Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Mechanisms underlying variations in excitation–contraction coupling across the mouse left ventricular free wall

Ca²⁺ release during excitation–contraction (EC) coupling varies across the left ventricular free wall. Here, we investigated the mechanisms underlying EC coupling differences between mouse left ventricular epicardial (Epi) and endocardial (Endo) myocytes. We found that diastolic and systolic [Ca²⁺]_i was higher in paced Endo than in Epi myocytes. Our data indicated that differences in action potential (AP) waveform between Epi and Endo cells only partially accounted for differences in [Ca²⁺]_i. Rather, we found that the amplitude of the [Ca²⁺]_i transient, but not its trigger – the Ca²⁺ current – was larger in Endo than in Epi cells. We also found that spontaneous Ca²⁺ spark activity was about 2.8-fold higher in Endo than in Epi cells. Interestingly, ryanodine receptor type 2 (RyR2) protein expression was nearly 2-fold higher in Endo than in Epi myocytes. Finally, we observed less Na⁺–Ca²⁺ exchanger function in Endo than in Epi cells, which was associated with decreased Ca²⁺ efflux during the AP; this contributed to higher diastolic [Ca²⁺]_i and SR Ca²⁺ in Endo than in Epi cells during pacing. We propose that transmural differences in AP waveform, SR Ca²⁺ release, and Na⁺–Ca²⁺ exchanger function underlie differences in [Ca²⁺]_i and EC coupling across the left ventricular free wall.     

Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 m m lactic acid nor increasing the [K⁺] of the bath solution from 5 to 10 m m had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 m m [K⁺]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca²⁺] ([Ca²⁺]_i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca²⁺]_i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K⁺] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca²⁺ release from the sarcoplasmic reticulum.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

Possible mechanisms of the concentration-dependent action of substance P to induce histamine release from rat peritoneal mast cells and the effect of extracellular calcium on mast-cell activation

Rat peritoneal mast cells purified on a Percoll gradient were loaded with the fluorescent Ca²⁺ indicator fura-2 and were challenged with different concentrations of substance P (SP), and intracellular calcium concentrations ([Ca²⁺]_i) were measured by a spectrofluorometric assay. SP at 5 × 10⁻⁶ mol/1 and 10⁻⁵ mol/1 caused a significant histamine release with a significant increase in [Ca²⁺]_i in a dose-dependent manner. However, SP at 10⁻⁸-10⁻⁶ mol/1 did not induce either histamine release or increase in [Ca²⁺]_i. Extracellular calcium at 0.9 mM inhibited the histamine release with a significant reduction of [Ca²⁺]ⁱ compared with that of the cells in a nominally calcium-free condition. These results indicate that the action of SP on rat mast cells relies upon [Ca²⁺]_i to induce histamine release.     

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl− transport in biliary epithelial cells through a PKCζ-dependent pathway

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl⁻ and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca²⁺]_i and membrane Cl⁻ permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow-stimulated ATP release was dependent on [Ca²⁺]_i, exhibited desensitization with repetitive stimulation, and was regulated by PKCζ. In conclusion, both human and rat biliary cells exhibit flow-stimulated, PKCζ-dependent, ATP release, increases in [Ca²⁺]_i and Cl⁻ secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (−40 mos m ) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca²⁺]_i and CBF, as well as potentiating ACh-stimulated rises in [Ca²⁺]_i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X₄ purinergic receptor. A hypo-osmotic stress (−40 mosM) transiently increased [Ca²⁺]_i and potentiated the ACh-stimulated [Ca²⁺]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca²⁺-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca²⁺]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X₄.     

Spatial Segregation and Interaction of Calcium Signalling Mechanisms in Rat Hippocampal CA1 Pyramidal Neurons

Postsynaptic [Ca²⁺]_i increases result from Ca²⁺ entry through ligand-gated channels, entry through voltage-gated channels, or release from intracellular stores. We found that these sources have distinct spatial distributions in hippocampal CA1 pyramidal neurons. Large amplitude regenerative release of Ca²⁺ from IP₃-sensitive stores in the form of Ca²⁺ waves were found almost exclusively on the thick apical shaft. Smaller release events did not extend more than 15 μm into the oblique dendrites. These synaptically activated regenerative waves initiated at points where the stimulated oblique dendrites branch from the apical shaft. In contrast, NMDA receptor-mediated increases were observed predominantly in oblique dendrites where spines are found at high density. These [Ca²⁺]_i increases were typically more than eight times larger than [Ca²⁺]_i from this source on the main aspiny apical shaft. Ca²⁺ entry through voltage-gated channels, activated by backpropagating action potentials, was detected at all dendritic locations. These mechanisms were not independent. Ca²⁺ entry through NMDA receptor channels or voltage-gated channels (as previously demonstrated) synergistically enhanced Ca²⁺ release generated by mGluR mobilization of IP₃.     

Simultaneous imaging of Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum

Electrical rhythmicity in smooth muscle cells is essential for the movement of the gastrointestinal tract. Interstitial cells of Cajal (ICC) lie adjacent to smooth muscle layers and are implicated as the pacemaker cells. However, the pace making mechanism remains unclear. To study the intercellular interaction during electrical rhythm generation, we visualized changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle cells and myenteric ICC within segments of mouse ileum loaded with a fluorescent Ca²⁺ indicator, fluo-3. We observed rhythmic [Ca²⁺]_i changes in longitudinal smooth muscle cells travelling rapidly through the smooth muscle cell layer. Between the rhythmic Ca²⁺ transients, we found brief Ca²⁺ transients localized to small areas within smooth muscle cells. The amplitude but not the periodicity of rhythmic [Ca²⁺]_i transients in both cell types was partially inhibited by nicardipine, an L-type Ca²⁺ channel antagonist, suggesting that the rhythmic [Ca²⁺]_i transients reflect membrane potential depolarizations corresponding to both slow waves and triggered Ca²⁺ spikes. Longitudinal smooth muscle cells and myenteric ICC showed synchronous spontaneous [Ca²⁺]_i transients in eight out of 21 ileac preparations analysed. In the remaining preparations, the synchrony between ICC and smooth muscle cells was absent, although the rhythmicity of the smooth muscle cells was not disturbed. These results suggest that myenteric ICC may play multiple roles including pace making for physiological bowel movement.     

Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte

G-protein-coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non-excitable rat megakaryocyte as a model system, we now show that P2Y receptor-evoked Ca²⁺ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to depolarization was always larger than the maximal reduction in [Ca²⁺]_i following an equivalent amplitude hyperpolarization. Significant [Ca²⁺]_i increases were observed in response to small amplitude (<5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor-evoked Ca²⁺ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca²⁺]_i increases. Furthermore, elevation of the temperature to physiological levels (36°C) resulted in a more sustained depolarization-evoked Ca²⁺ increase compared with more transient or oscillatory responses at 20–24°C. The ability of signalling via a G-protein-coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non-excitable tissues.     

HLA-DR molecules enhance signal transduction through the CD3/Ti complex in activated T cells

Abstract: Crosslinking HLA-DR molecules by monoclonal antibodies (mAb) induces protein tyrosine phosphorylation and results in a secondary elevation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in activated human T cells. Here we have studied the effect of DR on CD3-induced signal transduction in allospecific T-cell clones and T-leukemia (HUT78) cells. Co-crosslinking of DR with CD3 produced an enhanced [Ca²⁺]_i response compared to that seen with CD3 alone. In contrast, CD2 responses were not enhanced by co-crosslinking with DR. Co-crosslinking CD45 in a tri-molecular complex of CD45, CD3, and DR completely abrogated the enhancing effects of DR on CD3-induced [Ca²⁺]_i responses. In contrast, the enhancing effect of co-crosslinking CD4 on CD3 responses was not inhibited by co-crosslinking CD45. Thus, the DR-mediated accessory signals appear to be regulated differently from those provided by CD4 accessory molecules. The present data confirm, at the level of second messengers, recent findings suggesting that DR molecules have accessory functions in CD3/Ti-mediated T-cell responses.     

Increased Ca2+ influx through Na+/Ca2+ exchanger during long-term facilitation at crayfish neuromuscular junctions

Intense motor neuron activity induces a long-term facilitation (LTF) of synaptic transmission at crayfish neuromuscular junctions (NMJs) that is accompanied by an increase in the accumulation of presynaptic Ca²⁺ ions during a test train of action potentials. It is natural to assume that the increased Ca²⁺ influx during action potentials is directly responsible for the increased transmitter release in LTF, especially as the magnitudes of LTF and increased Ca²⁺ influx are positively correlated. However, our results indicate that the elevated Ca²⁺ entry occurs through the reverse mode operation of presynaptic Na⁺/Ca²⁺ exchangers that are activated by an LTF-inducing tetanus. Inhibition of Na⁺/Ca²⁺ exchange blocks this additional Ca²⁺ influx without affecting LTF, showing that LTF is not a consequence of the regulation of these transporters and is not directly related to the increase in [Ca²⁺]_i reached during a train of action potentials. Their correlation is probably due to both being induced independently by the strong [Ca²⁺]_i elevation accompanying LTF-inducing stimuli. Our results reveal a new form of regulation of neuronal Na⁺/Ca²⁺ exchange that does not directly alter the strength of synaptic transmission.     

Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) originating from voltage- or ligand-gated influx and by ligand- or Ca²⁺-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca²⁺ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca²⁺ pumps and Ca²⁺ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca²⁺]_i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca²⁺]_i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca²⁺ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca²⁺ influx, but also as an intracellular Ca²⁺ source that can be mobilized by a signal cascade originating at activated synapses.     

Metabolic Regulation of Ca2+ Release in Permeabilized Mammalian Skeletal Muscle Fibres

In the present study, the link between cellular metabolism and Ca²⁺ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca²⁺ release from the sarcoplasmic reticulum were detected with fluo-3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential-sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca²⁺] (with the Ca²⁺ indicator mag-rhod-2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca²⁺ release events were readily observed. The addition of l -glutamate or pyruvate reversibly decreased the frequency of Ca²⁺ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca²⁺] and promoted spontaneous Ca²⁺ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca²⁺ release events was not accompanied by a rise in global [Ca²⁺]_i. Our results suggest that mitochondria exert a negative control over Ca²⁺ signalling in skeletal muscle by buffering Ca²⁺ near Ca²⁺ release channels.