Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin

The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.     

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.     

Two different but converging messenger pathways to intracellular Ca(2+) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate

Hormones and neurotransmitters mobilize Ca(2+) from the endoplasmic reticulum via inositol trisphosphate (IP(3)) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca(2+) responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca(2+) spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca(2+) signals. We show that IP(3), cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca(2+) spiking by activating common oscillator units composed of IP(3) and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP(3) receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP-ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated specifically by cyclic ADP-ribose receptors. In the apical pole there is a common early activation site for Ca(2+) release, indicating that the three types of Ca(2+) release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.     

Bombesin-induced cytosolic Ca2+ spiking in pancreatic acinar cells depends on cyclic ADP-ribose and ryanodine receptors

Different hormones and neurotransmitters, using Ca2+ as their intracellular messenger, can generate specific cytosolic Ca2+ signals in different parts of a cell. In mouse pancreatic acinar cells, cytosolic Ca2+ oscillations are triggered by activation of acetylcholine (ACh), cholecystokinin (CCK) and bombesin receptors. Low concentrations of these three agonists all induce local Ca(2+)spikes, but in the case of bombesin and CCK these spikes can also trigger global Ca2+ signals. Here we monitor cytosolic Ca2+ oscillations induced by low (2-5 pM) concentrations of bombesin and show that, like ACh- and CCK-induced oscillations, the bombesin-elicited responses are inhibited by ryanodine(50 microM). We then demonstrate that, like CCK- but unlike ACh-induced oscillations, the responses to bombesin are abolished by intracellular infusion of the cyclic ADP ribose (cADPr) antagonist 8-NH2-cADPr (20 microM). We conclude that in mouse pancreatic acinar cells, bombesin, CCK and ACh all produce local Ca2+ spikes by recruiting common oscillator units composed of ryanodine and inositol trisphosphate receptors. However, bombesin and CCK also recruit cADPr receptors, which may account for the global Ca2+ signals that can be evoked by these two agonists. Our new results indicate that each Ca2+ -mobilizing agonist, acting on mouse pancreatic acinar cells, recruits a unique combination of intracellular Ca2+ channels.     

Glucose regulation of free Ca(2+) in the endoplasmic reticulum of mouse pancreatic beta cells

Free Ca(2+) was measured in organelles of individual mouse pancreatic beta cells loaded with the low affinity indicator furaptra. After removal of cytoplasmic indicator by controlled digitonin permeabilization the organelle Ca(2+) was located essentially in the endoplasmic reticulum (ER), >90% being sensitive to inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPases. The Ca(2+) accumulation in the ER of intact beta cells depended in a hyperbolic fashion on the glucose concentration with half-maximal and maximal filling at 5.5 and >20 mM, respectively. Also elevation of cytoplasmic Ca(2+) by K(+) depolarization significantly enhanced the Ca(2+) accumulation. In permeabilized beta cells 1-3 mM ATP caused rapid Ca(2+) filling of the ER reaching almost 500 microM. At 50 nM, Ca(2+) ER became half-maximally filled at 45 microM ATP, whereas only 3.5 microM ATP was required at 200 nM Ca(2+). Inositol 1,4,5-trisphosphate induced a rapid release of about 65% of the ER Ca(2+), and its precursor phosphatidylinositol 4,5-bisphosphate was found to slowly mobilize 75% by another mechanism. It is concluded that glucose is an efficient stimulator of Ca(2+) uptake in the ER of pancreatic beta cells both by increasing ATP and cytoplasmic Ca(2+). Because physiological concentrations of cytoplasmic ATP are in the mM range, Ca(2+) sequestration can be anticipated to be modulated by factors reducing its ATP sensitivity.     

Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis

Using double-barreled, Ca2(+)-sensitive microelectrodes, we have examined the characteristics of the Ca2+ release by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca2+ in each layer was directly measured under physiological conditions using theta-tubing, double-barreled, Ca2(+)-sensitive microelectrodes in which one barrel was filled with the Ca2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3 microns apart) enabling us to measure the kinetics of both the highly localized intracellular Ca2+ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER-enriched layer exhibited the largest release of Ca2+ in a dosage-dependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca2+ in a dosage-independent manner. The removal of released Ca2+ took place within approximately 1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca2+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)P3-sensitive Ca2+ store in the egg of Xenopus laevis.     

Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Translocation of Ca2+ into mitochondria from a functionally discrete portion of the nonmitochondrial store

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.     

Calcium signal compartmentalization

Cytosolic calcium signals are produced by suddenly increasing the concentration of free calcium ions (Ca2+). This can occur by opening channels permeable to Ca2+ either in the surface cell membrane or in the membranes of intracellular organelles containing high Ca2+ concentrations. Ca2+ signals can control several different processes, even in the same cell. In pancreatic acinar cells, for example, Ca2+ signals do not only control the normal secretion of digestive enzymes, but can also activate autodigestion and programmed cell death. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited. The mechanisms responsible for Ca2+ signal compartmentalization are now largely known and will be described on the basis of recent studies of Ca2+ transport pathways and their regulation in pancreatic acinar cells. It turns out that the Ca2+ handling as well as the structural characteristics of the endoplasmic reticulum (ER) and the mitochondria are of particular importance. Using a variety of Ca(2+)-sensitive fluorescent probes placed in different sub-cellular compartments in combination with local uncaging of caged Ca2+, many new insights into Ca2+ signal generation, compartmentalization and termination have recently been obtained.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca²⁺ stores that release Ca²⁺ in response to the second messengers IP₃ and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca²⁺ released from acidic organelles by NAADP subsequently recruits IP₃ or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca²⁺ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca²⁺ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca²⁺-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca²⁺ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca²⁺ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca²⁺ oscillations and waves.     

Immunocytochemistry of calciosomes in liver and pancreas

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.     

STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.     

Genetic analysis of lysosomal trafficking in Caenorhabditis elegans

The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.     

Endoplasmic reticulum calcium signaling in nerve cells

The endoplasmic reticulum (ER) is an important organelle involved in various types of signaling in nerve cells. The ER serves as a dynamic Ca2+ pool being thus involved in rapid signaling events associated with cell stimulation by either electrical (action potential) or chemical (neurotransmitters) signals. This function is supported by Ca2+ release channels (InsP3 and ryanodine receptors) and SERCA Ca2+ pumps residing in the endomembrane. In addition the ER provides a specific environment for the posttranslational protein processing and transport of various molecules towards their final destination. In parallel, the ER acts as a "calcium tunnel," which facilitates Ca2+ movements within the cell by avoiding cytoplasmic routes. Finally the ER appears as a source of numerous signals aimed at the nucleus and involved in long-lasting adaptive cellular responses. All these important functions are controlled by intra-ER free Ca2+ which integrates various signaling events and establishes a link between fast signaling, associated with ER Ca2+ release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. Disruption of ER Ca2+ homeostasis triggers several forms of cellular stress response and is intimately involved in neurodegeneration and neuronal cell death.     

Islet oxygen consumption and insulin secretion tightly coupled to calcium derived from L-type calcium channels but not from the endoplasmic reticulum

The aim of the study was to test whether the source of intracellular calcium (Ca2+) is a determinant of beta cell function. We hypothesized that elevations in cytosolic Ca2+ caused by the release of Ca2+ from the endoplasmic reticulum (ER) have little physiologic impact on oxygen consumption and insulin secretion. Ca2+ release from the ER was induced in isolated rat islets by acetylcholine and response of oxygen consumption rate (OCR), NAD(P)H, cytosolic Ca2+, and insulin secretory rate (ISR) were measured. Glucose increased all four parameters, and thereafter acetylcholine further increased cytosolic Ca2+, OCR, and ISR. To assess the contribution of Ca2+ release from the ER in mediating the effects of acetylcholine, ER Ca2+ stores were first emptied by inhibiting the sarcoendoplasmic reticulum Ca2+-ATPase, which subsequently reduced the effect of acetylcholine on cytosolic Ca2+ but not its effects on OCR or ISR. As predicted, OCR and ISR were acutely sensitive to changes in L-type Ca2+ channel activity; nimodipine completely inhibited glucose-stimulated ISR and suppressed OCR by 36%, despite only inhibiting cytosolic Ca2+ by 46%. Moreover, in the presence of nimodipine and high glucose, acetylcholine still elevated cytosolic Ca2+ levels above those observed in the presence of high glucose alone but did not significantly stimulate ISR. In conclusion, Ca2+ flux through L-type Ca2+ channels was tightly coupled to changes in OCR and ISR. In contrast, the results obtained support the notion that Ca2+ release from the ER has little or no access to the intracellular machinery that regulates OCR and ISR.     

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.     

Calcium signalling mechanisms in endoplasmic reticulum activated by inositol 1,4,5-trisphosphate and GTP

Ca2+ signals are known to mediate an array of cellular functions including secretion, contraction, and conductivity changes. In spite of the obvious role of Ca2+ in signalling, the control of Ca2+ within cells is known to be a complex phenomenon involving a number of distinct active and passive transport systems functioning within different organelles. Inositol 1,4,5-trisphosphate (IP3) is now established as a central mediator of Ca2+ mobilization, and the endoplasmic reticulum (ER) has been considered to be the site of action of IP3. However, this role has been ascribed almost by default to the ER, based on the knowledge that IP3 functions to release Ca2+ from an intracellular, nonmitochondrial, Ca2+-pumping organelle. Our interest has been to ascertain by what mechanism IP3 activates Ca2+ movements, at what intracellular locations it functions, and how the size and replenishment of the IP3-sensitive Ca2+ pool occurs. During the course of such studies, another mechanism inducing profound movements of Ca2+ within cells was identified. This process is activated by a highly sensitive and specific guanine nucleotide regulatory mechanism, which, while inducing fluxes of Ca2+ that resemble the action of IP3 under certain conditions, has now been determined to involve a quite distinct mechanism. The characteristics of this mechanism are described below. Although involving a very different Ca2+ translocation process to that activated by IP3, several important conclusions have been drawn on the relationship between IP3- and GTP-activated Ca2+ movements leading us to believe that the latter may have a regulatory role in controlling the size and/or entry of Ca2+ into the IP3-sensitive Ca2+ pool.