Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

Ribosome-free Terminals of Rough ER Allow Formation of STIM1 Puncta and Segregation of STIM1 from IP3 Receptors

Store-operated Ca²⁺ entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium [1]. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels ([2], [3], [4], [5], [6], [7] and [8]; reviewed in [9]). In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP₃) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca²⁺ loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex [5] and [10], and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca²⁺ release and Ca²⁺ reloading) and that any potential interference between Ca²⁺ release and Ca²⁺ influx is minimized by the spatial separation of the two processes.     

A Simulation Study of Calcium Dynamics Features Caused by Exchange between the Cytosol and Organellar Stores of Neurons

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron. The fragments contained the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER (model 2), functioning as calcium stores. With simulating single excitatory synaptic actions, we examined the dependence of the dynamics of intracellular Ca²⁺ levels on the maximum rate of Ca²⁺ exchange between the cytosol and these stores, as well as on the intensity of the diffusion flow into adjacent organelle-free regions. The plasma membrane of the compartment had ion channels (including those of the synaptic current) and the calcium pump characteristic of the mentioned neurons. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular environment, and organellar stores, as well as the diffusion process. In model 1, the mitochondria exchanged Ca²⁺ with the cytosol through the uniporter and sodium-calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also included. In model 2, the ER membrane had the calcium pump, leak channels, and channels of calcium-induced and inositol-3-phosphate-dependent Ca²⁺ release. The stores (mitochondria or ER) occupied 36% of the total volume of the compartment. An increase in the maximum rate of calcium exchange with the stores led to a proportional decrease in the peak Ca²⁺ concentrations in the cytosol ([Ca²⁺]_i), more pronounced in the case of the ER; the Ca²⁺ concentration in both types of stores increased significantly. Due to the higher storage rate, the ER was able to absorb several times greater amounts of Ca²⁺ than the mitochondria did. With smaller diffusion flux (e.g., similarly to the case of diffusion from a larger-sized head into the neck of the dendritic spine), the intensity of cytosolic transients increased at fixed kinetics of flux exchange with the stores. Therefore, the organellar stores can significantly modulate not only the intensity but also the time course of changes in the intracellular Ca²⁺ levels.     

Mutual influence of plasma membrane structures and intracellular calcium stores on the formation of calcium signals in primary nociceptive neurons

Participation of different calcium-regulating mechanisms in the formation of intracellular calcium signals in rat primary sensory neurons was studied using two-wavelength fluorescent microscopy. Mitochondria were shown to be the most powerful intracellular calcium-regulating structures in the investigated neurons. These organelles were involved in the modulation of calcium signals induced either by Ca²⁺ entry from the extracellular medium or by Ca²⁺ release from endoplasmic reticulum (ER). Analysis of the mitochondrial calcium exchange showed that the efficiency of mitochondria depended on whether calcium entered the cytosol from ER or from the extracellular solution. Depletion of ER by activation of ryanodine-sensitive, inositol-3-phosphate-sensitive receptors of ER or by activation of the leak channels via the block of ATPases in ER activated the store-operated calcium entry from the extracellular medium to cytosol. The kinetics of the rising phase of these Ca²⁺ transients depended on the way of ER depletion. This allows suggesting the existence of different activation mechanisms for the studied signals. The block of the mitochondrial calcium uniporter resulted in a rapid recovery of the intracellular calcium concentration after the Ca²⁺ transient induced by store-operated calcium influx. We conclude that mitochondrial calcium uptake can prevent calcium-dependent inactivation of store-operated calcium channels.     

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

Keeping zombies alive: The ER-mitochondria Ca2+ transfer in cellular senescence

Cellular senescence generates a permanent cell cycle arrest, characterized by apoptosis resistance and a pro-inflammatory senescence-associated secretory phenotype (SASP). Physiologically, senescent cells promote tissue remodeling during development and after injury. However, when accumulated over a certain threshold as happens during aging or after cellular stress, senescent cells contribute to the functional decline of tissues, participating in the generation of several diseases. Cellular senescence is accompanied by increased mitochondrial metabolism. How mitochondrial function is regulated and what role it plays in senescent cell homeostasis is poorly understood. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca²⁺) storage organelle in mammalian cells, through special domains known as mitochondria-ER contacts (MERCs). In this domain, the release of Ca²⁺ from the ER is mainly regulated by inositol 1,4,5-trisphosphate receptors (IP3Rs), a family of three Ca²⁺ release channels activated by a ligand (IP3). IP3R-mediated Ca²⁺ release is transferred to mitochondria through the mitochondrial Ca²⁺ uniporter (MCU), where it modulates the activity of several enzymes and transporters impacting its bioenergetic and biosynthetic function. Here, we review the possible connection between ER to mitochondria Ca²⁺ transfer and senescence.Understanding the pathways that contribute to senescence is essential to reveal new therapeutic targets that allow either delaying senescent cell accumulation or reduce senescent cell burden to alleviate multiple diseases.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Endoplasmic reticulum Ca2+ store: regulation of Ca2+ release and reuptake by intracellular and extracellular Ca2+ in pancreatic acinar cells

We investigated the effect of cytosolic and extracellular Ca2+ on Ca2+ signals in pancreatic acinar cells by measuring Ca2+ concentration in the cytosol([Ca2+]c) and in the lumen of the ER([Ca2+]Lu). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released Ca2+ mainly from the basolateral ER-rich part of the cell. The rate of Ca2+ release from the ER was highly sensitive to the buffering of [Ca2+]c whereas ER Ca2+ refilling was enhanced by supplying free Ca2+ to the cytosol with [Ca2+]c clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM Ca2+. Elevation of extracellular Ca2+ to 10 mM from 1 mM raised resting [Ca2+]c slightly and often generated [Ca2+]c oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular Ca2+-sensing receptors linked to phospholipase C that mobilize Ca2+ from the ER, exposure of cells to 10 mM Ca2+ did not decrease [Ca2+]Lu but rather raised it. From these findings we conclude that 1) ER Ca2+ release is strictly regulated by feedback inhibition of [Ca2+]c, 2) ER Ca2+ refilling is determined by the rate of Ca2+ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular Ca2+-induced [Ca2+]c oscillations appear to be triggered not by activation of extracellular Ca2+-sensing receptors but by the ER sensitised by elevated [Ca2+]c and [Ca2+]Lu.     

A bidomain threshold model of propagating calcium waves

We present a bidomain fire-diffuse-fire model that facilitates mathematical analysis of propagating waves of elevated intracellular calcium (Ca²⁺) in living cells. Modeling Ca²⁺ release as a threshold process allows the explicit construction of traveling wave solutions to probe the dependence of Ca²⁺ wave speed on physiologically important parameters such as the threshold for Ca²⁺ release from the endoplasmic reticulum (ER) to the cytosol, the rate of Ca²⁺ resequestration from the cytosol to the ER, and the total [Ca²⁺] (cytosolic plus ER). Interestingly, linear stability analysis of the bidomain fire-diffuse-fire model predicts the onset of dynamic wave instabilities leading to the emergence of Ca²⁺ waves that propagate in a back-and-forth manner. Numerical simulations are used to confirm the presence of these so-called ‘tango waves’ and the dependence of Ca²⁺ wave speed on the total [Ca²⁺].      

Nonuniformity of calcium efflux from pancreatic acinar cells and its analysis by mathematical model of calcium diffusion and buffering in extracellular solution

We studied spatial and temporal patterns of Ca²⁺ extrusion from pancreatic acinar cells evoked by acetylcholine(ACh)-induced activation of plasma membrane calcium pumps. Using a modification of an earlier developed model, we estimated the time course of extracellular calcium concentration changes near the basal pole of a cell in the case, when calcium ions are released from the same site on the cell surface, and in the case when they are extruded from the apical pole and diffuse to the basal one. It is concluded that at the first stage of ACh-induced Ca²⁺ extrusion the appearance of Ca²⁺ elevation near the basal pole of the cells cannot be explained as a result of diffusion, but is mainly determined by Ca²⁺ efflux from this pole. The results also show that there are plasma membrane calcium pumps in both apical and basal parts of pancreatic acinar cells, but the activity of the pumps is substantially higher in the apical region.     

Calcium Elevation in Mitochondria Is the Main Ca2+ Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

We have investigated in detail the role of intra-organelle Ca²⁺ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca²⁺ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca²⁺ responses because of the release of Ca²⁺ from both ER and acidic stores. Ca²⁺ responses to menadione were accompanied by elevation of Ca²⁺ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca²⁺ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca²⁺ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca²⁺ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca²⁺ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca²⁺ with pericam. We conclude that elevated Ca²⁺ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.Apoptosis, a mechanism of programmed cell death, usually occurs through intrinsic or extrinsic apoptotic pathways. The caspases involved in apoptosis can be split into two groups, the initiator caspases such as caspase-9 and effector caspases such as caspase-3. Effector caspases are activated by initiator caspases and mediate many of the morphological cellular changes associated with apoptosis (1).Calcium is an important signaling ion involved in the regulation of many physiological as well as pathological cellular responses (2). In the pancreas, we have shown that Ca²⁺ signals elicit enzyme secretion (3), apoptosis (4–6), and pathological intracellular activation of digestive enzymes (7). As such, there must be mechanisms in place by which the cell can differentiate between apoptotic and non-apoptotic Ca²⁺ signals.The spatiotemporal pattern of calcium signaling is crucial for the specificity of cellular responses. For example, repetitive cytosolic calcium spikes confined to the apical region of the pancreatic acinar cell are elicited by physiological stimulation with acetylcholine (ACh) or cholecystokinin (CCK) and result in physiological secretion of zymogen granules (8, 9). However, a sustained global increase in free cytosolic Ca²⁺ induced by supramaximal stimulation with CCK, which resembles prolonged hyperstimulation of pancreatic acinar cells in the pathophysiology of acute pancreatitis, can lead to premature activation of digestive enzymes and vacuole formation within the cell (10–12). Alternatively, global repetitive calcium spikes induced in the pancreatic acinar cell in response to oxidant stress can lead to induction of the mitochondrial permeability transition pore (mPTP)⁴ and apoptosis (4, 5, 13).To understand the role of calcium in apoptosis, several investigators have examined the influence of intracellular stores on the molding of calcium signals that lead to cell death (14–16). It has been well established in a range of cell types that the endoplasmic reticulum (ER) is the major intracellular calcium store required for induction of apoptosis. Pinton et al. (17) have shown that decreasing ER Ca²⁺ concentration with tBuBHQ increased HeLa cell survival in response to oxidant stress induced by ceramide. Scorrano and Korsmeyer (18) also observed that double knock-out Bax and Bak (pro-apoptotic proteins) mouse fibroblasts displayed a reduced resting concentration of ER Ca²⁺ compared with wild type and were resistant to induction of apoptosis by various stimulants, including ceramide. These important studies strongly suggest that the concentration of Ca²⁺ in the ER is a critical determinant of cellular susceptibility to apoptotic stimuli in the cell types studied.A key event in early apoptosis is permeabilization of the mitochondrial membrane. The mPTP is a pore whose molecular composition is still debated (19). Activation of an open pore state can result in swelling of the mitochondrial matrix and release of the apoptogenic proteins from the intermembrane space (20).One important activator of the mPTP is Ca²⁺ (20–22), a function which implicates Ca²⁺ in the initiation of apoptosis (23, 24). Once Ca²⁺ is released from the ER into the cytoplasm, mitochondria take up part of the released Ca²⁺ to prevent propagation of large calcium waves (25–27). This influx is followed by calcium efflux from the mitochondria back into the cytosol (28, 29). An increase in mitochondrial Ca²⁺ concentration in response to physiological stimuli induces increased activity of the mitochondrial respiratory chain and the synthesis of ATP to meet with increasing energy demands on the cell. When mitochondria are exposed to a pathological overload of calcium, opening of the mPTP is triggered, leading to mitochondrial dysfunction and eventually cell death. The mechanism through which calcium can trigger mPTP opening is still unclear and may involve cyclophilin D (30) and voltage-dependent anion channel (31). The mitochondria are endowed with selective and efficient calcium uptake (a calcium-selective uniporter) and release mechanisms (Ca²⁺/Na exchanger, Ca²⁺/H⁺ exchanger, and mPTP) (16, 29, 32, 33).Oxidant stress is a well known inducer of apoptosis in several cell types (34) and is thought to play an important role in the pathogenesis of acute pancreatitis (35). We have used the quinone compound menadione to induce oxidative stress in the pancreatic acinar cell. Menadione is metabolized by flavoprotein reductase to semiquinone and then is oxidized back to quinone, resulting in generation of superoxide anion radicals, hydrogen peroxide, and other reactive oxygen species (ROS) (36). In vivo, menadione causes depolarization and swelling of the mitochondria (37). In pancreatic acinar cells, treatment with menadione not only produces an increase in ROS, but has also been found to evoke cytosolic Ca²⁺ responses, mPTP opening, activation of caspases and apoptotic cell death (4, 5). When cells were pretreated with the calcium chelator BAPTA-AM, menadione was unable to induce apoptosis, indicating that oxidant stress-induced apoptosis in the pancreatic acinar cell is highly calcium-dependent. Here we show that in pancreatic acinar cells, oxidative stress-induced apoptosis is strongly dependent on the Ca²⁺ concentration within mitochondria at the time of ROS production.     

Ca sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca²⁺ concentration is necessary and sufficient for this process. The predominant source of Ca²⁺ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca²⁺ to the cytosol. The ER store is (re)filled by the store-specific Ca²⁺-ATPase. Ultimately, the depleted ER is replenished by Ca²⁺ which enters from the extracellular space to the cytosol via store-operated Ca²⁺ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca²⁺ channels and plasma membrane Na⁺/Ca²⁺ exchangers are additional means for cytosolic Ca²⁺ entry. Cytosolic Ca²⁺ levels can be modulated by mitochondria, which can take up cytosolic Ca²⁺ via the Ca²⁺ uniporter and release Ca²⁺ into cytosol via the mitochondrial Na⁺/Ca²⁺ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca²⁺ sources generates cytosolic Ca²⁺ dynamics that can drive Ca²⁺-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Effects of piperine and deoxyschizandrin on synchronized Ca2+ oscillations in cultured hippocampal neuronal cells

It has been reported that piperine (PIP) and deoxyschizandrin (DS) can modulate synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks. We investigated the modulation effects of four different combinations of piperine and deoxyschizandrin on synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks. The results showed that all four combinations (PIP:DS 4.9:1.9, 2.45:2.85, 7.35:0.95, and 2.45:0.95 mg/L) inhibit Ca²⁺ oscillation intensity to a similar extent. However, the first three combinations had strong inhibitory effects on the frequency of Ca²⁺ oscillations whereas the last combination (2.45:0.95 mg/L) only slightly enhanced the frequency of Ca²⁺ oscillations. We propose an improved Chay’s model to explain the mechanism of the effects of piperine and deoxyschizandrin on synchronized Ca²⁺ oscillations in cultured hippocampal neuronal cells. We concluded that deoxyschizandrin modulated synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks bidirectionally and the effect depended on concentration. Deoxyschizandrin reduced voltage-gated sodium channel conductance and ATP-sensitive potassium channel conductance, and affected the rate of exchange of intracellular calcium and the pump activity of Ca²⁺-ATPase in the endoplasmic reticulum (ER). Piperine reduced the activity of calcium release in the ER, and reduced the pump activity of calcium in the cytomembrane or enhanced the pump activity of Ca²⁺-ATPase in the ER.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca²⁺ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

Comparative Model Analysis of Calcium Exchange between the Cytosol and Stores of Mitochondria or Endoplasmic Reticulum

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron containing the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER, (model 2) as the calcium stores. We investigated the dependence of the intracellular Ca²⁺ dynamics on geometrical sizes of calcium exchanging parts of the intracellular space and the difference between the kinetic characteristics of storing in two types of stores occupying different portions of the compartment volume. The plasma membrane of the compartment bore the ion channels, particularly those conducting excitatory synaptic current, and the calcium pump typical of this neuron type. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular medium, organelle stores, non-organelle endogenous buffers, and an exogenous buffer (fluorescent dye), and also the diffusion of Са²⁺ into adjacent regions of the dendrite. In model 1, the mitochondria exchanged Са²⁺ with the cytosol via the uniporter and sodium/calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also taken into account. In model 2, the ER membrane contained the calcium pump, channels of passive leak, and channels of calcium-induced and inositol-3-phosphate-dependent release of Са²⁺. Increases in the portion of the stores in the total volume of the compartment from 1 to 36% led to a proportional increase in the peak values of the cytosolic calcium concentration ([Ca²⁺] _i ); the concentration of Са²⁺ in the mitochondria ([Ca²⁺]_mit) or ER ([Ca²⁺]_ER) increased correspondingly. During generation of bell-shaped cytosolic calcium signals of equal intensity and duration, the ER (due to a greater rate of storing, as compared with that in the mitochondria) was able to uptake several times more Са²⁺ (four times at 36% filling of the volume by the organelles). It is suggested that the revealed different kinetic characteristics of Са²⁺ storing by different organelles are determined by the rates of binding to transport molecules present in the store membrane and, therefore, are defined by concentrations (surface densities) of these molecules and their saturation at certain levels of [Ca²⁺]i. It has been shown that the occupancy of the intracellular volume by organelle stores of any type is a structural factor, which is able to essentially modulate the values of Ca²⁺ concentration.     

Role of acidic stores in secretory epithelia

There is growing evidence that intracellular calcium plays a primary role in the pathophysiology of the pancreas in addition to its crucial importance in major physiological functions. Pancreatic acinar cells have a remarkably large amount of Ca²⁺ stored in both the endoplasmic reticulum (ER) and the acidic stores. The vast majority of the classical ER Ca²⁺ store is located in the basal part of the acinar cells with extensions protruding into the apical area, however, the acidic stores are exclusively located in the secretory granular area of the cells. Both types of Ca²⁺ store respond to all three intracellular Ca²⁺ messengers – inositol trisphosphate (InsP₃), cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). The two stores interact with each other via calcium-induced calcium release; however, they can be separated using pharmacological tools. The ER relies on sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) that can be blocked by the specific inhibitor thapsigargin. The acidic store requires a low pH that can be modified by blocking vacuolar H⁺-ATPase.     

Pharmacological dose of melatonin reduces cytosolic calcium load in response to cholecystokinin in mouse pancreatic acinar cells

Intracellular Ca²⁺ overload has been considered a common pathological precursor of pancreatic injury. In this study, the effects of melatonin on Ca²⁺ mobilization induced by cholecystokinin octapeptide (CCK-8) in freshly isolated mouse pancreatic acinar cells have been examined. Changes in intracellular free Ca²⁺ concentration were followed by single cell fluorimetry. For this purpose, cells were loaded with the Ca²⁺-sensitive fluorescent dye fura-2-acetoxymethyl ester. In order to evaluate the contribution of Ca²⁺ transport at the plasma membrane, at the endoplasmic reticulum (ER) or at the mitochondria, cells were incubated with CCK-8 alone or in combination with LaCl₃, thapsigargin (Tps), or FCCP to, respectively, uncouple Ca²⁺ transport at these localizations. The experiments were performed in the absence or in the presence of melatonin in combination with the stimuli mentioned. Our results show that the total Ca²⁺ mobilization evoked by CCK-8 was attenuated by a 30 % in the presence of 100 µM melatonin compared with the responses induced by CCK-8 alone. Upon inhibition of Ca²⁺ transport into the ER by Tps, Ca²⁺ mobilization was also reduced in the presence of melatonin. In the presence of LaCl₃ plus melatonin, the total Ca²⁺ mobilization induced by CCK-8 was significantly decreased, compared with the response obtained without melatonin but in the presence of LaCl₃. No major differences were found when the cells were incubated with CCK-8 or Tps alone or in combination with LaCl₃ plus melatonin and FCCP, compared with the responses obtained in the absence of FCCP. The initial Ca²⁺ release from intracellular stores evoked by CCK-8 or Tps was not significantly reduced in the presence of melatonin. The effect of melatonin could be explained on the basis of a stimulated Ca²⁺ transport out of the cell through the plasma membrane and by a stimulation of Ca²⁺ reuptake into the ER. Accumulation of Ca²⁺ into mitochondria might not be a major mechanism stimulated by melatonin. We conclude that melatonin alleviates intracellular Ca²⁺ accumulation, a situation potentially leading to cell damage in the exocrine pancreas.     

Bcl-2 Modulates Endoplasmic Reticulum and Mitochondrial Calcium Stores in PC12 Cells

Endoplasmic reticulum (ER) and mitochondria are intracellular organelles and their interactions are directly involved in different processes such as Ca²⁺ signaling in cell survival and death mechanisms. Bcl-2 is an anti-apoptotic protein intrinsically related to ER and mitochondria, modulating Ca²⁺ content in these organelles. We investigated the effects of Bcl-2 overexpression on ER and mitochondrial Ca²⁺ dynamics in PC12 cells. Bcl-2 overexpressing and control cells were loaded with Fura 2/AM and stimulated with different drugs. Results showed that in Bcl-2 cells, ACh induced a lower Ca²⁺ response compared to control. Ca²⁺ release induced by TG was decreased in Bcl-2 cells, however, it was greater in Caff induced Ca²⁺ rise. In addition, FCCP induced a higher Ca²⁺ release in Bcl-2 cells. These results suggest that Bcl-2 overexpression modulate the ER Ca²⁺ pools differently and the release of ER Ca²⁺ may increase mitochondrial Ca²⁺ accumulation. These alterations of intracellular Ca²⁺ stores are important mechanisms for the control of Ca²⁺ signaling.