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The endoplasmic reticulum: One continuous or several separate Ca2+ stores?
Abstract
The Ca2+ store and sink in the endoplasmic reticulum (ER) is important for Ca2+ signal integration and for conveyance of information in spatial and temporal domains. Textbooks regard the ER as one continuous network, but biochemical and biophysical studies revealed apparently discrete ER Ca2+ stores. Recent direct studies of ER lumenal Ca2+ movements show that this organelle system is one continuous Ca2+ store, which can function as a Ca2+ tunnel. The concept of a fully connected ER network is entirely compatible with evidence indicating that the distribution of Ca2+-release channels in the ER membrane is discontinuous with clustering in certain localities.
... Replenishment and discharge of [Ca 2+ ] ER require sarcoendoplasmic reticulum Ca 2+ ATPases (SERCA) and type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) function during [Ca 2+ ] i oscillations. Addition of SERCA inhibitors (thapsigargin or cyclopiazonic acid (CPA)) could perturb Ca 2+ oscillations by reducing basal [Ca 2+ ] ER levels and holding the recovery back [21,23,24]. ...
... Replenishment and discharge of [Ca 2+ ] ER require sarco-endoplasmic reticulum Ca 2+ ATPases (SERCA) and type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) function during [Ca 2+ ] i oscillations. Addition of SERCA inhibitors (thapsigargin or cyclopiazonic acid (CPA)) could perturb Ca 2+ oscillations by reducing basal [Ca 2+ ] ER levels and holding the recovery back [21,23,24]. ...
... IP3R1, tetrameric Ca 2+ channels located on the membrane of ER, is responsible for the majority of [Ca 2+ ] i increases associated with Oncotarget 10 www.impactjournals.com/oncotarget fertilization [23,24]. Such channel is composed of a channel pore formed by six transmembrane regions in C-terminal followed a small cytosolic tail, the coupling domain in intermediate region, and a ligand-binding domain in large cytosolic N-terminal region [97,98]. ...
Under the guidance and regulation of hormone signaling, large majority of mammalian oocytes go through twice cell cycle arrest-resumption prior to the fertilized egg splits: oocyte maturation and egg activation. Cytosolic free calcium elevations and endoplasmic reticulum calcium store alternations are actively involved in triggering the complex machineries and events during oogenesis. Among these, calcium influx had been implicated in the replenishment of endoplasmic reticulum store during oocyte maturation and calcium oscillation during egg activation. This process also drove successful fertilization and early embryo development. Storeoperated Ca²⁺ entry, acts as the principal force of calcium influx, is composed of STIM1 and Orai1 on the plasma membrane. Besides, transient receptor potential channels also participate in the process of calcium inwards. In this review, we summarize the recent researches on the spatial-temporal distribution of store-operated calcium entry components and transient receptor potential channels. Questions about how these channels play function for calcium influx and what impacts these channels have on oocytes are discussed. At the time of sperm-egg fusion, sperm-specific factor(s) diffuse and enable eggs to mount intracellular calcium oscillations. In this review, we also focus on the basic knowledge and the modes of action of the potential sperm factor phospholipase C zeta, as well as the downstream receptor, type 1 inositol 1,4,5-trisphosphate receptor. From the achievement in the previous several decades, it is easy to find that there are too many doubtful points in the field that need researchers take into consideration and take action in the future.
... Previous studies have found the ER functions as a sink for Ca 2+ that enters cells via channels and as a store for Ca 2+ that is released into the cytosol [15]. Excessive calcium mobilization from the ER to the cytoplasm causes activation of caspase 1, pyroptosis, and proinflammatory cytokine secretion [16]. ...
... In addition, the ER functions as a sink for calcium that enters cells via channels and as a store for calcium that is released into the cytosol [15]. Therefore, to explore whether the increased calcium during H/R is related to the ER, we treated liver macrophages with H/R in vitro and then the inhibition of inositol 1,4,5-trisphosphate receptor type 1 (ITPR1, also known as IP3R, the primary calcium release channel of the ER) [26] by siRNA or by a pharmacological inhibitor (2-APB) blocked the calcium release channel of the ER. ...
Objectives:
Although a recent study reported that stimulator of interferon genes (STING) in macrophages has an important regulatory effect on liver ischemia-reperfusion injury (IRI), the underlying mechanism of STING-dependent innate immune activation in liver macrophages (Kupffer cells, KCs) remains unclear. Here, we investigated the effect of STING on liver macrophage pyroptosis and the associated regulatory mechanism of liver IRI.
Methods:
Clodronate liposomes were used to block liver macrophages. AAV-STING-RNAi-F4/80-EGFP, an adenoassociated virus (AAV), was transfected into the portal vein of mice in vivo, and the liver IRI model was established 14 days later. In vitro, liver macrophages were treated with STING-specific siRNA, and a hypoxia-reoxygenation (H/R) model was established. The level of STING was detected via Western blotting (WB), RT-PCR, and immunostaining. Liver tissue and blood samples were collected. Pathological changes in liver tissue were detected by hematoxylin and eosin (H&E) staining. Macrophage pyroptosis was detected by WB, confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and enzyme-linked immunosorbent assay (ELISA). The calcium concentration was measured by immunofluorescence and analyzed with a fluorescence microplate reader.
Results:
The expression of STING increased with liver IRI but decreased significantly after the clodronate liposome blockade of liver macrophages. After knockdown of STING, the activation of caspase 1-GSDMD in macrophages and liver IRI was alleviated. More interestingly, hypoxia/reoxygenation (H/R) increased the calcium concentration in liver macrophages, but the calcium concentration was decreased after STING knockdown. Furthermore, after the inhibition of calcium in H/R-induced liver macrophages by BAPTA-AM, pyroptosis was significantly reduced, but the expression of STING was not significantlydecreased.
Conclusions:
Knockdown of STING reduces calcium-dependent macrophage caspase 1-GSDMD-mediated liver IRI, representing a potential therapeutic approach in the clinic.
... These storages accumulate, release and buffer Ca 2+ ions constantly, during specific cellular events. The Mitochondria and the Endoplasmic Reticulum (ER) are the main ones [21,17], so in what follows are introduced the main features of these two organelles, but also the Golgi Apparatus and the Nucleus can be involved too in different scenarios [33,29,35]. All of these organelle can absorb high levels of ions in order to keep low the cytoplasmic concentration. ...
... An important role in sequestering calcium ions from the cytosol and on intracellular signaling is covered by the Endoplasmic Reticulum and the Mitochondria, which are typically located in close proximity to each other [29,28,22]. The first one is normally filled with ions that can be released to generate or reinforce Ca 2+ signals, the last ones is normally empty and can store large amounts of ions that can be buffered, used to increase the energy production or even released in the cytosol. ...
A number of techniques have been recently proposed to implement molecular communication, a novel method which aims to implement communication networks at the nanoscale, known as nanonetworks. A common characteristic of these techniques is that their main resource consists of molecules, which are inherently discrete. This paper presents DIRECT, a novel networking model which differs from conventional models by the way of treating resources as discrete entities; therefore, it is particularly aimed to the analysis of molecular communication techniques. Resources can be involved in different tasks in a network, such as message encoding, they do not attenuate in physical terms and they are considered 100% reusable. The essential properties of DIRECT are explored and the key parameters are investigated throughout this paper.
... The so-called Ca 2+ tunnel experiments (Mogami et al. 1997) showed that the ER could be refilled, after ACh-elicited emptying, from a point source at the base of an isolated acinar cell by a thapsigargin-sensitive process, and that re-stimulation with ACh would again cause a primary [Ca 2+ ] i rise in the apical pole, more than 10 μm away from the Ca 2+ entry point at the base, and without any discernible rise in [Ca 2+ ] i during the refilling period. A few years later, we were able to demonstrate directly that a high ACh concentration caused a major reduction in [Ca 2+ ] in the intracellular stores in the basal part of the cells (dominated by ER), but not in the apical part (dominated by ZGs), in spite of the fact that [Ca 2+ ] i rose primarily in the apical pole (Park et al. 2000;Petersen et al. 2001). It was also shown that the whole of the ER, including the fine extensions and terminals in the apical pole, is functionally connected, and that Ca 2+ diffuses easily inside the lumen of the ER (Park et al. 2000;Petersen et al. 2001). ...
... A few years later, we were able to demonstrate directly that a high ACh concentration caused a major reduction in [Ca 2+ ] in the intracellular stores in the basal part of the cells (dominated by ER), but not in the apical part (dominated by ZGs), in spite of the fact that [Ca 2+ ] i rose primarily in the apical pole (Park et al. 2000;Petersen et al. 2001). It was also shown that the whole of the ER, including the fine extensions and terminals in the apical pole, is functionally connected, and that Ca 2+ diffuses easily inside the lumen of the ER (Park et al. 2000;Petersen et al. 2001). These studies indicated that physiological stimuli, such as ACh, primarily release Ca 2+ from the ER, and that the bulk of the Ca 2+ comes from basal stores. ...
Acute pancreatitis is a human disease in which the pancreatic pro-enzymes, packaged into the zymogen granules of the acinar cells, become activated and cause auto-digestion. The main causes of pancreatitis are alcohol abuse and biliary disease. A considerable body of evidence indicates that the primary event initiating the disease process is excessive release of Ca2+ from intracellular stores, followed by excessive entry of Ca2+ from the interstitial fluid. However, Ca2+ release and subsequent entry are also precisely the processes that control physiological secretion of the digestive enzymes in response to stimulation via the vagal nerve or the hormone cholecystokinin. The spatial and temporal Ca2+ signal patterns in physiology and pathology, as well as the contributions from different organelles in the different situations, are therefore critical issues. There has recently been significant progress in our understanding of both physiological stimulus-secretion coupling and the pathophysiology of acute pancreatitis. Very recently, a promising potential therapeutic development has occurred with the demonstration that blockade of Ca2+ release-activated Ca2+ currents in pancreatic acinar cells offers remarkable protection against Ca2+ overload, intracellular protease activation and necrosis evoked by a combination of alcohol and fatty acids, which is a major trigger of acute pancreatitis.
... The lumen of the ER of neurones and non-neuronal cells forms a continuous, aqueous space in which Ca 2+ and small molecules, (e.g. fluorescent dyes) can readily diffuse (9)(10)(11)(12)(13)(14). The functional properties of the ER are also extraordinarily heterogeneous depending on spatial distribution of various enzymatic cascades (4,(15)(16)(17). ...
... Activation of ER Ca 2+ channels results in Ca 2+ release, which contributes to [Ca 2+ ] i elevation, whereas SERCA-dependent Ca 2+ uptake assists termination of cytosolic Ca 2+ signals. In addition, the continuous luminal space can act as a travelling route for free Ca 2+ ions ("Ca 2+ tunnels"), thus bypassing cytosolic Ca 2+ buffers and preventing mitochondrial Ca 2+ uptake or loss of Ca 2+ over the plasma membrane (6,9,11,22,23). ...
... The morphology of the ER, especially in neural cells is highly heterogeneous with strong plasticity tailored to various chemical and dynamic tasks in different areas of the cell, as well as between different cell types . On the other hand, in skeletal muscle the RyR1 of the SR has a specialized arrangement in order to ensure a quick delivery of the Ca 2+ needed for muscle contraction in response to membrane depolarization (Petersen et al., 2001, Berridge, 2002. ...
The precise spatio-temporal characteristics of subcellular calcium (Ca²⁺) transients are critical for the physiological processes. Here we report a green Ca²⁺ sensor called “G-CatchER⁺” using a protein design to report rapid local ER Ca²⁺ dynamics with significantly improved folding properties. G-CatchER⁺ exhibits a superior Ca²⁺ on rate to G-CEPIA1er and has a Ca²⁺-induced fluorescence lifetimes increase. G-CatchER⁺ also reports agonist/antagonist triggered Ca²⁺ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER⁺-JP45), we report a rapid local Ca²⁺ release that is likely due to calsequestrin. Transgenic expression of G-CatchER⁺ in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca²⁺ dynamics. G-CatchER⁺ will be an invaluable tool to examine local ER/SR Ca²⁺ dynamics and facilitate drug development associated with ER dysfunction.
... The ER is physically and functionally linked to other organelles such as mitochondria and lysosomes, establishing close contact sites and possessing a rapid effect on their physiological function [155]. The ER functions as a sink for Ca 2+ that enters cells via the channels, and also a store for Ca 2+ that is released into the cytosol [156]. It is generally accepted that the ER has one continuous Ca 2+ store, but emerging evidence reveals several apparently discrete ER Ca 2+ stores [157]. ...
The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome senses pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), and activates caspase-1, which provokes release of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 as well as pyroptosis to engage in innate immune defense. The endoplasmic reticulum (ER) is a large and dynamic endomembrane compartment, critical to cellular function of organelle networks. Recent studies have unveiled the pivotal roles of the ER in NLRP3 inflammasome activation. ER–mitochondria contact sites provide a location for NLRP3 activation, its association with ligands released from or residing in mitochondria, and rapid Ca2+ mobilization from ER stores to mitochondria. ER-stress signaling plays a critical role in NLRP3 inflammasome activation. Lipid perturbation and cholesterol trafficking to the ER activate the NLRP3 inflammasome. These findings emphasize the importance of the ER in initiation and regulation of the NLRP3 inflammasome.
... Third, STIM1-mediated SOCE in filopodia would favor the formation of ER-plasma membrane junctions, thought to be important for growth cone consolidation and extension (Davenport et al., 1996). Last, tubular ER is recognized as a wholly interconnected and continuous membrane in eukaryotes (Terasaki et al., 1994;Petersen et al., 2001;Wu et al., 2017). Such a calcium-rich network of organelles would facilitate the conduction of calcium signals from filopodia throughout the growth cone via ER micro-networks (Choi et al., 2006). ...
The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin- 3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.
... The diffusion coefficient D I is set to 0.05 unless otherwise noted. The role played by the distribution and the geometry of the ER in glial cells has been under debate with evidence supporting varying degrees of ER interaction between domains of the astrocyte (Blaustein and Golovina, 2001;Petersen et al., 2001;Levine and Rabouille, 2005). To be consistent with these recent findings, we start by setting D E = 0, but later consider the influence of non-zero values. ...
Intracellular Ca2+ dynamics in astrocytes can be triggered by neuronal activity and in turn regulate a variety of downstream processes that modulate neuronal function. In this fashion, astrocytic Ca2+ signaling is regarded as a processor of neural network activity by means of complex spatial and temporal Ca2+ dynamics. Accordingly, a key step is to understand how different patterns of neural activity translate into spatiotemporal dynamics of intracellular Ca2+ in astrocytes. Here, we introduce a minimal compartmental model for astrocytes that can qualitatively reproduce essential hierarchical features of spatiotemporal Ca2+ dynamics in astrocytes. We find that the rate of neuronal firing determines the rate of Ca2+ spikes in single individual processes as well as in the soma of the cell, while correlations of incoming neuronal activity are important in determining the rate of “global” Ca2+ spikes that can engulf soma and the majority of processes. Significantly, our model predicts that whether the endoplasmic reticulum is shared between soma and processes or not determines the relationship between the firing rate of somatic Ca2+ events and the rate of neural network activity. Together these results provide intuition about how neural activity in combination with inherent cellular properties shapes spatiotemporal astrocytic Ca2+ dynamics, and provide experimentally testable predictions.
... Although the ER normally appears to be one, continuously connected Ca 2+ pool (i.e. not containing physically separated compartments) [68,69], heterogeneous distribution of ER Ca 2+ has been reported in many different cell types under various conditions [66,67,[70][71][72]. Moreover, the Ca 2+ ionophore ionomycin may induce ER fragmentation [68,73]. ...
Endoplasmic reticulum (ER) Ca2+ depletion activates the unfolded protein response (UPR), inhibits bulk autophagy and eventually induces cell death in mammalian cells. However, the extent and duration of ER Ca2+ depletion required is unknown. We instigated a detailed study in two different cell lines, using sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors to gradually reduce ER Ca2+ levels in a controlled manner. Remarkably, UPR induction (as assessed by expression analyses of UPR-regulated proteins) and autophagy inhibition (as assessed by analyses of effects on starvation-induced bulk autophagy) required substantially higher drug concentrations than those needed to strongly decrease total ER Ca2+ levels. In fact, even when ER Ca2+ levels were so low that we could hardly detect any release of Ca2+ upon challenge with ER Ca2+ purging agents, UPR was not induced, and starvation-induced bulk autophagy was still fully supported. Moreover, although we observed reduced cell proliferation at this very low level of ER Ca2+, cells could tolerate prolonged periods (days) without succumbing to cell death. Addition of increasing concentrations of extracellular EGTA also gradually depleted the ER of Ca2+, and, as with the SERCA inhibitors, EGTA-induced activation of UPR and cell death required higher EGTA concentrations than those needed to strongly reduce ER Ca2+ levels. We conclude that ER Ca2+ depletion-induced effects on UPR, autophagy and cell death require either an extreme general depletion of ER Ca2+ levels, or Ca2+ depletion in areas of the ER that have a higher resistance to Ca2+ drainage than the bulk of the ER.
... In many non-excitable cells, intracellular Ca 2+ signals respond to extracellular agonist stimulation in an oscillatory, rather than sustained, manner [1][2][3]. It is well accepted that two distinct intracellular Ca 2+ pools, the inositol-1, 4, 5-trisphosphate (InsP 3 )-sensitive and ryanodine-sensitive pools, participate in the genesis of oscillatory Ca 2+ signals [4]. In 1986, Putney presented a model for capacitive calcium (Ca 2+ ) entry conveying that depletion of endoplasmic reticulum-stored Ca 2+ leads to activation of plasma membrane Ca 2+ channels that mediate influx of Ca 2+ from the extracellular space into cells [5], in a process called store-operated Ca 2+ entry (SOCE). ...
Cellular Ca2+ signals play a critical role in cell physiology and pathology. In most non-excitable cells, store-operated Ca2+ entry (SOCE) is an important mechanism by which intracellular Ca2+ signaling is regulated. However, few drugs can selectively modulate SOCE. 2-Aminoethoxydiphenyl borate (2APB) and its analogs (DPB162 and DPB163) have been reported to inhibit SOCE. Here, we examined the effects of another 2-APB analog, DPB161 on SOCE in acutely-isolated rat submandibular cells. Both patch-clamp recordings and Ca2+ imaging showed that upon removal of extracellular Ca2+ ([Ca2+]o=0), rat submandibular cells were unable to maintain ACh-induced Ca2+ oscillations, but restoration of [Ca2+]o to refill Ca2+ stores enable recovery of these Ca2+ oscillations. However, addition of 50 μM DPB161 with [Ca2+]o to extracellular solution prevented the refilling of Ca2+ store. Fura-2 Ca2+ imaging showed that DPB161 inhibited SOCE in a concentration-dependent manner. After depleting Ca2+ stores by thapsigargin treatment, bath perfusion of 1 mM Ca2+ induced [Ca2+]i elevation in a manner that was prevented by DPB161. Collectively, these results show that the 2-APB analog DPB161 blocks SOCE in rat submandibular cells, suggesting that this compound can be developed as a pharmacological tool for the study of SOCE function and as a new therapeutic agent for treating SOCE-associated disorders.
... The high mobility of Ca 2+ in the ER lumen was demonstrated directly by experiments in which changes in [Ca 2+ ] ER at various locations in the ER could be monitored after a highly localized uncaging of caged Ca 2+ in the ER lumen (Park et al. 2000). These experiments showed that after a local Ca 2+ uncaging event, rises in [Ca 2+ ] ER were observed quickly over considerable distances (more than 10 μm away from the site of uncaging) and that the whole of the ER was re-equilibrated with regard to [Ca 2+ ] ER within a few seconds (less than the time interval between cytosolic Ca 2+ spikes during physiological Ca 2+ signalling) (Park et al. 2000;Petersen et al. 2001). ...
Ca(2+) signalling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium-binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca(2+) concentration. In electrically non-excitable cells, for example epithelial cells, this is achieved by primary release of Ca(2+) from the endoplasmic reticulum via Ca(2+) release channels placed close to the physiological target. Because any rise in the cytosolic Ca(2+) concentration activates Ca(2+) extrusion, and in order for cells not to run out of Ca(2+) , there is a need for compensatory Ca(2+) uptake from the extracellular fluid. This Ca(2+) uptake occurs through a process known as store-operated Ca(2+) entry. Ideally Ca(2+) entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca(2+) tunneling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca(2+) entering via store-operated Ca(2+) channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept. This article is protected by copyright. All rights reserved.
... In the ER, the distribution of Ca 2+ channels in the ER membrane is discontinuous, with clustering in certain locations and different Ca 2+ concentrations in sub-compartments [56][57][58] . However, the ER luminal Ca 2+ movements show that this organelle system is one continuous Ca 2+ store with a Ca 2+ tunnel effect 59,60 . ...
The Bestrophin family has been characterized as Cl− channels in mammals and Na+ channels in bacteria, but their exact physiological roles remian unknown. In this study, a natural C-terminally truncated variant of mouse Bestrophin 3 (Best3V2) expression in myoblasts and muscles is demonstrated. Unlike full-length Best3, Best3V2 targets the two important intracellular Ca stores: the lysosome and the ER. Heterologous overexpression leads to lysosome swelling and renders it less acidic. Best3V2 overexpression also results in compromised Ca2+ release from the ER. Knocking down endogenous Best3 expression in myoblasts makes these cells more excitable in response to Ca2+ mobilizing reagents, such as caffeine. We propose that Best3V2 in myoblasts may work as a tuner to control Ca2+ release from intracellular Ca2+ stores.
... Subsurface cisterns are present underneath BK α clusters in both Purkinje cells and dentate gyrus granule cells (Kaufmann et al., 2009(Kaufmann et al., , 2010. These cisterns are apical extensions of smooth (ie, ribosome free) endoplasmic reticulum closely apposed against the plasma membrane that may serve as a source of local Ca 2+ sparks for coordinated opening of clustered BK channels (Petersen, Tepikin, & Park, 2001). BK channels located in these domains may become activated during AP-stimulated release of Ca 2+ from internal stores and thereby exert negative feedback on Ca 2+ entry through Ca V channels, in a manner reminiscent of their role in smooth muscle cells in response to Ca 2+ sparks elicited from sarcoplasmic reticulum stores (Brenner, Perez, et al., 2000;Burdyga & Wray, 2005;Ledoux, Werner, Brayden, & Nelson, 2006; see Chapter "BK Channels in the Vascular System" by Krishnamoorthy-Natarajan and Koide). ...
Large conductance Ca2+- and voltage-activated K+ (BK) channels are widely distributed in the postnatal central nervous system (CNS). BK channels play a pleiotropic role in regulating the activity of brain and spinal cord neural circuits by providing a negative feedback mechanism for local increases in intracellular Ca2 + concentrations. In neurons, they regulate the timing and duration of K+ influx such that they can either increase or decrease firing depending on the cellular context, and they can suppress neurotransmitter release from presynaptic terminals. In addition, BK channels located in astrocytes and arterial myocytes modulate cerebral blood flow. Not surprisingly, both loss and gain of BK channel function have been associated with CNS disorders such as epilepsy, ataxia, mental retardation, and chronic pain. On the other hand, the neuroprotective role played by BK channels in a number of pathological situations could potentially be leveraged to correct neurological dysfunction.
... Ca 2+ distribution within the ER is not homogeneous (Petersen et al., 2001) and its regulation across the ER membrane is facilitated by three classes of well-studied proteins: (1) Ca 2+ release channels -inositol-1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs), (2) Ca 2+ pumps -sarco-endoplasmic reticulum Ca 2+ -ATPases (SERCAs) that uptake Ca 2+ from the cytosol to the ER lumen, and (3) Ca 2+ binding proteins (Clapham, 2007;Mekahli et al., 2011;Pozzan et al., 1994;Prins and Michalak, 2011), Figure 1 and Table 1. ...
A number of chronic metabolic pathologies, including obesity, diabetes, cardiovascular disease, asthma, and cancer, cluster together to present the greatest threat to human health. As research in this field has advanced, it has become clear that unresolved metabolic inflammation, organelle dysfunction, and other cellular and metabolic stresses underlie the development of these chronic metabolic diseases. However, the relationship between these systems and pathological mechanisms is poorly understood. Here we discuss the role of cellular Ca(2+) homeostasis as a critical mechanism integrating the myriad of cellular and subcellular dysfunctional networks found in metabolic tissues such as liver and adipose tissue in the context of metabolic disease, particularly in obesity and diabetes.
Copyright © 2015 Elsevier Inc. All rights reserved.
... The smooth ER is the site where enzymes involved in lipid biosynthesis are localized. In some cells as muscle cells, regions of the smooth ER are the site of calcium storage and are called the sarcoplasmic reticulum (Petersen et al., 2001). ...
... It is interesting to note that even though there is continuity between the ER and nuclear envelope membranes, their protein composition appears to be different. The ER itself is not uniform with respect to channel, pump and protein distribution (reviewed by Petersen et al., 2001); for example, calsequestrin was located in the ER lumen and was enriched within small vacuoles that were also equipped with SERCAs (sarco/endoplasmic reticular Ca 2+ -ATPases). Some, but not all, of these vacuoles (calciosomes) contained IP 3 Rs (Volpe et al., 1991). ...
... At the end of the 90's it was acknowledged that the endoplasmic reticulum (ER) forms a continuous network of tubes and sacs that extends from the nuclear envelope out to the cell periphery (Petersen OH et al, 2001;Spacek J et al, 1997;Subramanian K et al,1997;Terasaki M et al,1994). This was shown through EM reconstructions (Spacek J et al, 1997) and diffusion of dye along internal membranes (Terasaki M et al,1994) to show the ER continuity in neurons, across the axon, soma, dendrites and the spine apparatus at the dendritic spines. ...
... Evidence has accumulated that Ca 2ϩ plays essential roles in vesicular traffic between the ER and the Golgi (Beckers and Balch, 1989;Pind et al., 1994;Ivessa et al., 1995;Ahluwalia et al., 2001;Chen et al., 2002;Hasdemir et al., 2005). Consistent with this, both the ER and the Golgi are intracellular Ca 2ϩ stores (Pinton et al., 1998;Petersen et al., 2001), and the cytoplasmic Ca 2ϩ concentration is reported to be relatively high around these organelles (Wahl et al., 1992). However, Ca 2ϩ -binding proteins of which functions are directly regulated by Ca 2ϩ in this trafficking pathway have been mostly unidentified. ...
The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.
... Therefore, the various Ca 2 -releasing messengers could be produced inside the nucleus to regulate release of Ca 2 from the nuclear envelope. In the intact cell, the Ca 2 store in the nuclear envelope is part of the unified and lumenally continuous ER store (Petersen et al., 2001), but this report on isolated nuclei reveals that the local control of Ca 2 release can operate in a distinct manner. Although the local Ca 2 spiking in the apical secretory pole region of the pancreatic acinar cells (as well as the global Ca 2 -induced Ca 2 waves) depends on cooperative interaction of IP 3 and ryanodine receptors (Cancela et al., 2000; ), these receptors can function independently in the nucleus to release Ca 2 into the nucleoplasm. ...
Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.
... In mammalian cells, COPII vesicles that bud from the ER do not directly fuse to the Golgi apparatus; instead, they are thought to tether to each other to form pre-Golgi intermediates, also termed vesicular tubular clusters (VTCs) or ER-Golgi intermediate compartment (ERGIC). The lumen of the ER and Golgi contain a high concentration of free Ca 2+ due to Ca 2+ -ATPase pump activity [34], [35], and the cytoplasmic Ca 2+ concentration immediately adjacent to the membranes of these organelles is reported to be relatively high, possibly because of nonspecific basal leakage of luminal Ca 2+ [36], [37]. However, since Ca 2+ -ATPases are not incorporated into COPII vesicles, pre-Golgi intermediates exhibit lower Ca 2+ signatures [38]. ...
Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.
George Palade's pioneering electron microscopical studies of the pancreatic acinar cell revealed the intracellular secretory pathway from the rough endoplasmic reticulum at the base of the cell to the zymogen granules in the apical region. Palade also described for the first time the final stage of exocytotic enzyme secretion into the acinar lumen. The contemporary studies of the mechanism by which secretion is acutely controlled, and how the pancreas is destroyed in the disease acute pancreatitis, rely on monitoring molecular events in the various identified pancreatic cell types in the living pancreas. These studies have been carried out with the help of high-resolution fluorescence recordings, often in conjunction with patch clamp current measurements. Through such studies we have gained much detailed information about the regulatory events in the exocrine pancreas in health as well as disease, and new therapeutic opportunities have been revealed.
It is generally accepted that the endoplasmic reticulum (ER) has one continuous lumen and operates as one unified Ca2+ store, but new data from Chen-Engerer et al. in hippocampal neurons now challenge this idea and indicate that receptors for inositol trisphosphate (IP3) and ryanodine may be located in two functionally distinct Ca2+ stores. Are both these stores in the ER?
Cortical spreading depolarization waves, the cause underlying migraine aura, are also the markers and mechanism of pathology in the acutely injured human brain. Propagation of spreading depolarization wave uniquely depends on the interaction between presynaptic and postsynaptic glutamate N-methyl-d-aspartate receptors (NMDARs). In the normally perfused brain, even a single wave causes a massive depolarization of neurons and glia, which results in transient loss of neuronal function and depression of the ongoing electrocorticographic activity. Endoplasmic reticulum is the cellular organelle of particular importance for modulation of neurotransmission. Neuronal endoplasmic reticulum structure is assumed to be persistently continuous in neurons, but is rapidly lost within 1 to 2 min of global cerebral ischaemia, i.e. the organelle disintegrates by fission. This phenomenon appears to be timed with the cardiac arrest-induced cortical spreading depolarizations, rather than ensuing cell death. To what extent NMDAR-dependent processes may trigger neuronal endoplasmic reticulum fission and whether fission is reversible in the normally perfused brain is unknown. We used two-photon microscopy to examine neuronal endoplasmic reticulum structural dynamics during whisker stimulation and cortical spreading depolarizations in vivo. Somatosensory stimulation triggered loss of endoplasmic reticulum continuity, a likely outcome of constriction and fission, in dendritic spines within less than 10 s of stimulation, which was spontaneously reversible and recovery to normal took 5 min. The endoplasmic reticulum fission was inhibited by blockade of NMDAR and Ca2+/calmodulin-dependent protein kinase II (CaMKII) activated downstream of the NMDARs, whereas inhibition of guanosine triphosphate hydrolases hindered regain of endoplasmic reticulum continuity, i.e. fusion. In contrast to somatosensory stimulation, endoplasmic reticulum fission during spreading depolarization was widespread and present in dendrites and spines, and was preceded by dramatic rise in intracellular Ca2+. The endoplasmic reticulum fission during spreading depolarization was more persistent, as 1 h after the depolarization cortical neurons still exhibited loss of endoplasmic reticulum continuity. Notably, endoplasmic reticulum fission was accompanied with loss of electrocorticographic activity, whereas subsequent regain of synaptic function paralleled the organelle fusion. Furthermore, blocking CaMKII activity partly rescued endoplasmic reticulum fission and markedly shortened the recovery time of brain spontaneous activity. Thus, prevention of endoplasmic reticulum fission with CaMKII inhibitors may be a novel strategy to rescue brain function in patients with migraine and a promising therapeutic avenue in the acutely injured brain.
The role of calcium for the maintenance of cellular activity was observed by Ringer [55] as early as 1883. Some years later, Stiles reported that calcium activates smooth muscle contraction [61]. The key role of calcium in intracellular functions was not perceived until 70 years later by Heilbrunn in the United States (1947) [39] and by Kamada in Japan (1943) [43].
The literature data and our own data on the synaptic plasticity and remodeling of synaptic organelles in the central nervous system are reviewed. Modern techniques of laser scanning confocal microscopy and serial thin sectioning for in vivo and in vitro studies of dendritic spines, including the relationship between morphological changes and the efficacy of synaptic transmission, are discussed using, in particular, a model of long-term potentiation. The organization of dendritic spines and postsynaptic densities of different categories as well as the role of filopodia in spine genesis were analyzed. It was shown that the method of serial ultrathin sections is the most effective for unbiased quantitative stereological analysis and 3D reconstructions. By using the refined method of serial ultrathin sections with subsequent three-dimensional reconstructions, the presence of giant mitochondria in hippocampal neuronal dendrites was demonstrated. It was shown that smooth endoplasmic reticulum forms a unified continuum with the outer membrane of the mitochondrial envelope within dendrites. It was suggested that this continuum provides calcium tunneling, which makes possible intracellular signal transduction during synaptic transmission. Evidence is presented indicating the presence of gap junctions (< >) in the synapses of mammalian brain, as well as between glial processes, and between glial cells and neurons. Our data and the data of other authors show that glial cell processes form a structural and functional glial network, which modulates the functioning of the neuronal network. The connection of dendritic spines with the glial network is shown on 3D reconstructions by analyzing the neuropil volume in CA1 hippocampal area of ground squirrels in three functional states: normothermia, provoked arousal, and hibernation when brain temperature falls below 6degreesC. The own data of the authors are discussed indicating the formation of more than five presynaptic boutons (multiple synapses) on both CA1 mushroom-like dendrite spines and CA3 thorny excrescences. On the basis of the analysis, new ideas of the organization and functioning of synapses were suggested.
In mouse pancreatic acinar cells, the hormone cholecystokinin (CCK) and the neurotransmitter acetylcholine (ACh) are the most important secretagogues and both of them elicit specific Ca2+ signatures (Fig. 1) [1– 4]. At low physiological concentration, CCK evokes a mixture of short-lasting local Ca2+ spikes in the secretory pole of the cell and long-lasting global Ca2+ spikes, whereas ACh at low concentration elicits local Ca2+ spikes in the secretory pole of the cell. At higher concentrations, CCK and ACh evoke global Ca2+ transients. Despite intensive research, the mechanisms underlying the complex Ca2+ oscillations remain unclear. It has been shown that heparin, an IP3-receptor antagonist, blocked the Ca2+ response elicited by ACh and CCK [5– 6]. These pharmacological data suggested that the IP3 receptors are involved in the secretagogue-evoked Ca2+ spikes and a two-pool model has been proposed for both ACh and CCK. In this model IP3 induces primary Ca2+ release, which then releases Ca2+ from an IP3-insensitive pool by a Ca2+-induced Ca2+ release (CICR) process [1,5]. However, this two-pool model relies on IP3 generation by CCK and ACh, which is not supported by biochemical data on the entire pancreatic acinar cell population [7]. In contrast to ACh, a physiological concentration of CCK does not generate detectable IP3.
Dr. Jaakko Saraste, Department of Anatomy and Cell Biology, University of Bergen, Årstadveien 19, N-5009 Bergen/Norway, Fax: +47 5558 6360.
The importance of calcium signaling in cell health and disease is the major driving force in current research of intracellular calcium homeostasis. Ca ²⁺ release from the endoplasmic reticulum ( ER ) and other calcium stores seems to be the crucial factor in the activation of many cellular functions. Significant changes in ER Ca ²⁺ content and dynamics have been implicated in the activation of the ER stress response, abnormal autophagy, and cell death which leads to a variety of pathological conditions. For example, in acute pancreatitis, an inflammatory disease of the exocrine pancreas caused primarily by bile stones or alcohol, excessive intracellular calcium overload due to Ca ²⁺ release from internal stores followed by store operated Ca ²⁺ entry ( SOCE ) leads to the premature activation of digestive proenzymes within pancreatic acinar cells. Recent data show that SOCE channel blockers are capable of substantially reducing the intracellular Ca ²⁺ overload and subsequent cell necrosis without major alteration of ER Ca ²⁺ content. We also demonstrate here that indirect ER measurements can be misleading and only direct intra‐ ER Ca ²⁺ monitoring offers reliable conclusions. In this respect, it is essential to summarize the methods available and provide examples of direct measurements of free Ca ²⁺ concentration [Ca ²⁺ ] in the ER lumen in pancreatic acinar cells. This article is aimed at highlighting the major techniques for monitoring ER Ca ²⁺ with reference to their advantages, limitations, and views for future improvements. WIREs Membr Transp Signal 2014,3:63–71. doi: 10.1002/wmts.106
For further resources related to this article, please visit the WIREs website .
Conflict of interest: The authors have declared no conflicts of interest for this article.
Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.
Acute pancreatitis is a sudden, severe inflammation of the pancreas which may eventually lead to life-threatening complications associated with digestive necrosis of the gland. Premature, intracellular activation of zymogens is believed to be responsible for the onset of acute pancreatitis; however, the underlying mechanisms remain unidentified. Alcohol is one of the most common risk factors in the development of this condition and may promote its deleterious effects by directly sensitizing the pancreatic acini to CCK, an important gastrointestinal peptide hormone and a digestive enzyme regulator which physiologically activates Ca2+ channels, thus serving an integral role in the physiological regulation of digestive enzyme secretion by pancreatic acinar cells. Nicotine exposure presents an accessory agonist and predisposing factor which may render the pancreas more susceptible to the injurious effects of alcohol-induced gland deficiency. It is important to evaluate the possible cumulative effect of these two high-risk factors in the pathophysiology of acute pancreatitis. The present study addresses the modulatory effects of ethanol and/or nicotine on CCK-evoked Ca2+ alterations and premature intracellular trypsin activation in murine pancreatic acinar cells. Our results indicate that alterations in Ca2+ signaling are often associated with pH modifications and may occasionally complement the conversion of trypsinogen to active trypsin in a time- and concentration dependent manner. We found corroborative evidence that ethanol affects cell signaling and sensitizes cells to the effects of CCK stimulation. Moreover, we found that the initiation of premature trypsin activity does not always succeed changes in [Ca2+]i. We also found evidence to support one existing pathophysiological theory which states that changes in [Ca2+]i are not a necessary indicator of premature trypsin activation. This initiating mechanism pathway may serve to complicate the development of therapeutic treatment and would suggest that other intrinsic factors may play a role in premature intracellular trypsin activation. These results not only indicate that CCK, ethanol and/or nicotine can trigger Ca2+ and pH responses, which may lead to autoactivation of digestive enzymes, but more importantly, may provide further evidence that Ca2+ is not the only initiating indicator of acute pancreatitis. Understanding the underlying cellular mechanism(s) by which ethanol and nicotine modulate pancreatic acinar cell activity and lead to premature intracellular activation of zymogens is paramount in design considerations which would identify pharmaceuticals effective in preventative and therapeutic treatment for acute pancreatitis.
In exocrine acinar cells, Ca2+-activated Cl channels in the apical membrane are essential for fluid secretion, but it is unclear whether such channels are important for Cl uptake at the base. Whole-cell current recording, combined with local uncaging of caged Ca2+, was used to reveal the Cl channel distribution in mouse pancreatic acinar cells, where 90% of the current activated by Ca2+ in response to acetylcholine was carried by Cl. When caged Ca2+ in the cytosol was uncaged locally in the apical pole, the Cl- current was activated, whereas local Ca2+ uncaging in the basal or lateral areas of the cell had no effect. Even when Ca2+ was uncaged along the whole inner surface of the basolateral membrane, no Cl- current was elicited. There was little current deactivation at a high cytosolic Ca2+ concentration ([Ca2+]c), but at a low [Ca2+]c there was clear voltage-dependent deactivation, which increased with hyperpolarization. Functional Ca2+-activated Cl- channels are expressed exclusively in the apical membrane and channel opening is strictly regulated by [Ca2+]c and membrane potential. Ca2+-activated Cl- channels do not mediate Cl- uptake at the base, but acetylcholine-elicited local [Ca2+]c spiking in the apical pole can regulate fluid secretion by controlling the opening of these channels in the apical membrane.
The acquisition of cell motility is a required step in order for a cancer cell to migrate from the primary tumor and spread to secondary sites (metastasize). For this reason, blocking tumor cell migration is considered a promising approach for preventing the spread of cancer. However, cancer cells just as normal cells can migrate by several different modes referred to as "amoeboid," "mesenchymal," and "collective cell." Under appropriate conditions, a single cell can switch between modes. A consequence of this plasticity is that a tumor cell may be able to avoid the effects of an agent that targets only one mode by switching modes. Therefore, a preferred strategy would be to target mechanisms that are shared by all modes. This chapter reviews the evidence that Ca(2+) influx via the mechanosensitive Ca(2+)-permeable channel (MscCa) is a critical regulator of all modes of cell migration and therefore represents a very good therapeutic target to block metastasis.
Living tissues are complex organizations of individual cells and to perform their specific functions the activity of each cell within the tissue must be regulated in a coordinated manner. The mechanisms through which this regulation occurs can be equally complex, but a common way to exert control is via neural transmission or hormonal stimulation. Irrespective of the organization of the extracellular control system, the regulatory signals need to be translated into an intracellular messenger that can modulate the cellular processes. Again, there are a variety of intracellular messengers that achieve this aim, including cAMP, cGMP and NO, but here we focus on the calcium ion as the internal messenger. The objective of this article is to provide an overview of the basic mechanisms of how Ca 2+ serves as a signaling messenger. For greater detail, the reader must refer to the many extensive reviews (for example, Berridge et al., 2003; Berridge et al., 2002). The details of the individual mechanisms are extremely important since they can confer specificity on the signaling model. As a result, model simulations of Ca 2+ signaling are most useful when the model is designed for a specific cell type and sufficient experimental detail can be incorporated.
Using a Ca2+-sensitive fluorescent indicator, Fura-2/AM, and a metallochromic dye, arsenazo, we measured the intracellular concentration of Ca2+ ([Ca2+]i
) and the content of total calcium in isolated acinar cells of the rat submandibular salivary gland. It was shown that the influence of a mercaptide-forming compound, sodium p-chloromercuribenzoate (pChMB), increased both the [Ca2+]i
and content of total calcium but did not change the intensity of exocytosis. Such a situation is probably related to the fact that pChMB inhibits plasmalemmal Ca2+-ATPase (PMCA). The absence of changes in the exocytotic activity can be explained as follows: the influence of a pChMB-induced significant increase in the [Ca2+]i
is neutralized due to the functioning of Ca2+-ATPases of the endoplasmic reticulum (SERCA), which pump Ca2+ into the store. Incubation of a microsomal fraction with pChMB resulted in suppression of the specific PMCA and SERCA activities with apparent constants of inhibition (I
50) 245 and 52 μM, respectively. Dithiothreitol (DTT, 0.1 mM) increased the PMCA and SERCA activities (probably facilitating the access of substrate to the active centers of ATPases at the expense of a decrease in the number of disulfide bonds, which is followed by changes in the conformation of intracellular hydrophilic loops of their molecules). Dithiothreitol also recovered the suppression of PMCA and SERCA activities induced by pre-incubation with pChMB (by 45 and 32%, respectively); these activities did not, however, reach the initial levels. A probable interpretation of this fact is that DTT shields from the action of pChMB only superficial but not sterically less accessible SH groups. Limited proteolysis of the microsomes by α-chymotrypsin decreased the specific PMCA and SERCA activities by 16 and 60%, respectively. Incubation of the microsomes in an α-chymotrypsin-containing medium (15 sec) with subsequent addition of 150 μM pChMB exerted almost no influence on the PMCA activity, whereas the SERCA activity dramatically increased (by 146%). This fact allows us to suggest that α-chymotrypsin is capable of eliminating the inhibitory effect of pChMB on the SERCA activity; the mechanism of this effect remains unknown. Therefore, functionally important SH groups are present in the catalytic and active centers of both PMCA and SERCA; superficial SH groups dominate in the PMCA molecules, whereas SERCA is controlled by more deeply localized SH groups.
Intracellular Ca
2+ oscillations most often appear as regular spikes. However, complex Ca
2+ oscillations in the form of bursting or chaos have also been observed. These oscillations most probably originate from the interplay between multiple regulatory mechanisms. From an experimental point of view, the mechanism underlying these types of Ca
2+ oscillations remain poorly understood. In this review, we first briefly review the various types of regulatory mechanism known to regulate Ca
2+ oscillations, together with the related theoretical models. We show that the interplay between the activation of an InsP3-metabolizing enzyme ( InsP
3 3-kinase) by Ca
2+ and Ca
2+-induced Ca
2+ release is a plausible explanation for at least some complex Ca
2+ oscillations. This regulation indeed provides a mechanism for the self-modulation of the InsP
3 signal, thus leading to a phenomenon analogous to the periodic forcing of an autonomous oscillator. We also briefly assess the role of luminal Ca
2+ in the onset of Ca
2+ oscillations, as well as the link between bistability and Ca
2+ oscillations.
Calcium ions are the most ubiquitous and pluripotent signalling molecules, which regulate a wide array of physiological and pathological reactions. The specific system, controlling cellular Ca2+ homeostasis appeared very early in the evolution, being initially survival system preventing Ca2+-mediated cell damage. Subsequently, the steep Ca2+ gradients maintained by Ca2+ homeostatic molecular cascades became the basis for Ca2+ signalling. This signalling system utilises Ca2+ channels and transporters localised in plasmalemma and intracellular membranes to create highly organised and compartmentalised cytosolic Ca2+ fluctuations occurring within the spatial and temporal domains. Changes in cytosolic Ca2+ concentrations regulate a multitude of Ca2+-dependent proteins, which serve as “Ca2+ sensors” and thus the effectors of Ca2+ signalling system.
Cell death following cerebral ischemia is mediated by a complex pathophysiologic interaction of different mechanisms. In this Chapter we will outline the basic principles as well as introduce in vitro and in vivo models of cerebral ischemia. Mechanistically, excitotoxicity, peri-infarct depolarization, inflammation and apoptosis seem to be the most relevant mediators of damage and are promising targets for neuroprotective strategies.
Ca2+ is an important regulator of many cell functions including proliferation, apoptosis, movements, secretion, contraction, excitation, and differentiation. The regulation of these different cell functions is encoded by the specific temporal and spatial distribution of Ca2+ signals. In degenerative diseases mutations can lead to changes in cell functions in the worst case to apoptosis. Thus analysis of signals arising as changes in intracellular free Ca2+ represent an important step towards the understanding of mutation-dependent or environmental impact into cell function. The classic approach to study changes in intracellular free Ca2+ is the measurement of intracellular Ca2+ by using Ca2+-sensitive fluorescence dyes in conjunction with fluorescence microscopy as a method called Ca2+ imaging.
In this chapter the basic method and a short theoretical background will be provided to perform Ca2+-imaging experiments. As a model cultured retinal pigment epithelial cells will be used. The basic steps of the method are the loading of the cells with the fluorescence dye by incubation with a membrane permeable ester of the dye. The next step would be the application of an agonist which can be further analyzed by blockers of enzymes or by manipulating the different Ca2+-storing compartments which contribute to changes in intracellular free Ca2+. At the end of an experiment an on-cell type of calibration will be performed to calculate the underlying concentration of intracellular free Ca2+. Furthermore, the successful calibration of an experiment can be used as a measure of a reliable experiment. In addition to that, three examples for basic experiments will be given which can lead to a first insight into the mechanism underlying changes in cytosolic free Ca2+as a second messenger.
Identification and visualization of specific cells and cellular structures in the retina are fundamental for understanding the visual process, retinal development, disease progression, and therapeutic intervention. The increased usage of transgenic and naturally occurring mutant mice has further emphasized the need for retinal cell-specific imaging. Immunofluorescence microscopy of retinal cryosections and whole mount tissue labeled with cell-specific markers has emerged as the method of choice for identifying specific cell populations and mapping their distribution within the retina. In most cases indirect labeling methods are employed in which lightly fixed retinal samples are first labeled with a primary antibody targeted against a cell-specific protein of interest and then labeled with a fluorescent dye-tagged secondary antibody that recognizes the primary antibody. The localization and relative abundance of the protein can readily be imaged under a conventional fluorescent or confocal scanning microscope. Immunofluorescence labeling can be adapted for imaging more than one protein antigen through the use of multiple antibodies and different, nonoverlapping fluorescent dyes. A number of well-characterized immunochemical markers are now available for detecting photoreceptors, bipolar cells, amacrine cells, horizontal cells, Müller cells, and retinal pigment epithelial cells in the retina of mice, and other mammals.
The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+-dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+-signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell.
The endoplasmic reticulum (ER) is a complex and highly dynamic three-dimensional intracellular membranous system, which acts
as a dynamic calcium store in the majority of eukaryotic cells. The special arrangement of intra-ER Ca2+ buffers, characterized by low affinity for Ca2+, in combination with SERCA pump activity keeps intraluminal Ca2+ ([Ca2+]L) at ∼0.1–0.8 mM (Cell Calcium 38:303–310, 2005), thus creating a steep electrochemical gradient aimed at the cytosol. Activation
of ER Ca2+ channels results in Ca2+ release, which contributes to [Ca2+]i elevation, whereas SERCA-dependent Ca2+ uptake assists termination of cytosolic Ca2+ signals. In addition, the continuous luminal space can act as a travelling route for free Ca2+ ions (“Ca2+ tunnels”), thus bypassing cytosolic Ca2+ buffers and preventing mitochondrial Ca2+ uptake or loss of Ca2+ over the plasma membrane. Furthermore, changes in [Ca2+]L regulate ER-resident chaperones, responsible for postranslational protein processing. Thus, [Ca2+]L integrates various signalling events and establishes a link between fast signalling, associated with the ER Ca2+release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. This paper overviews
modern techniques for the imaging of [Ca2+]L using synthetic fluorescent Ca2+ dyes. The methods for ER dye loading, with a particular emphasis on employment of ER targeted esterases (the Targeted-Esterase
induced Dye loading, TED) to increase specific accumulation of the probes within the ER lumen are described in detail.
Key wordsCalcium imaging-Calcium indicator-Esterase-Endoplasmic reticulum-Protein targeting-Neurons
The endoplasmic reticulum (ER) is the main intracellular agonist-sensitive Ca2+ store, and is involved in the regulation of a wide range of cellular functions depending on cytosolic Ca2+. In addition, it has recently been recognized that Ca2+ regulates also processes occurring in the ER lumen, such as protein synthesis and trafficking, and cellular responses to
stress. Accordingly, perturbation of ER Ca2+ homeostasis appears to be a key component in the development of several pathological situations. In this chapter, after providing
an overview of the Ca2+ signaling components of the ER, we briefly summarize their role in basic pathophysiological processes and specific diseases.
Diabetic neuropathy is a frequent complication of diabetes mellitus, for which no adequate clinical treatment is currently available. One of the main reasons for the absence of effective treatment of this disease is that information on how metabolic, vascular, and other abnormalities involved in the pathogenesis of diabetic neuropathy lead to dysfunction of nerve cells and pathways remains insufficient. Recent studies demonstrated that substantial abnormalities of calcium homeostasis in input neurons of the somatosensory nociceptive system are associated with many symptoms of diabetic neuropathy. Although proof of the causal linkage between calcium abnormalities and neuropathic complications is not conclusive, current research in neuroscience mostly indicates that such a linkage exists. Practically all known modifications of synaptic transmission in both central and peripheral nervous systems result from calcium-dependent modifications of the molecular players involved in this transmission. This is why the main goal of our review is to analyze in detail the fundamental cellular and molecular calcium-regulating mechanisms that are deteriorated in diabetes. As an important end-point of the proposed review, the capability of a widely used calcium channel blocker, nimodipine, to correct cytosolic and endoplasmic reticulum calcium abnormalities in neurons of the dorsal root ganglia and spinal dorsal horn and possible curative value of this agent in diabetic neuropathy are discussed.
For many years, the endoplasmic reticulum (ER) was considered to be involved in rapid signalling events due to its ability to serve as a dynamic calcium store capable of accumulating large amounts of Ca2+ ions and of releasing them in response to physiological stimulation. Recent data significantly increased the importance of the ER as a signalling organelle, by demonstrating that the ER is associated with specific pathways regulating long-lasting adaptive processes and controlling cell survival. The ER lumen is enriched by enzymatic systems involved in protein synthesis and correcting post-translational folding of these proteins. The processes of post-translational protein processing are controlled by a class of specific enzymes known as chaperones, which in turn are regulated by the free Ca2+ concentration within the ER lumen ([Ca2+]L). At the same time, a high [Ca2+]L determines the ability of the ER to generate cytosolic Ca2+ signals. Thus, the ER is able to produce signals interacting within different temporal domains. Fast ER signals result from Ca2+ release via specific Ca2+-release channels and from rapid movements of Ca2+ ions within the ER lumen (calcium tunneling). Long-lasting signals involve Ca2+-dependent regulation of chaperones with subsequent changes in protein processing and synthesis. Any malfunctions in the ER Ca2+ homeostasis result in accumulation of unfolded proteins, which in turn activates several signalling systems aimed at appropriate compensatory responses or (in the case of severe ER dysregulation) in cellular pathology and death (ER stress responses). Thus, the Ca2+ ion emerges as a messenger molecule, which integrates various signals within the ER: fluctuations of the [Ca2+]L induced by signals originating at the level of the plasmalemma (i.e., Ca2+ entry or activation of the metabotropic receptors) regulate in turn protein synthesis and processing via generating secondary signalling events between the ER and the nucleus.
Two recombinant aequorin isoforms with different Ca²⁺ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca²⁺ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca²⁺]er or [Sr²⁺]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca²⁺] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca²⁺ concentration, the [Ca²⁺]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca²⁺]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr²⁺ was used as a Ca²⁺ surrogate), the bulk of the organelle was shown to accumulate free [cation²⁺]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca²⁺], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca²⁺ or Sr²⁺, is presented. Moreover, a few other key problems concerning the ER Ca²⁺ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca²⁺]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca²⁺] the cation is simultaneously accumulated; (b) stimulation of Ca²⁺ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation²⁺]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca²⁺], observed when the cells are incubated in Ca²⁺-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).
To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.
In this study the effects of A23187 and thapsigargin on the degradation of T-cell antigen receptor-beta (TCR-beta) and CD3-delta in the endoplasmic reticulum have been studied. Preliminary experiments showed that these drugs had different effects on the secretory pathway. Depletion of cellular calcium pools by incubation of cells with A23187 in calcium-free medium blocked transport between the endoplasmic reticulum and the Golgi apparatus whereas thapsigargin caused a modest increase in transport. When added to cells transfected with TCR-beta or CD3-delta the drugs caused an immediate stimulation of proteolysis of presynthesized protein and at maximum effective concentrations caused a 3-fold increase in the rate of degradation. They did not affect the lag period of 1 h which precedes degradation of newly synthesized proteins. Chelation of cytosolic calcium also accelerated degradation, suggesting that depletion of calcium from the endoplasmic reticulum was the main stimulus of proteolysis and that increased degradation was not caused by a transient increase in cytosolic calcium levels. The selectivity of degradation in the endoplasmic reticulum was maintained. A23187 had no effect on the stability of CD3-gamma nor co-transfected epsilon-beta dimers. Calcium depletion increased the overall rate of degradation in the endoplasmic reticulum and increased the rate of proteolysis of an "anchor minus" beta chain. The results suggested that proteolysis within the endoplasmic reticulum may be regulated by the high concentrations of Ca2+ which are stored in the organelle. Ca2+ may be required for protein folding. Calcium depletion may have caused the beta and delta chains to adopt a conformation that was more susceptible to proteolysis. Alternatively, calcium depletion may have disrupted the lumenal content of the endoplasmic reticulum and increased the access of proteases to potential substrates.
To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.
Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.
Ca2+ mobilization by hormones, ionomycin, and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) were studied to determine whether Ca2+ release is a continuous or a quantal process. Hormone-mediated Ca2+ release occurs only during the first 2-4 s of stimulation. Stimulation of acini with a maximal hormone concentration following stimulation with a submaximal concentration resulted in free cytosolic Ca2+ concentration ([Ca2+]i) increase and 45Ca efflux. The peak [Ca2+]i increase induced by a maximal concentration of agonist was nearly constant when cells were prestimulated with a submaximal dose for 1-15 min. Submaximal hormone concentrations release only a fraction of intracellular 45Ca2+, after which intracellular Ca2+ content remains constant. The partially released stores remain depleted until cell stimulation is terminated, at which time the stores reload with Ca2+. For comparison, increasing concentrations of ionomycin resulted in increasing rates of Ca2+ release. Each ionomycin concentration released all the Ca2+ from intracellular stores. We therefore conclude that hormone-evoked Ca2+ release is a quantal rather than a continuous process. In permeabilized cells, increasing concentrations of Ins-1,4,5-P3 resulted in an increased fraction of Ca2+ release. No submaximal Ins-1,4,5-P3 concentration was capable of releasing all the Ins-1,4,5-P3-mobilizable Ca2+. Therefore, it appears that the quantal properties of hormone-evoked Ca2+ release reflect the quantal properties of Ins-1,4,5-P3-mediated Ca2+ release from intracellular stores.
Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.
Calsequestrin (CS) is the protein responsible for the high-capacity, moderate affinity binding of Ca2+ within the terminal cisternae of the sarcoplasmic reticulum, believed up to now to be specific for striated muscle. The cells of two nonmuscle lines (HL-60 and PC12) and of two rat tissues (liver and pancreas) are shown here to express a protein that resembles CS in many respects (apparent mass and pH-dependent migration in NaDodSO4/PAGE; blue staining with StainsAll dye; Ca2+ binding ability) and is specifically recognized by affinity-purified antibodies against skeletal muscle CS. In these cells, the CS-like protein is shown by immunofluorescence and immunogold procedures to be localized within peculiar, heretofore unrecognized structures distributed throughout the cytoplasm. These structures appear to be discrete organelles, which we propose to be named "calciosomes." By cell fractionation (Percoll gradient and free-flow electrophoresis), the CS-like protein of HL-60 cells is shown to copurify with the markers of the inositol 1,4,5-trisphosphate (Ins-P3)-sensitive Ca2+ store, whereas the markers of other organelles (endoplasmic reticulum, Golgi complex, mitochondria, endosomes) and of the plasma membrane do not. Calciosome might thus be the intracellular target of Ins-P3--i.e., the source of the Ca2+ redistributed to the cytosol following receptor-triggered generation of the messenger.
Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions.
The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca2+ pumps and characterizing their role in initiation and propagation of Ca2+ waves. Specific and polarized expression of Ca2+ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca2+ release with ionomycin on Ca2+ release evoked by agonists and Ca2+ uptake induced by antagonists. Despite their polarized expression, Ca2+ uptake by SERCA pumps and Ca2+ efflux by PMCA resulted in uniform reduction in [Ca2+]i. Surprisingly, inhibition of the SERCA pumps, but not Ca2+ release by ionomycin, eliminated the distinct initiation sites and propagated Ca2+ waves, leading to a uniform increase in [Ca2+]i. In addition, inhibition of SERCA pumps reduced the rate of Ca2+ release from internal stores. The implication of these findings to rates of Ca2+ diffusion in the cytosol, compartmentalization of Ca2+ signaling complexes, and mechanism of Ca2+ wave propagation are discussed.
Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.
Glial cells respond to various electrical, mechanical, and chemical stimuli, including neurotransmitters, neuromodulators, and hormones, with an increase in intracellular Ca2+ concentration ([Ca2+]i). The increases exhibit a variety of temporal and spatial patterns. These [Ca2+]i responses result from the coordinated activity of a number of molecular cascades responsible for Ca2+ movement into or out of the cytoplasm either by way of the extracellular space or intracellular stores. Transplasmalemmal Ca2+ movements may be controlled by several types of voltage- and ligand-gated Ca(2+)-permeable channels as well as Ca2+ pumps and a Na+/Ca2+ exchanger. In addition, glial cells express various metabotropic receptors coupled to intracellular Ca2+ stores through the intracellular messenger inositol 1,4,5-triphosphate. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca2+ responses. Such agonist specificity may provide a means for intracellular and intercellular information coding. Calcium signals can traverse gap junctions between glial cells without decrement. These waves can serve as a substrate for integration of glial activity. By controlling gap junction conductance, Ca2+ waves may define the limits of functional glial networks. Neuronal activity can trigger [Ca2+]i signals in apposed glial cells, and moreover, there is some evidence that glial [Ca2+]i waves can affect neurons. Glial Ca2+ signaling can be regarded as a form of glial excitability.
Although fluctuations in cytosolic Ca2+ concentration have a crucial role in relaying intracellular messages in the cell, the dynamics of Ca2+ storage in and release from intracellular sequestering compartments remains poorly understood. The rapid release of stored Ca2+ requires large concentration gradients that had been thought to result from low-affinity buffering of Ca2+ by the polyanionic matrices within Ca2+-sequestering organelles. However, our results here show that resting luminal free Ca2+ concentration inside the endoplasmic reticulum and in the mucin granules remains at low levels (20-35 microM). But after stimulation, the free luminal [Ca2+] increases, undergoing large oscillations, leading to corresponding oscillations of Ca2+ release to the cytosol. These remarkable dynamics of luminal [Ca2+] result from a fast and highly cooperative Ca2+/K+ ion-exchange process rather than from Ca2+ transport into the lumen. This common paradigm for Ca2+ storage and release, found in two different Ca2+-sequestering organelles, requires the functional interaction of three molecular components: a polyanionic matrix that functions as a Ca2+/K+ ion exchanger, and two Ca2+-sensitive channels, one to import K+ into the Ca2+-sequestering compartments, the other to release Ca2+ to the cytosol.
In the classical view, transmission of signals across synapses in the mammalian brain involves changes in the membrane potential of the postsynaptic cell. The use of high-resolution cellular imaging has revealed excitatory synapses at which postsynaptic, transient alterations in calcium ion concentration are tightly associated with electrical responses. Here, by investigating the synapse between parallel glutamatergic fibres and Purkinje cells in the mouse cerebellum, we identify a class of postsynaptic responses that consist of transient increases in dendritic Ca2+ concentration but not changes in somatic membrane potential. Our results indicate that these synaptic Ca2+ transients are mediated by activation of metabotropic glutamate-responsive mGluR1-type receptors and require inositol-1,4,5-trisphosphate-mediated Ca2+ release from intradendritic stores. The new type of synaptic response is restricted to postsynaptic microdomains, which range, depending on the frequency of stimulation, from individual spines to small spinodendritic compartments. Thus, the synaptic Ca2+-release signal may be one of the critical cues that determine the input specificity of long-term depression, a well-established form of activity-dependent plasticity at these synapses.
The endoplasmic reticulum (ER) is the major compartment for the processing and quality control of newly synthesized proteins. Green fluorescent protein (GFP) was used as a noninvasive probe to determine the viscous properties of the aqueous lumen of the ER. GFP was targeted to the ER lumen of CHO cells by transient transfection with cDNA encoding GFP (S65T/F64L mutant) with a C-terminus KDEL retention sequence and upstream prolactin secretory sequence. Repeated laser illumination of a fixed 2-μm diameter spot resulted in complete bleaching of ER-associated GFP throughout the cell, indicating a continuous ER lumen. A residual amount (<1%) of GFP-KDEL was perinuclear and noncontiguous with the ER, presumably within a pre- or cis-Golgi compartment involved in KDEL-substrate retention. Quantitative spot photobleaching with a single brief bleach pulse indicated that GFP was fully mobile with a t1/2 for fluorescence recovery of 88 ± 5 ms (SE; 60× lens) and 143 ± 8 ms (40×). Fluorescence recovery was abolished by paraformaldehyde except for a small component of reversible photobleaching with t1/2 of 3 ms. For comparison, the t1/2 for photobleaching of GFP in cytoplasm was 14 ± 2 ms (60×) and 24 ± 1 ms (40×). Utilizing a mathematical model that accounted for ER reticular geometry, a GFP diffusion coefficient of 0.5-1 × 10−7 cm2/s was computed, 9-18-fold less than that in water and 3-6-fold less than that in cytoplasm. By frequency-domain microfluorimetry, the GFP rotational correlation time was measured to be 39 ± 8 ns, ∼2-fold greater than that in water but comparable to that in the cytoplasm. Fluorescence recovery after photobleaching using a 40× lens was measured (at 23°C unless otherwise indicated) for several potential effectors of ER structure and/or lumen environment: t1/2 values (in ms) were 143 ± 8 (control), 100 ± 13 (37°C), 53 ± 13 (brefeldin A), and 139 ± 6 (dithiothreitol). These results indicate moderately slowed GFP diffusion in a continuous ER lumen.
Agonist-evoked cytosolic Ca(2+) spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca(2+) concentration using simultaneous imaging of Ca(2+)-sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca(2+)-releasing messenger inositol 1,4, 5-trisphosphate (IP(3)), the local repetitive Ca(2+) responses in the granule area were transformed into a global rise in the cellular Ca(2+) concentration. In the absence of IP(3), CCCP had no effect on the cytosolic Ca(2+) levels. Antimycin and antimycin + oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca(2+) diffusion from the primary Ca(2+) release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca(2+) signals spread all over the cytosol.
Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.
Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.
Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions.
• The droplet technique was used in this study to measure total calcium loss from pancreatic acinar cells due to calcium extrusion. The calcium binding capacity of the cytosol (kc) was measured as the ratio of the decrease in the total calcium concentration of the cytosol of the cell (Δ[Ca]c) and the synchronously occurring decrease in the free calcium ion concentration in the cytosol (Δ[Ca2+]c). The calcium dependency of the calcium binding capacity was determined by plotting values of kc against the corresponding [Ca2+]c. The rise in the cytosolic Ca2+ concentration of pancreatic acinar cells was triggered by stimulation with a supramaximal dose of cholecystokinin (CCK). The recovery of [Ca2+]c during continued exposure to the agonist was due to calcium extrusion from the cell. • The calcium binding capacity was about 1500-2000 for the [Ca2+]c range 150-500 nM. The mechanism of buffering was not investigated in this study. The calcium binding capacity of the cytosol did not vary significantly with [Ca2+]c in this range. The CCK-evoked decrease in the total calcium concentration in the lumen of the endoplasmic reticulum (ER) can be estimated from our data, taking into account previously published values for the volume of the ER in pancreatic acinar cells. Comparing the decrease in the total ER calcium concentration with our recently reported values for agonist-induced reductions in the free Ca2+ concentration inside the ER, we estimate that the calcium binding capacity of the ER is approximately 20. In pancreatic acinar cells we have therefore found a difference of two orders of magnitude in the efficiency of calcium buffering in the cytosol and the ER lumen.
The nuclear envelope has a relatively small volume, but is connected up to the vastly larger endoplasmic reticulum. The Ca2+ concentration in the lumen of the interconnected nuclear envelope and endoplasmic reticulum network is in the resting state maintained at a level of more than 100 μM. There are specific Ca2+ release channels present in the inner nuclear membrane that can be activated by inositol trisphosphate or cADP ribose. The system, therefore, allows selective release of Ca2+ into the nucleoplasm which could be important for the control of specific types of gene expression.
Ca2+ within intracellular stores has been proposed to act with cytosolic inositol 1,4,5-trisphosphate (InsP3) to cause opening of the integral Ca2+ channel of the InsP3 receptor, leading to mobilization of intracellular Ca2+ stores [FEBS Lett. 263:5-9 (1990)]. We have tested that suggestion in saponin-permeabilized rat hepatocytes by manipulating the Ca2+ content of the stores and then determining their sensitivity to InsP3, while keeping the cytosolic Ca2+ concentration constant. Stores depleted of Ca2+ by incubation with ionomycin were significantly less sensitive to InsP3, an effect thought likely to result from the decrease in luminal free Ca2+ concentration rather than from direct effects of ionomycin on InsP3 binding or Ca2+ permeability. The luminal free Ca2+ concentration of stores loaded in the presence of pyrophosphate appeared to be substantially reduced, and again there was a significant inverse correlation between the estimated free Ca2+ concentration of the stores and their sensitivity to InsP3. By following the kinetics of 45Ca2+ uptake into empty stores in the presence of inositol trisphosphorothioate, a stable InsP3 analogue, we demonstrated that stores respond to inositol trisphosphorothioate only after their luminal free Ca2+ concentration exceeds a critical level. We conclude that InsP3 and luminal Ca2+ together regulate Ca2+ mobilization from intracellular stores, and we discuss some of the implications of this interaction for the complex Ca2+ signals evoked by extracellular stimuli.
Specific inhibitors of the endoplasmic-reticulum Ca2+ pump will deplete intracellular stores and are therefore useful to study the role of store depletion on plasma-membrane Ca2+ permeability. We now report that the Ca(2+)-pump inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) reduces the passive Ca2+ leak from the internal stores in permeabilized A7r5 vascular smooth-muscle cells. This aspecific effect occurred at concentrations that are normally used to empty the stores in intact cells. Cyclopiazonic acid exerted a similar, although less pronounced effect, while thapsigargin did not affect the passive Ca2+ leak. The inositol 1,4,5-trisphosphate-mediated Ca2+ release was not affected. tBuBHQ and cyclopiazonic acid cannot therefore be used as specific tools to probe the mechanism of receptor-mediated Ca2+ entry.
1. Histamine-stimulated mobilization of intracellular Ca2+ stores was monitored in intact and permeabilized populations of HeLa cells using both the fluorescent Ca2+ indicator Fura-2 and 45Ca2+ measurements. Digital video imaging of Fura-2-loaded cells was used to measure the intracellular calcium concentration ([Ca2+]i) of single cells. 2. In populations of HeLa cells, histamine caused a concentration-dependent increase in cytoplasmic [Ca2+]. The initial transient increase was independent of extracellular Ca2+ (Ca2+o) and was followed by a sustained increase that was abolished by removal of Ca2+o. 3. In Ca(2+)-free medium ([Ca2+]o < 1 microM), a maximal histamine concentration (25 microM) caused a transient increase in [Ca2+]i, and a subsequent challenge with histamine failed to evoke a further response indicating that the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores had been completely emptied. Lower concentrations of histamine (0.5-10 microM) caused smaller, concentration-dependent increases in [Ca2+]i that were also transient. After exposure to these low histamine concentrations, where [Ca2+]i returned to baseline within 2 min, addition of a higher histamine concentration evoked a further increase in [Ca2+]i. The second increase in [Ca2+]i was inversely proportional to the increase caused by the first exposure to histamine, indicating that Ca2+ released in the initial response was not substantially resequestered into histamine-sensitive stores. 4. Single HeLa cells challenged with low concentrations of histamine in Ca(2+)-free medium responded with transient increases in [Ca2+]i, but individual cells differed in their sensitivity with 51% of cells responding to 1 microM, and 98% responding to 25 microM-histamine. 5. When single cells in Ca(2+)-free medium were challenged with stepwise increases in histamine concentration, they responded to each step with a transient [Ca2+]i increase after which [Ca2+]i returned to baseline within 1 min. Prolonging the interval between histamine additions by up to 25 min did not affect the [Ca2+]i increase evoked by a subsequent histamine addition. 6. Unidirectional 45Ca2+ efflux from saponin-permeabilized HeLa cells showed that, under conditions that prevented Ca2+ resequestration, submaximal concentrations of InsP3 rapidly emptied only a fraction of the InsP3-sensitive Ca2+ stores. The failure of low InsP3 concentrations to fully mobilize the InsP3-sensitive Ca2+ stores was not a consequence of InsP3 degradation. 7. We conclude that within single HeLa cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to InsP3, and the fraction of Ca2+ stores mobilized by InsP3 increases as the InsP3 concentration increases.
The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.
Recent imaging experiments have revealed the distinct spatial dynamics of second-messenger actions. In general, actions of Ca2+ tend to be local, whereas those of other messengers such as inositol 1,4,5-trisphosphate (IP3) and cAMP are long range. In pancreatic acinar cells, IP3 generated at the base can diffuse across the cell and evoke a spatially confined Ca2+ signal in the apical pole, triggering enzyme and fluid secretion. Similar mechanisms might also operate in other cell types. We propose that the distinct dynamics of messengers might be relevant to neuronal function: IP3 and cAMP could convey signals over long distances along neurites, and serve as mediators for association and co-operation, for example, during learning.
Hepatocytes respond to inositol 1,4,5-trisphosphate (InsP3)-linked agonists with frequency-modulated oscillations in the intracellular free calcium concentration ([Ca2+]i), that occur as waves propagating from a specific origin within each cell. The subcellular distribution and functional organization of InsP3-sensitive Ca2+ pools has been investigated, in both intact and permeabilized cells, by fluorescence imaging of dyes which can be used to monitor luminal Ca2+ content and InsP3-activated ion permeability in a spatially resolved manner. The Ca2+ stores behave as a luminally continuous system distributed throughout the cytoplasm. The structure of the stores, an important determinant of their function, is controlled by the cytoskeleton and can be modulated in a guanine nucleotide-dependent manner. The nuclear matrix is devoid of Ca2+ stores, but Ca2+ waves in the intact cell propagate through this compartment. The organization of [Ca2+]i signals has also been investigated in the perfused liver. Frequency-modulated [Ca2+]i oscillations are still observed at the single cell level, with similar properties to those in the isolated hepatocyte. The [Ca2+]i oscillations propagate between cells in the intact liver, leading to the synchronization of [Ca2+]i signals across part or all of each hepatic lobule.
1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura-2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)
1. Intracellular free calcium concentration ([Ca2+]i) in isolated non-identified Helix pomatia neurones has been clamped at different physiologically significant levels by a feedback system between the fluorescent signal of fura-2 probe loaded into the cell and ionophoretic injection of Ca2+ ions through a CaCl2-loaded microelectrode. The membrane potential of the neurone has also been clamped using a conventional two-microelectrode method. 2. Special measurements have shown that the transport indices of injecting microelectrodes filled with 50 mM CaCl2 are quite variable (0.11 +/- 0.06, mean +/- S.D.). However, for each electrode the transport indices remained stable during several injection trials into a solution drop having the size of a neurone. The spread of calcium ions from the tip of the microelectrode across the cytosol of the neurone terminated within 2-4 s. The spatial difference in [Ca2+]i at this time did not exceed 10%. 3. Clamping of [Ca2+]i at a new increased level was accompanied by a transient of the Ca(2+)-injecting current. To increase [Ca2+]i by 0.1 microM, the amount of calcium ions injected during this stage had to be 36 +/- 20 microM Ca2+ per cell volume. Obviously, this transient represents the filling of a fast cytosolic buffer which has to be saturated to reach a new increased level of [Ca2+]i. It was followed by a steady component of Ca(2+)-injecting current, which was quite low (corresponding to injection of 0.39 +/- 0.20 microM s-1 for a 0.1 microM change of [Ca2+]i). This may represent the functioning of Ca(2+)-eliminating systems and corresponds to a similar amount of Ca2+ extruded from the cytoplasm. 4. Changes in the injection current also developed when Ca2+ influx through the membrane was triggered by the activation of voltage-gated calcium channels. The amount of Ca2+ entering the cell during the first seconds of depolarization to--15 mV was equal to 0.59 +/- 0.31 microM s-1 per cell volume. 5. No activation of Ca(2+)-dependent potassium current was observed during the changes in [Ca2+]i to levels exceeding the basal one by several times. Obviously, to activate this current, a much stronger increase in [Ca2+]i is needed in the immediate vicinity of the corresponding channels.
1. The ability of Purkinje cells to rapidly buffer depolarization-evoked intracellular calcium changes (delta [Ca2+]i) was estimated by titrating the endogenous buffer against incremental concentrations of the Ca(2+)-sensitive dye fura-2. 2. In cells from 15-day-old rats, pulse-evoked delta [Ca2+]i were stable during the loading with 0.5 mM fura-2 through the patch pipette. In cells from 6-day-old rats, delta [Ca2+]i decreased by approximately 50% during equivalent experiments. This decrease was not related to changes in Ca2+ influx, since the integral of the Ca2+ currents remained constant throughout the recording. 3. Experiments with high fura-2 concentrations (1.75-3.5 mM) were performed in order to obtain for each cell the curve relating delta [Ca2+]i to fura-2 concentration. From this relationship, values for the Ca2+ binding ratio (the ratio of buffer-bound Ca2+ changes over free Ca2+ changes) were calculated. 4. In Purkinje cells from 15-day-old rats, the Ca2+ binding ratio was approximately 2000, an order of magnitude larger than that of other neurones and neuroendocrine cells studied to date. This Ca2+ binding ratio was significantly smaller (approximately 900) in Purkinje cells from 6-day-old rats. 5. We propose that the large Ca2+ binding ratio of Purkinje cells is related to the presence of large concentrations of Ca2+ binding proteins and that these cells regulate their ability to handle Ca2+ loads during development through changes in the concentration of Ca2+ binding proteins.
Intracellular Ca2+ store depletion induces Ca2+ entry across the plasma membrane, allowing the store to recharge. In our experiments, Ca2+ stores in pancreatic acinar cells were depleted by acetylcholine (ACh) stimulation in Ca2+-free solution. Thereafter, Ca2+ entry was only allowed through a CaCl2-containing pipette attached to the basal membrane. Recharging intracellular Ca2+ stores via a patch pipette occurred without a rise in the cytosolic Ca2+ concentration and depended on the operation of a thapsigargin-sensitive Ca2+ pump. After a period of focal Ca2+ entry, ACh could again evoke a rise in the cytosolic Ca2+ concentration, and this rise always started in the apical secretory pole. Recharging the apical Ca2+ store therefore depends on Ca2+ flow through a tunnel from the basal to the secretory pole, and the endoplasmic reticulum Ca2+ pump is essential for this process.
The organization of calcium (Ca2+) stores in the sarcoplasmic and endoplasmic reticulum (S-ER) is poorly understood. The dynamics of the storage and release of calcium in the S-ER of intact, cultured astrocytes and arterial myocytes were studied with high-resolution imaging methods. The S-ER appeared to be a continuous tubular network; nevertheless, calcium stores in the S-ER were organized into small, spatially distinct compartments that functioned as discrete units. Cyclopiazonic acid (an inhibitor of the calcium pump in the S-ER membrane) and caffeine or ryanodine unloaded different, spatially separate compartments. Heterogeneity of calcium stores was also revealed in cells activated by physiological agonists. These results suggest that cells can generate spatially and temporally distinct calcium signals to control individual calcium-dependent processes.
The spatial organization of endoplasmic reticulum (ER) and nuclear envelope (NE) calcium stores is important for the regulation of localized calcium signals and sustained calcium gradients. Here, we have used a lumenal GFP fusion protein and shown that, in resting cells, large molecules can rapidly diffuse across the cell within the lumenal storage space defined by the ER and NE membranes. Increases in cytosolic calcium concentration reversibly fragmented ER tubules and prevented lumenal diffusion. However, the integrity of the NE was maintained, and a significant fraction of NE lumenal protein accumulated in an NE-associated vesicle. These dynamic properties of ER-NE calcium stores provide insights into the spatiotemporal control of calcium signaling.
The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.
Brain ageing is associated with a marked decline in mental faculties. One hypothesis postulates that sustained changes in the regulation of intracellular Ca2+ concentration, [Ca2+]i, are the major cause of neuronal degeneration. This 'calcium hypothesis' is supported by demonstrations of the impairment in aged neurones of molecular cascades that regulate [Ca2+]i. However, the number of direct measurements of [Ca2+]i in senescent neurones is limited, and the hypothesis cannot be regarded as fully confirmed. Furthermore, physiological brain ageing, at least in certain regions, need not necessarily be a degenerative process accompanied by neuronal loss. Pharmacological manipulation of Ca2+ entry has been shown to be effective in increasing some aspects of cognitive function of the aged brain. Therefore, further exploration of Ca2+ homeostasis and signalling might reveal the mechanisms involved in the age-dependent decline in neuronal performance, and might aid the search for new therapeutic treatments.
The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution
in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts
were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol
1,4,5-triphosphate (IP3)–gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles
in regulation of Ca2+ signaling.
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In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.
A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.
The second messenger inositol-1,4,5-trisphosphate (InsP3) releases Ca2+ from intracellular Ca2+ stores by activating specific receptors on the membranes of these stores. In many cells, InsP3 is a global signalling molecule that liberates Ca2+ throughout the cytoplasm. However, in neurons the situation might be different, because synaptic activity may produce InsP3 at discrete locations. Here we characterize InsP3 signalling in postsynaptic cerebellar Purkinje neurons, which have a high level of InsP3 receptors. We find that repetitive activation of the synapse between parallel fibres and Purkinje cells causes InsP3-mediated Ca2+ release in the Purkinje cells. This Ca2+ release is restricted to individual postsynaptic spines, where both metabotropic glutamate receptors and InsP3 receptors are located, or to multiple spines and adjacent dendritic shafts. Focal photolysis of caged InsP3 in Purkinje cell dendrites also produces Ca2+ signals that spread only a few micrometres from the site of InsP3 production. Uncaged InsP3 produces a long-lasting depression of parallel-fibre synaptic transmission that is limited to synapses where the Ca2+ concentration is raised. Thus, in Purkinje cells InP3 acts within a restricted spatial range that allows it to regulate the function of local groups of parallel-fibre synapses.
We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).
The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.
In the nervous system, Ca2+ signalling is determined primarily by voltage-gated Ca2+-selective channels in the plasma membrane, but there is increasing evidence for involvement of intracellular Ca2+ stores in such signalling. It is generally assumed that neurotransmitter-elicited release of Ca2+ from internal stores is primarily mediated by Ins(1,4,5)P3, as originally discovered in pancreatic acinar cells. The more-recently discovered Ca2+-releasing messenger, cyclic ADP-ribose (cADPR), which activates ryanodine receptors, has so far only been implicated in a few cases, and the possible importance of another Ca2+-releasing molecule, nicotinic acid adenine dinucleotide phosphate (NAADP), has been ignored. Recent investigations of the action of the brain-gut peptide cholecystokinin on pancreatic acinar cells have indicated that NAADP and cADPR receptors are essential for Ca2+ release. Tools are available for testing the possible involvement of NAADP and cADPR in neurotransmitter-elicited intracellular Ca2+ release, and such studies could reveal complex mechanisms that control this release in the nervous system.
In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.
Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.