Ca2+ sparks in skeletal muscle

The study of Ca²⁺ sparks has led to extensive new information regarding the gating of the Ca²⁺ release channels underlying these events in skeletal, cardiac and smooth muscle cells, as well as the possible roles of these local Ca²⁺ release events in muscle function. Here we review basic procedures for studying Ca²⁺sparks in skeletal muscle, primarily from frog, as well as the basic results concerning the properties of these events, their pattern and frequency of occurrence during fiber depolarization and the mechanisms underlying their termination. Finally, we also consider the contribution of different ryanodine receptor (RyR) isoforms to Ca²⁺ sparks and the number of RyR Ca²⁺ release channels that may contribute to the generation of a Ca²⁺ spark. Over the decade since their discovery, Ca²⁺ sparks have provided a wealth of information concerning the function of Ca²⁺ release channels within their intracellular environment.     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.     

Electrophysiological and other aspects of excitation-contraction coupling and uncoupling in mammalian airway smooth muscle

In this article the electrophysiological events which are believed to underly agonist-induced contraction and relaxation of airway smooth muscle are reviewed, with special emphasis on the indispensable role of the Ca ion. The contribution made by Na, K, Ca and Cl to, and the role that the electrogenic Na:K-dependent ATPase plays in, the maintenance of the resting membrane potential in both normal and sensitised airway smooth muscle cells is described together with the permeability changes that occur in the plasmalemma in response to excitatory and inhibitory agonists. In addition, the currently available evidence for the existence of potential-sensitive and receptor-operated Ca channels in respiratory smooth muscle, and how such channels may be involved in the regulation of airway calibre, is critically assessed.     

Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit

The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.     

Dysferlin and the plasma membrane repair in muscular dystrophy

Muscular dystrophy covers a group of genetically determined disorders that cause progressive weakness and wasting of the skeletal muscles. Dysferlin was identified as a gene mutated in limb-girdle muscular dystrophy (type 2B) and Miyoshi myopathy. The discovery of dysferlin revealed a new family of proteins, known as the ferlin family, which includes four different genes. Recent work suggests the function of dysferlin in membrane repair and demonstrates that defective membrane repair is a novel mechanism of muscle degeneration. These findings reveal the importance of a basic cellular function in skeletal muscle and a new class of muscular dystrophy where the defect lies in the maintenance, not the structure, of the plasma membrane. Here, we discuss the current knowledge of dysferlin function in the repair of the plasma membrane of the skeletal muscle cells.     

Activation of the fast calcium input in the denervated smooth muscle of the cat nictitating membrane

The excitation and contraction features of innervated and sympathetically denervated smooth muscle strips from cat's nictitating membrane have been studied by single sucrose gap arrangement. Increasing of smooth muscle cells sensitivity to drugs were accompanied by elevation of membrane response and the ability to generation of action potentials. Action potentials have been induced by agonists or high potassium concentration in external solution and spontaneously. In innervated muscle action potentials have been evoked as a result of depolarization by high potassium concentration of TEA blockade of potassium conductance. Induced and spontaneously generated action potentials were blocked by organic and inorganic antagonists of potential dependent Ca++ channels. In Ca-free solution action potentials were absent but might be supported by Ba++. Decrease of Na+ had no effect on smooth muscle excitability. It is supposed that activation of potential depended Ca++ channels in smooth muscle cells with pharmaco-mechanical coupling are under influence of sympathetic nerves.     

Effects of long term denervation on smooth muscle of the chicken expansor secundariorum

Summary Denervation of the expansor secundariorum muscle of the adult and 2 week chicken, by sectioning the brachial plexus, resulted in an approximate twofold increase in dry weight over 8 weeks. Unlike skeletal muscle, no ultrastructural changes were exhibited by the smooth muscle cells for a period of up to 5 months post denervation. No evidence of hypertrophy of the individual muscle cells was observed, but following colchicine treatment a definite increase in the number of mitotic figures was noted within muscle bundles indicating that the increase in dry weight of the expansor muscle is due to hyperplasia of the smooth muscle cells. The results are discussed in relation to in vitro studies of the interaction of sympathetic nerves with smooth muscle.     

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.     

Regulation of oxygen sensing by ion channels.

O(2) sensing is of critical importance for cell survival and adaptation of living organisms to changing environments or physiological conditions. O(2)-sensitive ion channels are major effectors of the cellular responses to hypoxia. These channels are preferentially found in excitable neurosecretory cells (glomus cells of the carotid body, cells in the neuroepithelial bodies of the lung, and neonatal adrenal chromaffin cells), which mediate fast cardiorespiratory adjustments to hypoxia. O(2)-sensitive channels are also expressed in the pulmonary and systemic arterial smooth muscle cells where they participate in the vasomotor responses to low O(2) tension (particularly in hypoxic pulmonary vasoconstriction). The mechanisms underlying O(2) sensing and how the O(2) sensors interact with the ion channels remain unknown. Recent advances in the field give different support to the various current hypotheses. Besides the participation of ion channels in acute O(2) sensing, they also contribute to the gene program developed under chronic hypoxia. Gene expression of T-type calcium channels is upregulated by hypoxia through the same hypoxia-inducible factor-dependent signaling pathway utilized by the classical O(2)-regulated genes. Alteration of acute or chronic O(2) sensing by ion channels could participate in the pathophysiology of human diseases, such as sudden infant death syndrome or primary pulmonary hypertension.     

Properties of sympathetic neuromuscular transmission and smooth muscle cell membranes in vascular beds.

In vascular smooth muscle tissues, the cycle of contraction-relaxation is mainly regulated by the cytosolic Ca, and many other factors, such as substances released from endothelial cells and perivascular nerve terminals (mainly sympathetic nerves). In this article, we introduce regional differences in specific features of ionic channels in vascular smooth muscle membranes (mainly on features of Ca, Na and K channels) in relation to mobilization of the cytosolic Ca. In many vascular tissues, neurotransmitters released from sympathetic nerve terminals activate post-junctional receptors, and subsequently modify ion channels (receptor-activated cation channel and voltage-dependent Ca channel), whereas in some tissues, ionic channels are not modified by receptor activations (pharmaco-mechanical coupling). However, activation of receptors, with or without modulation of ionic channels, regulates the cytosolic Ca through synthesis of second messengers. In addition, receptors distributed on prejunctional nerve terminals positively or negatively regulate the release of transmitters. Roles of neurotransmitters (mainly ATP and noradrenaline) are also discussed in relation to the generation of excitatory junction potentials.     

Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

血管重构及其药物治疗

血管重构是血管为适应内外环境变化而发生的结构和功能改变。这个过程包括血管壁细胞的增生、肥大、凋亡、细胞迁移、细胞外基质的产生及降解等细胞生物学变化。血管重构可分为肥厚性血管重构与非肥厚性血管重构,两者以一定比例共存于同一血管上。非肥厚性血管重构的主要特征是血管外径明显减少、内径明显减少,中膜横截面积不变,血管平滑肌细胞发生重排;肥厚性血管重构的主要特征是内径明显减少,中膜横截面积增加,血管平滑肌细胞增生。血管重构既是高血压和动脉粥样硬化等相关疾病恶化的重要病理基础或也是此类疾病发生发展的病因。药物治疗不仅要缓解或消除相关疾病的症状,更重要的是逆转或减轻血管重构,保护靶器官。     

Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1^Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures.     

膜片钳技术在动脉粥样硬化研究中的应用

膜片钳技术是一种先进的电生理技术,在生命科学研究中已得到了广泛的应用.最近几年已把它运用于研究动脉粥样硬化血管平滑肌细胞离子通道电生理特性的改变.研究发现血管平滑肌细胞的凋亡与K+通道活动增加有关,在动脉粥样硬化发生与发展过程中大电导型钙激活钾通道起着重要的功能作用.某些药物影响动脉粥样硬化血管平滑肌细胞离子通道而发挥作用.膜片钳技术给动脉粥样硬化发病机理研究带来了新的亮点.     

In vivo TRPC functions in the cardiopulmonary vasculature

Cardiovascular diseases are the leading cause of death in the industrialized countries. The cardiovascular system includes the systemic blood circulation, the heart and the pulmonary circulation providing sufficient blood flow and oxygen to peripheral tissues and organs according to their metabolic demand. This review focuses on three major cell types of the cardiovascular system: myocytes of the heart as well as smooth muscle cells and endothelial cells from the systemic and pulmonary circulation. Ion channels initiate and regulate contraction in all three cell types, and the identification of their genes has significantly improved our knowledge of signal transduction pathways in these cells. Among the ion channels expressed in smooth muscle cells, cation channels of the TRPC family allow for the entry of Na(+) and Ca(2+). Physiological functions of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in the cardiovascular system, dissected by down-regulating channel activity in isolated tissues or by the analysis of gene-deficient mouse models, are reviewed. Possible functional roles and physiological regulation of TRPCs as homomeric or heteromeric channels in these cell types are discussed. Moreover, TRP channels may also be responsible for pathophysiological processes of the cardiovascular system like hypertension as well as cardiac hypertrophy and increased endothelial permeability.     

Transient receptor potential (TRP) channels as potential drug targets in respiratory disease

Calcium-permeable channels have traditionally been thought of as therapeutic targets in excitable cells. For instance, voltage-operated Ca2+ channels in neurones and smooth muscle cells for neurological and cardiovascular diseases although calcium-permeable channels are also functionally important in electrically non-excitable cells. In the lung, calcium channels play a pivotal role in the activation of all the cell types present, whether resident cells such as airway smooth muscle cells and macrophages or migratory cells such as neutrophils or lymphocytes.Previously, research in this area has been hindered by the lack of obvious molecular identity. More recently, the emergence of the transient receptor potential (TRP) cation family has yielded promising candidates which may underpin the different receptor-operated calcium influx pathways. The challenge now, is to ascribe function to the TRP channels expressed in each cell type as a first step in identifying which TRP channels may be potential drug targets for asthma and chronic obstructive pulmonary disease (COPD) (Fig. 1).     

Ryanodine receptors in muscarinic receptor-mediated bronchoconstriction

Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RyR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RyR3 is more centrally localized. Moreover, RyR1 and/or RyR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RyR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RyR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma.