Calcium sparks in smooth muscle

Local intracellular Ca²⁺transients, termed Ca²⁺ sparks, are caused by thecoordinated opening of a cluster of ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum ofsmooth muscle cells. Ca²⁺ sparks are activated byCa²⁺ entry through dihydropyridine-sensitivevoltage-dependent Ca²⁺ channels, although the precisemechanisms of communication of Ca²⁺ entry toCa²⁺ spark activation are not clear in smooth muscle.Ca²⁺ sparks act as a positive-feedback element to increasesmooth muscle contractility, directly by contributing to the globalcytoplasmic Ca²⁺ concentration([Ca²⁺]) and indirectly by increasingCa²⁺ entry through membrane potential depolarization,caused by activation of Ca²⁺ spark-activatedCl channels. Ca²⁺ sparks also have aprofound negative-feedback effect on contractility by decreasingCa²⁺ entry through membrane potential hyperpolarization,caused by activation of large-conductance, Ca²⁺-sensitiveK⁺ channels. In this review, the roles of Ca²⁺sparks in positive- and negative-feedback regulation of smooth musclefunction are explored. We also propose that frequency and amplitudemodulation of Ca²⁺ sparks by contractile and relaxantagents is an important mechanism to regulate smooth muscle function.

     

Calcium signalling in smooth muscle

Calcium signalling in smooth muscles is complex, but our understanding of it has increased markedly in recent years. Thus, progress has been made in relating global Ca2+ signals to changes in force in smooth muscles and understanding the biochemical and molecular mechanisms involved in Ca2+ sensitization, i.e. altering the relation between Ca2+ and force. Attention is now focussed more on the role of the internal Ca2+ store, the sarcoplasmic reticulum (SR), global Ca2+ signals and control of excitability. Modern imaging techniques have shown the elaborate SR network in smooth muscles, along with the expression of IP3 and ryanodine receptors. The role and cross-talk between these two Ca(2+) release mechanisms, as well as possible compartmentalization of the SR Ca2+ store are discussed. The close proximity between SR and surface membrane has long been known but the details of this special region to Ca2+ signalling and the role of local sub-membrane Ca2+ concentrations and membrane microdomains are only now emerging. The activation of K+ and Cl- channels by local Ca2+ signals, can have profound effects on excitability and hence contraction. We examine the evidence for both Ca2+ sparks and puffs in controlling ion channel activity, as well as a fundamental role for Ca2+ sparks in governing the period of inexcitability in smooth muscle, i.e. the refractory period. Finally, the relation between different Ca2+ signals, e.g. sparks, waves and transients, to smooth muscle activity in health and disease is becoming clearer and will be discussed.     

Calcium signaling in smooth muscle

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).     

Calcium release in smooth muscle

In smooth muscle, maintenance of the contractile response is due to Ca2+ influx through two types of Ca2+ channel, a voltage-dependent Ca2+ channel and a receptor-linked Ca2+ channel. However, a more transient contraction can be obtained by release of Ca2+ from a cellular store, possibly the sarcoplasmic reticulum. In spike generating smooth muscle (e.g., guinea-pig taenia caeci), spike discharges may trigger the release of cellular Ca2+ by activating a Ca2+-induced Ca2+ release mechanism. Caffeine directly activates this mechanism in the absence of a triggered Ca2+ influx. In contrast to this, maintained depolarization may not only release but also refill the Ca2+ store. Drug-receptor interactions also release Ca2+ from a cellular store. This release may be elicited with inositol trisphosphate produced by receptor-linked phosphoinositide turnover. In non-spike generating smooth muscle (e.g., rabbit thoracic aorta), maintained membrane depolarization does not release but, instead, fills the Ca2+ store. However, caffeine and receptor-agonists release the Ca2+ store - possibly by activating the Ca2+-induced Ca2+ release mechanism and phosphoinositide turnover, respectively. The Ca2+ store in smooth muscle is filled by Ca2+ entry through voltage dependent Ca2+ channels and also by resting Ca2+ influx in the absence of receptor-agonists. The Ca2+ entering the cells through these pathways may be accumulated by the Ca2+ store and may activate the contractile filaments.     

Calcium sparks in skeletal muscle fibers

Ca(2+) sparks monitor transient local releases of Ca(2+) from the sarcoplasmic reticulum (SR) into the myoplasm. The release takes place through ryanodine receptors (RYRs), the Ca(2+)-release channels of the SR. In intact fibers from frog skeletal muscle, the temporal and spatial properties of voltage-activated Ca(2+) sparks are well simulated by a model that assumes that the Ca(2+) flux underlying a spark is 2.5 pA (units of Ca(2+) current) for 4.6 ms (18 degrees C). This flux amplitude suggests that 1-5 active RYRs participate in the generation of a typical voltage-activated spark under physiological conditions. A major goal of future experiments is to estimate this number more precisely and, if it is two or more, to investigate the communication mechanism that allows multiple RYRs to be co-activated in a rapid but self-limited fashion.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

Calcium diffusion in uterine smooth muscle sheets

The potassium contracture in the longitudinal muscle of estrogen- treated rat uterus was kinetically investigated. The rates of tension development after Ca addition and relaxation after Ca removal were measured under the high-potassium depolarization. Both rates decreased with an increase in preparation thickness. The relaxation rate had only a slight dependence on temperature. On the contrary, both relaxation and contraction rates in a contraction induced by an electrical stimulation strongly depended on temperature, but not on preparation size. These results suggest that the Ca diffusion process in the extracellular space is the rate-limiting step in relaxation of Ca- dependent contracture under potassium depolarization. The diffusion model, in which the effect of the unstirred layer was considered, could quantitatively explain the experimental results. The apparent diffusion coefficient in the muscle sheet was estimated to be approximately 3 x 10(-7) cm2/s. The difference from that in aqueous solution is discussed.     

血管平滑肌细胞的钙动力学

血管平滑肌细胞外的Ca~(2+)通过多种通道进入细胞内。Ca~(2+)通道的本质是镶嵌在膜脂质双分子层中的糖蛋白,神经介质和药物可影响Ca~(2+)通道的功能。靠近胞膜的肌质网和胞膜内侧面的高亲和性Ca~(2+)结合位点是血管平滑肌细胞内储存和释放Ca~(2+)的主要部位。胞浆[Ca~(2+)]增高后在钙调蛋白的介导下引起血管收缩。高血压等血管性疾病的发生与其平滑肌细胞的钙动力学异常有关。     

Calcium regulation of muscle contraction.

Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.     

Calcium binding proteins in smooth muscle plasma membrane

Rat myometrium plasma membrane showed a number of 45Ca-binding proteins as identified by gel electrophoresis. An attempt was made to identify these either by studying the inhibition of this binding by several ions or by studying binding of these proteins to calmodulin, A9 an antibody against skeletal muscle Ca-binding proteins and Stains-all. On the basis of the molecular weight, calmodulin binding and La-sensitivity of Ca-binding, the Ca-binding protein at 137 +/- 2 kDa has been identified as the Ca-pump. This protein as judged from Coomassie blue staining forms a very small percentage of the proteins present in the plasma membrane.     

Calcium oscillations in human mesenteric vascular smooth muscle

Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca²⁺]_i oscillations in human mesenteric artery smooth muscle. In arterial segments preloaded with Fluo-4 AM and mounted on a myograph on the stage of a confocal microscope, we observed PE-induced oscillations in [Ca²⁺]_i, which initiated and maintained vasoconstriction. These oscillations present some variability, possibly due to compromised health of the tissue. This view is corroborated by our ultrastructural analysis of the cells, in which we found only (5 ± 2)% plasma membrane-sarcoplasmic reticulum apposition, markedly less than measured in healthy tissue from laboratory animals. We also partially characterized the oscillations by using the inhibitory drugs 2-aminoethoxydiphenyl borate (2-APB), cyclopiazonic acid (CPA) and nifedipine. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca²⁺ and inositol 1,4,5-trisphosphate receptor (IP3R) in the maintenance of the Ca²⁺ oscillations observed in human blood vessels.     

Calcium signaling in insulin action on striated muscle

Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca²⁺ mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca²⁺ into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP₃R). In skeletal muscle cells, a rapid, insulin-triggered Ca²⁺ release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca²⁺]_i trough L-type Ca²⁺ channels activation. In both cell types, a relatively slower Ca²⁺ release also occurs through IP₃R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca²⁺ released from IP₃R of skeletal muscle also promotes mitochondrial Ca²⁺ uptake. We review here these actions of insulin on intracellular Ca²⁺ channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca²⁺ release from the SER.     

Calcium in excitation--contraction coupling of frog skeletal muscle

A principal step in the process leading to muscle contraction is the intracellular release of Ca2+. We have detected and compared some physical and chemical events that reflect Ca2+ release in contracting frog skeletal muscle cells, described the effects of some agents that are believed to alter intracellular Ca2+ release during contraction, and speculated about the role of Ca2+ release in influencing some of the mechanical properties of frog muscle. The specific physical features recorded were changes in striation spacing, myofibrillar orientation, and force development. The chemical feature was the relative change in intracellular [Ca2+] recorded as light emission from cells microinjected with the Ca2+-sensitive protein aequorin. The presence or absence of a correlation among these variables has been used (i) to evaluate the action of some agents thought to change intracellular Ca2+ release in excitation--contraction (E--C) coupling, (ii) to further substantiate the effects of cell length on Ca2+ release, and (iii) to examine some details of models for E--C coupling. The results showed that potentiating agents enhance and prolong intracellular Ca2+ release without changing the rate of Ca2+ removal during E--C coupling. This extra Ca2+ does not produce the same effect on contractions at all lengths. Contractility is inversely related to cell length, and Ca2+-induced activation is normally less than maximum not only at short lengths but also at optimal striation spacings.