrane SM, and, is more probable than all through calcium-dependent and ATP-sensitive components. Potencial-dependent control of a muscle tone comes true through change of efficacy of an operation from a branch of the calcium signal system and calcium pompes at submaximal concentrations of free calcium in citosolium.     

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.     

Isotonic relaxation of control and sensitized airway smooth muscle

Smooth muscle relaxation has most often been studied in isometric mode. However, this only tells us about the stiffness properties of the bronchial wall and thus only about wall capacitative properties. It tells us little about airflow. To study the latter, which of course is the meaningful parameter in regulation of ventilation and in asthma, we studied isotonic shortening of bronchial smooth muscle (BSM) strips. Failure of BSM to relax could be another important factor in maintaining high airway resistance. To analyze relaxation curves, we developed an index of isotonic relaxation, t1/2(P, lCE), which is the half-time for relaxation that is independent of muscle load (P) and of initial contractile element length (lCE). This index was measured in curves of relaxation initiated at 2 s (normally cycling crossbridges) and at 10 s (latch-bridges). At 10 s no difference was seen for adjusted t1/2(P, lCE) between curves obtained from control and sensitized BSM, (8.38 +/- 0.92 s vs. 7.78 +/- 0.93 s, respectively). At 2 s the half-time was almost doubled in the sensitized BSM (6.98 +/- 0.01 s (control) vs. 12.74 +/- 2.5 s (sensitized)). Thus, changes in isotonic relaxation are only seen during early contraction. Using zero load clamps, we monitored the time course of velocity during relaxation and noted that it varied according to 3 phases. The first phase (phase i) immediately followed cessation of electrical field stimulation (EFS) at 10 s and showed almost the same velocity as during the latter 1/3 of shortening; the second phase (phase ii) was linear in shape and is associated with zero load velocity, we speculate it could stem from elastic recoil of the cells' internal resistor; and the third phase (phase iii) was convex downwards. The zero load velocities in phase iii showed a surprising spontaneous increase suggesting reactivation of the muscle. Measurements of intracellular calcium (Fura-2 study) and of phosphorylation of the 20 kDa myosin light chain showed simultaneous increments, indicating phase iii represented an active process. Studies are under way to determine what changes occur in these 3 phases in a sensitized muscle. And of course, in the context of this conference, just what role the plastic properties of the muscle play in relaxation requires serious consideration.     

Molecular mechanisms of regulation of the contractile apparatus of smooth muscles]

This review describes the main ultrastructural features, molecular organization and regulation of smooth muscle contractile machinery. Possible molecular mechanisms of smooth muscle sensitization to Ca2+, Ca(2+)-independent contraction and latch state are discussed.     

Chronic inflation of ferret lungs with CPAP reduces airway smooth muscle contractility in vivo and in vitro.

The mechanical stress imposed on the lungs during breathing is an important modulator of airway responsiveness in vivo. Our recent study demonstrated that continuous positive airway pressure applied to the lungs of nonanesthetized, tracheotomized rabbits for 4 days decreased lower respiratory system responsiveness to challenge with ACh (Xue Z, Zhang L, Ramchandani R, Liu Y, Antony VB, Gunst SJ, Tepper RS. J. Appl Physiol 99: 677-682, 2005). In addition, airway segments excised from the lungs of these animals and studied in vitro exhibited reduced contractility. However, the mechanism for this reduction in contractility was not determined. The stress-induced decrease in airway responsiveness could have resulted from alterations in the excitation-contraction coupling mechanisms of the smooth muscle cells, or it might reflect changes in the structure and/or composition of the airway wall tissues. In the present study, we assessed the effect of prolonged chronic stress of the lungs in vivo on airway smooth muscle force generation, myosin light chain phosphorylation, and airway wall structure. To enhance the potential development of stress-induced structural changes, we applied mechanical stress for a prolonged period of time of 2-3 wk. Our results demonstrate a direct connection between the decreased airway responsiveness caused by chronic mechanical stress of the lungs in vivo and a persistent decrease in contractile protein activation in the airway smooth muscle isolated from those lungs. The chronic stress also caused an increase in airway size but no detectable changes in the composition of the airway wall.