兔膈肌骨骼肌型DHPRα1亚单位和RyRs的mRNA与蛋白表达测定

本文测定了兔膈肌钙释放单位中骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲｓ的ｍＲＮＡ与蛋白表达水平,探讨了兔膈肌钙释放单位的结构组成特征。采用ＲＴ－ＰＣＲ、原位杂交和免疫组织化学技术,分别测定兔膈肌骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲ１、ＲｙＲ２及ＲｙＲ３的ｍＲＮＡ与蛋白表达。结果显示,兔膈肌可见较高水平的骨骼肌型α１－亚单位和ＲｙＲ１ｍＲＮＡ与蛋白表达;较低水平的ＲｙＲ３ ｍＲＮＡ与蛋白表达。表明兔膈肌     

不同频率电刺激膈神经导致隔肌肌浆网Ca^2＋—ATPase和Ca^2＋释放—摄取动力学改变

研究不同频率慢性电刺激（CES）后兔膈肌肌浆网（SR）Ca^2 -ATPase活性以及SRC^2 摄取－释放动力学对不同频率CES的活应性变化,建立不同频率CES组,用定磷法测定SR Ca^2 -ATPaes活性,用Fura-2荧光法测定SR Ca^2 摄取－释放动力学,与对照组比较,慢性低频电刺激10Hz和20Hz组的SR Ca^2 －ATPase活性明显降低（P＜0．01）,Ca^2 释放－摄动力学也显著降低（P＜0．01）,慢性高频电刺激50Hz和100Hz组的SRCa^2 -ATPase活性则显著升高（P＜0．01）,Ca^2 释放－摄取动力学亦明显升高（P＜0．01）,实验提示,ECS后不同频率CES导致膈肌SRCa^2 -ATPase,Ca^2 摄取－释放动力学产生不同的适应性变化,对不同功能状态的膈应用不同频谱的慢性电刺激可能具有重要的临床意义。     

兔膈肌力学模式对慢性电刺激的适应性改变和细胞外Ca2+变化的影响研究

目的 :研究兔膈肌肌条力学对不同频率慢性电刺激 (CES)的适应性变化特征和细胞外Ca2 变化对其力学特征的影响。方法 :测定正常对照组和CES组的颤搐收缩张力 (Pt)、峰值张力时间 (TPT)、1/ 2松驰时间 (1/ 2RT)、强直颤搐收缩张力 (Po)、疲劳指数 (FI)和疲劳恢复指数 (FRI) ;观察在无Ca2 Hank’s液和标准Hank’s液时肌条收缩张力消失和恢复的时间差异。结果 :①同对照组作比较 ,10Hz和 2 0Hz组的Pt、Po、Pt/Po明显降低 (P <0 .0 1) ,TPT和 1/ 2Rt明显延长 (P <0 .0 1) ,FI和FRI明显下降 (P <0 .0 1)。 5 0Hz和 10 0Hz组出现完全相反的效应 (P<0 .0 1)。②细胞外Ca2 变化对CES各组肌条收缩张力的降低和恢复均有明显的影响 ,但以 10Hz和 2 0Hz组尤为显著 ,其收缩张力在较短的时间内明显降低 (P <0 .0 5 )。结论 :①膈肌肌条力学模式在不同频率CES后呈现出明显的频率依赖性 ;②细胞外Ca2 变化对慢性低频电刺激后的膈肌肌条力学的影响明显增加     

兔膈肌肌纤维亚型和代谢酶活性对不同频率CES的适应性变化

目的:探讨不同频率慢性电刺激对膈肌肌纤维亚型、肌球蛋白重链(MHC)亚型和代谢酶活性的适应性变化的影响.方法:分别用还原型辅酶Ⅰ四唑氮还原法、SDS-PAGE法和酶组织化学染色法观察、测定慢性电刺激后兔膈肌纤维类型、肌球蛋白重链(MHC)和NADHD等九种代谢酶活性变化.结果:①同对照组和慢性高频电刺激50Hz和100 Hz组比较,10 Hz和20 Hz慢性低频电刺激组膈肌Ⅰ型纤维,Ⅰ型MHC显著增加(P＜0.01);ⅡB型纤维和ⅡB型MHC显著减少(P＜0.01);慢性高频电刺激50Hz和100 Hz组则出现完全相反的变化(P＜0.01).②同对照组和慢性高频电刺激50 Hz和100 Hz组比较,慢性低频电刺激10 Hz和20 Hz组LDH和a-GPDHD活性明显降低(P＜0.01),MDH、SDH、GDH、G-6-PD、NADHD和NADPHD活性显著升高(P＜0.01);慢性高频电刺激50Hz和100 Hz组则出现完全相反的变化(P＜0.01).结论:肌纤维与代谢酶模式的适应性变化有明显的频率依赖性.     

兴奋-收缩偶联中DHPR与RyR的相互作用

兴奋-收缩偶联（E—C coupling）依赖纽胞膜二氢吡啶受体（DHPR）／L型电压门控Ca^2＋通道和肌浆网兰诺定受体（RyR）／Ca^2＋释放通道的相互作用。在骨骼肌细胞中,DHPR与RyRl在结构上二机械偶联,不依赖细胞外Ca^2＋即可激活RyRl;在心肌细胞中,去极化激活DHPR,细胞外Ca^2＋内流,内流的Ca^2＋通过钙诱导钙释放（CICR）机制激活RyR2。最近的研究表明,DHPR与RyR之间的信号转导通常是双向的。DHPR与RyR机械和化学的双向偶联机制调节这两种Ca^2＋通道的效率、精确度和活性。     

气道平滑肌细胞Ryanodine受体亚型mRNA的表达及其在哮喘中的变化

目的:测定正常大鼠气道平滑肌细胞(ASMCs)中Ryanodine受体(Ryanodine receptor RyR)各亚型mRNA的表达以及在哮喘中的变化.方法:采用胶原酶消化法培养大鼠ASMCs,RT-PCR测定受体各亚型mRNA的表达.卵蛋白致敏并激发大鼠制备慢性哮喘模型,RT-PCR测定各受体mRNA表达水平的变化.结果:大鼠ASMCs中可见RyR1、RyR2和RyR3三种RyR亚型mRNA共表达,扩增片段测序结果与GenBank中登录的大鼠RyR1-3序列完全一致.制备的慢性哮喘模型中,病理切片显示平滑肌层及黏膜层明显增厚,RyR1的表达较对照组有明显的上调P＜0.05.结论:大鼠气道平滑肌细胞中RyR三种亚型共表达,提示存在着复杂的细胞内钙调节机制;并且RyR1在慢性哮喘的发生过程中可能起了一定的作用.     

不同频率电刺激膈神经导致膈肌肌浆网Ca~(2 )-ATPase和Ca~(2 )释放-摄取动力学改变

研究不同频率慢性电刺激（ＣＥＳ）后兔膈肌肌浆网（ＳＲ）Ｃａ２＋－ＡＴＰａｓｅ活性以及ＳＲ Ｃａ２＋摄取－释放动力学对不同频率ＣＥＳ的适应性变化。建立不同频率ＣＥＳ组;用定磷法测定ＳＲ Ｃａ２＋－ＡＴＰａｓｅ活性;用Ｆｕｒａ－２荧光法测定ＳＲ　Ｃａ２＋摄取－释放动力学。与对照组比较,慢性低频电刺激１０ Ｈｚ和２０Ｈｚ组的ＳＲ　Ｃａ２＋－ＡＴＰａｓｅ活性明显降低（Ｐ＜０．０１）,Ｃａ２＋释放－摄取动力学也显著降低（Ｐ＜０．０１）;慢性高频电刺激５０ Ｈｚ和１００Ｈｚ组的ＳＲ Ｃａ２＋－ＡＴＰａｓｅ活性则显著升高（Ｐ＜０．０１）,Ｃａ２＋释放－摄取动力学亦明显升高（Ｐ＜０．０１）。实验提示,ＣＥＳ后不同频率ＣＥＳ导致膈肌ＳＲＣａ２＋－ＡＴＰａｓｅ、Ｃａ２＋摄取－释放动力学产生不同的适应性变化;对不同功能状态的膈肌应用不同频谱的慢性电刺激可能具有重要的临床意义。     

不同功能状态的兔骨骼肌ryanodine受体的原子力显微镜研究

用原子力显微镜观察了外源性磷脂对兔骨骼肌ryanodine受体/钙释放通道(RyR1)结构的影响, 并且测定了有外源性磷脂的RyR1在不同功能状态时高度与弹性的变化. 结果表明: 有外源性磷脂的RyR1能更好地保持其完整的结构; AMP和Ca²⁺共同作用使其Young模量值增加, 而表观高度不变. 但是, 当用其他激动剂或抑制剂作用时, RyR1高度和Young模量值没有显著变化.     

Ryanodine受体间相互作用及其与钙释放功能的关系

在真核生物和原核生物的生物膜上都存在由同种受体蛋白相互连接在一起形成的紧密二维排列。最近的模型计算表明这种排列方式可能是一种新型信号转导机制的结构基础,相邻受体可通过功能上的耦联优化信号处理性能。Ryanodine受体（ryanodine receptor,RyR）／钙释放通道通常在肌肉的肌浆网膜上形成二维晶格排列,该蛋白成为研究受体二维排列及其生理功能的一个很好的模型。本文综述了近几年在RyR相互作用及其二维排列工作模式和生理功能研究方面的进展,着重介绍了我们实验室利用新方法对RyR相互作用及其调控进行的研究工作。我们研究中发现了RyR功能状态对其相互作用的调控,本文对据此提出的RyR二维排列的“动态耦联模型”及其可能的生理功能进行了详细讨论。     

增龄对大鼠膈肌Glut-1和Glut-4 mRNA表达的影响

目的研究大鼠膈肌Glut-1和Glut-4 mRNA的增龄变化情况,进一步探讨增龄对呼吸肌代谢及功能变化的机制。方法雄性健康清洁级SD大鼠24月龄7只、3月龄12只,应用RT-PCR方法检测大鼠膈肌Glut-1、Glut-4 mRNA表达。结果老年组大鼠Glut-1、Glut-4 mRNA表达较年轻组表达明显升高。结论大鼠膈肌不同于单纯骨骼肌和心肌,Glut-1、Glut-4 mRNA的表达具有特殊的增龄趋势。     

Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle

Excitation-contraction (e-c) coupling in muscle relies on the interaction between dihydropyridine receptors (DHPRs) and RyRs within Ca(2+) release units (CRUs). In skeletal muscle this interaction is bidirectional: alpha(1S)DHPRs trigger RyR1 (the skeletal form of the ryanodine receptor) to release Ca(2+) in the absence of Ca(2+) permeation through the DHPR, and RyR1s, in turn, affect the open probability of alpha(1S)DHPRs. alpha(1S)DHPR and RyR1 are linked to each other, organizing alpha(1S)-DHPRs into groups of four, or tetrads. In cardiac muscle, however, alpha(1C)DHPR Ca(2+) current is important for activation of RyR2 (the cardiac isoform of the ryanodine receptor) and alpha(1C)-DHPRs are not organized into tetrads. We expressed RyR1, RyR2, and four different RyR1/RyR2 chimeras (R4: Sk1635-3720, R9: Sk2659-3720, R10: Sk1635-2559, R16: Sk1837-2154) in 1B5 dyspedic myotubes to test their ability to restore skeletal-type e-c coupling and DHPR tetrads. The rank-order for restoring skeletal e-c coupling, indicated by Ca(2+) transients in the absence of extracellular Ca(2+), is RyR1 > R4 > R10 > R16 > R9 > RyR2. The rank-order for restoration of DHPR tetrads is RyR1 > R4 = R9 > R10 = R16 > RyR2. Because the skeletal segment in R9 does not overlap with that in either R10 or R16, our results indicate that multiple regions of RyR1 may interact with alpha(1S)DHPRs and that the regions responsible for tetrad formation do not correspond exactly to the ones required for functional coupling.     

Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.     

The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail

Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.     

Accessibility of targeted DHPR sites to streptavidin and functional effects of binding on EC coupling

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca(2+) channel and as the voltage sensor for excitation-contraction (EC coupling), triggering Ca(2+) release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR alpha(1S) or beta(1a) subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the approximately 60-kD streptavidin molecule had access to the beta(1a) N and C termini and to the alpha(1S) N terminus and proximal II-III loop (residues 671-686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the alpha(1S) C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the beta(1a) N or C terminus, or to the alpha(1S) N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal alpha(1S) II-III loop abolished such contractions, without affecting agonist-induced Ca(2+) release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the alpha(1S) II-III loop are necessary for EC coupling in skeletal muscle.     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.     

Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.     

兔膈肌力学模式对慢性电刺激的适应性改变和细胞外Ca …

目的：研究兔膈肌肌条力学对不同频率慢性电刺激（ＣＥＳ）的适应性变化特征和细胞外Ｃａ＾２＋变化夺其力学特征的影响。方法：测定正常对照组和ＣＥＳ组的颤搐收缩张力（Ｐｔ）、峰值张力时间（ＴＰＴ）、１／２松弛时间（１／２ＲＴ）、强直颤搐收缩张力（Ｐｏ）、疲劳指数（ＦＩ）和疲劳恢复指数（ＦＲＩ）;观察在无Ｃａ＾２＋Ｈａｎｋ’ｓ液和标准Ｈａｎｋ’ｓ液时肌条收缩张力消失和恢复的时间差异。结果：①同对照组作比较,     

Deconvolution of confocal images of dihydropyridine and ryanodine receptors in developing cardiomyocytes.

Colocalization of dihydropyridine (DHPR) and ryanodine (RyR) receptors, a key determinant of Ca(2+)-induced Ca2+ release, was previously estimated in 3-, 6-, 10-, and 20-day-old rabbit ventricular myocytes by immunocytochemistry and confocal microscopy. We now report on the effects of deconvolution (using a maximum-likelihood estimation algorithm) on the calculation of colocalization indexes. Clusters of DHPR and RyR can be accurately represented as point sources of fluorescence, which enables a model of their relative distributions to be constructed using images of point spread functions to simulate their fluorescence inside a cell. This model was used to investigate the effects of deconvolution on colocalization as a function of separation distance. Deconvolution resulted in significant improvements in both axial and transverse resolutions, producing significant increases in clarity. Comparisons of intensity profiles (full-width half-maximum) pre- and postdeconvolution showed decreased dispersion of the fluorescent signal and a corresponding decrease in false colocalization as determined by fluorescence modeling. This hypothesis was extended to physiological data previously collected. The number of colocalized voxels was quantified after deconvolution, and the degree of colocalization of DHPR with RyR decreased significantly after deconvolution in all age groups: 3 days (62 +/- 2% before deconvolution, 43 +/- 3 after deconvolution) to 20 days old (79 +/- 1% before deconvolution, 63 +/- 2% after deconvolution). The data demonstrate that confocal images should be deconvolved before any quantitative analysis, such as colocalization index determination, to minimize the detrimental effects of out-of-focus light in coincident voxels.     

Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C). The interaction is restricted to the CB and IQ motifs involved in the calmodulin-mediated Ca(2+)-dependent inactivation of the DHPR, suggesting functional interactions between the two channels.