Myosin phosphorylation, agonist concentration and contraction of tracheal smooth muscle

Myosin phosphorylation plays an important part in excitation--contraction coupling in smooth muscle. Phosphorylation by a Ca2+, calmodulin-dependent kinase stimulates the actin-activated Mg2+-ATPase activity of smooth muscle myosin, suggesting that myosin phosphorylation regulates smooth muscle contraction. This hypothesis is supported by evidence that myosin is phosphorylated during contraction and dephosphorylated during relaxation of intact smooth muscles stimulated with a single agonist concentration. However, there is little information regarding the response to stimulation with various agonist concentrations. As the dose-response relationships for phosphorylation and tension should be similar if myosin phosphorylation does, in fact, regulate smooth muscle contraction, we studied myosin phosphorylation in tracheal smooth muscle stimulated with a broad range of concentrations of the cholinergic agonist, methacholine. The results of these experiments are consistent with the hypothesis that myosin phosphorylation regulates smooth muscle contraction but they indicate a relatively complex relationship between myosin phosphorylation and the generation of isometric tension.     

平滑肌肌球蛋白轻链激酶对肌球蛋白的非钙依赖性磷酸化

发现肌球蛋白轻链激酶(MLCK)不仅以钙依赖性的方式,同时也以非钙依赖性的方式参与对肌球蛋白轻链的磷酸化。该非钙依赖性磷酸化的特征是:需要高浓度,但受温度升高、反应时间延长、离子浓度升高的影响远小于钙依赖性磷酸化。研究结果提示肌球蛋白轻链激酶对肌球蛋白的非钙依赖性磷酸化可能是一种新的机制,并与平滑肌张力的维持有关。     

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.     

膀胱逼尿肌的组织及生理学概述

膀胱逼尿肌在组织结构、超微结构、收缩功能、舒张功能、自律性及电生理学方面具有自身的特点.在组织结构上,通过平滑肌肌束之间的交叉聚集使膀胱逼尿肌成为一个统一的整体;在超微结构上,通过逼尿肌肌细胞之间的中间连接使平滑肌肌束成为一个有机的组合;在收缩功能上,膀胱逼尿肌的特点是自发性收缩和神经性收缩的动态平衡;在舒张功能上,膀胱逼尿肌的特点是应力性舒张和神经性舒张的协调一致;在自律性方面,膀胱逼尿肌具有产生自发性动作电位的能力以及触发自主性收缩的能力;在电生理方面,膀胱逼尿肌的肌膜去极化引起的动作电位通过作用于钙通道形成Ca2+内流,实现了兴奋与收缩之间的耦联.     

阿司匹林对在体及离体气管平滑肌的影响

目的:研究阿司匹林对在体及离体气管平滑肌的影响.方法:观察不同剂量的阿司匹林对乙酰胆碱/磷酸组织胺诱发在体及离体豚鼠气管平滑肌收缩的影响.结果:阿司匹林对乙酰胆碱/磷酸组织胺诱发的体内外气管平滑肌收缩的对抗作用具有明显的剂量差异,即随着剂量增加,阿司匹林对抗气管平滑肌收缩的作用越来越弱,而沙丁胺醇且有很好的扩张气管平滑肌的作用.结论:适宜浓度的阿司匹林具有扩张气管平滑肌的作用.     

肌肉收缩过程中ATP浓度的变化

ATP是肌肉收缩的直接能源,在整个肌肉收缩过程中起着关键的作用.从能量循环的观点出发,用化学动力学方法对肌肉收缩过程中ATP的浓度变化进行了研究.结果表明ATP的浓度围绕平衡点周期性的振荡.对理论结果的生物学意义进行了讨论.     

Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) can stimulate skinned smooth and skeletal muscle to contract by initiating Ca2+ release from the sarcoplasmic reticulum. Whether this process is an integral component of the in vivo muscle activation mechanism was tested by releasing InsP3 rapidly within skinned muscle fibers of rabbit main pulmonary artery and frog semitendinosus. InsP3 was liberated on laser pulse photolysis of a photolabile but biologically inactive precursor of InsP3 termed caged InsP3. Caged InsP3 is a mixture of compounds in which InsP3 is esterified with 1(2-nitrophenyl)diazoethane (probably at the P4- or P5-position). Photochemical release of InsP3 induced a full contraction in both muscles at physiological free Mg2+ concentrations, but only in the smooth muscle were the InsP3 concentration (0.5 microM) and the activation rate compatible with the in vivo physiological response. Endogenous InsP3-specific phosphatase activity was present in smooth muscle and had about 35-fold greater activity than that in the skeletal-muscle preparation. Caged InsP3 was not susceptible to phosphatases in either preparation.     

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.     

Vasoregulation by the beta1 subunit of the calcium-activated potassium channel

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.     

甲状旁腺素(PTH)对氯化钾引起大白鼠输精管收缩的舒张作用

本研究探讨甲状旁腺素(PTH)对输精管平滑肌的舒张作用及其机制。不同剂量的氯化钾(20、40、60mM)引起大白鼠输精管的收缩,同样可被牛型—PTH(1—34)或人型—PTH(1—34)所舒张,而且均呈现剂量的依从关系。异丙肾上腺素舒张输精管的作用可被心得安所阻滞,但心得安或酚妥拉明均不抑制甲状旁腺素舒张输精管的作用,提示甲状旁腺素和异丙肾上腺素对输精管的舒张作用是通过不同的受体实现的。不同剂量的氯化钙引起输精管不同张力强度的收缩,甲状旁腺素对这种外源性钙引起的收缩呈现明显的抑制作用,说明甲状旁腺素有减少钙离子进入平滑肌组织的作用。     

川楝素对骨骼肌、平滑肌和心肌收缩的作用

川楝素能轻度可逆地增强箭毒化及去神经骨骼肌由直接刺激引起的收缩反应.当用Sr~(2+)代替溶液中的Ca~(2+)时,这种正肌力作用仍能出现.川楝素对豚鼠平滑肌的自发收缩亦有增强作用,但川楝素对大鼠心肌由直接电刺激引起的收缩反应则具有负肌力作用.     

Slow inward calcium currents have no obvious role in muscle excitation-contraction coupling

It has been proposed that an influx of calcium ions into twitch muscle fibres during an action potential might initiate contraction. However, when external Ca2+ is lowered to 10(-8) M with EGTA, the fibres can produce normal twitches for many minutes. Nevertheless, a clear Ca2+ influx during contraction has been demonstrated, and it has been found that phasic skeletal muscle has an inward calcium current (ICa) which can give rise to calcium spikes. In certain conditions, a reduction in external Ca2+ with 80-90 mM EGTA results in reversible blockade of excitation-contraction (e-c) coupling, leading some authors to suggest that extracellular Ca2+ moved into the myoplasm due to ICa may be involved in the e-c coupling mechanism that triggers contraction. This proposition was further supported by the localization of ICa in the T-system, which circumvented the problem of the delay due to calcium diffusion from the surface membrane. We have now investigated whether ICa has a clear role in initiating or sustaining contractions in twitch muscle fibres. Our approach was to decrease or eliminate ICa with the calcium-blocking agent diltiazem (Herbesser) and to see how the twitch, tetanic and potassium-contracture tensions were affected. We found that ICa could be decreased or cancelled with the calcium-blocking agent, but that the same concentration of the drug potentiated the twitch, tetanus and contractures. We conclude, therefore, that ICa has no role in e-c coupling. A preliminary report of these results has been presented elsewhere.     

肌醇磷脂降解物在大鼠输精管平滑肌收缩中的作用

探讨肌醇磷脂代谢及其降解产物在大鼠输精管平滑肌收缩中的作用．用32P对输精管平滑肌进行标记，用薄层层析进行脂类分类，用放射自显影方法对磷脂进行定量研究，结果表明去甲肾上腺素能促进大鼠输精管平滑肌PI、PC、PA的代谢，PKC的大分子抑制剂和氯丙嗪不完全地抑制其代谢，Ca2+对上述反应有加速作用．本文提示去甲肾上腺素通过促进PI的代谢激活PKC，进而引起平滑肌的收缩，存在于输精管平滑肌中的PKC大分子蛋白抑制剂起着重要的调节作用．     

Noradrenaline contracts arteries by activating voltage-dependent calcium channels

Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.     

苦物质引起去甲肾上腺素预收缩的大鼠主动脉平滑肌舒张的作用机理研究

为研究苦物质舒张预收缩的大鼠胸主动脉平滑肌的作用机制,以测定肌张力为主要手段,结合多种细胞信号通路阻断剂研究了苦物质的作用通路.结果表明:苦物质对于去甲肾上腺素介导的大鼠胸主动脉的收缩具有高效、快速的舒张作用,此舒张作用与IP3受体和L型钙通道的阻断密切相关.故以氯喹和苦精为代表的苦物质因对血管平滑肌具有良好的舒张作用,有望成为针对高血压等血管相关疾病的新的治疗药物.     

Membrane currents that govern smooth muscle contraction in a ctenophore

Ctenophores are transparent marine organisms that swim by means of beating cilia; they are the simplest animals with individual muscle fibres. Predatory species, such as Beroe ovata, have particularly well-developed muscles and are capable of an elaborate feeding response. When Beroe contacts its prey, the mouth opens, the body shortens, the pharynx expands, the prey is engulfed and the lips then close tightly. How this sequence, which lasts 1 s, is accomplished is unclear. The muscles concerned are structurally uniform and are innervated at each end by a neuronal nerve net with no centre for coordination. Isolated muscle cells studied under voltage-clamp provide a solution to this puzzle. We find that different groups of muscle cells have different time-dependent membrane currents. Because muscle contraction depends upon calcium entry during each action potential, these different currents produce different patterns of contraction. We conclude that in a simple animal such as a ctenophore, a sophisticated set of membrane conductances can compensate for the absence of an elaborate system of effectors.     

Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase

The presence of actin and myosin in non-muscle cells suggests that they may be involved in a wide range of cellular contractile activities. The generally accepted view is that interaction between actin and myosin in these cells and in vertebrate smooth muscle, is regulated by the level of phosphorylation of the 20,000-molecular weight (MW) light chain. In the absence of calcium, this light chain is not phosphorylated and the myosin cannot interact with actin. Calcium activates a specific calmodulin-dependent kinase which phosphorylates the light chain, initiating actin-myosin interaction. Although most studies on the role of phosphorylation have concentration on the regulation of actin-activated myosin Mg-ATPase activity, phosphorylation of the light chain also seems to control the assembly of smooth muscle myosin into filaments. Using purified smooth muscle light chain kinase, we have confirmed this observation. We report here studies of myosins isolated from the two non-muscle sources, thymus cells and platelets. We observed that these myosins are assembled into filaments at physiological ionic strength and Mg-ATP concentrations, only when the 20,000-MW light chain is phosphorylated.     

Probing the calcium-induced conformational transition of troponin C with site-directed mutants

The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC). TnC consists of two spatially independent domains, each of which contains two metal ion binding sites. Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes. Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges. Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development. Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension. Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88. In separate experiments we have replaced each of these residues by lysines. The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding. This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule. Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered. Thus, these residues may participate in the contraction signal transmission.     

云南松松塔正丁醇提取物对大鼠离体子 宫平滑肌的影响

目的研究云南松松塔正丁醇提取物对大鼠离体子宫平滑肌的作用。方法通过制备大鼠离体子宫标本,观察云南松松塔正丁醇提取物对大鼠离体子宫平滑肌自发收缩活动,催产素及乙酰胆碱诱发收缩活动的影响。结果云南松松塔正丁醇提取物可使正常大鼠自发收缩和催产素及乙酰胆碱诱发收缩的频率减慢、张力减小、活动力减弱。结论云南松松塔正丁醇提取物对大鼠离体子宫平滑肌的收缩活动具有抑制作用,其机制可能跟阻断催产素受体和M受体有关。     

Active transport of Ca2+ by an artificial photosynthetic membrane

Transport of calcium ions across membranes and against a thermodynamic gradient is essential to many biological processes, including muscle contraction, the citric acid cycle, glycogen metabolism, release of neurotransmitters, vision, biological signal transduction and immune response. Synthetic systems that transport metal ions across lipid or liquid membranes are well known, and in some cases light has been used to facilitate transport. Typically, a carrier molecule located in a symmetric membrane binds the ion from aqueous solution on one side and releases it on the other. The thermodynamic driving force is provided by an ion concentration difference between the two aqueous solutions, coupling to such a gradient in an auxiliary species, or photomodulation of the carrier by an asymmetric photon flux. Here we report a different approach, in which active transport is driven not by concentration gradients, but by light-induced electron transfer in a photoactive molecule that is asymmetrically disposed across a lipid bilayer. The system comprises a synthetic, light-driven transmembrane Ca2+ pump based on a redox-sensitive, lipophilic Ca2+-binding shuttle molecule whose function is powered by an intramembrane artificial photosynthetic reaction centre. The resulting structure transports calcium ions across the bilayer of a liposome to develop both a calcium ion concentration gradient and a membrane potential, expanding Mitchell's concept of a redox loop mechanism for protons to include divalent cations. Although the quantum yield is relatively low (approximately 1 per cent), the Ca2+ electrochemical potential developed is significant.