Oxidative-induced membrane damage in diabetes lymphocytes: Effects on intracellular Ca2 + homeostasis

Oxidative stress is linked to several human diseases, including diabetes. However, the intracellular signal transduction pathways regulated by reactive oxygen species (ROS) remain to be established. Deleterious effects of ROS stem from interactions with various ion transport proteins such as ion channels and pumps, primarily altering Ca^{2 +} homeostasis and inducing cell dysfunction. This study characterized the Ca^{2 +} transport system in lymphocytes of patients with type-2 diabetes, evaluating the possible correlation between cell modifications and the existence of specific oxidative stress damage. Lymphocytes from type-2 diabetes patients displayed oxidative stress features (accumulation of some ROS species, membrane peroxidation, increase in protein carbonyls, increase in SOD and Catalase activity) and Ca^{2 +} dyshomeostasis (modified voltage-dependent and inositol 1,4,5-triphosphate-mediated Ca^{2 +} channel activities, decrease in Ca^{2 +} pumps activity). The data support a correlation between oxidative damage and alterations in intracellular Ca^{2 +} homeostasis, possibly due to modification of the ionic control in lymphocytes of type-2 diabetes patients.     

Wavelet Transform-Based De-Noising for Two-Photon Imaging of Synaptic Ca2+ Transients

Postsynaptic Ca²⁺ transients triggered by neurotransmission at excitatory synapses are a key signaling step for the induction of synaptic plasticity and are typically recorded in tissue slices using two-photon fluorescence imaging with Ca²⁺-sensitive dyes. The signals generated are small with very low peak signal/noise ratios (pSNRs) that make detailed analysis problematic. Here, we implement a wavelet-based de-noising algorithm (PURE-LET) to enhance signal/noise ratio for Ca²⁺ fluorescence transients evoked by single synaptic events under physiological conditions. Using simulated Ca²⁺ transients with defined noise levels, we analyzed the ability of the PURE-LET algorithm to retrieve the underlying signal. Fitting single Ca²⁺ transients with an exponential rise and decay model revealed a distortion of τ_rise but improved accuracy and reliability of τ_decay and peak amplitude after PURE-LET de-noising compared to raw signals. The PURE-LET de-noising algorithm also provided a ∼30-dB gain in pSNR compared to ∼16-dB pSNR gain after an optimized binomial filter. The higher pSNR provided by PURE-LET de-noising increased discrimination accuracy between successes and failures of synaptic transmission as measured by the occurrence of synaptic Ca²⁺ transients by ∼20% relative to an optimized binomial filter. Furthermore, in comparison to binomial filter, no optimization of PURE-LET de-noising was required for reducing arbitrary bias. In conclusion, the de-noising of fluorescent Ca²⁺ transients using PURE-LET enhances detection and characterization of Ca²⁺ responses at central excitatory synapses.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

Stimulus-Dependent Regulation of Nuclear Ca2+ Signaling in Cardiomyocytes: A Role of Neuronal Calcium Sensor-1

In cardiomyocytes, intracellular calcium (Ca²⁺) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca²⁺levels ([Ca²⁺]) also occur in the nucleus, the precise mechanism of nuclear [Ca²⁺] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca²⁺] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca²⁺ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca²⁺ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca²⁺ transients are slower than cytoplasmic Ca²⁺ transients, and chelating cytoplasmic Ca²⁺ abolished nuclear Ca²⁺ transients, suggesting that nuclear Ca²⁺ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca²⁺] compared to cytoplasmic [Ca²⁺]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca²⁺ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca²⁺ transient amplitude was significantly lower in myocytes lacking neuronal Ca²⁺ sensor-1 (NCS-1), a Ca²⁺ binding protein implicated in IP₃R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP₃R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca²⁺] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca²⁺ signal regulation.     

Stochastic reaction-diffusion modeling of calcium dynamics in 3D dendritic spines of Purkinje cells

Calcium (Ca²⁺) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression and long-term potentiation at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP₃)-induced Ca²⁺ release. These Ca²⁺ transients happen in response to stimuli from parallel fibers (PFs) from granule cells and climbing fibers (CFs) from the inferior olivary nucleus. These events occur at low numbers of free Ca²⁺, requiring stochastic single-particle methods when modeling them. We use the stochastic particle simulation program MCell to simulate Ca²⁺ transients within a three-dimensional Purkinje cell dendritic spine. The model spine includes the endoplasmic reticulum, several Ca²⁺ transporters, and endogenous buffer molecules. Our simulations successfully reproduce properties of Ca²⁺ transients in different dynamical situations. We test two different models of the IP₃ receptor (IP₃R). The model with nonlinear concentration response of binding of activating Ca²⁺ reproduces experimental results better than the model with linear response because of the filtering of noise. Our results also suggest that Ca²⁺-dependent inhibition of the IP₃R needs to be slow to reproduce experimental results. Simulations suggest the experimentally observed optimal timing window of CF stimuli arises from the relative timing of CF influx of Ca²⁺ and IP₃ production sensitizing IP₃R for Ca²⁺-induced Ca²⁺ release. We also model ataxia, a loss of fine motor control assumed to be the result of malfunctioning information transmission at the granule to Purkinje cell synapse, resulting in a decrease or loss of Ca²⁺ transients. Finally, we propose possible ways of recovering Ca²⁺ transients under ataxia.     

A critical appraisal of the role of intracellular Ca2+-signaling pathways in Kawasaki disease

Kawasaki disease is a multi-systemic vasculitis that generally occurs in children and that can lead to coronary artery lesions. Recent studies showed that Kawasaki disease has an important genetic component. In this review, we discuss the single-nucleotide polymorphisms in the genes encoding proteins with a role in intracellular Ca²⁺ signaling: inositol 1,4,5-trisphosphate 3-kinase C, caspase-3, the store-operated Ca²⁺-entry channel ORAI1, the type-3 inositol 1,4,5-trisphosphate receptor, the Na⁺/Ca²⁺ exchanger 1, and phospholipase Cß4 and Cß1. An increase of the free cytosolic Ca²⁺ concentration is proposed to be a major factor in susceptibility to Kawasaki disease and disease outcome, but only for polymorphisms in the genes encoding the inositol 1,4,5-trisphosphate 3-kinase C and the Na⁺/Ca²⁺ exchanger 1, the free cytosolic Ca²⁺ concentration was actually measured and shown to be increased. Excessive cytosolic Ca²⁺ signaling can result in hyperactive calcineurin in T cells with an overstimulated nuclear factor of activated T cells pathway, in hypersecretion of interleukin-1ß and tumor necrosis factor-α by monocytes/macrophages, in increased urotensin-2 signaling, and in an overactivation of vascular endothelial cells.     

Passive Ca overload in H9c2 cardiac myoblasts: Assessment of cellular damage and cytosolic Ca transients

Increase of resting Ca²⁺ levels and amplitude of vasopressin-induced Ca²⁺ transients were observed when cells in serum-free medium were exposed to 5 mM Ca²⁺ for 2 h. Small effect on cell viability was also observed. A rapid cytotoxic effect was developed in the presence of 10 mM Ca²⁺ and absence of serum. However, cells exposed to 10 mM Ca²⁺ in the presence of serum were protected from damage for at least 2 days. Resting Ca²⁺ levels and cytosolic Ca²⁺ transients in serum-containing medium with 10 mM Ca²⁺ displayed lower increases and a tendency to recover control values. When serum was absent, cells preincubated with 10 mM Ca²⁺ were more sensitive to thapsigargin-induced damage than cells preincubated with lower Ca²⁺. The sensitivity was similar when serum was present. Tolerance to high Ca²⁺ in the presence of serum was linked to potentiation of the mitochondrial Ca²⁺ entry to decrease the sarcoplasmic reticulum Ca²⁺ overload.     

Phospholipase C-η2 is activated by elevated intracellular Ca(2+) levels

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [³H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na⁺/H⁺ antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na⁺/Ca²⁺-exchange. Direct activation of PLCη2 by < 1 μM Ca²⁺ was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca²⁺ due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca²⁺-binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca²⁺. This suggests that the C2 domain is not responsible for Ca²⁺ activation. Collectively, this work highlights an important new component of the Ca²⁺ signalling toolkit and given its sensitivity to Ca²⁺, this enzyme is likely to facilitate the amplification of intracellular Ca²⁺ transients and/or crosstalk between Ca²⁺-storing compartments in vivo.     

Extracellular ATP-induced nuclear Ca transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic β-cells

Extracellular ATP (eATP) induces an intracellular Ca²⁺ transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca²⁺ release from intracellular stores in mouse pancreatic β-cells. Using laser scanning confocal microscopy, Ca²⁺ indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca²⁺ release in pancreatic β-cell nuclei. eATP induced a higher nuclear Ca²⁺ transient in pancreatic β-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca²⁺ transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca²⁺-ATPase (PMCA) or the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) by LaCl₃ or by replacement of Na⁺ with N-Methyl-Glucosamine. eATP-induced nuclear Ca²⁺ transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic β-cells. These results indicate that eATP triggers nuclear Ca²⁺ transients by mobilizing a nuclear Ca²⁺ store via nuclear IP3Rs.     

cADPR stimulates SERCA activity in Xenopus oocytes

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca²⁺ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca²⁺ following Ca²⁺ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP₃), and by Ca²⁺ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca²⁺ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH₂-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca²⁺ sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca²⁺.     

Carbachol- and KCl-induced changes in phosphoinositide metabolism and free calcium in guinea pig cerebral cortex synaptosomes

Phosphoinositide (PI) and calcium metabolism were studied in guinea pig cerebral cortex synaptosomes. Mass amounts of inositol and inositol monophosphates, and the levels of free intrasynaptosomal calcium ([Ca²⁺]_i) were measured after KCl (60 mM), after a direct cholinergic agonist carbachol (CA, 1mM), and after their combination. Inositol, inositol-1-phosphate (Ins1P), inositol-4-phosphate (Ins4P) and [Ca²⁺]_i were measured with and without 10 mM LiCl in the incubation medium. CA-induced cholinergic stimulation elevated synaptosomal Ins4P levels by 40% but did not affect Ins1P or [Ca²⁺]_i. On the contrary, KCl elevated Ins1P by 50% and [Ca²⁺]_i by 40% above the resting level, and decreased inositol by 20%, whereas no alterations in Ins4P occurred. CA did not modify the response of KCl, but KCl abolished the elevation of Ins4P by CA. LiCl attenuated KCl-induced elevation of Ins1P but amplified the CA-induced elevation of Ins4P. The elevation of presynaptic [Ca²⁺]_i was accompanied by accumulation of Ins1P but not that of Ins4P. Hence, the present results suggest that presynaptic cholinergic stimulation and KCl-induced depolarization may activate different degradation pathways of inositolphosphate metabolism.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson''s disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.     

Facilitated Hyperpolarization Signaling in Vascular Smooth Muscle-overexpressing TRIC-A Channels

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca²⁺ release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca²⁺ release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca²⁺ sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca²⁺ transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca²⁺ sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca²⁺ sparks were facilitated and cell surface Ca²⁺-dependent K⁺ channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca²⁺ channels were inactivated, and thus, the resting intracellular Ca²⁺ levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca²⁺ signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.     

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca~(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca~(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca~(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca~(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca~(2+) transients were generated from day 1 onward. That ATP was inducing Ca~(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca~(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Agonist-Specific Calcium Signaling and Phosphoinositide Hydrolysis in Human SK-N-MCIXC Neuroepithelioma Cells

Abstract: Fura-2 digital imaging microfluorimetry was used to evaluate the Ca²⁺ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca²⁺_i) but of different magnitudes (ET-1 = ATP> NE). The Ca²⁺ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca²⁺ influx. The Ca²⁺ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca²⁺ signal, which was characteristic of the response to Oxo-M, was observed in <40% of cells stimulated with ET-1 and absent from Ca²⁺ signals elicited after NE addition. Removal of extracellular Ca²⁺ enhanced the rate of decay of Ca²⁺ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca²⁺ influx. In the absence of extracellular Ca²⁺, NE elicited asynchronous multiple Ca²⁺ transients. In either the absence or presence of extracellular Ca²⁺,>94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were ～74 and ～48%. Sequential addition of agonists to cells maintained in a Ca²⁺-free buffer indicated that each ligand mobilized Ca²⁺ from a common intracellular pool. When monitored as a release of a total inositol phosphate fraction, all four agonists elicited similar (four- to sixfold) increases in phosphoinositide hydrolysis. However, the addition of ET-1 or ATP resulted in larger increases in the net formation of inositol 1,4,5-trisphosphate than did either Oxo-M or NE. These results indicate that, in SK-N-MCIXC cells, the characteristics of both Ca²⁺ signaling and inositol phosphate production are agonist specific.     

Dopaminergic Neurotoxicants Cause Biphasic Inhibition of Purinergic Calcium Signaling in Astrocytes

Dopaminergic nuclei in the basal ganglia are highly sensitive to damage from oxidative stress, inflammation, and environmental neurotoxins. Disruption of adenosine triphosphate (ATP)-dependent calcium (Ca²⁺) transients in astrocytes may represent an important target of such stressors that contributes to neuronal injury by disrupting critical Ca²⁺-dependent trophic functions. We therefore postulated that plasma membrane cation channels might be a common site of inhibition by structurally distinct cationic neurotoxicants that could modulate ATP-induced Ca²⁺ signals in astrocytes. To test this, we examined the capacity of two dopaminergic neurotoxicants to alter ATP-dependent Ca²⁺ waves and transients in primary murine striatal astrocytes: MPP⁺, the active metabolite of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Both compounds acutely decreased ATP-induced Ca²⁺ transients and waves in astrocytes and blocked OAG-induced Ca²⁺ influx at micromolar concentrations, suggesting the transient receptor potential channel, TRPC3, as an acute target. MPP⁺ inhibited 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca²⁺ transients similarly to the TRPC3 antagonist, pyrazole-3, whereas 6-OHDA only partly suppressed OAG-induced transients. RNAi directed against TRPC3 inhibited the ATP-induced transient as well as entry of extracellular Ca²⁺, which was augmented by MPP⁺. Whole-cell patch clamp experiments in primary astrocytes and TRPC3-overexpressing cells demonstrated that acute application of MPP⁺ completely blocked OAG-induced TRPC3 currents, whereas 6-OHDA only partially inhibited OAG currents. These findings indicate that MPP⁺ and 6-OHDA inhibit ATP-induced Ca²⁺ signals in astrocytes in part by interfering with purinergic receptor mediated activation of TRPC3, suggesting a novel pathway in glia that could contribute to neurotoxic injury.     

Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity Calcium Ruby and its dextran conjugate

The limited choice and poor performance of red-emitting calcium (Ca²⁺) indicators have hampered microfluorometric measurements of the intracellular free Ca²⁺ concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca²⁺ indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH₂ linker arm. The low-affinity variant (K_D,Ca ～30 μM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca²⁺ transients. While Calcium Ruby is a mitochondrial Ca²⁺probe, its conjugation, via the NH₂ tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca²⁺ probe with an affinity matched to microdomain Ca²⁺ signals. As an example, we show depolarization-evoked Ca²⁺ signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca²⁺ measurements.