Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨ_m) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨ_m. Mitochondrial density was highest in the perinuclear region, whereas ΔΨ_m tended to be higher in peripheral mitochondria. Spontaneous ΔΨ_m fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca²⁺, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca²⁺ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca²⁺ load or metabolic impairment abolished ΔΨ_m fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨ_m heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨ_m fluctuations are an indication of mitochondrial viability and are triggered by local Ca²⁺ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

Effect of mitochondria poisoning by FCCP on Ca2+ signaling in mouse skeletal muscle fibers

We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca²⁺ signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca²⁺]_i and MagFluo-4AM to measure Ca²⁺ transients. Exposure to FCCP (2 μM, 2 min) caused a continuous increase in [Ca²⁺]_i at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca²⁺ transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca²⁺]_i = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca²⁺ release. FCCP diminished the rate but not the amount of Ca²⁺ released, indicating that depletion of Ca²⁺ stores did not cause the decrease in Ca²⁺ transient amplitude. Ca²⁺ transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca²⁺]_i induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca²⁺ in the FCCP effect on transient amplitude.     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

Quantal ATP release in rat β-cells by exocytosis of insulin-containing LDCVs

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic β-cells expressing P2X₂ receptors. Stimulation of exocytosis evoked rapidly activating and deactivating ATP-dependent transient inward currents (TICs). The unitary charge (q) of the events recorded at 0.2 μM [Ca²⁺]_i averaged 4.3 pC. The distribution of the ³√q of these events could be described by a single Gaussian. The rise times averaged ∼5 ms over a wide range of TIC amplitudes. In β-cells preloaded with 5-hydroxytryptamine (5-HT; accumulating in insulin granules), ATP was coreleased with 5-HT during >90% of the release events. Following step elevation of [Ca²⁺]_i to ∼5 μM by photo release of caged Ca²⁺, an increase in membrane capacitance was observed after 33 ms, whereas ATP release first became detectable after 43 ms. The step increase in [Ca²⁺]_i produced an initial large TIC followed by a series of smaller events that echoed the changes in membrane capacitance (ΔC _m). Mathematical modeling suggests that the large initial TIC reflects the superimposition of many unitary events. Exocytosis, measured as ΔC _m or TICs, was complete within 2 s after elevation of [Ca²⁺]_i with no sign of endocytosis masking the capacitance increase. The relationship between total charge (Q) and ΔC _m was linear with a slope of ∼1.2 pC/fF. The latter value predicts a capacitance increase of 3.6 fF for the observed mean value of q, close to that expected for exocytosis of individual insulin granules. Our results indicate that measurements of ATP release and ΔC _m principally (≥85–95%) report exocytosis of insulin granules.     

Factors affecting SOCE activation in mammalian skeletal muscle fibers

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca²⁺ entry, SOCE. Sarcoplasmic reticulum (SR) Ca²⁺ stores were depleted by repetitive exposures to high K⁺ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca²⁺. SR Ca²⁺ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca²⁺ (5 mM) increased basal [Ca²⁺]_i under two modalities. In mode 1 [Ca²⁺]_i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca²⁺]_i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca²⁺ entry. In nine FCCP-poisoned fibers, the initial rate of Ca²⁺ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca²⁺, we show that during depletion [Ca²⁺]_mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca²⁺]_i, associated with SOCE activation, diminishes upon external Na⁺ withdrawal. Na⁺ entry through the SOCE pathway and activation of the reversal of Na⁺/Ca²⁺ exchanger could explain this SOCE modulation by Na⁺.     

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release

Electrophysiological measurements of cell capacitance (C _m) and biochemical assays of [³H] serotonin ([³H]5-hydroxytryptamine or [³H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced C _m changes (ΔC _m), indicative of exocytosis, with the same voltage and Ca²⁺ dependency as the inward Ca²⁺ currents (I _Ca). Ba²⁺ was able to substitute for Ca²⁺ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca²⁺-versus Ba²⁺-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [³H]5-HT to study hormone release from cell populations. [³H]5-HT was released in a depolarization-, Ca²⁺- and time-dependent manner. Ba²⁺ also substituted for Ca²⁺ in depolarization-induced [³H]5-HT release. Thapsigargin, used to deplete Ca²⁺ stores, had no effects on Ca²⁺-triggered C _m increases, but Ca²⁺-triggered [³H]5-HT release was abolished. Ba²⁺-triggered [³H]5-HT release, however, was only slightly affected by Ca²⁺ store depletion. Ba²⁺ was found to act directly as a secretagogue of [³H]5-HT in intact cells, but not in C _m measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action. Received: 18 October 1995/Received after revision and accepted: 9 January 1996     

H2O2-mediated modulation of cytosolic signaling and organelle function in rat hippocampus

Reactive oxygen species (ROS) released from (dys-)functioning mitochondria contribute to normal and pathophysiological cellular signaling by modulating cytosolic redox state and redox-sensitive proteins. To identify putative redox targets involved in such signaling, we exposed hippocampal neurons to hydrogen peroxide (H₂O₂). Redox-sensitive dyes indicated that externally applied H₂O₂ may oxidize intracellular targets in cell cultures and acute tissue slices. In cultured neurons, H₂O₂ (EC₅₀ 118 μM) induced an intracellular Ca²⁺ rise which could still be evoked upon Ca²⁺ withdrawal and mitochondrial uncoupling. It was, however, antagonized by thapsigargin, dantrolene, 2-aminoethoxydiphenyl borate, and high levels of ryanodine, which identifies the endoplasmic reticulum (ER) as the intracellular Ca²⁺ store involved. Intracellular accumulation of endogenously generated H₂O₂—provoked by inhibiting glutathione peroxidase—also released Ca²⁺ from the ER, as did extracellular generation of superoxide. Phospholipase C (PLC)-mediated metabotropic signaling was depressed in the presence of H₂O₂, but cytosolic cyclic adenosine-5′-monophosphate (cAMP) levels were not affected. H₂O₂ (0.2–5 mM) moderately depolarized mitochondria, halted their intracellular trafficking in a Ca²⁺- and cAMP-independent manner, and directly oxidized cellular nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH₂). In part, the mitochondrial depolarization reflects uptake of Ca²⁺ previously released from the ER. We conclude that H₂O₂ releases Ca²⁺ from the ER via both ryanodine and inositol trisphosphate receptors. Mitochondrial function is not markedly impaired even by millimolar concentrations of H₂O₂. Such modulation of Ca²⁺ signaling and organelle interaction by ROS affects the efficacy of PLC-mediated metabotropic signaling and may contribute to the adjustment of neuronal function to redox conditions and metabolic supply. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Florian J. Gerich and Frank Funke contributed evenly to this study.     

The antioxidant Trolox restores mitochondrial membrane potential and Ca2+-stimulated ATP production in human complex I deficiency

Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca²⁺ content of the endoplasmic reticulum (ER_Ca) and bradykinin(Bk)-induced increases in cytosolic and mitochondrial free Ca²⁺ ([Ca²⁺]_C; [Ca²⁺]_M) and ATP ([ATP]_C; [ATP]_M) concentration. Here, we determined the mitochondrial membrane potential (Δψ) in patient skin fibroblasts and show significant correlations with cellular ROS levels and ER_Ca, i.e., the less negative Δψ, the higher these levels and the lower ER_Ca. Treatment with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) normalized Δψ and Bk-induced increases in [Ca²⁺]_M and [ATP]_M. These effects were accompanied by an increase in ER_Ca and Bk-induced increase in [Ca²⁺]_C. Together, these results provide evidence for an integral role of increased ROS levels in complex I deficiency and point to the potential therapeutic value of antioxidant treatment. Felix Distelmaier and Henk-Jan Visch contributed equally to this paper.     

Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells

Sustained increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) is a critical early signal from T-cell receptor (TCR/CD3). In general, Ca²⁺-release activated Ca²⁺ channels (CRAC) are responsible for the Ca²⁺ influx and Δ[Ca²⁺]_c after TCR/CD3 stimulation. However, T cells also express Ca²⁺-permeable nonselective cation channels such as TRPM2 and TRPC. Gd³⁺ is a relatively selective blocker for CRAC at micromolar concentrations. Here, Jurkat T cells were used to investigate the Gd³⁺-resistant Ca²⁺ influx (Δ[Ca²⁺]_c,Gd) induced by concanavalin A (ConA, 1 μg/ml), a widely used mitogenic agent for T cells, or by anti-CD3 Ab (αCD3). αCD3-induced Δ[Ca²⁺]_c was partly (~60%) inhibited by 1 μM Gd³⁺ while thapsigargin-induced Δ[Ca²⁺] was almost completely abolished. ConA-induced Δ[Ca²⁺] was mostly inhibited by 1 μM Gd³⁺ during the early phase (<30 s of ConA application) and became resistant during the late phase (>2 min). Induction of Δ[Ca²⁺]_c,Gd by αCD3 and ConA was inhibited by 2-aminoethoxydiphenyl borate (2-APB) and by N-(p-amylcinnamoyl) anthranilic acid, indicating that TRPM2 and TRPC are involved in this process. Treatment with Pyr-3, a TRPC3-specific inhibitor, potently suppressed Δ[Ca²⁺]_c,Gd by αCD3 (IC₅₀, 0.16 μM). Patch clamp experiments demonstrated that the TRPM2 channels were activated by ConA, and the TRPC-like channels were activated by αCD3. Our present study suggests that TRPM2 and TRPC3 are activated by ConA and TCR/CD3, respectively, in Jurkat T cells and are responsible for the induction of Δ[Ca²⁺]_c,Gd.     

Alterations in calcium homeostasis reduce membrane excitability in amphibian skeletal muscle

The effects of alterations in intracellular calcium homeostasis on surface membrane excitability were investigated in resting Rana temporaria sartorius muscle. This was prompted by initial results from a fatiguing stimulation protocol study that demonstrated a fibre subpopulation in which action potential generation in response to a standard 1.5 V electrical stimulus failed despite mean membrane potentials [E _m, −69±2.3 mV (n=14)] compatible with spike firing in a control set of quiescent muscle fibres. Intracellular micro-electrode recordings showed a similar reversible loss of excitability, attributable to an increased threshold, despite only small (7.1±1.8 mV) positive changes in E _m after approximately 60-min exposures to nominally 0 Ca²⁺ Ringer solutions in which Ca²⁺ was replaced by Mg²⁺. This effect was not reproduced by addition of Mg²⁺ to the Ringer solution and persisted under conditions of Cl⁻ deprivation. The effects of three pharmacological agents, cyclopiazonic acid (CPA), caffeine and 4-chloro-m-cresol (4-CmC), each known to deplete store Ca²⁺ and increase cytosolic Ca²⁺ through contrasting mechanisms without influencing E _m, were then investigated. All three agents produced a more rapid, but nevertheless still reversible, loss of membrane excitability than in 0 Ca²⁺ Ringer solution alone. This reduction in membrane excitability persisted in fibres studied in solutions containing a normal [Ca²⁺] following prior depletion of store Ca²⁺ using CPA- and 4-CmC-containing solutions. These novel findings suggest that sarcoplasmic reticulum Ca²⁺ content profoundly influences surface membrane excitability, thereby providing a potential mechanism by which spike firing fails in well-polarised fibres during fatigue.The authors Usher-Smith and Xu were equal contributors to this paper.     

Spectrofluorometric analysis of length-dependent conformational changes in cardiac troponin C

Length modulation of cardiac muscle is manifested in the Frank–Starling relation of the heart. Recently, it has been shown that length-dependent changes in SH reactivity of cardiac troponin C (cTnC) occurred in association with cross-bridge attachment and Ca²⁺. However, the presence of two SH groups (Cys-35 and Cys-84) in the regulatory region of cTnC complicates efforts to detect conformational changes. In this study skinned porcine cardiac fibers were reacted with 7-diethylamino-3-[4′maleimidylphenyl]-4-methylcoumarin (CPM). Alkaline urea gel electrophoresis, along with protein elution, was used to isolate filament bound cTnC. Analysis of fluorescence measurement showed that there is a Ca²⁺-increased fluorescence for CPM-labeled cTnC in long fibers (sarcomere length = 2.2 ∼ 2.5 μm) but not in short fibers (sarcomere length = 1.6 ∼ 1.8 μm). In addition, the labeled cTnC was measured for the fluorescence decrease over time by adding a non-fluorescence energy acceptor, 4-dimethylaminophenylazophenyl-4′maleimide (DABMI), in the presence and absence of Ca²⁺. Fluorescence quenching by DABMI is not affected by Ca²⁺ in long fibers but it is significantly increased in short fibers. However, the fibers maintained in the relaxed state with 5 mM MgATP and 1 mM Vanadate showed no length effect on the CPM-labeled cTnC in terms of the Ca²⁺-mediated changes in fluorescence spectrum and in fluorescence quenching by DABMI. All together, our results suggest that the relative reactivities of Cys-35 and Cys-84 vary with sarcomere length. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca²⁺ indicator dye, fura-2 to investigate the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_o) on sarcoplasmic reticulum (SR) Ca²⁺ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca²⁺]_o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca²⁺ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca²⁺]_o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca²⁺]_o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca²⁺ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca²⁺ entry across the sarcolemma during this period. We conclude that in low [Ca²⁺]_o, short stimulation interval may limit Ca²⁺ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.     

Protective effect of oxymatrine on chronic rat heart failure

Oxymatrine is one of the alkaloids extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with anti-inflammatory, immune reaction inhibiting, antiviral, and hepatocyte and antihepatic fibrosis protective activities. However, the effect of oxymatrine on heart failure is not yet known. In this study, the effect of oxymatrine on heart failure was investigated using a Sprague-Dawley rat model of chronic heart failure. Morphological findings showed that in the group treated with 50 and 100 mg/kg of oxymatrine; intermyofibrillar lysis disappeared, myofilaments were orderly, closely and evenly arranged; and mitochondria contained tightly packed cristae compared with the heart failure group. We investigated the cytosolic Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content, and assessed the expression of ryanodine receptor (RyR2), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (dihydropyridine receptor, DHPR). We found that the cytosolic Ca²⁺ transients were markedly increased in amplitude in the medium- (ΔF/F ₀ = 26.22 ± 2.01) and high-dose groups (ΔF/F ₀ = 29.49 ± 1.17) compared to the heart failure group (ΔF/F ₀ = 12.12 ± 1.35, P < 0.01), with changes paralleled by a significant increase in the SR Ca²⁺ content (medium-dose group: ΔF/F ₀ = 32.20 ± 1.67, high-dose group: ΔF/F ₀ = 32.57 ± 1.29, HF: ΔF/F ₀ = 17.26 ± 1.05, P < 0.01). Moreover, we demonstrated that the expression of SERCA2a and cardiac DHPR was significantly increased in the medium- and high-dose group compared with the heart failure rats. These findings suggest that oxymatrine could improve heart failure by improving the cardiac function and that this amelioration is associated with upregulation of SERCA2a and DHPR.     

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis

 We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca²⁺ indicators in response to large (50–200 μM), short duration (<100 ns), free [Ca²⁺] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca²⁺). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca²⁺ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca²⁺] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca²⁺ signaling events and the use of flash photolysis in cells and model systems. Received: 17 March 1997 / Received after revision and accepted: 16 May 1997     

Real-time measurements of calcium dynamics in neurons developing in situ within zebrafish embryos

 We have developed a non-invasive technique to measure intracellular calcium ([Ca²⁺]_i) in neurons growing within intact embryos of the zebrafish (Danio rerio). A single blastomere was injected with a calcium-sensitive fluorescent dye (Calcium Green dextran) between the 32- and 128-cell stage and the embryo imaged between 16 h and 20 h postfertilisation using laser scanning confocal microscopy. Labelled nerve cells from embryos preinjected with dye and dissociated at 16 h showed a fluorescence increase (66±22%; n=11) in response to depolarisation with KCl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca²⁺]_i changes in vivo. Imaging of dye loaded cells over 30-min periods in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescence changes distinguishable from noise. However, a subset of neurons within the ventral spinal cord exhibited spontaneous, repetitive [Ca²⁺]_i oscillations which may have a functional significance during neuronal development. Received: 16 December 1997 / Received after revision: 10 April 1998 / Accepted: 15 April 1998     

Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions

The present study investigated mitochondrial Ca²⁺ uptake and release in intact living skeletal muscle fibres subjected to bouts of repetitive activity. Confocal microscopy was used in conjunction with the Ca²⁺-sensitive dye Rhod-2 to monitor changes in mitochondrial Ca²⁺ in single Xenopus or mouse muscle fibres. A marked increase in the mitochondrial Ca²⁺ occurred in Xenopus fibres after 10 tetani applied at 4 s intervals. The mitochondrial Ca²⁺ continued to increase with increasing number of tetani. After the end of tetanic stimulation, mitochondrial Ca²⁺ declined to 50% of the maximal increase within 10 min and thereafter took up to 60 min to return to its original value. Depolarization of the mitochondria with FCCP greatly attenuated the rise in the mitochondrial Ca²⁺ evoked by repetitive tetanic stimulation. In addition, FCCP slowed the rate of decay of the tetanic Ca²⁺ transient which in turn led to an elevation of resting cytosolic Ca²⁺. Accumulation of Ca²⁺ in the mitochondria was accompanied by a modest mitochondrial depolarization. In contrast to the situation in Xenopus fibres, mitochondria in mouse toe muscle fibres did not show any change in the mitochondrial Ca²⁺ during repetitive stimulation and FCCP had no effect on the rate of decay of the tetanic Ca²⁺ transient. It is concluded that in Xenopus fibres, mitochondria play a role in the regulation of cytosolic Ca²⁺ and contribute to the relaxation of tetanic Ca²⁺ transients. In contrast to their important role in Xenopus fibres, mitochondria in mouse fast-twitch skeletal fibres play little role in Ca²⁺ homeostasis.     

ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile

Here, we describe novel mechanisms limiting a toxic cytosolic Ca²⁺ rise during adenosine 5′-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca²⁺ signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca²⁺ extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca²⁺ influx. Alternative inhibition of Ca²⁺ extrusion by carboxyeosin had a much smaller effect on Ca²⁺ influx. The coordinated metabolic inhibition of Ca²⁺ influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca²⁺ oscillations, which was due to the inhibition of Ca²⁺ release from internal stores. This could be particularly important for limiting Ca²⁺ toxicity during periods of hypoxia. In contrast, metabolic control of Ca²⁺ influx and Ca²⁺ release from internal stores spectacularly failed to prevent large toxic Ca²⁺ responses induced by bile acids—activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca²⁺ release from internal stores nor Ca²⁺ influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

严重烧伤早期心肌[Ca2+]m变化及其机制研究

目的:探讨严重烧伤早期心肌线粒体Ca²⁺浓度([Ca²⁺]_m)的动态变化规律及其发生机制。方法:复制30%Ⅲ°烫伤大鼠模型,测定伤后1、3、6、12、24h大鼠心肌[Ca²⁺]_m,同时检测影响[Ca²⁺]_m的相关指标—胞浆Ca^2＋浓度(_c)及线粒体Ca^2＋转运速率。结果:烧伤后1、3、6h[Ca²⁺]_m依次升高,12、24h较6h虽有所下降,但仍高于正常对照组;_c除伤后1h无明显变化外,其余各时相点变化趋势与[Ca²⁺]_m相同,且伤后[Ca²⁺]_m与_c呈显著正相关,相关系数为0.9177(P＜0.01)。伤后1h心肌线粒体Ca^2＋摄取速率明显升高,而Ca^2＋释放速率无明显改变,但3、6、12、24h心肌线粒体Ca^2＋摄取速率与Ca^2＋释放速率均显著降低,且烧伤后3、6、12、24h[Ca²⁺]_m分别与线粒体Ca^2＋释放速率呈明显负相关。结论:烧伤后心肌线粒体存在明显的Ca^2＋超载和转运紊乱。     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996