However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2–5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform . This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.Single mechanically skinned extensor digitorum longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca A similar conclusion was reached in a recent study on mouse skeletal fibres, subject to 1–7 days culture and consequent dedifferentiation 2+ sparks was initially low, but increased >10-fold by days 5–7. This marked increase in spark frequency coincided with morphological changes, including disorganization of the t-tubule network, suggesting a causal relationship. Further work revealed that suppression of Ca2+ sparks does not occur in dysgenic myotubes (i.e. lacking DHPRs), suggesting that in resting myotubes, the DHPRs within each CRU exert a tonic inhibitory influence on associated RyRs A number of previous studies have investigated the relationship between the t-tubule network and RyR1 function in cultured skeletal myotubes. Early experiments on primary cultured mouse myotubes revealed that spontaneous Ca2+ spark activity, it is not clear to what extent these findings translate to adult muscle, where there are clear differences in signalling pathways and the expression levels of key proteins such as RyR3 2+ sparks, while over expression of RyR1 did not 2+ sparks can be explained partly by the relatively low level of RyR3 expression, compared with developing myotubes. However, these findings do not exclude the possibility of a tonic inhibitory influence on RyR1 conferred by the t-tubule/DHPR in resting adult muscle.While these studies on cultured cells suggest a role for the t-tubule/DHPR in the suppression of Ca2+ release mechanism to be initiated by field stimulation. Using confocal microscopy, we showed that a proportion of skinned fibres exhibit localised regions of SR Ca2+ release failure, where the t-tubule network failed to re-seal. These fibres were used as a model to compare properties of RyR1 (agonist sensitivity and Ca2+ spark frequency) in adjacent regions of the cell with or without a functional t-system.In the present study, mechanically skinned skeletal fibres from adult rats were used to investigate the influence of functional, polarised t-tubules on RyR1. Following skinning, the t-tubules re-seal and repolarise, allowing the physiological SR Camilieu. Following skinning, the t-tubules re-seal and repolarise, allowing the physiological depolarisation-induced Ca2+ release process to be initiated by field stimulation Rats (200–300 g) were humanely killed in accordance with UK legislation (Schedule 1 procedure) Single extensor digitorium longus (EDL) muscle fibres were mechanically skinned under oil and then perfused with solutions approximating to the intracellular +, 37; K+, 126; phosphocreatine, 10; EGTA, 0.05; HEPES, 90; fluo-3, 0.02. The free [Mg2+] of this solution was adjusted to 1 mM by addition of MgO. The free [Ca2+] was ∼60 nM. In most experiments 50 μM n-benzyl-p-toluene sulphonamide (BTS) was added to inhibit Ca2+-activation of the myofilaments 2+ release or the occurrence of localised Ca2+ release failure. Corrections for ionic strength, details of pH measurement, allowance for EGTA purity and the principles of the calculations are described elsewhere 2+ release was induced by rapid addition of a solution with 20 mM caffeine, no added Mg2+. However, while the applied solution contains has no added Mg2+, there is some mixing with the control solution , which means that the actual [Mg2+] at the preparation is ∼20 μM . Consequently, we have referred to this throughout as application of 20 mM caffeine, low Mg2+.All chemicals were purchased from Sigma unless stated otherwise. For most experiments, a basic solution was prepared containing (mM): HDTA, 50; ATP, 8; NaThe apparatus used for mounting the preparation and application of solutions is described in detail elsewhere 2+ release from the SR was detected using a Bio-rad Cellmap confocal scanhead attached to the side port of the Nikon TE3000 microscope (excitation 488 nm). In some experiments, sequential frames were collected in x–y mode before, during and after brief (300–400 ms) tetanic responses initiated by applying suprathreshold square-wave stimuli  at 50 Hz. A relatively slow x–y frame rate  was used to minimize noise on the Ca2+ signal. However, the collection rate was sufficient to ensure that one to two frames were obtained during the peak of the peak of each tetanic response.Ca2+, the dye is at or near full saturation within the t-tubules (free [Ca2+] is ∼1 mM), allowing regions of the cell with re-sealed t-tubules to be identified using confocal microscopy. In control experiments, this method was compared with an alternative procedure, in which the dye is introduced into the t-tubules before skinning We have previously reported that (i) when skinned fibres are bathed in a solution with either fluo-3 or fluo-5N, the dye progressively accumulates within re-sealed t-tubules and associated vesicles and (ii) dye transport into the t-tubules can be inhibited using probenecid p < 0.05) levels were calculated using a Student's t-test for unpaired observations and the data are presented as mean ± standard error (S.E.M.).Significance  illustrates the maximum length of fibre that could be observed in a single frame under 40× magnification. Although this varies depending on optical arrangement and fibre orientation, this is typically ∼200–300 μm.While studies involving force measurements on mechanically skinned skeletal muscle fibres typically use sections 2–3 mm in length, confocal imaging systems allow only a fraction of the preparation to be viewed at one time. x–y images were obtained at 6.2 Hz. This relatively low frame rate optimised the signal-noise-ratio, while ensuring that at least one frame was obtained during the peak of each tetanic response. The graph illustrates the changes in [Ca2+] during the period of stimulation, where each point was obtained by integrating a single x–y frame. The individual x–y frames obtained during the peak of each transient show that the tetanic responses are reproducible and that Ca2+ release is typically uniform in nature. Also shown is a series of sequential x–y frames obtained during a single tetanic response (right) and a transverse line scan image (below) obtained during a tetanic response.2+] was typically uniform in randomly selected regions of each fibre, spatial non-uniformities were observed in approximately 20% of fibres. This is shown in 2+ throughout the fibre, during the peak of the tetanic responses. Pronounced “notches” in the fluorescence profile are apparent, corresponding to regions of the fibre where field stimulation has failed to elicit SR Ca2+ release. As shown in the expanded sections, these localised regions of SR Ca2+-release failure are variable in size and shape, but have sharply defined boundaries. The pattern of Ca2+ release was not altered by changing the polarity of the electrodes, or by increasing the stimulation voltage (not shown), suggesting that Ca2+ release failure did not reflect an inadequate depolarisation stimulus.While the profile of tetanic . Control experiments showed that the dye was not saturated during a maximal SR Ca2+ release.The presence of readily identifiable regions of skinned fibres lacking re-sealed t-tubules was used to study the functional relationship between the DHPR/t-tubule network and associated RyRs located within the junctional SR. In 2+ release including (1) a decrease in free [Mg2+] to 20 μM, (2) 500 μM 4-CMC or (3) 20 μM ryanodine. In each fibre, although each intervention released less Ca2+ than t-tubule depolarisation, the spatial distribution of Ca2+ was similar. As considered above, regions of the cell in which the t-tubule network has apparently failed to re-seal following skinning, exhibit a lower sensitivity to RyR1 agonists. This might reflect the polarization status of the t-tubule membrane in re-sealed regions, e.g. (1) if the configuration of the DHPRs in regions of the cell with polarised t-tubules differs from that in regions with un-sealed (depolarised) t-tubules, such that a higher sensitivity to RyR1 agonists is conferred or (2) if re-sealed t-tubules are subject to spontaneous, subthreshold fluctuations in membrane potential, which leads to a reduced inhibitory influence of the DHPRs on associated RyRs.2+ release identified (upper panel). As in previous examples, only the region exhibiting depolarisation-induced Ca2+ release (middle) responded to 5 mM caffeine (right). The same fibre was then perfused with a solution in which K-HDTA was substituted with Na-HDTA in order to induce sustained depolarisation of the t-tubules (lower panel). Following Na-substitution, the fibre was unresponsive to field stimulation, consistent with sustained depolarisation of the t-tubule network . However, on subsequent addition of 5 mM caffeine, the pattern of Ca2+ release remained similar to that observed under control conditions . This suggests that the higher sensitivity to caffeine in regions responsive to field stimulation is unrelated to the polarisation status of the re-sealed t-tubules. Again, addition of 20 mM caffeine/low Mg2+ induced a uniform release throughout the fibre , demonstrating that RyRs remain functional throughout the preparation and that the SR [Ca2+] content is relatively uniform. Similar results were obtained in five other preparations.The role of t-tubule polarisation was investigated using the protocol shown in 2+ release to be identified. As in previous examples, addition of 5 mM caffeine induced Ca2+ release only in the region sensitive to field-stimulation, while 20 mM caffeine/low Mg2+ induced a uniform SR Ca2+ release throughout the preparation (upper panel). The preparation was then returned to a control solution before exposure to 50 μg/ml saponin for 10 min (middle panel). The initial exposure to saponin induced a detectable release of Ca2+ due to selective permeabilization (and depolarisation) of the re-sealed t-tubule network. Following removal of saponin, neither substitution of K-HDTA with Na-HDTA nor field stimulation induced a detectable rise in [Ca2+], consistent with permeabilization of the t-tubule network. However, after exposure to saponin, submaximal concentrations of caffeine (2 or 5 mM) induced a uniform release of Ca2+ throughout the fibre (lower panel). Exposure to 20 mM caffeine/low Mg2+ induced a maximal release of Ca2+, which was comparable to that obtained prior to saponin treatment, suggesting that saponin treatment did not affect the SR Ca2+ content or the distribution of Ca2+ within the SR network. Similar results were obtained in four other preparations.In the experiment shown in 2+ release following exposure to saponin is that the responsiveness of RyR1 may be influenced by the presence of Ca2+ within the re-sealed t-tubule, which is lost on permeabilization. Therefore, a further series of experiments was carried out in which cells were initially exposed to a Ca2+-free Tyrode's solution (containing 5 mM EGTA). Following skinning, the free [Ca2+] within the t-tubule was calculated to be ∼1 nM (based on 10 μM contamination), and the presence of millimolar EGTA within the t-tubules ensured that the [Ca2+] remained low for a prolonged period, despite any slow accumulation of Ca2+ from the bathing solution. In most of these preparations, field stimulation continued to release substantial Ca2+ from the SR, allowing identification of localised regions exhibiting Ca2+ release failure  and 5 mM EGTA, before skinning . While quiescent, the cell also exhibited Ca2+ sparks and longer embers, similar to those reported in intact and skinned skeletal cells. However, these events were predominantly restricted to the region of the cell responsive to field stimulation. As described previously −, which normally serves to stabilize the membrane potential, some skinned cells exhibit spontaneous depolarisations, resulting in repeated twitch-like responses. An example of this is shown in 2+ transients arose spontaneously at irregular intervals. In this case the upper half of the scan line was positioned through a region producing spontaneous Ca2+ transients, while the lower half was quiescent and insensitive to field stimulation (not shown). Again, spontaneous Ca2+ sparks were apparent, but only at high frequency in the region of the fibre exhibiting spontaneous Ca2+ transients.2+ release events (upper panel). Field stimulation resulted in a homogeneous rise in [Ca2+] throughout the fibre, indicating that the presence of functional, polarised t-tubules throughout. The fibre was then exposed to a Na-HDTA solution to induce sustained depolarisation of the t-tubule network. Thereafter, both spontaneous Ca2+ release events were markedly reduced in frequency and field stimulation failed to elicit a response. 2+ release events. In each cell, half of the longitudinal scan line was positioned in a region responsive to field stimulation and the other half in an unresponsive region. The ordinate shows the total number of spontaneous Ca2+ release events in four fibres at the position indicated either before (black bars) or after depolarisation of the t-tubule network with Na-HDTA (open bars). All distances are relative to the boundary between responsive and unresponsive regions of the cell. The data show that in cells exhibiting spontaneous Ca2+ sparks and embers in regions responsive to field stimulation, sustained depolarisation of the t-tubule membrane reduces the frequency of spontaneous Ca2+ release events to a low level, which is not significantly different to that in the unresponsive regions.2+ release  was similar in regions with or without re-sealed t-tubules  2+ following activation of RyR1 2+] that occurs in the vicinity of the RyR on application of caffeine. However, in control experiments using fluo-5N to measure Ca2+ within the t-tubules, submaximal levels of caffeine (≤5 mM applied with 1 mM Mg2+) did not induce sufficient SR Ca2+ depletion to activate SOCE . Furthermore, we show that Mg2+ can replace Ca2+ in the t-tubule network and confer higher agonist sensitivity in regions with re-sealed t-tubules  Ca-tubules . As Mg2+2+ to the DHPR results in increased agonist sensitivity of the RyR1 a via a long-range allosteric mechanism. Early studies addressing the role of extracellular Ca2+ in excitation contraction coupling are consistent with this potential mechanism: First, although Ca2+ entry is not essential for excitation–contraction coupling in skeletal muscle, the binding of extracellular Ca2+ to the DHPR is required for normal activation and other divalent ions can substitute for Ca2+ at this site Second, studies on intact skeletal preparations from normal muscle and muscle susceptible to malignant hyperthermia have shown that withdrawal of extra Ca2+ reduces the sensitivity of RyR1 to caffeine 2+ or other divalent ions in the extracellular solution, consistent with involvement of an extracellular divalent binding site. Third, while little trans-sarcolemmal Ca2+ entry occurs during muscle activation, fibres from malignant hyperthermia susceptible muscle often fail to respond to triggering levels of halothane in the absence of extracellular Ca2+2+ is associated with a conformational state of the DHPR, which increases the agonist sensitivity of the RyR1.An alternative possibility is simply that the binding of extracellular Ca2+ sparks arose spontaneously or could be induced by a brief pulse of Na-HDTA to induce partial depolarisation of the t-tubule network , which normally serves to stabilize the membrane potential in intact cells In approximately 10% of cells, spontaneous Ca network . These e-tubules . This suansients , it seem2+2+]) during fatiguing stimulation is dependent upon the presence of extracellular Ca2+2+ entry pathway 2+ may affect the development of fatigue by acting on RyR1 indirectly via, a mechanism that involves occupation of an extracellular divalent ion binding site, most likely associated with the DHPR.The present study demonstrates that extracellular divalent ions influence the gating properties of RyR1 and its sensitivity to activation by pharmacological agents. This finding may have relevance to studies on muscle from patients with malignant hyperthermia, where direct activation of RyR1 by triggering anaesthetics does not occur in the absence of extracellular Ca


Though the mechanism of extra-matrix Ca2+ dependency of mitochondrial Ca2+ uptake has been well characterized both experimentally and mathematically, the mechanism of membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake has not been completely characterized. In this paper, we perform a quantitative reevaluation of a previous biophysical model of mitochondrial Ca2+ uniporter that characterized the possible mechanism of ΔΨ dependency of mitochondrial Ca2+ uptake. Based on a model simulation analysis, we show that model predictions with a variant assumption , that provides the best possible description of the ΔΨ dependency, are highly sensitive to variation in matrix [Ca2+], indicating limitations in the variant assumption (Case 2) in providing physiologically plausible description of the observed ΔΨ dependency. This sensitivity is attributed to negative estimate of a biophysical parameter that characterizes binding of internal Ca2+ to the uniporter. Reparameterization of the model with additional nonnengativity constraints on the biophysical parameters showed that the two variant assumptions (Case 1 and Case 2) are indistinguishable, indicating that the external and internal Ca2+ binding constants for the uniporter may be equal (Case 1). The model predictions in this case are insensitive to variation in matrix [Ca2+] but do not match the ΔΨ dependent data in the domain ΔΨ≤120 mV. To effectively characterize this ΔΨ dependency, we reformulate the ΔΨ dependencies of the rate constants of Ca2+ translocation via the uniporter by exclusively redefining the biophysical parameters associated with the free-energy barrier of Ca2+ translocation based on a generalized, non-linear Goldman-Hodgkin-Katz formulation. This alternate uniporter model has all the characteristics of the previous uniporter model and is also able to characterize the possible mechanisms of both the extra-matrix Ca2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake. In addition, the model is insensitive to variation in matrix [Ca2+], predicting relatively stable physiological operation. The model is critical in developing mechanistic, integrated models of mitochondrial bioenergetics and Ca2+ handling.Mitochondrial Ca Mitochondrial Ca2+ homeostasis is critical for metabolic regulation, mitochondrial function/dysfunction, and cell physiology/pathophysiology 2+ uptake via the uniporter is essential for developing mechanistic, integrated models of mitochondrial bioenergetics and Ca2+ handling that can be helpful in understanding the mechanisms by which Ca2+ plays a role in mediating signaling pathways between cytosol and mitochondria and modulating mitochondrial energy metabolism in health and disease Mitochondrial Ca2+ uptake depends on the catalytic properties of the uniporter and also on the electrochemical gradient of Ca2+ across the inner mitochondrial membrane (IMM), which has been extensively studied both experimentally 2+ dependency of mitochondrial Ca2+ uptake has been well characterized, the mechanism of membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake has not been completely characterized.The kinetics of mitochondrial Ca2+ uniporter  under two different kinetic assumptions  were formulated to characterize the extra-matrix Ca2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake via the uniporter 2+ dependent data 2+ uptake.In the development of our recent model of the uniporter 2+] was unknown from the measurements. For model parameterization, matrix [Ca2+] was fixed at 250 nM. Although the two variant models under Case 2 were able to adequately describe all the available experimental data with appropriate model perturbations as provided by the experimental protocols, it was unknown whether physiological variation of matrix [Ca2+], as seen in the intact myocyte, have significant impacts on the estimates of model parameters and model predicted trans-matrix Ca2+ fluxes via the uniporter. Therefore, it is important to test the robustness of the estimates of model parameters and model predictions subject to such physiological variation.The four variant models of the uniporter 2+] (ranging from 100 nM to 500 nM), suggesting that the model parameter estimates under Case 2 would vary significantly to variation in matrix [Ca2+], and hence can not be robust. This indeed indicates that the Case 2, in which the Ca2+ binding constants for the uniporter at the inside and outside of the IMM are distinct, is physiologically implausible, and hence can not be a feasible representation of the observed ΔΨ dependency of the uniporter-mediated mitochondrial Ca2+ uptake. Furthermore, the Case 2 is associated with negative estimates of the biophysical parameter αx (with αe = 0 fixed)  are indistinguishable from each other, indicating that the external and internal Ca2+ binding constants for the uniporter may be equal (Case 1). The model predictions in this case are insensitive to variation in matrix [Ca2+], but do not match the ΔΨ dependent data In the present paper, we attempt to provide a quantitative reevaluation of our previous model of the uniporter xed) see , which i2+ uptake via the uniporter in the entire ΔΨ domain for which data are available kin and kout of Ca2+ translocation in our previous model of the uniporter βe and βx associated with the free-energy barrier of Ca2+ translocation based on a generalized, non-linear GHK (Goldman-Hodgkin-Katz) formalism  under both the kinetic assumptions (Case 1 and Case 2) is chosen, because both the fully and partial cooperativity binding models (Model 1 and Model 2) are indistinguishable from the available experimental data This section presents the detailed simulation analyses of our previous model of mitochondrial Ca2+ uptake based on our previous model (Model 1) of mitochondrial Ca2+ uniporter The simulation analyses of mitochondrial Ca2+ influx via the uniporter were measured in suspensions of energized mitochondria purified from rat hearts and rat livers following additions of varying levels of extra-matrix Ca2+   (2+] was fairly unknown. Our previous model of the uniporter 2+] of 250 nM. 2+] on the estimates of model parameters and model predicted Ca2+ fluxes via the uniporter.In the experiments of Scarpa and Graziotti ctively) . In the  = 0 mM) ; ΔΨ was 2+ fluxes via the uniporter (lines) as functions of extra-matrix [Ca2+] (ΔΨ = 190 mV) and ΔΨ  over a range of matrix [Ca2+] along with the experimental data 2+] was varied from 100 nM to 500 nM. The simulations corresponding to the model fits to the data are based on matrix [Ca2+] of 250 nM. 2+ fluxes via the uniporter as a function of extra-matrix [Ca2+] for a range of ΔΨ (100 mV to 210 mV) and as a function of ΔΨ for a range of extra-matrix [Ca2+] (10 µM to 150 µM), with matrix [Ca2+] fixed at 250 nM.Specifically, 2+] fixed at 250 nM is able to fit well to all of the available experimental data 2+]  , it is unlikely that physiological variation of matrix [Ca2+] would have any appreciable effects on the experimental measurements and model predictions on Ca2+ fluxes via the uniporter as well as on the estimates of the uniporter model parameters. Furthermore, it is unlikely that in the experiments of Scarpa and colleagues 2+] would have been precisely maintained at 250 nM. Therefore, the present model simulation analyses suggest that the model parameter estimates under Case 2 would vary considerably with variation in matrix [Ca2+] as well as with different initial guesses for the parameters, compared to that under Case 1. In the other words, the model parameter estimates under Case 2 would be ambiguous and not unique (robust), and are expected to be different for different matrix [Ca2+] and different initial guesses for the parameters. Given matrix [Ca2+], the initial guesses for the parameters need to be close to the optimal parameter estimates for the optimization algorithm to converge to the optimal parameter estimates. In this case, the sensitivities of the model to variations in matrix [Ca2+] would also be different for different model parameter estimates with similar fittings of the model to the experimental data.It is apparent from the model simulation analyses in αx (with αe = 0 fixed), which is found to be contributing to the high sensitivities of the model predictions to variation in matrix [Ca2+] and stiff gradients of Ca2+ uptake profiles to variation in ΔΨ. The Ca2+ uptake profiles under Case 2 attaining saturation for a lower level of extra-matrix [Ca2+] is attributed to the lower estimates of 2+] to the uniporter , as in our previous paper on the uniporter 2+ dependent kinetic data of Scarpa and Graziotti α, βe, and βx are arbitrarily chosen to satisfy the two kinetic and thermodynamic constraints of Eq. (S7) as well as to fit these data sets, as these parameters can not be accurately estimated from these data sets in which ΔΨ is constant. These extra-matrix Ca2+ dependent data sets, however, provide accurate estimates of the binding constants α, βe, and βx are estimated from the ΔΨ dependent kinetic data of Wingrove et al. α, βe, and βx. In the final step, with the values of α, βe, and βx fixed, as estimated from the first and second steps, the rate constants 2+ in the experimental buffer).As shown in rter see . To recoαe = αx = α≈0, βe≈0.113, and βx≈0.887. The new model fittings to the data for both the cases (Case 1 and Case 2) are exactly same as those shown in 2+ binding constants for the uniporter were assumed to be distinct  of mitochondrial Ca2+ uniporter, in which αe = αx = α, and βe and βx are functions of ΔΨ and nH (see Eq. 7), are shown in 2+ dependent kinetic data of Scarpa and coworkers 2+ to the uniporter with arbitrary values of α, and nH. This estimated α, and nH) are estimated from the ΔΨ dependent kinetic data of Gunter and coworkers α, and nH fixed, the rate constant The simulation analyses of mitochondrial Ca2+ fluxes via the uniporter (lines) as functions of extra-matrix [Ca2+] (ΔΨ = 190 mV) and ΔΨ  with the variation in matrix [Ca2+] (100 nM to 500 nM) and their comparisons to the available experimental data 2+] = 250 nM. 2+ fluxes via the uniporter as a function of extra-matrix [Ca2+] for a range of ΔΨ (100 mV to 210 mV) and as a function of ΔΨ for a range of extra-matrix [Ca2+] (10 µM to 150 µM), obtained with matrix [Ca2+] = 250 nM. The left, middle, and right plots correspond to the model simulation analyses based on the experimental protocols of Scarpa and Graziotti 2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake via the uniporter in the entire ranges of extra-matrix [Ca2+] and ΔΨ for which data were available. In addition, this alternate uniporter model is insensitive to variation in matrix [Ca2+], making the model physiologically plausible. This characteristic of the model helps provide unique and accurate estimates of the model parameters with different matrix [Ca2+]. It is observed from 2+ uptake profiles, obtained from the present alternate uniporter model, do not have stiff gradients with respect to ΔΨ, and reach saturation for a higher level of extra-matrix [Ca2+], comparable to that obtained under Case 1 of our previous uniporter model  to the extra-matrix Cavely see . These dβe and βx that characterize the ΔΨ dependent factor E(ΔΨ) in the uniporter flux expressions , and hence the ΔΨ dependency of mitochondrial Ca2+ uptake via the uniporter βe and βx are analytical functions of ΔΨ (Eq. 7), while the dotted lines are based on our previous model of the uniporter βe and βx are constants and are numerically estimated. The results show that βe and βx based on the previous model abruptly change their respective values at ΔΨ = 0, resulting in non-differentiability of the factor E(ΔΨ) at ΔΨ = 0. In contrast, βe, βx, and E(ΔΨ) based on the present model are smooth functions of ΔΨ. The ΔΨ dependent factor E(ΔΨ) in the present model has a larger spread, resulting in a better fit of the model to the ΔΨ dependent data The ΔΨ dependencies of the biophysical parameters 2+ uniporter 2+ into energized (respiring) mitochondria, and hence plays an important role in mitochondrial Ca2+ homeostasis. Specifically, the present paper provides an alternate (improved) biophysical model of the uniporter that overcomes the limitations of our previous uniporter model by mechanistically recharacterizing the membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake via the uniporter. This alternate uniporter model is able to accurately characterize the possible mechanisms of both the extra-matrix Ca2+ and ΔΨ dependencies of the uniporter-mediated mitochondrial Ca2+ uptake +/Ca2+ exchanger 2+ handling that can be helpful in understanding the mechanisms by which Ca2+ plays a role in mediating signaling pathways between cytosol and mitochondria and modulating mitochondrial energy metabolism in health and disease.The major contributions of the present paper lies in the improvements of our previous biophysical model of mitochondrial Ca2+ translocation) 2+ to the uniporter, as depicted experimentally 2+ to the uniporter; Case 1 or Case 2: external and internal Ca2+ binding constants for the uniporter are equal or distinct) to several independent experimental data sets from the literature on the kinetics of Ca2+ fluxes via the uniporter 2+ dependent data of Scarpa and coworkers 2+ to the uniporter are distinct (Case 2). Therefore, the mechanistic formulation, thermodynamic feasibility, and ability to describe a large number of independent experimental data sets are some of the notable features of the model 2+ translocation, the present model has all the characteristics of the previous model.Our previous model of the uniporter 2+] was not measured. Our two variant models of the uniporter under two different cases 2+] of 250 nM. Although both the variant models under Case 2 were able to satisfactorily explain all the available experimental data with suitable model perturbations as provided by the experimental protocols, it was not realized whether physiological variation of matrix [Ca2+], as observed in the intact myocyte, will have any appreciable effect on the estimates of model parameters and model predicted trans-matrix Ca2+ fluxes via the uniporter. The present study provides a quantitative reevaluation of the previous uniporter model 2+] ranging from 100 nM to 500 nM. Based on this model simulation analysis, it is found that the two variant model predictions under Case 2 are highly sensitive to variation in matrix [Ca2+]   , attribu2+ uniporter 2+ binding constants for the uniporter were assumed distinct  , and the Eq. B7; . The altvailable . In addi2+ signaling 2+ in distinct region of the cells and maintain spatial Ca2+ concentration low even under strong global Ca2+ mobilization upon cell stimulation is critical for the Ca2+ sensitive signal transduction within the cell 2+ oscillations and waves, generated by the Ca2+ release from the endoplasmic reticulum 2+ oscillations, the mitochondrial Ca2+ uptake has also been shown to influence significantly the nature of Ca2+ oscillations 2+ entry 2+ uptake has considerable effect on the STIM1-Orai1-dependent store operated Ca2+ entry into endothelial cells 2+ uniporter will be crucial for developing an integrated model of intracellular and mitochondrial Ca2+ handling which can be helpful in understanding many aspects of signal transduction mechanisms.Recently, mitochondria have been recognized as one of the key organelles that actively involves in physiological Ca2+ uniporter that accurately characterizes the ΔΨ dependency of mitochondrial Ca2+ uptake via the uniporter kin and kout of Ca2+ translocation  formulation. The expressions for the binding constants Ke and Kx and the equilibrium constants Keq remain the same as in Eqs. (S4) and (S6) of 2+ and ΔΨ dependencies of the uniporter-mediated mitochondrial Ca2+ uptake 2+], making the model physiologically plausible.This section provides the derivation of the present alternate (improved) biophysical model of mitochondrial Ca Eq. S5,  in our pkin and kout of Ca2+ translocation via the uniporter (see Eq. S5) can be expressed in the following form kin and kout and Eq. (S4) for Ke and Kx into Eqs. (S1–S2), the uniporter flux expression is reduced toD = D1 for Model 1 (Eq. S9a) and D = D2 for Model 2 (Eq. S9b). In order to derive the functional form of 2+ transport via the uniporter 2+ transport via the uniporter in the absence of ΔΦ nH, and hence the two rate constants kin and kout in Eq. (1) are fully characterized by only three unknown parameters nH, in contrast to four unknown parameters βe and βx in the previous formulation  and D = D2 for Model 2 (Eq. S9b). By comparing the present uniporter model (Eq. 5) with the previous uniporter model (Eq. S8), we obtain the following functional relationship between the biophysical parameters αe, αx, βe, βx, and nH:βe and βx can be expressed in terms of ΔΦ asβe and βx in the present model of the uniporter become functions of ΔΨ, compared to the previous model of the uniporter βe and βx were constant with respect to ΔΨ. Furthermore, both the parameters are characterized by only one unknown parameter nH.Based on a generalized, non-linear GHK formulation, the rate constants tion see . In stan2+ fluxes via the uniporter subject to the constraints: αe = αx = α≥0 (Case 1). Thus, only four unknown parameters Nexp is the number of experiments and Ndata is the number of data points in a particular experiment, 2+ uptakes and EClick here for additional data file.Table S12+ uniporter.Estimated parameter values in the previous models of mitochondrial Ca(0.09 MB DOC)Click here for additional data file.


Calcium is a ubiquitous signal molecule that is involved in the control of proliferation and apoptosis. We aimed to investigate if the endoplasmic reticulum (ER) Ca2+-signaling was investigated using fluorescence microscopy and the expression of Ca2+-regulating proteins was assessed using Western Blot analysis.The intracellular Ca2+-content was reduced compared to NHBE. The reduced Ca2+-content correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP3R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. Lowering the ER Ca2+-content with CPA led to increased proliferation NHBE and lung cancer cells.In a Small Cell Lung Cancer (H1339) and an Adeno Carcinoma Lung Cancer (HCC) cell line but not in a Squamous Cell Lung Cancer (EPLC) and a Large Cell Lung Cancer (LCLC) cell line the ER Ca2+-homeostasis between lung cancer and NHBE cells could represent a new target for cancer treatments.The significant differences in Ca Lung cancer is the leading cause of cancer death in the industrial nations . Despite2+-concentration ([Ca2+]c) can result either from Ca2+-influx from the extracellular space or from Ca2+-release from internal Ca2+-stores, primarily the ER. Proteins involved in the Ca2+-release from the ER are the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR)  force calcium against the concentration gradient from the cytoplasm into the ER. Within the ER, calcium is buffered by calreticulin c, cells were loaded for 30 min at 37°C with the calcium indicator dye Fluor-4 AM  in supplemented Hanks Balanced Salt Solution (sHBSS) containing 0.2% Pluronic . After loading, the cells were incubated for at least 30 min in sHBSS to allow for complete dye deesterification and examined with a fluorescence microscope . Images were recorded in time lapse (1 frame/sec) using a digital CCD camera . For each image, regions of interest (ROIs) were defined in single cells, and the average fluorescence intensity of each ROI was measured. Final fluorescence values were expressed as a fluorescence ratio (F/Fo) normalized to the initial fluorescence (Fo). Each analysis was performed using custom written macros in the image analysis software "Scion".For quantification of changes in the  Abcam 1:500, SERCA3 ATPase antibody Abcam 1:200). Sheep anti-mouse IgG horseradish peroxidase linked whole antibodies  were used as secondary antibodies. β-actin served as a loading control. Antibody complexes were visualized using Hyperfilm ECL chemiluminescence  and evaluated using the "Image-J" analysis software.Protein expression was determined by immunoblotting with protein extracts prepared with the Compartmental Protein Extraction Kit according to the manufacturer's instructions . EGFR was used as control for plasma membrane contamination, which was found to be low with no differences between cell types. Protein concentrations of the extracts were quantified using the Bradford Protein Assay Kit . Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on a 6% separating and 4% stacking gel (for SERCA 2), on a 4% separating and 3% stacking gel  were performed using the "Sigma Stat" software . A 2+-influx in Ca2+-homeostasis in lung cancer cells, NHBE , H1339 , HCC (adeno carcinoma), EPLC 272 (squamous cell carcinoma) and LCLC (large cell lung carcinoma) cells were exposed to 1 mM ATP in the presence and the absence of extracellular calcium . The resulting increase in the [Ca2+]c was quantified using fluorescence microscopy. Baseline fluorescence values were similar in all cell lines (data not shown). In NHBE, H1339 and HCC cells, the ATP-induced Ca2+-increase was comparable with and without external calcium suggesting an insignificant role for Ca2+-influx  is not reliably feasible. Therefore, we used an indirect approach. SERCA were inhibited using 1 μM cyclopiazonic acid (CPA) leading to a net Ca2+-efflux out of the ER. The resulting increase in [Ca2+]c was used as an estimate of the [Ca2+]ER [2+-influx contributed to the ATP-induced Ca2+-increase (EPLC 272 and LCLC) the [Ca2+]ER was equal to the [Ca2+]ER in NHBE  [Ca2+]ER was lower than in NHBE. The SCLC line H1339 showed the lowest [Ca2+]ER . In contrast, IP3R showed substantial expression, which was higher in the lung cancer cell lines, and the highest in H1339 cells  and an additional small cell lung cancer cell line (DMI 53 pI). In both cell lines, the ATP-induced increase in [Ca2+]C was independent from Ca2+-influx from the extracellular space (data not shown). Treatment with CPA caused in NHBE cells and all lung cancer cell lines an increase in cell number compared with non-treated controls  the ER Ca2+-content was reduced compared to NHBE cells. The reduced Ca2+-content in H1339 and HCC cells correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP3R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. Reducing the ER Ca2+-content with CPA for 24 h led to an increased cell number.In this study, we showed that the contribution of CaThe origin of the various lung carcinomas is still controversially being discussed. While squamous cell lung carcinomas are believed to origin from metaplastic bronchial epithelium, many authors believe small cell lung carcinomas to origin from neuro-epithelial bodies. But, the origin of large cell carcinomas and adeno carcinomas is less clear. However, being forced to choose a "normal" tissue to compare the malignant cell lines with, we decided to use normal human bronchial epithelial cells as a reference knowing that this choice constitutes a compromise.2+-content. In heart failure, the ER Ca2+-content of cardiac myocytes has been found to be reduced due to altered expression of SERCA ER and proliferation, Legrand et al. showed that in prostate cancer cells an increased growth rate was correlated with higher [Ca2+]ER and increased SERCA 2 expression [2+]ER and decreased expression of SERCA 2b. Neuroendocrine differentiation of prostate cancer cells is considered to mark increased aggressiveness of cancer growth. Vanverberghe et al. showed that neuroendocrine differentiation in these cells was associated with apoptosis resistance probably due to decreased filling of the ER Ca2+-store caused by under-expression of SERCA 2 and calreticulin [2+]ER due to increased SERCA 2 expression [2+]c or sustained Ca2+-oscillations, decreased refilling of the ER because of SERCA inhibition, and enhanced store operated Ca2+-entry from the extracellular space [2+]ER, SERCA expression and tumor growth varies between studies, cell types and differentiation status. However, an altered ER Ca2+-homeostasis is obviously involved in malignant transformation. To our knowledge, this is the first report showing an altered ER Ca2+-homeostasis in lung cancer cells.Investigating the correlation of SERCA expression, ER is regarded as a pathophysiological mechanism in heart failure [2+-homeostasis become available for clinical use, the altered Ca2+-homeostasis of cancer cells may become a valuable target to improve therapeutic options in lung cancer.A decreased [Ca failure . Istarox failure . As subs2+-content was reduced compared to NHBE cells. The reduced Ca2+-content correlated with a reduced expression of SERCA 2 pumping calcium into the ER, an increased expression of IP3R releasing calcium from the ER, and a reduced expression of calreticulin buffering calcium within the ER. The differences in the intracellular Ca2+-homeostasis between lung cancer and normal bronchial epithelial cells may lay the basis for new diagnostic or therapeutical approaches.In our study, we showed that in H1339 and HCC cells the ER CaThe authors declare that they have no competing interests.2+-signaling and drafted the manuscript. JK carried out experiments on the Ca2+-signaling and Western Blot analysis. AT and RMH participated in the study design and revised the manuscript critically for important intellectual content.AB conceived the study, carried out experiments on the Ca


Mechanisms must exist to clear the ciliary Ca2+ before it reaches toxic levels. One such organelle has been well studied: the vertebrate olfactory cilium. Recent studies have suggested that clearance from the olfactory cilium is mediated in part by plasma membrane Ca2+-ATPase (PMCA).In any fine sensory organelle, a small influx of Ca2+ in single frog olfactory cilia. Ca2+ was allowed to enter isolated cilia, either through the detached end or through membrane channels. Intraciliary Ca2+ was monitored via the activity of ciliary Ca2+-gated Cl− channels, which are sensitive to free Ca2+ from about 2 to 10 µM. No significant effect of MgATP on intraciliary free Ca2+ could be found. Carboxyeosin, which has been used to inhibit PMCA, was found to substantially increase a ciliary transduction current activated by cyclic AMP. This increase was ATP-independent.In the present study, electrophysiological assays were devised to monitor cytoplasmic free Ca2+ clearance. It is concluded that PMCA in the cilium plays a very limited role in clearing the micromolar levels of intraciliary Ca2+ produced during the odor response.Alternative explanations are suggested for two previous experiments taken to support a role for PMCA in ciliary Ca However, Ca2+ becomes toxic at high levels, and several mechanisms are known that limit or reverse increases in cytoplasmic Ca2+2+ clearance can be an acute problem in any dendrite, cilium, or other cellular process with an enormous surface-to-volume ratio. In such a process, a small influx of extracellular Ca2+ could quickly elevate cytoplasmic Ca2+ to a toxic concentration. How such fine cellular compartments clear Ca2+ is not well understood.Calcium ion is widely used as in intracellular messenger. Many signal-transduction processes cause cytoplasmic Ca2+ clearance in fine organelles. Olfactory signal transduction occurs on these cilia, which extend from the tip of each olfactory receptor neuron into the mucus 2+ accumulates within the cilium during odor transduction. Transduction begins when an odorant molecule binds to a receptor on the membrane of a cilium. A G-protein-coupled cascade produces cyclic AMP (cAMP), which leads to a depolarization caused by a sequence of two ciliary membrane channels 2+ and Na+2+ then gates Cl− channels, which generate a further inward current via an efflux of Cl−2+ during the odor response range from 300 nM 2+ from the ciliary cytoplasm. However, there is little direct evidence that Ca2+ clearance is a function of the cilium itself.The vertebrate olfactory cilium is an attractive system for studying Ca2+-ATPase  as described elsewhere Electrical recordings were made from olfactory cilia of Northern grass frogs  solution contained (in mM): NaCl 110; KCl 5; K4BAPTA 2; K-HEPES 5; MgCl2 2; CaCl2 1; pH 7.2. The concentration of free Ca2+ in this bath was 0.1 µM. To make bath solutions with 7 µM free Ca2+, BAPTA was replaced with 2 mM dibromoBAPTA and CaCl2 was increased to 1.7 mM. To make bath solutions with 20 µM free Ca2+, BAPTA was replaced with 2 mM HEDTA and CaCl2 was increased to 1.2 mM. To make the bath with 300 µM free Ca2+, the 2 mM BAPTA was saturated by adding 2.3 mM CaCl2. Other bath solutions contained additions of ATP, ITP, or cAMP as indicated. Concentrations of free Ca2+ were estimated by the method of Bers The extracellular (pipette) solution contained (in mM): NaCl 115; KCl 3; Na-HEPES 5; MgClIn any given bath, the current-voltage relation usually stabilized within 15 s. This stability was verified before transferring the cilium to a new bath. Between tests in solutions expected to activate currents, the cilium was kept in an appropriate control bath for 2 min. Before testing for effects of ITP or ATP, the cilium was kept in a control bath containing the same nucleotide. Thus the nucleotide was already present in the cilium when the recordings commenced.v/v) was also included in all control baths in those studies.DibromoBAPTA was purchased from Molecular Probes/Invitrogen  and other reagents from Sigma-Aldrich . 5(6)-carboxyeosin diacetate was prepared as a 5 mM stock solution in DMSO and diluted from that to a final concentration of 0.01 to 50 µM. The same final concentration of DMSO  using pCLAMP 5.5 data-acquisition software . A small leak current measured in the control bath was subtracted when quantifying the recordings. This leak has not been subtracted from the recordings shown.2+-activated Cl− current, average current values during the initial plateau and final plateaus were calculated. The Ca2+-activated Cl− current accounts for the current between these plateaus. The onset time was arbitrarily defined as the time at which 10% of the Cl− current appeared. This was measured by interpolation after digitally smoothing the recording by averaging every 20 adjacent points.To measure the onset time of the Cat-test for repeated measures. Significance was claimed only when P<0.05.Effects of reagents were tested by comparing the currents measured in one cilium in each of two baths (e.g. with or without ATP). The significance of any difference between the two treatments was calculated with Student's 2+ from the cilium, it should be possible to demonstrate an ATP-dependent decrease in intraciliary Ca2+. To follow changes in intraciliary Ca2+, two assays were developed that use the ciliary Ca2+-gated Cl− channel as a sensor of free Ca2+. Its useful range as a sensor is about 2 to 10 µM If PMCA is able to clear Ca2+, allowing this solution to fill the cilium. The pipette containing the cilium was then transferred to a cytoplasmic bath with a high level of free Ca2+. Solutions on both sides of the ciliary membrane contained Cl−. As Ca2+ diffused into the cilium from the bath, some of the Ca2+ molecules gated Cl− channels. Since the inside of the cilium was held more negative than the equilibrium potential for Cl−, an inward Cl− current (i.e. a Cl− efflux) resulted. Recordings from two such experiments are shown in − current that developed beginning at t = 1.8 s.For the first assay, a pipette containing a cilium was held in a cytoplasmic bath containing 0.1 µM free Ca2+ from the cilium, one would expect to see a slower development of the Cl− current in the presence of 2 mM cytoplasmic MgATP than in its absence. The onset time of the Cl− current was measured in both the presence and absence of 2 mM MgATP as Ca2+ was allowed to diffuse into the cilium. There was no significant difference in the onset times . These conclusions held when baths containing any of three different concentrations of free Ca2+ were tested (2+ by nucleotides.If ATP were able to support the extrusion of 2 to 10 µM Cae tested . In the − channels during the odor response. An excised cilium was placed in a cytoplasmic bath containing cAMP. The extracellular (pipette) solution contained 1 mM Ca2+, and both solutions contained Cl−. Initially, the voltage was clamped at the reversal potential for the cAMP-activated current (0 mV). So although the CNG channels were gated by the cAMP, there was almost no net current at the start of the experiment. When the voltage was jumped to a negative potential, cations including Ca2+ carried an inward current through the CNG channels  might have competed with PMCA for Ca2+ and made detection of Ca2+ pumping more difficult. The test was repeated in baths with just 0.2 mM BAPTA. In this condition, addition of cAMP is unnecessary. Spontaneous gating of the CNG channels allows a Ca2+ influx sufficient to gate the Cl− channels 2+ buffering, the onset of the Cl− current was not significantly changed by the addition of ATP  − current (− current (not shown).PMCA is effective at much lower Ca2+-activated Cl−current could be found at either a half-maximal (5 µM) or saturating (300 µM) concentration of cytoplasmic free Ca2+ (Carboxyeosin (CE) is an inhibitor of PMCA ree Ca2+ . To ensu2+ that accumulates within the olfactory cilia during the odor response 2+ clearance, and (2) is this PMCA activity located in the cilium?Numerous studies suggest that PMCA may contribute to the clearance of Ca2+ following activation of the odor-transduction cascade 2+. If PMCA contributes to clearance of this Ca2+, then carboxyeosin (CE), an inhibitor of PMCA 2+. That in turn should prolong the Ca2+-activated Cl− component of the transduction current. CE (50 µM) was found to prolong the transduction current. In a second study, a lower concentration of CE (10 µM) also prolonged the current but to a lesser extent Recent physiological studies of single olfactory receptor neurons have yielded results consistent with a role for PMCA in clearing Ca2+ clearance may be further supported by testing specific peptide inhibitors of PMCA However, a second mechanism may have contributed to this result. CE has a surprising effect on the CNG transduction channels; at concentrations of 0.1 µM or higher, it substantially increases the current activated by subsaturating concentrations of cAMP . Such an2+ to remain elevated, thus increasing the duration of the transduction current. Again, though, it is likely that a second mechanism contributes to this result. Na+/Ca2+ exchange contributes to the extrusion of cytoplasmic Ca2+ following the odor response 2+ is extruded by exchange, cytoplasmic Na+ should accumulate within the cilium. It is possible that such an accumulation of Na+ might greatly reduce Na+/Ca2+ exchange unless the Na+,K+-ATPase can expel Na+ at a comparable rate +,K+-ATPase (as by elimination of ATP) might indirectly cause a shutdown of Na+/Ca2+ exchange, and this too could account for the dependence of Ca2+ clearance on ATP. A relation between Na+/Ca2+ exchange and Na+,K+-ATPase has been demonstrated in other systems +,K+-ATPase is present in olfactory cilia +,K+-ATPase. At present, the studies with CE and ATP It is also established that removal of cytoplasmic ATP prolongs the transduction current in olfactory receptor neurons 2+ from the cilium. Are the Ca2+ transporters located in the cilium itself? In models of the cilium, Ca2+ clearance is insufficient unless a Ca2+ transporter is assumed to operate in the ciliary membrane +/Ca2+ exchange occurs in the cilium 2+ may be cleared by diffusion into other cellular compartments. However, it appears that the free Ca2+ produced during a moderate odor response may extend just ∼2 µm from the site of odor binding 2+ buffers also help to limit free Ca2+. In some cellular compartments, Ca2+ can also be sequestered within endoplasmic reticulum or mitochondria A second question involves the cellular location of the mechanisms mediating clearance of Ca2+ concentration during the odor response range from 300 nM − channels are gated during a moderate response 2+ must exceed ∼2 µM during such a response, at least in local domains. In ciliary membrane vesicles, PMCA is half-maximally and maximally effective at free Ca2+ concentrations of 0.67 and 5 µM, respectively 2+ seen during the odor response. While examining cilia in isolation, though, I was unable to detect ATP-dependent expulsion of Ca2+ over the range of Ca2+ concentrations detectable by the assays (2 to 10 µM). The second assay (2+ into the cilium during the odor response. Recording from a single cilium has the advantage of eliminating transport activities in other cellular compartments. At the same time, it must be acknowledged that ciliary excision could allow the loss of cytoplasmic factors needed for normal physiological function.Estimates of the intraciliary free Cand assay  closely +/Ca2+ exchange may both be active at the levels of free Ca2+ generated during the odor response. However, due to its much lower capacity 2+ expulsion. In this case, the rate of Ca2+ expulsion by PMCA could be too small to be detected by the assays used here. PMCA in nonciliary compartments, which were not studied here, may also contribute to Ca2+ clearance.The results of the present study do not obviously conflict with previous reports. In the cilium, PMCA and Na


This regulation was not affected by RR, an inhibitor of the mitochondrial Ca2+ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca2+-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca2+. At micromolar concentrations, Ca2+ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca2+ regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca2+.We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca Aralar supplies OXPHOS with glutamate, a key mitochondrial substrate. In this study, we addressed the question whether OXPHOS can be directly activated by extramitochondrial Ca2+ and if so, whether aralar is involved in this regulation.Recent data suggest that the activity of the malate aspartate shuttle (MAS), including glutamate/aspartate carriers as aralar, is activated by extramitochondrial Ca2+ on OXPHOS of isolated rat brain mitochondria in a medium containing 150 nM free Ca2+ (Ca2+free), corresponding to basal levels of cytosolic Ca2+ under physiological conditions glu/mal was obtained .First, we investigated the influence of Caobtained . Howevera2+free . This Cae/malate , state 33glu/mal . Howevernate did . Fig. 1Dubstrate . We perf2+ activation  and the extent of Ca2+ stimulation . Neither parameter was affected by RR . Thus, Ca2+ influx into the mitochondrial matrix appears not to be required for state 3glu/mal stimulation and, hence, Ca2+ activation must be an extramitochondrial effect.Next, we investigated the kinetics of Cativation . Ca2+ wared Ca2+  in order2+ effects due to potential interactions of digitonin with mitochondrial membranes, we also varied the digitonin concentration used during the preparation of mitochondria to permeabilize synaptosomal membranes. Omitting digitonin did not cause any significant changes in the extent of extramitochondrial Ca2+ activation of state 3glu/mal . This application scheme resulted in enhanced maximum rates of uncoupled respiration in a Ca2+ -dependent manner  containing RR. With glutamate/malate as substrates, ADP (150 µM) caused an intermediate activation of phosphorylating respiration with a maximum rate of 50 nmol O2/mg/min . At Ca2+free levels up to 1.2 µM, mitochondrial Ca2+ accumulation was not detectable. Only after further Ca2+ additions did mitochondrial Ca2+ uptake become visible (not shown).In the next series of experiments, we determined the K2+ exerts a parallel activation of extramitochondrial ATPases and OXPHOS, thereby balancing exactly ATP consumption and production without major changes in ADP concentration 2+ transport into the mitochondrial matrix and subsequent activation of distinct intramitochondrial dehydrogenases 0F1ATPase 2+ is questionable in the light of following arguments. (i) Computer modeling of intramitochondrial Ca2+ activation of OXPHOS was unable to simulate the OXPHOS activation in response to physiological changes of work load in vivo2+ uniporter to Ca2+free (KM = 3.7±0.9 µM) should not allow an effective increase in intramitochondrial Ca2+ effectively under conditions of only slightly elevated Ca2+. Therefore, detectable mitochondrial Ca2+ uptake at nanomolar Ca2+ levels was explained by spatial heterogeneity of cytosolic Ca2+ concentration 2+2+ (S0.5 = 0.4 - 13 µM Ca2+free) 2+free levels for their activation compared with extramitochondrial Ca2+ activation of state 3glu/mal and OXPHOS. Thus, the function of mitochondrial Ca2+ uptake and accumulation appears rather to serve as reversible Ca2+ buffer, ensuring intracellular Ca2+ homeostasis, than to regulate state 3glu/mal and OXPHOS It is widely believed that increased cytosolic Ca2+ activation of state 3glu/mal and OXPHOS mediated by aralar. This finding is supported by several earlier observations. (i) RR inhibits cardiac function only slightly in vivo2+ uptake is not obligatory for stimulation of mitochondrial ATP production in vivo. (ii) In contrast, AOA, an inhibitor of MAS, attenuates the respiration of isolated synaptosomes This study reveals a novel mechanism of extramitochondrial Ca2+, beside its regulation of MAS 2+ is able to adjust the supply of OXPHOS with its main substrates precisely and reversibly, like a physiological “gas pedal”, acting in response to distinct, Ca2+-mediated cellular demands.Since pyruvate formation and aralar function are tightly interconnected in intact cells 2+.Taken together, our results imply a new and consistent feature of OXPHOS regulation in brain mitochondria in which the mitochondrial glutamate/aspartate carrier aralar controls mitochondrial substrate supply and OXPHOS according to the extramitochondrial level of Caet al., which includes permeabilization of synaptosomes with digitonin 2+ content was routinely diminished by extraction with nitriloacetic acid using the method of Brandt et al. Brain mitochondria  were isolated from 3–4-month-old Wistar WU rats  according to the protocol by Kudin 2PO4, 60 mM KCl, 5 mM MgCl2, and either 0 or 100 µM EGTA, pH 7.4. Ca2+free concentrations in the various media were measured with Fura-2 as described below. EGTA-free medium contained 0.6 µM Ca2+free. 100 µM EGTA medium contained 0.15 µM Ca2+free.Mitochondrial respiration was measured with a Clark-type oxygen electrode by means of high-resolution respirometry 2+ accumulation by isolated mitochondria (0.25 mg protein/ml) was monitored fluorimetrically in the presence of 0.5 µM Calcium Green-5N (Invitrogen) in a medium containing 120 mM mannitol, 40 mM MOPS, 5 mM KH2PO4 and 60 mM KCl. Measurements were performed in stirred and thermostatted (30°C) cells using a Carry Eclipse fluorimeter (Varian Deutschland GmbH) as described previously Ca2+ in EGTA medium was measured fluorimetrically with Fura-2 (10 µM) as described previously d) of the Ca2+-Fura-2 complex was determined experimentally under these conditions and was found to be 0.3 µM, which was similar to that found in a previous study CaMitochondrial protein concentrations were determined by the bicinchoninic acid assay


PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na+/Ca2+ exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.Mutations in  PINK1, DJ-I, and Omi/HtrA2, encode mitochondrially located proteins, further implicating mitochondrial dysfunction as the primary event sufficient to cause PD.Mitochondrial dysfunction has been implicated in a range of neurodegenerative diseases, in particular, Parkinson's disease (PD). PD is a common neurodegenerative disease characterized initially by loss of dopaminergic neurons in the substantia nigra (SN), and later, by more widespread nondopaminergic neuronal loss . The mol2+/calmodulin family. Studies both in vitro  a human dopaminergic neuroblastoma cell line (SH-SY5Y), and (2) human neurons derived from fetal mesencephalic stem cells that represent the closest in vitro model of primary human neurons. Finally, we corroborated our findings with another mammalian model: (3) primary cortical and midbrain neurons from a PINK1 knockout (KO) transgenic mouse . Stable m) is an indicator of mitochondrial state. To control for the effect of multidrug resistance pumps on loading of dyes and experimental results, cells were initially loaded in the presence of the MDR inhibitor cyclosporine H. No difference in loading or in results was detected, and therefore all further experiments were conducted without cyclosporine H.Mitochondrial membrane potential . Re-expression of WT PINK1, but not K219M-PINK1, rescued the TMRM signal in neuroblastoma cells (data not shown). PINK1 KD in human neurons is also associated with a significant lowering of the mean TMRM fluorescence signal by 16%  . In contrast, oligomycin caused marked mitochondrial depolarization in PINK1 KD/KO neurons , while subsequent inhibition of complex 1 by rotenone (5 μM) caused a rapid loss of potential Ci. Therem . The membrane-permeable succinate analog (5 mM methyl succinate) also increased basal Δψm in PINK1 KO neurons  to PINK1 KD neurons caused an increase in Δψm in PINK1 KD/KO cells, although the normal mechanisms that maintain Δψm may be restored in PINK1 KD neurons by increasing substrate supply to complex I and II.Thus, respiration is insufficient to maintain the Δψ2+ is a function of respiratory chain activity and the rate of substrate supply. We measured the resting level of NADH and FAD2+ autofluorescence and generated the “redox index,” a ratio of the maximally oxidized (response to 1 μM FCCP) and maximally reduced (response to 1 mM NaCN) signals. The redox index of NADH in the PINK1 KD cells was significantly more oxidized than in controls , compared to control mouse neurons, 23.4% ± 1.8%, n = 86; p < 0.001 for all type of the cells)  . Re-exprl levels D.The more oxidized redox level in PINK1 KD/KO cells may be due to a rate-limiting substrate supply . PreincuOxygen Consumption. The basal oxygen consumption  compared to control cells . Oligomycin 2 μg/ml inhibited the respiration coupled to oxidative phosphorylation in control cells , but not in PINK1 KD cells . FCCP (1 μM) accelerated respiration to maximal levels in control cells, but to a lesser extent in PINK1 KD cells (3.98 ± 0.19 versus 1.14 ± 0.089 nmol/O2/min/106). These data suggest a generalized impairment of respiration in PINK1 KD cells.sumption G was sig2/min/106 cells to 0.81 ± 0.09 nmol/O2/min/106 cells; n = 4 experiments; p < 0.001). Of note, the presence of pyruvate also changed the response of the cells to oligomycin. Maximal respiration induced by FCCP was also significantly higher following addition of pyruvate in PINK1 KD cells . Application of methyl succinate significantly stimulated cell respiration increasing the maximal rate of respiration . The rate of glucose uptake in mouse PINK1 KO neurons  was significantly slower compared to WT (n = 122) neurons  . The mitochondrial depolarization was prevented by preincubation with the mPTP inhibitor cyclosporin A (CsA)  Aii. In h neurons Bi and a  0.5 μM; C.2+]c and Δψm. Application of 100 μM ATP to neuroblastoma cells induced a [Ca2+]c signal in both control . Calcium-induced depolarization in PINK1 KD neuroblastoma cells was prevented by preincubation of cells with 0.5 μM CsA .Neuroblastoma cells were loaded with fluo-4 and TMRM (redistribution mode) for simultaneous measurement of c increase in WT mouse cortical neurons with no associated change in Δψm , whereas PINK1 KD neurons exhibited a rise in [Ca2+]c, followed by a stepwise mitochondrial depolarization, where each step coincided with each UV induced [Ca2+]c rise  in order to generate a standard calcium signal free from variations in calcium influx/release. UV induced flash photolysis produced a rapid m suggested a possible impairment of calcium efflux from the mitochondria, mediated by the Na+/Ca2+ exchanger. Preincubation of control neurons with a specific inhibitor of the Na+/Ca2+ exchanger, 10 μM CGP-37157, converted the signal in control neurons to one identical to PINK1 KD neurons mimicking the [Ca2+]m overload and inducing mitochondrial depolarization , and permeabilized in pseudo-intracellular solution containing 20 μM digitonin . As the cells are permeabilized, this protocol excludes any difference in intracellular Na+ concentration or pH as the basis for altered mitochondrial Ca2+ handling. In control neurons, flash photolysis induced a rise in [Ca2+]m, with a concomitant reduction in the [Na+ ]m. As demonstrated in 2+ peak is followed by Ca2+ recovery coinciding with Na+ increase, reflecting the activity of the Na+/Ca2+ exchanger (antiporter). In PINK1 KD neurons each UV-induced Ca2+ rise induced a stepwise increase in [Ca2+]m without recovery (n = 58), associated with reduced Na+ influx compared to control cells  Na+ to the medium in the presence of 7 μM ruthenium red (inhibitor of Ca2+ uniporter), inducing Ca2+ efflux from the mitochondria  to deplete Ca2+ from the ER in Ca2+free medium. Application of ionomycin eliminates any remaining [Ca2+] gradient between all membranes. Following thapsigargin, the sole remaining gradient is the mitochondria-cytosolic [Ca2+] gradient which may be quantified by measurements of releasable Ca2+ (2+]m in PINK1 KO mouse neurons and astrocytes was much higher than in control cells (PINK1 KO neurons 196.5 ± 14.5% (n = 69) of WT neurons (n = 88); p < 0.05; PINK1 KO astrocytes 148.6 ± 11.2% (n = 88) of control neurons (n = 74), p < 0.05; Figures 2+]m was also higher in PINK1 KD human neurons compared to control neurons (PINK1 KD 183.7 ± 11.4% (n = 67) of control (n = 45), p < 0.001; ble Ca2+  The resp2+]c signal (as shown before) and also stimulated ROS production in control cells . In PINK1 KD/KO cells 50 mM KCl showed a small nonsignificant activation of ROS production . Both the basal overproduction of ROS, as well as the KCl-stimulated ROS production, was blocked by the NADPH oxidase (NOX) inhibitors DPI (0.5 μM) or AEBCF  or the mitochondrially targeted equivalent MitoSOX. The basal cytosolic ROS (cROS) production in PINK1 KO mouse neurons was significantly higher than in WT neurons  in human PINK1 KD neuroblastoma cells (n = 2 clones). Reduction of NOX2 expression was confirmed by qRT-PCR (data not shown). Physiologically, application of the main activator of NOX, PMA, further confirmed the presence/absence of the enzyme in ROS production. In NOX2-siRNA PINK1 KD cells, the basal ROS production was significantly reduced compared to PINK1 KD cells treated with scrambled siRNA  production was also significantly higher in PINK1 KD/KO cells than in controls . The resulting reduction of ROS generation in PINK1 KO cells significantly increased the rate of glucose uptake from 41.7% ± 3.9% to 79.1% ± 5.8% of controls, n = 56; p < 0.05 (2-induced Δψm depolarization (data not shown).p < 0.05 E. Preincp < 0.05 E. Furthep < 0.05 G. Howeve2+]m, and a reduced Ca2+ capacity, secondary to dysfunction of the Na+/Ca2+ exchanger. Secondary effects of raised [Ca2+]c, such as activation of NADPH oxidase, cause increased ROS production, which results in impairment of glucose uptake, a reduction in substrate availability, a lower level of respiration, and a lower basal Δψm. These processes and their consequences are outlined in Overall, these results suggest that the major consequence of PINK1 deficiency is an increase in [CaThe first and most robust animal models of Parkinson's disease have been toxin-based models using the complex 1 inhibitors rotenone and MPTP . Such mo1F0-ATPase in order to maintain their Δψm  following an increase in [Ca2+]m there was no mitochondrial efflux of Ca2+ and concomitant influx of Na+ , (2) application of external Na+ was able to activate the Na+/Ca2+ exchanger in control neurons but not in PINK1 KD neurons, and (3) there was no apparent abnormality of calcium influx in PINK1 KD/KO cells, To date, the existence of the calcium uniporter and antiporters has been established only functionally. The Na+/Ca2+ antiporter was first recognized in heart and brain mitochondria  siRNA, and ROS scavengers enabled us to distinguish the primary abnormalities from the secondary consequences of ROS production in PINK1 KD/KO neurons. Reduction of ROS production was able to reverse the impaired glucose uptake in PINK1-deficient cells, and thus oxidative stress plays an important role in causing the impaired respiration seen in PINK1 KD/KO. Provision of substrates was also able to overcome the impaired respiration and mitochondrial depolarization. However neither reduction of ROS nor substrate provision was able to affect mitochondrial calcium overload or calcium-induced mitochondrial depolarization in PINK1 KD/KO cells.Oxidative stress has long been implicated in sporadic PD: there is evidence of elevated levels of lipid peroxidation markers  and protein nitration in the substantia nigra and in Lewy bodies of patients with PD . Howeverpiration . In our piration . In addipiration . As calcpiration , we postc release and neuronal apoptosis. Neurons of the substantia nigra, where there is increased oxidative stress and large calcium influxes, will be particularly susceptible to mitochondrial apoptosis via these mechanisms.In summary, we have characterized the mitochondrial pathophysiology that occurs due to PINK1 loss of function and have attempted to define mechanisms by which PINK1 deficiency renders neurons vulnerable. Within mitochondria there is considerable crosstalk between the bioenergetic function and calcium homeostasis. Our data suggest that as a result of PINK1 deficiency there is primarily an impairment of mitochondrial calcium efflux resulting in mitochondrial calcium overload. This induces a rise in ROS that may further impair calcium efflux and also inhibit glucose uptake, resulting in reduced substrate delivery and impaired respiration. Ultimately, the synergistic action of increased mROS and mitochondrial calcium overload induces opening of the PTP. Opening of the PTP, occurring either as an early event  or as a late event, has several consequences that exacerbate the mitochondrial pathophysiology and promote cell death: (1) PTP opening will further increase ROS production in the mitochondria, (2) PTP opening will result in reduced rate of maximal respiration through pyridine nucleotide depletion , and (3)The generation, characterization, and maintenance of the neuroblastoma, human neuron, and mouse neuronal cultures are described briefly in the 2+]c and [Ca2+]m, cells were loaded for 30 min at room temperature with 5 μM of either fura-2 AM or fluo-4 AM in combination with 5 μM of either X-Rhod-1 AM or Rhod-5n, and 0.005% pluronic acid in a HEPES-buffered salt solution (HBSS) composed of 156 mM NaCl, 3 mM KCl, 2 mM MgSO4, 1.25 mM KH2PO4, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES (pH adjusted to 7.35 with NaOH). For flash photolysis experiments, caged Ca2+, 10 μM o-nitrophenyl EGTA, AM  was loaded at the same time as the other indicators, and Ca2+-free medium containing 0.5 mM EGTA was used. For experiments using permeabilized cells, neurons were exposed to 20 μM digitonin in a “pseudo-intracellular” solution consisting of 135 mM KCl, 10 mM NaCl, 20 mM HEPES, 5 mM pyruvate, 5 mM malate, 0.5 mM KH2PO4, 1 mM MgCl2, 5 mM EGTA, and 1.86 mM CaCl2 (to yield a free [Ca2+] of ∼100 nM).For measurements of i and ΔΨm were monitored in single cells using excitation light provided by a Xenon arc lamp, the beam passing sequentially through 10 nm band pass filters centered at 340, 380, and 490 nm housed in computer-controlled filter wheel . Emitted fluorescence light was reflected through a 515 nm long-pass filter to a cooled CCD camera  and digitized to 12 bit resolution . All imaging data were collected and analyzed using software from Andor . Wherever possible, we have normalized the signals between resting level (set to 0) and a maximal signal generated in response to the uncoupler FCCP .Fluorescence measurements were obtained on an epifluorescence inverted microscope equipped with a ×20 fluorite objective. [CaConfocal images were obtained using a Zeiss 510 uv-vis CLSM equipped with a META detection system and a 40× oil immersion objective. The 488 nm Argon laser line was used to excite fluo-4 fluorescence, which was measured using a band-pass filter from 505 to 550 nm. Illumination intensity was kept to a minimum (at 0.1%–0.2% of laser output) to avoid phototoxicity, and the pinhole was set to give an optical slice of ∼2 μm. TMRM was excited using the 543 nm laser line and fluorescence measured using a 560 nm long-pass filter. For HEt, MitoSOX, X-Rhod, or Rhod-5n measurements the 543 nm laser line and 560 nm long-pass filter were used. NADH autofluorescence was excited at 351 and measured at 375–470 nm. 2-NDBG was excited at 458 nm, and fluorescence was measured at 520 nm. All data presented were obtained from at least five coverslips and two to three different cell preparations.7cells were suspended in respiration medium (HBSS) with 10 mM D-glucose in a Clark-type oxygen electrode thermostatically maintained at 37°C. The oxygen electrode was calibrated with air-saturated water, assuming 406 nmol O atoms/ml at 37°C. Oxygen consumption was measured over 10 min with addition of oligomycin  and 0.5 μM FCCP. All data were obtained using a MacLab system with Chart recording software.To measure respiration rate in intact cells, approximately 1 × 10∗p < 0.05, ∗∗p < 0.005. For all graphs, error bars represent mean ± SEM.Data were generated from a minimum of three independent experiments, using a minimum of 20 cells per experiment and replication in two to three different clones for PINK1 KD. Statistical analysis and exponential curve fitting were performed using Origin 7  software. Data were analyzed by parametric Student's t tests and significance expressed:


The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential.The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca Post-menopausal and age-related osteoporosis afflicts large segments of the population and can markedly increase skeletal fragility. Bone fractures that occur as a consequence substantially increase health care expenditures and raise levels of morbidity. While strategies that prevent further loss of bone exist, options that compensate for bone loss accrued over age are less numerous. Physical exercise holds promise in this realm. However, in part due to deficits in how cells within bone respond to skeletal loading, readily complied exercise has proved ineffective in enhancing bone mass in the elderly. In this study, we examined whether the ability of physical exercise to increase bone mass can be restored at advanced age. To this end, we developed a computational model describing how a specific aspect (or pathway) activated in bone cells by skeletal loading may be altered with age. Our model proved successful in describing age-related pathway alterations and identified specific deficits that were amenable to therapeutic manipulation. We subsequently discovered that when an extremely inexpensive, currently approved pharmaceutical is used as a supplement, bone response to skeletal loading was completely rescued in aged animals. We believe that this result provides the rationale to consider this approach as a means to increasing bone mass in the elderly. Mechanical stimuli are anabolic for bone and hold promise to counteract skeletal fragility associated with bone loss pathologies during brief stimuli are focal events that guide unique downstream bone adaptation. A variety of second messengers  are acutely activated by mechanical stimuli. Of these, the Ca2+ ion second messenger system may be unique in its specificity given that all important aspects of mechanical stimuli that influence bone formation in vivo  have been observed to provoke highly specific real-time Ca2+ oscillations in bone cells in vitro 2+ signaling disrupts mechano-responsive gene expression 2+ signaling within the bone cell network 2+ ion system, activation of a variety of transcription factors  provides putative links between cell responses on the order of seconds to cell function over successive days of the week. Of these, the nuclear factor of activated T-cell family of transcription factors (NFAT c1–c4) may be unique. Specifically, NFAT activation dynamics (within minutes) is remarkably specific to Ca2+ amplitudes and frequencies known to influence distinct downstream cell functions, including proliferation, differentiation and apoptosis 2+/NFAT signaling and age-related alterations in bone cells in vivo may prove useful. However, it is not currently possible to experimentally quantify real-time activation of Ca2+/NFAT signaling in situ within bone.To begin to explore underlying potential for age-related deficits in mechanotransduction function, we first focused upon observations that a single bout of mechanical stimuli (∼100 s) is sufficient to influence bone matrix secretion up to a week later 2+ signaling induced in simple networks of osteocytic cells subject to mechanical stimuli using a numerical/computational technique called agent-based modeling (ABM) 2+ signaling in cell ensembles). Based in part upon our original framework 2+/NFAT signaling in situ within bone's cellular syncytium and its relation to osteoblastic relative mineral apposition rates induced by mechanical loading  induced in vivo by a variety of mechanical stimuli in young and senescent female C57BL/6 mice 2+/NFAT pathway incorporated sufficient parametric complexity to simulate rp.BFR induced by a variety of loading protocols in young adult 2+ oscillations in individual cells . Post-hoc analysis of ABM simulations suggested that ‘knock-out’ of the remaining parameters de-couples Ca2+ oscillation from induced strains (Tεx = 0), inhibits secondary Ca2+ transients (R0 = 0), decreases Ca2+ induced dephosphorylation of NFAT (α = 0) and renders mineral apposition rates into a binary ‘on/off’ state in osteoblasts . ‘Knock-out’ of parameters resulted in simulations that were significantly different from both the experimental rp.BFR data p≤0.02;  [12], , i(t), emerged via an interplay between Ca2+ influx due to loading induced strains at the location of the osteocytic cell (εi(t)), cell-cell gap junctional exchange of Ca2+ ions and efflux from ER Ca2+ stores as follows:εxT is the parameterized threshold strain magnitude required to maximize the amplitude of Ca2+ oscillations in mechanosensory osteocytes , in is the number of cells (osteocytes and/or precursor cells) functionally coupled to the ith cell, j is an element of the cell set in, CnT represents threshold ‘incoming’ Ca2+ ion levels required to initiate oscillations in the recipient cells (specified via preliminary simulations to be 2.5%), and 0ER and 0R are the parameterized maximal Ca2+ store capacity and maximal store recovery rates, respectively. The specific mathematical formulations for osteocyte sigmoidal dose-response relation to strain (eqn 1b) were based upon observations of Ca2+ oscillations induced in bone cells by mechanical stimuli 2+ ion propagation between cells based upon reports 2+ store depletion (eqn 1d) is similar to previously 2+ store recovery upon cellular quiescence (eqn 1d), models the action of the SERCA pump working against a Ca2+ ion concentration gradient and is similar to literature reports The ABM is a biophysical model that relies on experimentally identified ‘building blocks’ to algorithmically describe how activation of the Caeoblasts . In the 2+ oscillations in osteocytic networks hold potential to regulate surface osteoblastic cells 2+ oscillations upon NFAT transcription factor dynamics. Specifically, downstream of increased intracellular Ca2+ oscillations, cytoplasmic NFAT dephosphorylation and nuclear translocation in surface precursor cells was modeled to be reflective of a ‘memory’ of Ca2+ oscillation characteristics such as amplitude, frequency and durations, and included an implicit consideration of negative feedback elements 2+ oscillations. The quantity of NFAT translocated into the ith cell's nucleus (boutt) was modeled simply as:2+ signaling and the NFAT pathway code for highly specific cell proliferation, differentiation and apoptotic events nxNFAT is the maximal nuclear NFAT DNA binding capacity, and xr.MAR, the maximal relative mineral apposition rate by osteoblasts. The mathematical form (eqn 3) reflects considerations that transcriptional regulation is likely to be a threshold-driven process Given observations that CaεxT, 0ER, 0R, α, nxNFAT, xr.MAR prior to execution of the simulations). To initialize the ABM (at t = 0), strains induced by loading at osteocyte cell locations were 0  = 0), and the stores of all cells were assumed to be filled to capacity  = ER0). Based upon preliminary studies, we simulated intracellular Ca2+ oscillations for an extra 200 seconds beyond cessation of a given loading bout to permit the unfolding of cell-cell cross talk, and NFAT dephosphorylation for an extra 100 s beyond cessation of Ca2+ oscillations to more fully account for prior Ca2+ oscillation histories.Simulations with the ABM involved solving equations (1–3), given osteocyte level strains derived over each second of a loading bout in the context of the ABM's 6 independent parameters  at the initiation of the simulations then predicts mean values i(θμ) = rp.BFRi for each loading protocol ‘i’. Under the normal error model, the log-likelihood function for the ABM is then ij’‘y is the rp.BFR measured in the jth animal for the ith loading protocol, μ(θ) is the vector of means σ2 is the vector of population variances protocolN’ is the number of independent loading protocols, ‘ain’ is the number of animals that underwent the ith loading protocol, ‘an’ is the total number of experimental observations. The maximum likelihood estimate (MLE), for the vector of means, μ(θ) and variances σ2, is simply the set of model parameters that maximizes L(μ(θ), σ2). For a given vector of mean parameters μ(θ) of σ2, maximization with respect to the variances σ2 is achieved by setting:2). Using this procedure, separate ABM models were calibrated to the rp.BFR data obtained from young animals subjected to 10 protocols and senescent animals subjected to 7 protocols  Is there evidence against the null hypothesis that each of the ABM parameters takes value zero ? (d) Is there evidence in the observed data against the null hypothesis that the population values of the ABM parameters are the same for young and senescent animals? (e) If null hypothesis in (d) is rejected, is there evidence against the null hypotheses that specific model parameters or combination of parameters are unchanged by age? (f) Do the calibrated ABMs make predictions about the results of hypothetical interventions that would restore specific ABM parameters in senescent animals to their young values? (g) Do ABM simulations of loading induced rp.BFR, upon complete restoration of parameters significantly degraded by aging, predict data from senescent animals subject to loading supplemented with CsA? We examined these questions using a series of likelihood ratio tests (within MATLAB) and factorial ANOVAs (within SPSS) where appropriate; please see supporting information for additional details  received mechanical loading using the noninvasive murine tibia loading device Protocol S1Consolidated (‘zipped’) file that includes a ‘readme’, c++ code and necessary input files for compiling and executing the code representing the developed model, and executable files compiled for both Macintosh OSX (Leopard) and windows PC based computer platforms.(5.17 MB ZIP)Click here for additional data file.Table S1Animal specific peak longitudinal normal strains (mean+S.E.) and rp.BFR (mean+S.E.) induced in vivo in young adult animals by 10 independent loading protocols, and in senescent animals by 7 independent loading protocols. Note that these data represent mean values after removal of outliers (mean ± 2×S.D.). This resulted in the removal of 1 data point in young adult animals (from n = 70) and of 4 data points in senescent animals (from n = 56).(0.07 MB DOC)Click here for additional data file.Table S2Definitions for notations and symbols used in manuscript.(0.09 MB DOC)Click here for additional data file.Text S1Methods describing previous data set used to calibrate model and statistical analysis.(0.12 MB DOC)Click here for additional data file.