Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling.

BACKGROUND INFORMATION: The IP(3)R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca(2+) release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP(3)R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP(3)R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. RESULTS: In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP(3)R1 increased the rate of thapsigargin-mediated Ca(2+) leak and decreased the rate of Ca(2+) uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca(2+) stores. We detected the truncated IP(3)R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP(3)R1 blocked sperm factor-induced Ca(2+) oscillations and induced an apoptotic phenotype. CONCLUSIONS: In the present study, we show that caspase-3-mediated truncation of IP(3)R1 enhanced the Ca(2+) leak from the ER. We suggest a model in which, in normal conditions, the increased Ca(2+) leak is largely compensated by enhanced Ca(2+)-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca(2+) leak acts as a feed-forward mechanism to divert the cell into apoptosis.     

Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.

We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.     

Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate- mediated Ca(2+) release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.     

The variety of cytosolic calcium responses and possible roles of PLC and PKC

A model of ligand-induced intracellular calcium (Ca2+) responses incorporating phospholipase C (PLC) and protein kinase C (PKC) is developed for the purpose of understanding the mechanisms underlying the observed temporal patterns of intracellular calcium (Ca(i)2+) under sustained agonist stimulation. Some studies have suggested that inhibition of ligand receptors and PLC by PKC could generate sinusoidal Ca2+ oscillations, while PKC-independent Ca2+-induced Ca2+ release (CICR) via IP(3)-gated Ca2+ channels on the endoplasmic reticulum (ER) is believed to be responsible for baseline spiking. However, some evidence also indicates that baseline spiking can be observed under high-PKC activity, or under low-PKC activity with low agonist stimulus, as well. Insight into the basis of these observations regarding the role of PKC in Ca(i)2+ response patterns can be gained by developing and analyzing a mathematical model of Ca(i)2+ responses. We do this herein and find that (1) interaction of CICR and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump is enough to generate both types of Ca(i)2+ oscillations, (2) there exist four possible Ca(i)2+ response patterns under sustained agonist stimulus: a sub-threshold response (SR), baseline spiking, sinusoidal oscillations (SO) and transient with plateau, and (3) the IP(3) concentration, which is controlled by the strength of the interaction between PKC and PLC, can be used to predict the Ca(i)2+ response patterns. From this analysis we conclude that the different patterns of Ca(i)2+ oscillations can be understood as a generic consequence of the interactions between CICR via the IP(3)-gated Ca(2+) channels in response to changes in the level of IP(3), and re-uptake into the ER/SR via the SERCA pump. PKC, in conjunction with PLC, can act as a switch between different Ca(i)2+ response patterns by modulating the cytosolic IP(3) level, which determines the Ca(i)2+ patterns.     

Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.

Alterations in intracellular Ca(2+) homeostasis and cytochrome c release from mitochondria have been implicated in the regulation of apoptosis, but the relationship between these events remains unclear. Here we report that enforced expression of either Bax or Bak via adenoviral gene delivery results in the accumulation of the proteins in the endoplasmic reticulum (ER) and mitochondria, resulting in early caspase-independent BCL-2-sensitive release of the ER Ca(2+) pool and subsequent Ca(2+) accumulation in mitochondria. The inhibition of ER-to-mitochondrial Ca(2+) transport with a specific inhibitor of mitochondrial Ca(2+) uptake attenuates cytochrome c release and downstream biochemical events associated with apoptosis. Bax and Bak also directly sensitize mitochondria to cytochrome c release induced by immediate emptying of ER Ca(2+) pool. Our results demonstrate that the effects of the "multidomain" proapoptotic BCL-2 family members Bak and Bax involve direct effects on the endoplasmic reticular Ca(2+) pool with subsequent sensitization of mitochondria to calcium-mediated fluxes and cytochrome c release. These effects modulate the kinetics of cytochrome c release and apoptosis.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

A mathematical model predicts that calreticulin interacts with the endoplasmic reticulum Ca(2+)-ATPase.

A robust mathematical model developed from single cell calcium (Ca(2+)) dynamics has enabled us to predict the consequences of over-expression of endoplasmic reticulum-located chaperones. Model predictions concluded that calreticulin interacts with the lumenal domain of the sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) pump, altering pump affinity for Ca(2+) (K(1/2) switches from 247 to 431 nM) and hence generating Ca(2+) oscillations. Expression of calreticulin in the ER generated an average of six transient-decline oscillations during the Ca(2+) recovery phase, upon exposure to maximal levels of the agonist ATP. In contrast, normal cells produced a single Ca(2+) transient with few or no oscillations. By conditioning the model to experimental data, parameters for generation and decay of IP(3) and SERCA pump kinetics were determined. To elucidate the possible source of the oscillatory behavior three possible oscillators, 1) IP(3), 2) IP(3)R, and 3) SERCA pump, were investigated and parameters constrained by experimental data to produce the best candidate. Each of the three oscillators generated very good fits with experimental data. However, converting a normal exponential recovery to a transient-decline oscillator predicted that the SERCA pump is the most likely candidate for calreticulin-mediated Ca(2+) release, highlighting the role of this chaperone as a signal protein within the endoplasmic reticulum.     

Modeling the interrelations between the calcium oscillations and ER membrane potential oscillations

A refined electrochemical model accounting for intracellular calcium oscillations and their interrelations with oscillations of the potential difference across the membrane of the endoplasmic reticulum (ER) or other intracellular calcium stores is established. The ATP dependent uptake of Ca2+ from the cytosol into the ER, the Ca2+ release from the ER through channels following a calcium-induced calcium release mechanism, and a potential-dependent Ca2+ leak flux out of the ER are included in the model and described by plausible rate laws. The binding of calcium to specific proteins such as calmodulin is taken into account. The quasi-electroneutrality condition allows us to express the transmembrane potential in terms of the concentrations of cytosolic calcium and free binding sites on proteins, which are the two independent variables of the model. We include monovalent ions in the model, because they make up a considerable portion in the balance of electroneutrality. As the permeability of the endoplasmic membrane for these ions is much higher than that for calcium ions, we assume the former to be in Nernst equilibrium. A stability analysis of the steady-state solutions (which are unique or multiple depending on parameter values) is carried out and the Hopf bifurcation leading from stable steady states to self-sustained oscillations is analysed with the help of appropriate mathematical techniques. The oscillations obtained by numerical integration exhibit the typical spike-like shape found in experiments and reasonable values of frequency and amplitude. The model describes the process of switching between stationary and pulsatile regimes as well as changes in oscillation frequency upon parameter changes. It turns out that calcium oscillations can arise without a permanent influx of calcium into the cell, when a calcium-buffering system such as calmodulin is included.     

Modeling of Ca2+ flux in pancreatic beta-cells: role of the plasma membrane and intracellular stores

We have developed a detailed mathematical model of ionic flux in beta-cells that includes the most essential channels and pumps in the plasma membrane. This model is coupled to equations describing Ca2+, inositol 1,4,5-trisphosphate (IP3), ATP, and Na+ homeostasis, including the uptake and release of Ca2+ by the endoplasmic reticulum (ER). In our model, metabolically derived ATP activates inward Ca2+ flux by regulation of ATP-sensitive K+ channels and depolarization of the plasma membrane. Results from the simulations support the hypothesis that intracellular Na+ and Ca2+ in the ER can be the main variables driving both fast (2-7 osc/min) and slow intracellular Ca2+ concentration oscillations (0.3-0.9 osc/min) and that the effect of IP3 on Ca2+ leak from the ER contributes to the pattern of slow calcium oscillations. Simulations also show that filling the ER Ca2+ stores leads to faster electrical bursting and Ca2+ oscillations. Specific Ca2+ oscillations in isolated beta-cell lines can also be simulated.     

A mathematical model quantifying GnRH-induced LH secretion from gonadotropes

A mathematical model is developed to investigate the rate of release of luteinizing hormone (LH) from pituitary gonadotropes in response to short pulses of gonadotropin-releasing hormone (GnRH). The model includes binding of the hormone to its receptor, dimerization, interaction with a G protein, production of inositol 1,4, 5-trisphosphate, release of Ca(2+) from the endoplasmic reticulum, entrance of Ca(2+) into the cytosol via voltage-gated membrane channels, pumping of Ca(2+) out of the cytosol via membrane and endoplasmic reticulum pumps, and release of LH. Cytosolic Ca(2+) dynamics are simplified (i.e., oscillations are not included in the model), and it is assumed that there is only one pool of releasable LH. Despite these and other simplifications, the model explains the qualitative features of LH release in response to GnRH pulses of various durations and different concentrations in the presence and absence of external Ca(2+).     

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.     

Effects of aging on inositol 1,4,5-triphosphate-induced Ca(2+) release in unfertilized mouse oocytes

We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca(2+)-ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383-390). We undertook the present study to further examine the effects of oocyte aging on Ca(2+) release from the inositol 1,4,5-triphosphate (InsP(3))-sensitive Ca(2+) channels of the ER membrane, because not only Ca(2+) reuptake, but also Ca(2+) release from the ER, substantially affect Ca(2+) oscillations in fertilized oocytes. A transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was induced by photolysis of caged InsP(3) microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca(2+)](i) significantly decreased in the aged oocytes. Reduced ER Ca(2+) release in the aged oocyte may not be attributable to aging-related desensitization of the InsP(3)-sensitive Ca(2+) channels in the ER because concentrations of caged InsP(3) for half maximal [Ca(2+)](i) increase were identical for fresh and aged oocytes. The peak [Ca(2+)](i) response following administration of 5 microM thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca(2+) stores. We conclude from these results that reduction of Ca(2+) release from the InsP(3)-sensitive Ca(2+) stores in the aged oocyte arises from depletion of the ER Ca(2+) stores with aging. These aging-related changes in Ca(2+) release and reuptake may account for alterations in Ca(2+) oscillations in aged fertilized oocytes.     

Biophysical re-equilibration of Ca2+ fluxes as a simple biologically plausible explanation for complex intracellular Ca2+ release patterns

Physiological regulation of Ca(2+) release from the endoplasmic reticulum (ER) is critical for cell function. Recent direct measurements of free [Ca(2+)] inside the ER ([Ca(2+)](ER)) revealed that [Ca(2+)](ER) itself is a key regulator of ER Ca(2+) handling. However, the role of this new regulatory process in generating various patterns of Ca(2+) release remains to be elucidated in detail. Here, we incorporate the recently quantified experimental correlations between [Ca(2+)](ER) and Ca(2+) movements across the ER membrane into a mathematical model ER Ca(2+) handling. The model reproduces basic experimental dynamics of [Ca(2+)](ER). Although this was not goal in model design, the model also exhibits mechanistically unclear experimental phenomena such as "quantal" Ca(2+) release, and "store charging" by increasing resting cytosolic [Ca(2+)]. While more complex explanations cannot be ruled out, on the basis of our data we propose that "quantal release" and "store charging" could be simple re-equilibration phenomena, predicted by the recently quantified biophysical dynamics of Ca(2+) movements across the ER membrane.     

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.     

Current compensation in neuronal homeostasis

Ischemic injury of cells in the central nervous system is typically set in motion by influx of extracellular Ca(2+). In this issue of Neuron, Stys and colleagues propose that ischemic injury in spinal cord axons is partly the result of ryanodine receptor-mediated release of Ca(2+) from the endoplasmic reticulum (ER), a site of intracellular Ca(2+) storage.     

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.     

Modeling the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations

Calcium (Ca2+) oscillations play fundamental roles in various cell signaling processes and have been the subject of numerous modeling studies. Here we have implemented a general mathematical model to simulate the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations. In addition, we have compared two different models of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and their influences on intracellular Ca2+ oscillations. Store-operated Ca2+ entry following Ca2+ depletion of endoplasmic reticulum (ER) is an important component of Ca2+ signaling. We have developed a phenomenological model of store-operated Ca2+ entry via store-operated Ca2+ (SOC) channels, which are activated upon ER Ca2+ depletion. The depletion evokes a bi-phasic Ca2+ signal, which is also produced in our mathematical model. The IP3R is an important regulator of intracellular Ca2+ signals. This IP3 sensitive Ca2+ channel is also regulated by Ca2+. We apply two IP3R models, the Mak-McBride-Foskett model and the De Young and Keizer model, with significantly different channel characteristics. Our results show that the two separate IP3R models evoke intracellular Ca2+ oscillations with different frequencies and amplitudes. Store-operated Ca2+ entry affects the oscillatory behavior of these intracellular Ca2+ oscillations. The IP3 threshold is altered when store-operated Ca2+ entry is excluded from the model. Frequencies and amplitudes of intracellular Ca2+ oscillations are also altered without store-operated Ca2+ entry. Under certain conditions, when intracellular Ca2+ oscillations are absent, excluding store-operated Ca2+ entry induces an oscillatory response. These findings increase knowledge concerning store-operated Ca2+ entry and its impact on intracellular Ca2+ oscillations.     

Buffers and oscillations in intracellular Ca2+ dynamics

I model the behavior of intracellular Ca(2+) release with high buffer concentrations. The model uses a spatially discrete array of channel clusters. The channel subunit dynamics is a stochastic representation of the DeYoung-Keizer model. The calculations show that the concentration profile of fast buffer around an open channel is more localized than that of slow buffers. Slow buffers allow for release of larger amounts of Ca(2+) from the endoplasmic reticulum and hence bind more Ca(2+) than fast buffers with the same dissociation constant and concentration. I find oscillation-like behavior for high slow buffer concentration and low Ca(2+) content of the endoplasmic reticulum. High concentration of slow buffer leads to oscillation-like behavior by repetitive wave nucleation for high Ca(2+) content of the endoplasmic reticulum. Localization of Ca(2+) release by slow buffer, as used in experiments, can be reproduced by the modeling approach.