植物体内Ca2+信号转导过程的研究进展

Ca2+是高等植物细胞内普遍存在的一种信使分子,在植物体内起着非常广泛的作用,参与了植物体内多种刺激-反应的藕联过程。本文介绍了植物体内Ca2+转移系统,Ca2+信号的产生、终止和传递途径,Ca2+信号编码的多样性的最近研究进展。     

Ca2+及其信号转导途径与长时程增强的关系

Ca2 作为信号转导过程中的第二信使,参与机体的各种反应,尤其在神经突触可塑性方面,突触前后Ca2 浓度的变化发挥了重要的信息传递作用.Ca2 在长时程增强(long-term potentiation,LTP)过程中也发挥重要作用.它不仅是LTP产生的触发器,而且能通过激活下游的蛋白激酶、磷酸化ERK,以及活化即刻早期基因(IEGs)等促进基因转录和蛋白质合成,最终参与LTP的维持.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

钙-钙调素信号系统参与热激信号转导的研究

根据作者实验室的研究工作结合国内外的研究动态讨论热激信号转导的Ca2 -CaM途径。作者实验室的工作表明,钙一钙调素(Ca^2 -CaM)信号系统参与植物热激信号转导。激光共聚焦扫描显微镜的观察结果表明,37℃热激可引起小麦胞内自由Ca。’浓度迅速提高。在Ca^2 存在条件下,热激也引起小麦CaM基因CaM1-2表达及CaM蛋白含量增加。Ca^2 可促进小麦热激基因hsp26和mp70表达和热激蛋白合成,而Ca^2 螯合剂EGTA、Ca^2 通道阻断剂异搏定和LaCl3、CaM抑制剂W7、TFP和CPZ明显降低热激基因hsp26和mp70表达和热激蛋白合成。EGTA、异搏定、TFP或CPZ也阻止小麦耐热性的获得。小麦CaM基因与热激基因的表达动力学研究表明CaM位于热激信号转导的上游,而Ca^2 是启动热激反应的胞内关键因子。凝胶阻滞分析的结果表明,Ca^2 -CaM在热激信号转导中的作用是通过激活热激转录因子的DNA结合活性来实现的。根据大量实验证据,作者提出在植物细胞内存在一条新的热激信号转导途径——钙一钙调素途径。     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.     

The role of mitochondrial Ca2+ transport and matrix Ca2+ in signal transduction in mammalian tissues

The pyruvate, NAD(+)-isocitrate and 2-oxoglutarate dehydrogenases are key regulatory enzymes in intramitochondrial oxidative metabolism in mammalian tissues, and can all be activated by increases in Ca2+ in the micromolar range. There is now mounting evidence that hormones and other stimuli which act by increasing cytosolic Ca2+ also, as a result, cause increases in mitochondrial matrix Ca2+ and hence activation of these enzymes, suggesting that the primary physiological function of mitochondrial Ca2(+)-transport is to be involved in this relay mechanism. This may also explain how in such circumstances rates of ATP production may be increased to meet the greater demand, but without any decreases in ATP/ADP occurring.     

Physiological functions and Ca2+ signal transduction pathway exploring during molting of silkworm,Bombyx mori

Calcium ions are an important second messagers in the life of organisms, such as in silkworm growth and their developmental processes; these ions can trigger various effects, including the muscle excitation-contraction coupling mechanism, secretion of endocrine and exocrine glands, neuronal activities, midgut nutrients metabolisms, sensory system, enzymatic systems, and acceleration of metabolism activities. This paper reviewed the ecdysis process and mechanisms, where calcium plays an important role in larvae-to-larvae ecdysis and larvae-to-pupae and pupae-to-moth metamorphosis of silkworms; the paper focuses on the advanced achievements in the field of calcium actions during larvae-to-larvae and larvae-to-pupae changes. Calcium ion channels are shown to be the action mechanism of phospholipase C in the G protein cascade of the calcium signal transduction pathway, and the activators and inhibitors of these calcium channels are further discussed.     

Ca2+ signaling differentiation during oocyte maturation

Oocyte maturation is an essential cellular differentiation pathway that prepares the egg for activation at fertilization leading to the initiation of embryogenesis. An integral attribute of oocyte maturation is the remodeling of Ca2+ signaling pathways endowing the egg with the capacity to produce a specialized Ca2+ transient at fertilization that is necessary and sufficient for egg activation. Consequently, mechanistic elucidation of Ca2+ signaling differentiation during oocyte maturation is fundamental to our understanding of egg activation, and offers a glimpse into Ca2+ signaling regulation during the cell cycle.     

LET-23-mediated signal transduction during Caenorhabditis elegans development

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming α-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the “vulvaless” phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the “multivulva” phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development—larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways. © 1995 wiley-Liss, Inc.     

Ca2+ changes the force sensitivity of the hair-cell transduction channel

The mechanically gated transduction channels of vertebrate hair cells tend to close in approximately 1 ms after their activation by hair bundle deflection. This fast adaptation is correlated with a quick negative movement of the bundle (a "twitch"), which can exert force and may mediate an active mechanical amplification of sound stimuli in hearing organs. We used an optical trap to deflect bullfrog hair bundles and to measure bundle movement while controlling Ca(2+) entry with a voltage clamp. The twitch elicited by repolarization of the cell varied with force applied to the bundle, going to zero where channels were all open or closed. The force dependence is quantitatively consistent with a model in which a Ca(2+)-bound channel requires approximately 3 pN more force to open, and rules out other models for the site of Ca(2+) action. In addition, we characterized a faster, voltage-dependent "flick", which requires intact tip links but not current through transduction channels.     

Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).     

Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca²⁺–dependent manner with a K_d of 7.6 nM and a Ca²⁺ binding K_d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca²⁺. Increasing cytosolic Ca²⁺ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca²⁺/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca²⁺ can act in an integrated fashion to gate currents through these plant ion channels.     

A signal transduction pathway model prototype II: Application to Ca2+-calmodulin signaling and myosin light chain phosphorylation

An agonist-initiated Ca(2+) signaling model for calmodulin (CaM) coupled to the phosphorylation of myosin light chains was created using a computer-assisted simulation environment. Calmodulin buffering was introduced as a module for directing sequestered CaM to myosin light chain kinase (MLCK) through Ca(2+)-dependent release from a buffering protein. Using differing simulation conditions, it was discovered that CaM buffering allowed transient production of more Ca(2+)-CaM-MLCK complex, resulting in elevated myosin light chain phosphorylation compared to nonbuffered control. Second messenger signaling also impacts myosin light chain phosphorylation through the regulation of myosin light chain phosphatase (MLCP). A model for MLCP regulation via its regulatory MYPT1 subunit and interaction of the CPI-17 inhibitor protein was assembled that incorporated several protein kinase subsystems including Rho-kinase, protein kinase C (PKC), and constitutive MYPT1 phosphorylation activities. The effects of the different routes of MLCP regulation depend upon the relative concentrations of MLCP compared to CPI-17, and the specific activities of protein kinases such as Rho and PKC. Phosphorylated CPI-17 (CPI-17P) was found to dynamically control activity during agonist stimulation, with the assumption that inhibition by CPI-17P (resulting from PKC activation) is faster than agonist-induced phosphorylation of MYPT1. Simulation results are in accord with literature measurements of MLCP and CPI-17 phosphorylation states during agonist stimulation, validating the predictive capabilities of the system.