Presynaptic and postsynaptic actions of cadmium in cardiac muscle

A transmembrane flux of Ca2+ has been demonstrated in many nerve and muscle cells. In cardiac muscle, Ca2+ channels in the sarcolemma transfer sufficient Ca2+ to trigger and partially control tension development. This time- and voltage-dependent Ca2+ current is also important in the development of the pacemaker potential, or diastolic depolarization. In addition, transmitter release from autonomic nerve varicosities in the myocardium exhibits a strong dependence on external calcium concentration [( Ca2+]o). Agents that selectively alter either pre- or postsynaptic Ca2+ channels are therefore of considerable interest. Our results illustrate two distinct effects of Cd2+ in cardiac muscle. Data from conventional electrophysiological recordings from primary pacemaker cells within the rabbit sinoatrial node indicate that Cd2+ (10(-6)-10(-5) M) may selectively inhibit acetylcholine release. Voltage clamp measurements of transmembrane Ca2+ currents in single isolated bullfrog atrial cells show that Cd2+ (10(-4)-10(-3) M) is also a very potent inhibitor of postsynaptic Ca2+ channels; these effects of Cd2+ mimic those seen after [Ca2+]o removal.     

Calcium transport systems in cardiac sarcolemma and their regulation by the second messengers cyclic AMP and calcium-calmodulin

The purpose of this survey is to describe the importance of cyclic AMP and Ca2+-calmodulin as mediators of the effects of beta-adrenergic agonists on cardiac sarcolemma. First, the basic characteristics of the three sarcolemmal Ca2+-transporting systems, the slow Ca2+ channel, the Ca2+-pumping ATPase and the Na+/Ca2+ antiporter, are described. These different pathways for in- and outflux of Ca2+ play a crucial role in the excitation-contraction coupling and relaxation of heart muscle. Catecholamines in the myocardium cause an increase in the rate and extent of tension development during systole, and in the rate of relaxation during diastole. These functional changes may largely be brought about by cyclic AMP-induced phosphorylation of membrane proteins that increases both the probability of opening the slow Ca2+ channels and the rate of Ca2+ pumping ATPase. It is generally believed that the effects on Ca2+ transport systems are due to direct actions of beta-adrenergic agonists leading to an increased cytosolic Ca2+ level during systole. Indirectly, an increase in systolic Ca2+ can amplify the primary effect of catecholamine on the Ca2+ pumping ATPase and probably also on the Na+/Ca2+ antiporter through Ca2+-calmodulin-dependent phosphorylation of membrane proteins. The intimate involvement of calmodulin in the operation of several sarcolemmal Ca2+-transporting systems is discussed in the light of the unknown mechanism of action of the so-called Ca2+ channel blockers, a class of drugs that have a very important potential to provide information on the fundamental reaction steps in excitation-contraction coupling. Some of these drugs are potent inhibitors of Ca2+-calmodulin-regulated enzymes.     

T型钙通道对心脏功能的调节作用

T型钙通道存在于心血管、神经和内分泌系统.T型钙通道在心脏自律性,细胞生长和心脏重塑中起关键性的作用.心脏疾病时T型钙通道表达增多.因此T型钙通道在生理和病理生理情况下均可参与心脏功能的调节.随着对钙通道研究的日益加深,可望更深刻地了解T型钙通道并研制出新的钙通道拮抗剂,这对心脏疾病的治疗策略具有重要的意义.     

Mitochondria as regulators of stimulus-evoked calcium signals in neurons

An important challenge in the study of Ca2+ signalling is to understand the dynamics of intracellular Ca2+ levels during and after physiological stimulation. While extensive information is available regarding the structural and biophysical properties of Ca2+ channels, pumps and exchangers that control cellular Ca2+ movements, little is known about the quantitative properties of the transporters that are expressed together in intact cells or about the way they operate as a system to orchestrate stimulus-induced Ca2+ signals. This lack of information is particularly striking given that many qualitative properties of Ca2+ signals (e.g. whether the Ca2+ concentration within a particular organelle rises or falls during stimulation) depend critically on quantitative properties of the underlying Ca2+ transporters (e.g. the rates of Ca2+ uptake and release by the organelle). This monograph describes the in situ characterization of Ca2+ transport pathways in sympathetic neurons, showing how mitochondrial Ca2+ uptake and release systems define the direction and rate of net Ca2+ transport by this organelle, and how the interplay between mitochondrial Ca2+ transport and Ca+2 transport across the plasma membrane contribute to depolarization-evoked Ca2+ signals in intact cells.     

Oxidative stress and redox regulation of phospholipase D in myocardial disease

Oxidative stress may be viewed as an imbalance between reactive oxygen species (ROS) and oxidant production and the state of glutathione redox buffer and antioxidant defense system. Recently, a new paradigm of redox signaling has emerged whereby ROS and oxidants can function as intracellular signaling molecules, where ROS- and oxidant-induced death signal is converted into a survival signal. It is now known that oxidative stress is involved in cardiac hypertrophy and in the pathogenesis of cardiomyopathies, ischemic heart disease and congestive heart failure. Phospholipase D (PLD) is an important signaling enzyme in mammalian cells, including cardiomyocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA). Two mammalian PLD isozymes, PLD1 and PLD2 have been identified, characterized and cloned. The importance of PA in heart function is evident from its ability to stimulate cardiac sarcolemmal membrane and sarcoplasmic reticular Ca2+-related transport systems and to increase the intracellular concentration of free Ca2+ in adult cardiomyocytes and augment cardiac contractile activity of the normal heart. In addition, PA is also considered an important signal transducer in cardiac hypertrophy. Accordingly, this review discusses a role for redox signaling mediated via PLD in ischemic preconditioning and examines how oxidative stress affects PLD in normal hearts and during different myocardial diseases. In addition, the review provides a comparative account on the regulation of PLD activities in vascular smooth muscle cells under conditions of oxidative stress.     

Block by extracellular divalent cations of Drosophila big brain channels expressed in Xenopus oocytes

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain (bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca(2+) and Ba(2+) but not by Mg(2+) in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu(274)) and aspartate (Asp(253)) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu(71)) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu(71) to Asp introduced a new sensitivity to block by extracellular Mg(2+); substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca(2+), suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination.     

Depolarization of macrophage polykaryons in the absence of external sodium induces a cyclic stimulation of a calcium-activated potassium conductance

Macrophage polykaryons associated with the foreign body granuloma display several electrophysiological properties when studied with intracellular microelectrodes. One of the most evident properties is the slow hyperpolarization (2-5 s long, 10-60 mV amplitude), due to transient openings of Ca2+-dependent K+ channels, that is similar to those observed in macrophages. How this oscillation of membrane potential is triggered is not well known and the only way to repeatedly activate it under experimental control is through the intracellular injection of Ca2+. Although this technique is important for understanding the properties of the K+ channels, no information has been obtained about the way Ca2+ levels are raised and controlled in the cytosol. Slow hyperpolarizations can also be triggered by electrical stimulation, but reproducibility is low with cells bathed in physiological solutions. We then decided to investigate the effect of depolarization on the electrophysiological properties of macrophage polykaryons exposed to bathing solutions of several ionic compositions. We show in this paper that cell membrane depolarization induced by a long current pulse can trigger several patterns of membrane potential changes and that, in the absence of extracellular Na+, repetitive oscillations of decaying amplitudes are observed in almost all the cells. They are very similar to the slow hyperpolarizations, are dependent on the presence of extracellular Ca2+, and are blocked by quinine and D-600. Whole-cell patch clamp recording under voltage clamp conditions showed an outward current that oscillates and that also exhibits decaying amplitudes. The data presented here indicate that these oscillations are a consequence of the cyclic opening of the Ca2+-activated K+ channels and support the hypothesis that favors the participation of Ca2+ channels and of the Ca2+/Na+ exchange system in their triggering. These two mechanisms are not enough to explain either why the K+ channels close or why the membrane potential returns to the original level at the end of each cycle. The possibility of using these oscillations as a model to study the slow hyperpolarization is discussed.     

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.     

Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes

The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells.     

Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.     

The role of calcium in the response of cardiac muscle to stretch

This review focuses on the complex interactions between two major regulators of cardiac function; Ca2+ and stretch. Initial consideration is given to the effect of stretch on myocardial contractility and details the rapid and slow increases in contractility. These are shown to be related to two diverse changes in Ca2+ handling (enhanced myofilament Ca2+ sensitivity and increased intracellular Ca2+ transient, respectively). Interaction between stretch and Ca2+ is also demonstrated with respect to the rhythm of cardiac contraction. Stretch has been shown to alter action potential configuration, generate stretch-activated arrhythmias, and increase the rate of beating of the sino-atrial node. A variety of Ca(2+)-dependent mechanisms including attenuation of Ca2+ extrusion via Na+/Ca2+ exchange, Ca2+ entry through stretch-activated channels (SACs) and mobilisation of intracellular Ca2+ stores have been proposed to account for the effect of stretch on rhythm. Finally, the interaction between stretch and Ca2+ in the secretion of natriuretic peptides and onset of hypertrophy is discussed. Evidence is presented that Ca2+ (entering through L-type Ca2+ channels or SACs, or released from sarcoplasmic reticular stores) influences secretion of both atrial and B-type natriuretic peptide; there is data to support both positive and negative modulation by Ca2+. Ca2+ also appears to be important in the pathway that leads to expression of precursors of hypertrophic protein synthesis. In conclusion, two of the major regulators of cardiac muscle function, Ca2+ and stretch, interact to produce effects on the heart; in general these effects appear to be additive.     

Comparative analysis of the kinetic characteristics of L-type calcium channels in cardiac cells of hibernators.

An undefined property of L-type Ca2+ channels is believed to underlie the unique phenotype of hibernating hearts. Therefore, L-type Ca2+ channels in single cardiomyocytes isolated from hibernating versus awake ground-squirrels (Citellus undulatus) were compared using the perforated mode of the patch-clamp technique, and interpreted by way of a kinetic model of Ca2+ channel behavior based upon the concept of independence of the activation and inactivation processes. We find that, in hibernating ground-squirrels, the cardiac L-type Ca2+ current is lower in magnitude when compared to awake animals. Both in the awake or hibernating states, kinetics of L-type Ca2+ channels could be described by a d2f1(2)f2 model with an activation and two inactivation processes. The activation (or d) process relates to the movement of the gating charge. The slow (or f1) inactivation is associated with movement of gating charge and is current-dependent. The rapid (or f2) inactivation is a complex process which cannot be represented as a single-step conformational transition induced by the gating charge movement, and is regulated by beta-adrenoceptor stimulation. When compared to awake animals, the kinetic properties of Ca2+ channels from hibernating ground-squirrels differed in the following parameters: (1) pronounced shift (15-20 mV) toward depolarization in the normalized conductance of both inactivation components, and moderate shift in the activation component; (2) 1.5-2-fold greater time constants; and (3) two-fold greater activation gating charge. Thus, L-type Ca2+ channels apparently switch their phenotype during the hibernating transition. Stimulation of beta-adrenoceptors by isoproterenol, reversed the hibernating kinetic- (but not amplitude-) phenotype toward the awake type. Therefore, an aberrance in the beta-adrenergic system can not fully explain the observed changes in the L-type Ca2+ current. This suggests that during hibernation additional mechanisms may reduce the single Ca2+ channel-conductance and/or keep a fraction of the cardiac L-type Ca2+ channel population in a non-active state.     

Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences

Rabbit genomic DNA contains two genes that encode Ca2+ ATPases of fast twitch and of slow twitch (and cardiac) sarcoplasmic reticulum, respectively. The deduced amino acid sequences of the products of the two genes are highly conserved in putative Ca2+ binding regions, in sectors leading from cytoplasmic domains into transmembrane domains, and in transmembrane helices. A transport mechanism is proposed in which Ca2+ binds to negatively charged groups on amphipathic stalk sectors, becoming occluded during enzyme phosphorylation by bound ATP. Rotation of the stalk sectors is induced as the energy in the phosphorylated enzyme (E1P) is utilized in conformational changes leading to the low energy form, E2P. Rotation leads to disruption of high affinity Ca2+ binding sites and release of Ca2+ into a charge-lined membrane channel. Ca2+ then traverses the membrane by exchange diffusion.     

心肌细胞发育过程中胞浆内钙稳态的调控

Ca^2＋信号是细胞和各器官生长发育、行使其生理功能的基础,维持心肌细胞的钙稳态是保持正常心脏功能的先决条件。作为在胚胎发育过程中最早出现并行使功能的器官,胚胎期心脏的形态结构发生了明显的变化,泵血功能不断增强,以适应不断增强的机体的生理需求。从胚胎到成年,心肌细胞的功能有非常大的改变,各钙离子通道的表达也发生明显变化。因此,发育早期心肌细胞的钙稳态调控与成熟心肌细胞有明显的不同,在发育过程中引起细胞收缩的Ca^2＋来源也有明显的变化。随着分子和细胞生物学研究的发展,以及胚胎干细胞体外分化模型的应用,人们对心肌细胞发育过程中钙稳态的调控有了进一步的认识。本文综述了早期心肌细胞发育过程中胞浆内钙稳态的变化,总结了早期心肌细胞钙稳态调控机制的最新研究进展。     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ''two-pore'' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.     

Ca2+ current activation rate correlates with alpha 1 subunit density.

We report here that L-type Ca2+ channels activate rapidly in myotubes expressing current at high density and slowly in myotubes expressing current at low density. Partial block of the current in individual cells does not slow activation, indicating that Ca2+ influx does not link activation rate to current density. Activation rate is positively correlated with the density of gating charge (Qmax) associated with the L-type Ca2+ channels. The range of values for Qmax, and the relationship between activation rate and Qmax, are similar for myotubes expressing native or recombinant L-type Ca2+ channels, whereas peak Ca2+ current density is approximately 3-fold higher for native channels. Taken together, these results suggest that Ca2+ channel density can govern activation kinetics. Our findings have important important implications for studies of ion channel function because they suggest that biophysical properties can be significantly influenced by channel density, both in heterologous expression systems and in native tissues.     

Mathematical model of the neonatal mouse ventricular action potential

Therapies for heart disease are based largely on our understanding of the adult myocardium. The dramatic differences in action potential (AP) shape between neonatal and adult cardiac myocytes, however, indicate that a different set of molecular interactions in neonatal myocytes necessitates different treatment for newborns. Computational modeling is useful for synthesizing data to determine how interactions between components lead to systems-level behavior, but this technique has not been used extensively to study neonatal heart cell function. We created a mathematical model of the neonatal (day 1) mouse myocyte by modifying, on the basis of experimental data, the densities and/or formulations of ion transport mechanisms in an adult cell model. The new model reproduces the characteristic AP shape of neonatal cells, with a brief plateau phase and longer duration than the adult (action potential duration at 80% repolarization = 60.1 vs. 12.6 ms). The simulation results are consistent with experimental data, including 1) decreased density and altered inactivation of transient outward K+ currents, 2) increased delayed rectifier K+ currents, 3) Ca2+ entry through T-type as well as L-type Ca2+ channels, 4) increased Ca2+ influx through Na+/Ca2+ exchange, and 5) Ca2+ transients resulting from transmembrane Ca2+ entry rather than release from the sarcoplasmic reticulum (SR). Simulations performed with the model generated novel predictions, including increased SR Ca2+ leak and elevated intracellular Na+ concentration in neonatal compared with adult myocytes. This new model can therefore be used for testing hypotheses and obtaining a better quantitative understanding of differences between neonatal and adult physiology.     

Modeling Ca2+ signaling differentiation during oocyte maturation

Ca2+ is a fundamental intracellular signal that mediates a variety of disparate physiological functions often in the same cell. Ca2+ signals span a wide range of spatial and temporal scales, which endow them with the specificity required to induce defined cellular functions. Furthermore, Ca2+ signaling is highly plastic as it is modulated dynamically during normal physiological development and under pathological conditions. However, the molecular mechanisms underlying Ca2+ signaling differentiation during cellular development remain poorly understood. Oocyte maturation in preparation for fertilization provides an exceptionally well-suited model to elucidate Ca2+ signaling regulation during cellular development. This is because a Ca2+ signal with specialized spatial and temporal dynamics is universally essential for egg activation at fertilization. Here we use mathematical modeling to define the critical determinants of Ca2+ signaling differentiation during oocyte maturation. We show that increasing IP3 receptor (IP3R) affinity replicates both elementary and global Ca2+ dynamics observed experimentally following oocyte maturation. Furthermore, our model reveals that because of the Ca2+ dependency of both SERCA and the IP3R, increased IP3R affinity shifts the system's equilibrium to a new steady state of high cytosolic Ca2+, which is essential for fertilization. Therefore our model provides unique insights into how relatively small alterations of the basic molecular mechanisms of Ca2+ signaling components can lead to dramatic alterations in the spatio-temporal properties of Ca2+ dynamics.