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1.
We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner. 相似文献
2.
M C Glennon G S Bird H Takemura O Thastrup B A Leslie J W Putney 《The Journal of biological chemistry》1992,267(35):25568-25575
The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells. 相似文献
3.
The norepinephrine-sensitive Ca2+-storage site differs from the caffeine-sensitive site in vascular smooth muscle of the rat aorta 总被引:5,自引:0,他引:5
Using microfluorometry of quin 2, a Ca2+-sensitive dye, we characterized the release and uptake of Ca2+ by the norepinephrine-sensitive Ca2+-storage site and the caffeine-sensitive one. The norepinephrine-sensitive Ca2+-storage site was readily depleted in Ca2+-free medium and almost completely replenished by loading with 1.0 mM Ca2+ solution for 3 min, whereas the caffeine-sensitive site was scarcely affected. Furthermore, norepinephrine has little effect on the caffeine-sensitive Ca2+-storage site in Ca2+-free medium, and vice versa. We conclude that the location and mechanisms of release and uptake of Ca2+ of these two Ca2+-storage sites differ in the case of rat aortic vascular smooth muscle cells in primary culture. 相似文献
4.
We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP-dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole. ATP-dependent Ca2+ accumulation into non-vacuolar Ca(2+)-storing organelles was detected. This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P-type ATPases. The null mutation of cta3, a putative Ca2+ gene, [Ghislain, M., Goffeau, A., Halachmi, D. and Eilam, Y. (1990) J. Biol. Chem. 265, 18400-18407] strongly reduced the level of ATP-dependent Ca2+ uptake into non-vacuolar intracellular storing organelles. This result suggests that cta3 encodes an intracellular ATP-dependent Ca2+ pump. The residual ATP-dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca(2+)-ATPase encoded by a different gene. 相似文献
5.
Mitochondrial Ca(2+) and its relation with the contraction induced by phenylephrine was investigated. In normal Ca(2+), carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP) and oligomycin produced contraction similar to that promoted by phenylephrine. Phenylephrine-induced contraction was reduced by FCCP+oligomycin. In Ca(2+)-free, FCCP+oligomycin did not induce contraction. Response to FCCP+oligomycin was reduced upon Ca(2+) repletion and this response was lower than that to phenylephrine. Ca(2+) concentration was increased by FCCP+oligomycin. Since a profuse net of sarcoplasmic reticulum encloses mitochondria, a cross-talk between the two organelles may play an important role in the phenylephrine-induced contraction in presence of Ca(2+) encountered in both sarcoplasmic reticulum and extracellular medium of anococcygeus cells. 相似文献
6.
Calcium signaling and apoptosis 总被引:18,自引:0,他引:18
Hajnóczky G Davies E Madesh M 《Biochemical and biophysical research communications》2003,304(3):445-454
Ca(2+) is one of the key regulators of cell survival, but Ca(2+) can also induce apoptosis in response to a variety of pathological conditions. The pro-apoptotic effects of Ca(2+) are mediated by a diverse range of Ca(2+)-sensitive factors that are compartmentalized in various intracellular organelles including the ER, cytoplasm, and mitochondria. The Ca(2+) dynamics of these organelles appear to be modulated by the apoptosis-regulating Bcl-2 family proteins. In this paper, the recent progress of research on the mechanisms mediating the apoptosis-regulating effects of Ca(2+) and the interactions of Bcl-2 family proteins with the Ca(2+) storage organelles are discussed. 相似文献
7.
Calcium signal compartmentalization 总被引:3,自引:0,他引:3
Petersen OH 《Biological research》2002,35(2):177-182
Cytosolic calcium signals are produced by suddenly increasing the concentration of free calcium ions (Ca2+). This can occur by opening channels permeable to Ca2+ either in the surface cell membrane or in the membranes of intracellular organelles containing high Ca2+ concentrations. Ca2+ signals can control several different processes, even in the same cell. In pancreatic acinar cells, for example, Ca2+ signals do not only control the normal secretion of digestive enzymes, but can also activate autodigestion and programmed cell death. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited. The mechanisms responsible for Ca2+ signal compartmentalization are now largely known and will be described on the basis of recent studies of Ca2+ transport pathways and their regulation in pancreatic acinar cells. It turns out that the Ca2+ handling as well as the structural characteristics of the endoplasmic reticulum (ER) and the mitochondria are of particular importance. Using a variety of Ca(2+)-sensitive fluorescent probes placed in different sub-cellular compartments in combination with local uncaging of caged Ca2+, many new insights into Ca2+ signal generation, compartmentalization and termination have recently been obtained. 相似文献
8.
The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis. 相似文献
9.
Ca2+ microdomains and the control of insulin secretion 总被引:2,自引:0,他引:2
Nutrient-induced increases in intracellular free Ca(2+) concentrations are the key trigger for insulin release from pancreatic islet beta-cells. These Ca(2+) changes are tightly regulated temporally, occurring as Ca(2+) influx-dependent baseline oscillations. We explore here the concept that locally high [Ca(2+)] concentrations (i.e. Ca(2+) microdomains) may control exocytosis via the recruitment of key effector proteins to sites of exocytosis. Importantly, recent advances in the development of organelle- and membrane-targeted green fluorescent protein (GFP-) or aequorin-based Ca(2+) indicators, as well as in rapid imaging techniques, are providing new insights into the potential role of these Ca(2+) microdomains in beta-cells. We summarise here some of the evidence indicating that Ca(2+) microdomains beneath the plasma membrane and at the surface of large dense core vesicles may be important in the normal regulation of insulin secretion, and may conceivably contribute to "ATP-sensitive K(+)-channel independent" effects of glucose. We also discuss evidence that, in contrast to certain non-excitable cells, direct transfer of Ca(2+) from the ER to mitochondria via localised physical contacts between these organelles is relatively less important for efficient mitochondrial Ca(2+) uptake in beta-cells. Finally, we discuss evidence from single cell imaging that increases in cytosolic Ca(2+) are not required for the upstroke of oscillations in mitochondrial redox state, but may underlie the reoxidation process. 相似文献
10.
Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling. 相似文献
11.
Alvarez J 《Cell calcium》2012,51(3-4):331-337
Cellular Ca(2+)signaling results from a complex interplay among a variety of Ca(2+) fluxes going across the plasma membrane and across the membranes of several organelles, together with the buffering effect of large numbers of Ca(2+)-binding sites distributed along the cell architecture. Endoplasmic and sarcoplasmic reticulum, mitochondria and even nucleus have all been involved in cellular Ca(2+) signaling, and the mechanisms for Ca(2+) uptake and release from these organelles are well known. In neuroendocrine cells, the secretory granules also constitute a very important Ca(2+)-storing organelle, and the possible role of the stored Ca(2+) as a trigger for secretion has attracted considerable attention. However, this possibility is frequently overlooked, and the main reason for that is that there is still considerable uncertainty on the main questions related with granular Ca(2+) dynamics, e.g., the free granular [Ca(2+)], the physical state of the stored Ca(2+) or the mechanisms for Ca(2+) accumulation and release from the granules. This review will give a critical overview of the present state of knowledge and the main conflicting points on secretory granule Ca(2+) homeostasis in neuroendocrine cells. 相似文献
12.
The ability of Ca(2+), the simplest of all intracellular messengers, selectively to regulate so many cellular behaviours is due largely to the complex spatiotemporal organization of intracellular Ca(2+) signals. Most signalling pathways, including those that culminate in Ca(2+) signals, comprise sequences of protein-protein interactions linked by diffusible messengers. Using specific examples to illustrate key principles, we consider the roles of both components in defining the spatial organization of Ca(2+) signals. We discuss evidence that regulation of most Ca(2+) channels by Ca(2+) contributes to controlling the duration of Ca(2+) signals, to signal integration and, via Ca(2+)-induced Ca(2+) release, to defining the spatial spread of Ca(2+) signals. We distinguish two types of protein-protein interaction: scaffolds that allow rapid local transfer of diffusible messengers between signalling proteins, and interactions that directly transfer information between signalling proteins. Store-operated Ca(2+) entry provides a ubiquitous example of the latter, and it serves also to illustrate how Ca(2+) signals can be organized at different levels of spatial organization - from interactions between proteins to interactions between organelles. 相似文献
13.
Monteith GR 《Immunology and cell biology》2000,78(4):403-407
The role of Ca2+ in the regulation of the cell cycle has been investigated mostly in studies assessing global cytosolic free Ca2+. Recent studies, however, have used unique techniques to assess Ca2+ in subcellular organelles, such as mitochondria, and in discrete regions of the cytoplasm. These studies have used advanced fluorescence digital imaging techniques and Ca2+-sensitive fluorescence probes, and/or targeting of Ca2+-sensitive proteins to intracellular organelles. The present review describes the results of some of these studies and the techniques used. The novel techniques used to measure Ca2+ in microdomains and intracellular organelles are likely to be of great use in future investigations assessing Ca2+ homeostasis during the cell cycle. 相似文献
14.
Hepatocytes are highly differentiated and spatially polarised cells which conduct a wide range of functions, including intermediary metabolism, protein synthesis and secretion, and the synthesis, transport and secretion of bile acids. Changes in the concentrations of Ca(2+) in the cytoplasmic space, endoplasmic reticulum (ER), mitochondria, and other intracellular organelles make an essential contribution to the regulation of these hepatocyte functions. While not yet fully understood, the spatial and temporal parameters of the cytoplasmic Ca(2+) signals and the entry of Ca(2+) through Ca(2+)-permeable channels in the plasma membrane are critical to the regulation by Ca(2+) of hepatocyte function. Ca(2+) entry across the hepatocyte plasma membrane has been studied in hepatocytes in situ, in isolated hepatocytes and in liver cell lines. The types of Ca(2+)-permeable channels identified are store-operated, ligand-gated, receptor-activated and stretch-activated channels, and these may vary depending on the animal species studied. Rat liver cell store-operated Ca(2+) channels (SOCs) have a high selectivity for Ca(2+) and characteristics similar to those of the Ca(2+) release activated Ca(2+) channels in lymphocytes and mast cells. Liver cell SOCs are activated by a decrease in Ca(2+) in a sub-region of the ER enriched in type1 IP(3) receptors. Activation requires stromal interaction molecule type 1 (STIM1), and G(i2alpha,) F-actin and PLCgamma1 as facilitatory proteins. P(2x) purinergic channels are the only ligand-gated Ca(2+)-permeable channels in the liver cell membrane identified so far. Several types of receptor-activated Ca(2+) channels have been identified, and some partially characterised. It is likely that TRP (transient receptor potential) polypeptides, which can form Ca(2+)- and Na(+)-permeable channels, comprise many hepatocyte receptor-activated Ca(2+)-permeable channels. A number of TRP proteins have been detected in hepatocytes and in liver cell lines. Further experiments are required to characterise the receptor-activated Ca(2+) permeable channels more fully, and to determine the molecular nature, mechanisms of activation, and precise physiological functions of each of the different hepatocyte plasma membrane Ca(2+) permeable channels. 相似文献
15.
Although it has long been known that mitochondria possess a complex molecular repertoire for accumulating and releasing Ca2+, only in recent years has a large body of data demonstrated that these organelles promptly respond to Ca(2+)-mediated cell stimulations. In this contribution, we will review the principles of mitochondrial Ca2+ homeostasis and its signaling role in different physiological and pathological conditions. 相似文献
16.
Regulation of the concentration of Ca2+ in the apoplast of plants is essential in order to allow Ca(2+)-dependent processes, such as Ca(2+)-mediated signal transduction in stomatal guard cells, to function correctly. This is particularly important for plants growing with high levels of Ca2+ in the rhizosphere. Recently, we have shown that in two calcicoles, Leontodon hispidus L. and Centaurea scabiosa L., trichomes play a key role in this regulatory process. Ozone is known to have a marked effect on plant Ca2+ homeostasis. Therefore, we have examined the effect of this pollutant on the regulation by trichomes of apoplastic Ca2+ in the calcicole L. hispidus. Treatment with 100 nl l-1 ozone resulted in a reduction in stomatal conductance of approximately 25% in plants grown with 15 mM Ca2+ in the rhizosphere. Analysis of total Ca2+ levels revealed that these changes in stomatal behaviour reflect a decrease in the ability of trichomes to sequester Ca2+. The amount of Ca2+ present in the trichome tip cell was reduced by approximately 38%. This was accompanied by an increase in the levels of Ca2+ in the guard cells and other tissues of the leaf. These data suggest that ozone has a detrimental effect on the ability of trichomes to regulate the concentration of apoplastic Ca2+ in L. hispidus, resulting in altered stomatal behaviour, and hence gaseous exchange, possibly due to the disruption of guard-cell Ca(2+)-mediated signal transduction. This has important implications for the growth and survival of plants growing in Ca(2+)-rich soils. 相似文献
17.
Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion 总被引:6,自引:0,他引:6
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《The Journal of cell biology》1994,124(6):1061-1070
Transition of leukocyte function-associated antigen-1 (LFA-1), from an inactive into an activate state depends on the presence of extracellular Mg2+ and/or Ca2+ ions. Although Mg2+ is directly involved in ligand binding, the role of Ca2+ in LFA-1 mediated adhesion remained obscure. We now demonstrate that binding of Ca2+, but not Mg2+, directly correlates with clustering of LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-1-ligand interaction. Using a reporter antibody (NKI-L16) that recognizes a Ca(2+)-dependent epitope on LFA-1, we found that Ca2+ can be bound by LFA-1 with different strength. We noticed that weak binding of Ca2+ is associated with a dispersed LFA-1 surface distribution on T cells and with non- responsiveness of these cells to stimuli known to activate LFA-1. In contrast, stable binding of Ca2+ by LFA-1 correlates with a patch-like surface distribution and vivid ligand binding after activation of LFA- 1. Mg(2+)-dependent ligand binding does not affect binding of Ca2+ by LFA-1 as measured by NKI-L16 expression, suggesting that Mg2+ binds to a distinct site, and that both cations are important to mediate adhesion. Only Sr2+ ions can replace Ca2+ to express the L16 epitope, and to induce clustering of LFA-1 at the cell surface. We conclude that Ca2+ is involved in avidity regulation of LFA-1 by clustering of LFA-1 molecules at the cell surface, whereas Mg2+ is important in regulation of the affinity of LFA-1 for its ligands. 相似文献
18.
Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+i) in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca(2+)-sensitive dye fura-2 and organelle-specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co-localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura-2 in subcellular organelles, incomplete hydrolysis of fura-2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura-2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook: Cell 61:197-199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i. 相似文献
19.
Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca(2+) is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-x(L) on Ca(2+) overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-x(L) neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca(2+)-mediated mitochondrial multiple conductance channel opening. Bcl-x(L) neuroprotective actions were antagonized by the Bcl-x(L) inhibitor ABT-737 and potentiated by the Ca(2+) chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death. 相似文献
20.
A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves 总被引:1,自引:0,他引:1
A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump. 相似文献