两个品系豚鼠对化学介质诱导产生气道反应的差异性研究

目的　研究新育成的Zmu 1∶DHP豚鼠气道对外界化学介质诱导产生的反应性。为研究哮喘选择和提供具有较高敏感性的速发型过敏性动物模型。方法　采用雾化气体吸入法 ,按递增浓度 ,让动物吸入组胺及乙酰胆碱气体 ,记录豚鼠到达哮喘发作时的介质浓度和呼吸频率及幅度 ,评定对化学介质的敏感程度 ,同时用DHP品系豚鼠进行对照。结果　当 0 2 %组胺浓度雾化吸入时 ,Zmu 1∶DHP豚鼠哮喘发作的呼吸频率及每分钟通气量 ,显著大于DHP豚鼠 (P <0 0 5 ) ;当 0 4 %浓度时 ,前者的潮气量及每分钟通气量 ,比后者有增高的趋势 (P <0 0 5 ) ;当0 6 %浓度时 ,前者的潮气量及每分钟通气量 ,显著小于后者 (P <0 0 5 )。二个品系豚鼠吸入乙酰胆碱雾化气体后 ,无明显差异 (P >0 0 5 )。结论　在低浓度组胺雾化吸入时 ,Zmu 1∶DHP品系豚鼠产生哮喘的敏感性显著高于DHP豚鼠 ;高浓度时 ,气道可能因失敏而降低反应性     

黄体酮对离体家兔气管平滑肌收缩活动的影响

目的：研究黄体酮对离体家兔气管平滑肌收缩活动的影响。方法：采用家兔离体气管平滑肌标本,观察黄体酮对ACh及Ca-Cl2量效曲线的影响,同时观察在无钙液中加入CaCl2时单剂量Ach诱发标本双时相收缩反应的影响。结果：黄体酮能使ACh及CaCl2量效曲线明显压低,最大反应降低,且能够抑制ACh引起的第1时相收缩,对第Ⅱ时相收缩作用无明显影响。且黄体酮对ACh量效曲线的影响是上皮依赖性的。结论：黄体酮对离体气管平滑肌的松弛作用与抑制电压依赖性钙通道(PDC)和ACh引起的细胞内Ca^2 释放及上皮细胞的作用有关。     

旋割法制备气管螺旋条评价

目的通过改进螺旋剪法建立制备气管螺旋条的旋割法。方法 40只豚鼠,用旋割法和螺旋剪法制备离体豚鼠气管螺旋条,在Kreb＇s液中平衡孵育2 h后,以组胺histamine（浴槽浓度2.0×10-3g/L）和乙酰胆碱acetylcholine（浴槽浓度2.0×10-4g/L）引发气管螺旋条收缩,用BL420生物信号采集系统与张力传感器测定标本张力变化值。数据采用SPSS11.5软件在α=0.05的信度下进行t检验。结果 2 g负荷下,旋割法标本histamine引发收缩幅度是螺旋剪法制备标本的1.31倍,乙酰胆碱引发收缩幅度旋割法是螺旋剪法制备标本的1.208倍,经t检验,P〈0.05,差异均具有显著性;旋割法标本,histamine引发2 g负荷标本收缩幅度是1 g负荷的1.48倍,乙酰胆碱引发2 g负荷标本的收缩幅度是1 g负荷的1.38倍,经t检验,P〈0.05,差异均具有显著性;旋割法标本经hista-mine或acetylcholine激发收缩,洗净药物重复激发6次收缩幅度的RSD值分别为19.8%和19.1%,螺旋剪法标本经histamine或acetylcholine 6次重复引发诱发收缩幅度的RSD值分别35.3%和33.7%。结论与螺旋剪法制备气管螺旋条标本比较,旋割法制备螺旋条标本对收缩诱导剂histamine与acetylcholine的敏感性高,标本负荷以2 g较好,旋割法标本重复利用收缩幅度变化值较螺旋剪法标本小。     

血管平滑肌细胞表型转化在心血管疾病中作用的研究进展

血管平滑肌细胞(vascular smooth muscle cells,VSMCs)具有高度可塑性,病理条件下VSMCs发生由收缩型到合成型的表型转化,参与心血管疾病的发生发展。VSMCs表型转化是动脉粥样硬化、血管成形术后再狭窄、高血压、肺动脉高压等多种心血管疾病共同的病理基础。血小板源性生长因子、血管紧张素Ⅱ、转化生长因子β、活性氧簇及微小RNAs等均参与调节VSMCs的表型转化。本文就VSMCs表型转化的标志基因,VSMCs表型转化的分子调控机制及其与心血管疾病的关系作一综述。     

血清后人血管平滑肌细胞表型转化与microRNAs表达间关系的研究

目的:研究去血清诱导人血管平滑肌细胞发生表型转化与microRNAs表达间的关系。方法:采取人血管平滑肌细胞克隆株HITASY,培养人血管平滑肌细胞(VSMCs)。用去血清的方法处理人血管平滑肌细胞,使VSMCs由去分化表型向分化表型转化,通过蛋白印迹法观察平滑肌细胞中分化标记物蛋白的变化,同时用实时定量PCR法检测细胞中相关microRNA的表达。结果:①去血清处理组与无去血清处理组比较,平滑肌细胞分化标记物(SM-α-actin、calponin)表达显著性增加,而通过SM-α-actin、calponin的蛋白表达量显著增加,提示血管平滑肌细胞向分化表型转化(P0.05);②同时去血清处理组与无去血清处理组比较,Mir-649、Mir-944表达量显著增加,Mir-140、Mir-361表达量显著减少(P0.05)。结论:Mir-649、Mir-944、Mir-140、Mir-361在血管平滑肌细胞表型转化中有关键性作用。     

细胞代数和密度影响血管平滑肌细胞表型重塑和收缩反应性的机制

探讨细胞代数和密度对血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型重塑能力的影响及机制,观察血清饥饿诱导的不同代数和密度的VSMC骨架的组构特征及收缩反应性,检测细胞骨架中收缩蛋白的含量和比例变化。结果发现,低代数(3代)、高密度的VSMC经血清饥饿诱导后易于形成束状、极性排列的应力纤维,乙酰胆碱(Ach)刺激可产生明显的收缩反应。Western印迹显示,3代高密度VSMC中,平滑肌22α(SM22α)在F-肌动蛋白中的组成比例及其在F-/G-肌动蛋白的含量之比明显高于8代细胞。结果提示,SM22α在F-肌动蛋白中的分布比例可能决定了应力纤维的排布方式,是细胞获得收缩性的主要调节因素,在VSMC表型重塑过程中具有重要意义。     

肺泡巨噬细胞对豚鼠气道平滑肌张力的调控

经调理酵母多糖(OPZ)刺激的大鼠肺泡巨噬细胞培养上清液可松弛豚鼠离体气管肌条,上清液中PGE_1增加,表明PGE_1是肺泡巨噬细胞松弛气管肌条的介质之一。经OPZ激活的肺泡巨噬细胞培养上清液与豚鼠血小板作用后,其松弛效应被逆转为收缩效应,提示可能由于肺泡巨噬细胞分泌血小板活化因子激活血小板,使释放收缩介质所致。肺泡巨噬细胞借助所分泌的介质经常性地调节气道阻力,对肺通气具有保护意义。     

Ghrelin对豚鼠胃窦平滑肌细胞钙离子浓度的影响及与NO关系研究

目的:探讨Ghrelin对豚鼠胃窦平滑肌细胞内钙离子浓度的影响及其与一氧化氮(NO)的关系。方法:采用荧光免疫组化检测胃窦平滑肌细胞ghrelin受体(GHS-R)的表达;应用钙离子(Ca2+)指示剂Fluo-3/AM作为细胞内Ca2+的荧光探针,对负载培养的平滑肌细胞应用激光共聚焦显微镜技术,检测不同浓度ghrelin对平滑肌细胞内Ca2+荧光强度(FI)的影响,以及ghrelin受体阻断剂D-Lys3-GHRP-6、NO供体硝普钠(SNP),一氧化氮合酶(NOS)抑制剂N-硝基左旋精氨酸甲酯(L-NAME)对ghrelin调控Ca2+荧光强度的影响。结果:(1)豚鼠胃窦平滑肌细胞呈GHS-R免疫反应阳性表达.(2)随着ghrelin浓度升高(10-11,10-10,10-9,10-8,10-7mol/L),平滑肌细胞内Ca2+荧光强度逐渐升高,组间峰值(分别为54.7±11.5,58.1±5.7,64.8±6.6,84.9±7.1,95.7±10.5)和峰高(分别为1.8±0.3,2.1±0.8,5.3±1.3,28.9±4.2,37.6±3.7)均存在显著差异(P<0.05-0.01),即呈明显剂量依赖...     

肺泡巨噬细胞对豚鼠气道平滑肌神经兴奋的影响

以调理酵母多糖（ＯＰＺ）激活的大鼠肺泡巨噬细胞（ＡＭ）上清液,作用于受电场刺激的豚鼠单个气管环平滑肌标本。结果显示,对气道平滑肌（ＡＳＭ）胆碱能神经兴奋有易化作用,其机理可能是ＡＭ释放前列腺素类物质（ＰＧｓ）促进了接头前膜ＡＣｈ的释放。ＡＭ上清液对肾上腺素能和非肾上腺素能抑制神经活动没有明显影响,但使ＡＳＭ舒张恢复５０％时程（ＲＴ１／２）缩短,提示在病理情况下ＡＭ大量激活时,可导致气道口径缩小、阻力增大;这可能在哮喘的发病中起一定作用。     

花椒挥发油对离体豚鼠气管平滑肌作用的实验研究

目的研究花椒挥发油对组胺(Histamine,His)、乙酰胆碱(Acetylcholine,Ach)诱发的豚鼠离体气管条收缩作用的影响,以及作用的量效关系,为研发平喘类中药奠定基础。方法采用Medlab生物信号采集处理系统利用定量药理分析方法分别测定:(1)His的量效曲线;(2)Ach的量效曲线;(3)不同浓度的花椒挥发油对His诱发的豚鼠离体气管条收缩的影响;(4)不同浓度花椒挥发油对Ach诱发的豚鼠离体气管条收缩的影响。结果His、Ach在体外引起了豚鼠离体气管条的浓度依赖性的收缩,其中His与Ach在10-2mol/L时均可以使气管条收缩达到最大值,花椒挥发油对His、Ach诱发的支气管收缩均有抑制作用,在花椒挥发油浓度达0.48 g/L时抑制率为49%;但对Ach诱发的支气管收缩在剂量为0.48 g/L时抑制率仅达到26%,抑制作用明显弱于His;结论花椒挥发油在体外浓度为0.08~0.48 g/L时均能有效地抑制His、Ach诱发的支气管收缩,对His诱导的豚鼠气管平滑肌收缩的抑制作用强于Ach,提示花椒挥发油对过敏性刺激诱发的哮喘有较好的抑制作用,将为开发抗过敏性哮喘疾病的中药研发奠定基础。     

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.     

Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers

We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.     

Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Comparative statistical mechanics of myosin molecular motors in rat heart, diaphragm and tracheal smooth muscle

Purpose

Statistical mechanics establishes a link between microscopic properties of matter and its bulk properties. A. Huxley's equations (1957) [1] provide the necessary phenomenological formalism to use statistical mechanics.

Methods

We compared statistical mechanics in rat diaphragm in tetanus (tet; n = 10) and twitch (tw; n = 12) modes, in heart in twitch mode (n = 20), and in tracheal smooth muscle in tetanus mode (TSM; n = 10). This powerful tool makes it possible to determine: (i) statistical entropy (S) which is related to the dispersal of energy and represents a measure of the degree of disorder in muscular system; (ii) thermodynamic force A/T (chemical affinity A and temperature T); (iii) thermodynamic flow (υ); (iv) entropy production rate (A/T × υ), which quantifies irreversible chemical processes generated by myosin crossbridge (CB) molecular motors.

Results

All muscles studied operated near equilibrium, i.e., A << 2500 J/mol and in a stationary linear regime, i.e., A/T varied linearly with υ. The heart operated farther from equilibrium than both diaphragm (tet and tw) and TSM, as attested by its high entropy production rate. S was of the same order of magnitude in heart and TSM but lower in diaphragm (tet and tw).

Conclusion

CB kinetics derived from A. Huxley's equations conferred a characteristic profile in terms of statistical mechanics on each muscle type. All studied muscles differed in terms of statistical entropy, chemical affinity, and entropy production rate. Stimulation mode (tet and tw) modulated CB kinetics and statistical mechanics. All muscle types operated near equilibrium and in a stationary linear regime.     

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.     

Muscarinic agonists acting through M2 acetylcholine receptors stimulate the migration of an NO-sensitive guanylyl cyclase to the plasma membrane of bovine tracheal smooth muscle

Muscarinic agonists acting on bovine tracheal smooth muscle (BTSM) induce two separate cGMP signals, one at 20?sec associated with NO-sensitive-soluble-guanylyl-cyclase (NO-sGC) and another at 60?sec, linked to natriuretic-peptide-GC. The 20-sec-cGMP novel cascade starts with mAChRs, via unknown components, activates an NO-sGC. To unravel this cascade, in crude membranes isolated from intact BTSM strips exposed to muscarinic agonists, we detected GC activities increments at 20?sec and 60?sec. The 20-sec-GC is a NO-sensitive-GC, identified as α₁β₁-heterodimer. In reconstitution experiments with purified plasma membranes and cytosol, muscarinic agonists induced an NO-sGC migration in a dose-dependent manner, being inhibited by muscarinic antagonists displaying an M₂AChR profile and blocked by PTX, suggesting the involvement of G_o/G_i proteins. The NO-sGC related to migration was isolated and identified as an α₁β₁-heterodimer. This work shows that muscarinic agonists in BTSM induce a massive and selective α₁β₁-NO-sGC migration from cytoplasm to plasma membranes being responsible for the 20-sec-cGMP signal.     

Arrangement of smooth muscle cells and intramuscular septa in the taenia coli

Summary Bands of electron-dense material beneath the cell membrane of smooth muscle cells of the guinea-pig taenia coli provide attachment to thin myofilaments and to intermediate (10 nm) filaments; about 50% of the cell membrane is occupied by dense bands in muscle cells transversely sectioned at the level of their nucleus, and between 50 and 100% in smaller cell profiles nearer the cell's ends. In addition to the known cell-to-cell junctions (intermediate contacts), more complex apparatuses anchor muscle cells together, either end-to-end or end-to-side or side-to-side. They consist of elaborate folds, invaginations and protrusions accompanied by large amounts of basal lamina material. In the end-to-end anchoring apparatuses numerous finger-like and laminar processes from the two cells interdigitate. Other muscle cells have a star-shaped profile in the last few microns of their length, or show longitudinal invaginations occupied by a thickened basal lamina and occasionally by collagen fibrils. The septa of connective tissue extend only for a few hundred microns along the length of the taenia. In taeniae fixed in condition of mild stretch the muscle cells form an angle of about 5° with the septa. In muscles fixed during isotonic contraction the angle increases to about 20–22°, and in longitudinal sections the muscle cells appear arranged in a herring-bone pattern. The collagen concentration in the taenia coli is 4–6 times greater that in skeletal and cardiac muscles. These various structures are discussed in terms of their possible role in the mechanism of force transmission.I thank Mr. S.J. Sarsfield and Miss E.M. Franke for expert technical assistance, and Dr. Adam Yamey for much help in the experiments on collagen content. This work is supported by grants from the Medical Research Council     

Effect of motilin on the contractility of gastric smooth muscle via NO pathway in guinea pigs

This study investigates the gastroprokinetic effects of motilin and erythromycin A (EM-A) and its potential mechanism in guinea pigs Cavia porcellus in vitro. Guinea pig stomach strips were mounted under organ baths containing Krebs solution. Motilin,EM-A,Nω-Nitro-L-arginine (L-NNA),L-arginine (L-AA) were added to the bathing solution in a non-cumulative way. Then the effects of motilin and EM-A was studied during electrical field stimulation (EFS) in the absence and presence of L-NNA and L-AA in the gastri...     

Mechanisms of the contractile effect of the alcoholic extract of Aegle marmelos Corr. on isolated guinea pig ileum and tracheal chain

In the present work, the effect of the alcoholic extract of the leaves of Aegle marmelos Corr. on guinea pig isolated ileum and tracheal chain was investigated, as this plant is used traditionally to treat asthma and related afflictions. These effects were investigated using the isolated organ bath method. 1 mg/ml and 2 mg/ml doses of the alcoholic extract of this plant produced a positive relaxant effect in isolated guinea pig ileum and tracheal chain, respectively. In addition, they antagonized the contractions, which are produced by histamine. Because the alcoholic extracts elicited the antagonistic effect against histamine and also relaxed the histamine-induced contractions, it can be concluded that relaxations induced by A. marmelos in both guinea pig ileum and tracheal chain were due to the depression of H₁-receptors. Since we observed a complete relaxation of the guinea pig ileum and tracheal chain produced by the extract, we investigated its antagonistic effect against histamine. These results were due to the presence of one or more anti-histaminic constituents present in the alcoholic extract of this plant, therefore supporting to the traditional use of A. marmelos in asthmatic complaints.