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![Regulation of the Ca 2 + content of the endoplasmic reticulum The diagram summarizes results from several cell types to illustrate the many mechanisms that may be involved in regulating the luminal [Ca 2 + ] of the intracellular Ca 2 + stores, assumed to reside largely within the endoplasmic reticulum [8]. Changes in cytosolic [Ca 2 + ] affect the rate of Ca 2 + uptake, because most SERCA have Ca 2 + affinities similar to the normal cytosolic [Ca 2 + ] [8]. Our results suggest that luminal [Ca 2 + ] co-operatively stimulates the channel through which Ca 2 + passively leaks from the intracellular Ca 2 + stores. In other cell types, luminal Ca 2 + has also been proposed to inhibit the SERCA in a very co-operative fashion [27]. Finally, ryanodine and InsP 3 receptors are each sequentially stimulated and inhibited by cytosolic Ca 2 + and both may also be stimulated by luminal Ca 2 + .](profile/Colin-Taylor-7/publication/13567963/figure/fig2/AS:601676062924831@1520462160639/Regulation-of-the-Ca-2-content-of-the-endoplasmic-reticulum-The-diagram-summarizes.png)
Regulation of the Ca 2 + content of the endoplasmic reticulum The diagram summarizes results from several cell types to illustrate the many mechanisms that may be involved in regulating the luminal [Ca 2 + ] of the intracellular Ca 2 + stores, assumed to reside largely within the endoplasmic reticulum [8]. Changes in cytosolic [Ca 2 + ] affect the rate of Ca 2 + uptake, because most SERCA have Ca 2 + affinities similar to the normal cytosolic [Ca 2 + ] [8]. Our results suggest that luminal [Ca 2 + ] co-operatively stimulates the channel through which Ca 2 + passively leaks from the intracellular Ca 2 + stores. In other cell types, luminal Ca 2 + has also been proposed to inhibit the SERCA in a very co-operative fashion [27]. Finally, ryanodine and InsP 3 receptors are each sequentially stimulated and inhibited by cytosolic Ca 2 + and both may also be stimulated by luminal Ca 2 + .
Source publication
Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either ionomycin or thapsigargin, although both were required for complete inhibition. Unidirectional efflux of 45Ca2+ after removal of ATP from cells loaded to steady state (1.60+/-0.12 nmol/10(6) cells) was monoexpon...
Contexts in source publication
Context 1
... [25] and others [6,26] observed previously that a small fraction ( 10 %) of the Ca# + actively accumulated into intracellular stores by means of the SERCA was not readily released after inhibition of further Ca# + uptake. We confirmed that observation and now suggest that it results from a steeply co-operative effect of luminal Ca# + on the leak pathway, such that Ca# + becomes trapped within the stores when their luminal [Ca# + ] falls to a critical level ( Figure 5). This form of regulation may have important physiological consequences. ...
Context 2
... has, for example, been suggested [28,29] that the capacitative Ca# + -entry pathway may be activated only after near-complete depletion of the stores and, that in patch-clamped cells, InsP $ is more effective than thapsigargin in activating capacitative Ca# + entry [28]. Tight regulation of the luminal [Ca# + ] of the endoplasmic reticulum by, among other mechanisms, the effect of luminal Ca# + on the passive leak (Figure 5), may therefore serve both to maintain an appropriate environment within the endoplasmic reticulum and to ensure that capacitative Ca# + entry is not activated in- appropriately. ...
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We investigated why oscillations of intracellular Ca2+ concentrations ([Ca2+]i) in endothelial cells challenged by sub-maximal histamine run down in Ca(2+)-free medium despite stores retaining most of their Ca2+. One explantation is that only a small subpopulation of the Ca2+ stores oscillate and are completely emptied of Ca2+. To investigate if in...
The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, i...
Citations
... A similar calculation reported a τ leak of 138 s for primary human T cells [24]. Previous estimates of τ leak based on direct measurements of TG-induced ER Ca 2+ depletion using 45 Ca 2+ as well as the Ca 2+ indicators Mag-Fura-2, Fluo-5 N and D1ER indicated τ leak values of 115-500 s for various cells types [14,27,[55][56][57][58]. In conclusion, τ leak for NALM-6 cells fits within published data. ...
The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca2+ store in the cell, we characterised the Ca2+ homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca2+ leak from ER, Ca2+ storage capacity and store-operated Ca2+ entry were significantly enlarged in the secretory RPMI 8226 cell line. Using an immunoglobulin M heavy chain producing HeLa cell model, we found that the enlarged Ca2+ storage capacity and Ca2+ leak from ER are linked to ER expansion. Our data delineates a developmental remodelling of Ca2+ homeostasis in professional secretory cells in which a high Sec61-mediated Ca2+ leak and, thus, a high Ca2+ turnover in the ER is backed up by enhanced store-operated Ca2+ entry. This article is protected by copyright. All rights reserved.
... Daher strömt Calcium aus dem Hauptspeicherorganell über sogenannte Leckströme. Dieses Entweichen erfolgt jedoch nicht über einen der regulierten Calciumkanäle(Beecroft & Taylor 1998;Camello et al. 2002). Hierbei handelt es sich in der Regel um indirekte und unregulierte Ströme, die dem Konzentrationsgefälle folgen. ...
... Overall, we employ a linear-function representation of the concentration gradient between the ER lumen and cytosolic calcium for luminal calcium leakage into the cytosol, as follows: The ''baseline'' sub-and superscripts indicate that the terms here are at the initial values used for the respective concentrations and hence are constant. Beecroft (43) observed that ER calcium efflux through the leakage mechanism essentially shuts down when luminal calcium levels drop to~7% of baseline concentration. We represent this aspect of leakage via the f shutoff (Ca er ) function, which is a Heaviside step function. ...
Release of inflammatory mediators by mast cells in type 1 immediate-hypersensitivity allergic reactions relies on antigen-dependent increases in cytosolic calcium. Here, we used a series of electron microscopy images to build a 3D reconstruction representing a slice through a rat tumor mast cell, which then served as a basis for stochastic modeling of inositol-trisphosphate-mediated calcium responses. The stochastic approach was verified by reaction-diffusion modeling within the same geometry. Local proximity of the endoplasmic reticulum to either the plasma membrane or mitochondria is predicted to differentially impact local inositol trisphosphate receptor transport. The explicit consideration of organelle spatial relationships represents an important step toward building a comprehensive, realistic model of cellular calcium dynamics.
... Cependant, dans d'autres types cellulaires, la TG n'a pas d'effet sur la fuite de Ca 2+ induite par la suppression de androgènes cytosolique (Hofer et al. 1996;Beecroft and Taylor 1998). ...
Prostate cancer is the second cancer-related cause of decease. Nowadays, treatments aim at decreasing the androgen action on this organ. Unfortunately, with time, patients develop an androgen-independent cancer with fatal issue. Calcium (Ca2+), an ubiquitary second messenger, is involved in several processes such as apoptosis and proliferation. Endoplasmic Reticulum (ER) is a major actor in Ca2+ signalling. Thus, the study of ER channels is critical for developing new therapeutic strategies. Our work led to the identification of two new ER proteins: the translocon and TRPM8 channel (Transient Receptor Potential Melastatin 8). These two channels could represent fundamental elements of calcium signalisation by regulating Ca2+ concentration in the ER. Moreover, the study of Ca2+ signature evolution during prostate carcinogenesis led us to show that Orai1 (identified here as the main carrier of capacitative Ca2+ entry) expression is decreased in advanced prostate cancer. Reversely, TRPV6 (Transient Receptor Potential Vanilloid 6), a Ca2+ channel involved in constitutive Ca2+ entry, is over expressed in prostate cancer later stages. Thus, variations in these proteins expression could be responsible respectively for apoptosis resistance or an increase in prostate cancer cells proliferation. This might explain the evolution of prostate cancer to more aggressive stages.
... In the resting status, steady state ER Ca 2+ levels have been assumed by long time to reflect a balance between the active, MgATP-dependent inward transport and a passive backflux of the ion (e.g., [3][4][5][6][7][8][9]). However, the studies on the pathways/ channels involved in the passive Ca 2+ efflux-often referred to as (basal) Ca 2+ leak-from ER and sarcoplasmic reticulum (SR) have been shadowed by the large interest in channels of these organelles, which are involved in cell signalling and muscle contraction. ...
... The possibility that the pore/channel of the inactive MgATP-dependent Ca 2+ pump contribute to the efflux of Ca 2+ was unlikely. Actually the Ca 2+ pump blocker thapsi-gargin did not affect the rate of Ca 2+ efflux in liver microsomes (Table 1), even in the presence of ATP (Table 1), as previously observed in permeabilized hepatocytes [8]. ...
Steady-state levels of calcium ions in endoplasmic reticulum reflect a balance between active inward transport, mediated by MgATP-dependent Ca(2+) pumps, and passive backflux of the ions, through putative "leak channels". We have investigated the efflux of Ca(2+) from rat liver microsomal vesicles, passively pre-equilibrated in the presence radiolabelled Ca(2+). Similarly, we have also evaluated the efflux of a low-Mwt uncharged compound, i.e., sucrose. The results show that two major passive Ca(2+) efflux pathways exist. One appeared to involve the translocon pore, since it was stimulated by the translocon opener puromycin, and also allowed the passage of sucrose. Putative channels likely mediated the other one, since it required counter ion influx and was inhibited by Gd(3+) and La(3+). The latter pathway did not appear to involve inactive Ca(2+) pumps, Bcl2 proteins, or known channels, such as the InsP3 and ryanodine receptors. While sucrose efflux was highly represented in a rough microsomal subfraction--enriched in the translocon component Sec61alpha--the efflux of Ca(2+) was represented both in smooth and in rough microsomes. We conclude that the passive efflux of Ca(2+) from the (liver) ER could be mediated by both the translocon pore and putative Ca(2+) leak channels. However, the relative role of these Ca(2+) efflux pathways in the intact cell as well as the molecular nature of the Ca(2+) leak channel(s) remain to be clarified.
... We began with the assumption that ER calcium leakage balances SERCA activity at steady state. Beecroft et al. (23) and Bergling et al. (24) estimated the decay constant for ER leakage at 110-140 s. We initially employed the unidirectional model of SERCA activity, where the SERCA is assumed to operate only in one direction: uptake of calcium from the cytosol. ...
... We thus construct the leakage term to balance the SERCA value at baseline concentrations. Beecroft (23) observed that ER calcium efflux through the leakage mechanism essentially shuts down when luminal calcium levels drop to ;7% of baseline concentration. We represent this aspect of leakage via the f shutoff (Ca er ) function, which is a sigmoidal function crafted to smoothly shut down the leakage when the ER calcium reaches this observed threshold. ...
We describe a finite-element model of mast cell calcium dynamics that incorporates the endoplasmic reticulum's complex geometry. The model is built upon a three-dimensional reconstruction of the endoplasmic reticulum (ER) from an electron tomographic tilt series. Tetrahedral meshes provide volumetric representations of the ER lumen, ER membrane, cytoplasm, and plasma membrane. The reaction-diffusion model simultaneously tracks changes in cytoplasmic and ER intraluminal calcium concentrations and includes luminal and cytoplasmic protein buffers. Transport fluxes via PMCA, SERCA, ER leakage, and Type II IP3 receptors are also represented. Unique features of the model include stochastic behavior of IP3 receptor calcium channels and comparisons of channel open times when diffusely distributed or aggregated in clusters on the ER surface. Simulations show that IP3R channels in close proximity modulate activity of their neighbors through local Ca2+ feedback effects. Cytoplasmic calcium levels rise higher, and ER luminal calcium concentrations drop lower, after IP3-mediated release from receptors in the diffuse configuration. Simulation results also suggest that the buffering capacity of the ER, and not restricted diffusion, is the predominant factor influencing average luminal calcium concentrations.
... 2+ sequestering compartments TG treatment of cultured cells results in a rapid emptying of ER Ca 2+ stores (Islam and Berggren 1993). This passive Ca 2+ efflux, or leakage, from intracellular stores is poorly understood but is distinguished from Ca 2+ efflux mediated by IP 3 R and RyR (Beecroft and Taylor 1998). To determine whether the TG-R Ca 2+ sequestering compartment exhibits passive Ca 2+ efflux, we performed kinetic studies in which Ca 2+ accumulation was allowed to proceed for 90 min at which point 1 lM TG was added. ...
... Heterogeneous distribution of the TG-R pool could thus have contributed to the differential cytoplasmic calcium responses to TG and IP 3 mobilizing agonists reported in different cell lines (Razani-Boroujerdi et al. 1994). However, the reported sensitivity of salivary gland TG-R pools to IP 3 and cADPr (Ghosh et al. 1991; Beecroft and Taylor 1998) suggests that Ca 2+ release from this compartment may be differentially regulated in different organs. It is also possible that specific Ca 2+ release agents acting on the TG-R pool are yet to be fully appreciated (Hardy et al. 1995). ...
Acute Wernicke's encephalopathy (WE) is caused by profound vitamin B1 (thiamine) deficiency and commonly presents with the classic clinical triad of mental confusion, ataxia, and ophthalmoplegia. This characteristic presentation results from the propensity of acute thiamine deficiency to preferentially injure specific brain regions: the dorsomedial thalamus, periaqueductal gray, and mamillary bodies. In these regions, abnormal magnetic resonance signaling on conventional sequences has been well described; however, diffusion restriction has only recently been reported. The authors demonstrate diffusion-weighted imaging (DWI) abnormalities of the splenium of the corpus callosum in a patient with acute WE, which has not been reported previously, and suggest a potential pathological mechanism. With the recent addition of DWI, MRI is becoming more sensitive to the changes in acute WE. Furthermore, the use of apparent diffusion coefficient mapping to evaluate the extent of likely underlying cytotoxic injury may help determine long-term response to vitamin therapy and, thus, disability.
... The experimental relationships obtained when [Ca 2+ ] c is clamped (Fig. 1) are not incompatible with curves predicted by this equation, although it should be pointed out that as [Ca 2+ ] ER increases, it is difficult to judge whether the Ca 2+ leak continues to increase very gently (as predicted by Eq. (2)), or whether it truly saturates. The temperature-dependence of the Ca 2+ leak also indicates that it is mediated by a channel [69]. Thus, although the evidence in favour of channel-mediation is indirect [68] , it is perhaps the simplest explanation compatible with the currently available data. ...
The concentration of Ca2+ inside the lumen of endoplasmic reticulum (ER) regulates a vast array of spatiotemporally distinct cellular processes, from intracellular Ca2+ signals to intra-ER protein processing and cell death. This review summarises recent data on the mechanisms of luminal Ca2+-dependent regulation of Ca2+ release and uptake as well as ER regulation of cellular adaptive processes. In addition we discuss general biophysical properties of the ER membrane, as trans-endomembrane Ca2+ fluxes are subject to basic electrical forces, determined by factors such as the membrane potential of the ER and the ease with which Ca2+ fluxes are able to change this potential (i.e. the resistance of the ER membrane). Although these electrical forces undoubtedly play a fundamental role in shaping [Ca2+](ER) dynamics, at present there is very little direct experimental information about the biophysical properties of the ER membrane. Further studies of how intraluminal [Ca2+] is regulated, best carried out with direct measurements, are vital for understanding how Ca2+ orchestrates cell function. Direct monitoring of [Ca2+](ER) under conditions where the cytosolic [Ca2+] is known may also help to capture elusive biophysical information about the ER, such as the potential difference across the ER membrane.
... Ca 2+ efflux pathways distinguish TG-S and TG-R Ca 2+ sequestering compartments TG treatment of cultured cells results in a rapid emptying of ER Ca 2+ stores (Islam and Berggren 1993). This passive Ca 2+ efflux, or leakage, from intracellular stores is poorly understood but is distinguished from Ca 2+ efflux mediated by IP 3 R and RyR (Beecroft and Taylor 1998). To determine whether the TG-R Ca 2+ sequestering compartment exhibits passive Ca 2+ efflux, we performed kinetic studies in which Ca 2+ accumulation was allowed to proceed for 90 min at which point 1 lM TG was added. ...
... Heterogeneous distribution of the TG-R pool could thus have contributed to the differential cytoplasmic calcium responses to TG and IP 3 mobilizing agonists reported in different cell lines (Razani-Boroujerdi et al. 1994). However, the reported sensitivity of salivary gland TG-R pools to IP 3 and cADPr (Ghosh et al. 1991;Beecroft and Taylor 1998) suggests that Ca 2+ release from this compartment may be differentially regulated in different organs. It is also possible that specific Ca 2+ release agents acting on the TG-R pool are yet to be fully appreciated (Hardy et al. 1995). ...
Ca2+ uptake into the endoplasmic reticulum (ER) is mediated by Ca2+ ATPase isoforms, which are all selectively inhibited by nanomolar concentrations of thapsigargin. Using ATP/Mg2+-dependent 45Ca2+ transport in rat brain microsomes, tissue sections, and permeabilized cells, as well as Ca2+ imaging in living cells we distinguish two ER Ca2+ pools in the rat CNS. Nanomolar levels of thapsigargin blocked one component of brain microsomal 45Ca2+ transport, which we designate as the thapsigargin-sensitive pool (TG-S). The remaining component was only inhibited by micromolar thapsigargin, and thus designated as thapsigargin resistant (TG-R). Ca2+ ATPase and [32P]phosphoenzyme assays also distinguished activities with differential sensitivities to thapsigargin. The TG-R Ca2+ uptake displayed unique anion permeabilities, was inhibited by vanadate, but was unaffected by sulfhydryl reduction. Ca2+ sequestered into the TG-R pool could not be released by inositol-1,4,5-trisphosphate, caffeine, or cyclic ADP-ribose. The TG-R Ca2+ pool had a unique anatomical distribution in the brain, with selective enrichment in brainstem and spinal cord structures. Cell lines that expressed high levels of the TG-R pool required micromolar concentrations of thapsigargin to effectively raise cytoplasmic Ca2+ levels. TG-R Ca2+ accumulation represents a distinct Ca2+ buffering pool in specific CNS regions with unique pharmacological sensitivities and anatomical distributions.
... The extrapolated initial Ca# + contents of the stores were similar for cells in low and high Ca# + (108p1 % and 108p9 % respectively of their contents measured at 30 s) as were the Ca# + contents extrapolated to infinite time (22p2 % and 20p5 %). The incomplete loss of %&Ca#+ from preloaded stores after sustained inhibition of Ca# + uptake is consistent with previous work showing that luminal Ca# + regulates the basal leak of Ca# + from the intracellular stores of hepatocytes [15]. ...
... At 2 mC, the rate of %&Ca#+ efflux was much slower than at 37 mC, in keeping with previous work [15], and the rates were similar in CLM containing a [Ca# + ] c of approx. 40 nM or 100 µM (Figures 2A and 2B). ...
... 30 nM free [Ca# + ]), but Ca# + efflux was stimulated by Ca# + (Figure 2C). These results establish that under conditions where the SERCA has been completely inhibited [15], concentrations of Ca# + similar to those found in unstimulated cells, stimulate temperature-sensitive Ca# + efflux from the intracellular stores. ...
Members of both major families of intracellular Ca(2+) channels, ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, are stimulated by substantial increases in cytosolic free Ca(2+) concentration ([Ca(2+)]c). They thereby mediate Ca(2+)-induced Ca(2+) release (CICR), which allows amplification and regenerative propagation of intracellular Ca(2+) signals. In permeabilized hepatocytes, increasing [Ca(2+)]c to 10 microM stimulated release of 30+/-1% of the intracellular stores within 60 s; the EC(50) occurred with a free [Ca(2+)] of 170+/-29 nM. This CICR was abolished at 2 degrees C. The same fraction of the stores was released by CICR before and after depletion of the IP3-sensitive stores, and CICR was not blocked by antagonists of IP3 receptors. Ryanodine, Ruthenium Red and tetracaine affected neither the Ca(2+) content of the stores nor the CICR response. Sr(2+) and Ba(2+) (EC(50)=166 nM and 28 microM respectively) mimicked the effects of increased [Ca(2+)] on the intracellular stores, but Ni(2+) blocked the passive leak of Ca(2+) without blocking CICR. In rapid superfusion experiments, maximal concentrations of IP3 or Ca(2+) stimulated Ca(2+) release within 80 ms. The response to IP3 was complete within 2 s, but CICR continued for tens of seconds despite a slow [half-time (t(1/2))=3.54+/-0.07 s] partial inactivation. CICR reversed rapidly (t(1/2)=529+/-17 ms) and completely when the [Ca(2+)] was reduced. We conclude that hepatocytes express a novel temperature-sensitive, ATP-independent CICR mechanism that is reversibly activated by modest increases in [Ca(2+)], and does not require IP3 or ryanodine receptors or reversal of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase. This mechanism may both regulate the Ca(2+) content of the intracellular stores of unstimulated cells and allow even small intracellular Ca(2+) signals to be amplified by CICR.
